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II IBEROAMERICAN CONFERENCE ON BENEFICIAL PLANT - MICROORGANISM - ENVIRONMENT INTERACTIONS (IBEMPA) XIV NATIONAL MEETING OF THE SPANISH SOCIETY OF NITROGEN FIXATION (SEFIN) XXVI LATIN AMERICAN MEETING ON RHIZOBIOLOGY (ALAR) III SPANISH-PORTUGUESE CONGRESS ON NITROGEN FIXATION “Microorganisms for future agriculture" Organizers Spanish Society of Nitrogen Fixation (SEFIN) Latin American Society Rhizobiology (ALAR) University of Seville ISBN-10: 84-616-6794-8 ISBN-13: 978-84-616-6794-9 Sponsors AMC Chemical Bruker Catering Sevilla Coitand C. Viral SL Kimitec Laboratorios Biagro Metabolon OpGen ResBioAgro Sistemas genómicos Symborg Thermo Fisher Scientific Welcome address Dear colleagues, On behalf of the Organizing Committee for II IBEMPA Meeting, it is our great pleasure and honour to welcome you to the II Iberoamerican Conference on Beneficial Plant Microorganism Environment Interactions (IBEMPA), XIV National Meeting of the Spanish Society of Nitrogen Fixation (SEFIN), XXVI Latin American Meeting on Rhizobiology (ALAR) and III SpanishPortuguese Con gress on Nitrogen Fixation. This meeting will provide the opportunity to share and discuss latest scientific news on genomic, biochemical, ecological and agronomic aspects of diazotrofic microorganisms or beneficial bacteria and their interaction with plants. We hope you agree that the selected topics and speakers are of great interest and exceptional quality. The meeting program includes six sessions plus the opening and closing keynote conferences. Each session includes keynotes speakers, oral communications and poster presentations. Before the beginning and in parallel to the congress three satellite workshops and a meeting have also been organized: “Omic-technologies Workshop” and “Workshop FABATROPIMED” (1st September), “Inoculant production Workshop” (2nd September) and the meeting on "Microorganisms for Agriculture Future" (4th September). We encourage all delegates to participate actively on all these activities. Besides, several attractive social activities have b een programmed. We would like to thank all institutions and companies that contributed to the organization of this congress, very specially to the University of Sevilla, the Spanish Society of Nitrogen Fixation and the Latin American Society of Rhizobiology. We hope that all delegates will enjoy during their stay in Sevilla and will found an excellent atmosphere to discuss about scientific matters, new collaborations and future directions in the field of the beneficial microorganisms and their association with plants. We also hope that you discover the hospitality of the people of Sevilla and have some free time for visiting this beautiful city. We look forward to welcome you in Seville in September 2013, for what we hope will be an enjoyable meeting both scientifically and socially. Yours sincerely, Manuel Megías President of the Organizing Committee i Committees Honor Committee S.A.R. la Infanta Doña Elena. Presidente del Comité de Honor. Excmo. Sr. D. Miguel Arias Cañete. Ministerio de Agricultura, Alimentación y Medioambiente. Excma. Sra. Dª Carmen Vela Olmo. Ministerio de Economía y Competitividad. Secretaría de Estado de Investigación Desarrollo e Innovación Excmo. Sr. D. Luis Planas Puchades. Consejería Agricultura, Pesca y Medioambiente. Excmo. Sr. D. Antonio Ávila Cano. Consejería de Economía, Innovación, Ciencia y Empleo. Excmo. Sr. D. Juan Ignacio Zoido Álvarez. Alcaldía de Sevilla. Excmo. Sr. D. Fernando Rodríguez Villalobos. Diputación de Sevilla. Excmo. y Mgco. Sr. D. Antonio Ramírez de Arellano López. Universidad de Sevilla. Excmo. y Mgco. Sr. D. Juan Manuel Suárez Japón. UNIA. Sra. Dª Teresa Millán Valenzuela. Asociación Española de Leguminosas. Ilmo. Sr. D. José Luís García Palacios. Caja Rural del Sur. Ilmo. Sr. D. Antonio Vergel Román. COITAND. Sr. D. Joaquín Moya-Angeler Cabrera. CTA. Ilma. Sra. Dª María Cinta Castillo Jiménez. Doñana21. Sr. D. Francisco Casero Rodríguez. Valor Ecológico. Organizing Committee President: Dr. Manuel Megías. Universidad de Sevilla. España. Vice presidents: Dr. Eulogio Bedmar (President of SEFIN). Estación Experimental del Zaidín. CSIC. España. Dr. Alicia Arias (President of ALAR). Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Uruguay. Dr. Francisco Merchán. Universidad de Sevilla. España. Dr. María Jesús Delgado. Estación Experimental del Zaidín. CSIC. España. Dr. Dulce Nombre Rodríguez. IFAPA. Las Torres-Tomejil. España. Dr. F. Javier Ollero. Universidad de Sevilla. España. Dr. Teresa Cubo. Universidad de Sevilla. España. Treasurers: Secretary: Web-Master: ii Scientific Committee Dr. José E. Ruíz. Dr. M. Rosario Espuny. Dr. Eduardo Leidi. Dr. Federico Sánchez. Dr. Yaakov Okon. Dr. Esperanza Martínez. Dr. Doris Zúñiga. Dr. Emanuel de Souza. Dr. Marcia Toro. Dr. Eduardo Ortega. Dr. Maribel Parada. Dr. Fabio L. Olivares. Dr. Elena Beyhaut. Dr. Rafael Rivilla. Dr. Isabel V. Castro. Dr. María J. Soto. Dr. Raúl Rivas. Dr. Milagros León. Dr. Nuria Ferrol. Dr. Manuel Becana. Dr. Fernando González. Dr. José Antonio Herrera. Dr. José Antonio Lucas. Dr. Eloisa Pajuelo. Dr. Pedro F. Mateo. Universidad de Sevilla. España. Universidad de Sevilla. España. Instituto de Recursos Naturales y Agrobiología. CSIC. España. Instituto de Biotecnología. UNAM. Mexico. The Hebrew University. Jerusalem. Israel. Centro de Ciencias Genómicas. UNAM. México. Universidad Nacional Agraria La Molina. Perú. Universidad Federal de Parana. Brasil. Universidad Central de Venezuela. Venezuela. Universidad de la Habana. Cuba. Universidad de la Frontera. Chile. Universidad Estatal del Norte Fluminense. Brasil. INIA. Uruguay. Universidad Autónoma de Madrid. España. Instituto Nacional de Investigaciones Biológicas. Portugal. Estación Experimental del Zaidín. CSIC. España. Universidad de Salamanca. España. Universidad de la Laguna. España. Estación Experimental del Zaidín. CSIC. España. Estación Experimental de Aula Dei. CSIC. España. Universidad de León. España. Universidad de Granada. España. Universidad San Pablo CEU. España. Universidad de Sevilla y IFAPA-Las Torres-Tomejil. España. Universidad de Salamanca. España. Honor Scientific Committee Dr. José Olivares. Dr. Claudino Rodríguez-Barrueco. Dr. M. Rosario de Felipe. Dr. José M. Barea. Dr. Eugenio M. Ferreira. Dr. Roberto Racca. Dr. Alicias Godeas. Dr. Lillian Frioni. Dr. Carlos Labandera. Dr. Fabio Pedrosa. Dr. María Valdés. Dr. Eduardo Schroeder. Dr. Pedro Balatti. Dr. Gabriel Faveluques. Dr. João Ruy Jardim Freire. Dr. Avilio Antonio Franco. España. España. España. España. Portugal. Argentina. Argentina. Uruguay. Uruguay. Brasil. Mexico. Puerto rico. Argentina. Argentina. Brasil. Brasil. iii Local Organizing Committee Dr. Ignacio Rodríguez. Universidad de Sevilla. España. Dr. Pilar Tejero. Universidad de Sevilla. España. Dr. Ramón Bellogín. Universidad de Sevilla. España. Dr. Ana Buendía. Universidad de Sevilla. España. Dr. José María Vinardell. Universidad de Sevilla. España. Dr. Miguel Ángel Rodríguez-Carvajal. Universidad de Sevilla. España. Dr. Francisco Javier López-Baena. Universidad de Sevilla. España. Dr. Miguel Ángel Caviedes. Universidad de Sevilla. España. Dr. María Camacho. IFAPA-Las Torres-Tomejil. España. Dr. Carolina Sousa. Universidad de Sevilla. España. Dr. M. Carmen Márquez. Universidad de Sevilla. España. Omic-Tecnologies Workshop Organizing Committee Dr. Antonio M. Gil. Universidad de Sevilla. España. Dr. Francisco Martínez. Estación Experimental del Zaidín. CSIC. España. Dr. O. Mario Aguilar. Universidad Nacional de La Plata. Argentina. Inoculant Production Workshop Organizing Committee Dr. Francisco Temprano. IFAPA-Las Torres-Tomejil. España. Dr. Enrique Moretti. Laboratorios Biagro. Argentina. Dr. Mohammed Dary. ResBioAgro, SL. España. Dr. Antonio Lagares. BIOFAG. Universidad Nacional de La Plata. Argentina. Dr. Mariangela Hungria. EMBRAPA. Brasil. Dr. Juan Sanjuán. Estación Experimental del Zaidín. CSIC. España. iv General information Venue The meeting will be held in the Lecture Theatre of the Facultades de Ciencias del Trabajo y Derecho of the University of Seville (http://www.derecho.us.es) and Hotel NH Central Convenciones (http://www.nh-hoteles.es/nh/es/hoteles/espana/sevilla/nh-centralconvenciones.html) Language Spanish and Portuguese are the official languages of the congress. English is the scientific language. Registration and information desk The registration will be held on 2 September from 11:00 to 18:00 h. and during the meeting period at the desk located on the convention hall of the hotel. Information about the congress, social activities, etc. can also be obtained at the registration desk during the meeting. The official travel agency of the congress, CAC Travel ([email protected]), will be available at the registration desk for accommodation, touristic and travel information, etc. Lunch and coffee-breaks The lunch on 3, 5 and 6 September will be held at the hotel restaurant. These lunchs and coffee-breaks are included on the registration fee for meeting participants and accompanying persons. Oral presentations The keynote speakers have 20 minutes for their presentations plus 5 minutes for questions (25 minutes total), while the offered oral communications will be presented during 15 minutes plus 5 minutes for questions (20 minutes total). We encourage speakers to prepare their presentations according to the time assigned. Session Chairs have been asked to ensure that speakers restrict themselves to the allowed time; they have been advised to adhere most strictly to this policy. The meeting rooms are prepared with a PC (Powerpoint 2010) and projector. Macintosh is not available. Please note that we are unable to allow the use of personal laptops. Please bring your presentation in a memory stick (using the USB port in the computer) and load it on the meeting computer. Congress staff will be prepared to assist you. All presentations must be loaded preferably on Monday 2 September. Poster sessions Poster boards are located at Salón Marbella and Salón Málaga on the convention area of the hotel. Posters will be on display throughout Tuesday morning (3 September) up to late Thursday evening (5 September) with snacks and drinks. The dimensions of the poster are: 130 cm (height) by 90 cm (width). The organization will provide the materials necessary for mounting the posters on the boards. First authors of posters from Sessions I, V y VI should be present beside their own panel during the Poster Session on Tuesday 3 September (from 20:30 v to 22:00 h). First authors of posters from Sessions II, III and IV should be present beside their own panel during the Poster Session on Thursday 5 September (from 20:30 to 22:00 h). Poster discussion sessions will take place at the Salones Almenara, Almería and Alanda on the convention area of the hotel from 18:00 to 19:30 h, on Tuesday 3 September (Poster Sessions I, V y VI) and Thursday 5 September (Poster Sessions II, III and IV). Social events The social program includes: Musical performance at 19:40 h, on Monday 2 September following the opening ceremony and opening conference at the Lecture Theatre of the Facultades de Ciencias del Trabajo y Derecho of the University of Seville. Get Together at 21:30 h, on Monday 2 September at the Patios of the Facultades de Ciencias del Trabajo y Derecho of the University of Seville. Visit to the Guadalquivir marshes, on Wednesday 4 September. Buses will departure from Hotel NH Central Convenciones at 10:00 h (front door). It will include a visit to the rice fields of Seville (from 11:00 to 13:00 h) and tasting of typical products of the Guadalquivir marshes (from 13:00 to 15:30 h). Visit to the University of Seville (front door, c/ San Fernando), on Wednesday 4 September at 19:00 h. Tapas Tour, on Wednesday 4 September at 20:30 h. Closing dinner, on Friday 6 September at 22:00 h. at the “Hípica Pedro Macías” a riding centre served by Catering Sevilla in the arena of a bullring. (http://www.hipicapedromacias.es). It is not included with the registration. Its estimated price will be 45€. Buses will departure from Hotel NH Central Convenciones at 21:30 h. Accompanying persons The registration as accompanying participants includes Get together, lunches and coffee breaks, snakcs and drinks during poster sessions, Cultural visits and visit to the rice fields of the Guadalquivir marshes of Seville. Badge policy Delegates are requested to exhibit the congress badges all the time during the scientific and social events. Internet connection Wireless internet service will be available in the congress venue. vi Liability and insurance The organizers are not able to take responsibility whatsoever for injury or damage involving persons and property during the congress. Mobile phones Participants are requested to keep their mobile phones switched off in the session rooms. Time Sevilla time is the Central European time zone (Greenwich Mean Time + 2:00), i.e. one hour more than in Lisbon or London. Climate and clothing Sevilla enjoys a typically Mediterranean climate throughout the year. September in Sevilla is not as hot as August but is still extremely warm. The average daily temperature may reaches 32 °C (about 90 °F) at noon and 18 °C (64 °F) at night. The average daily sunshine is 9 hours a day with average rainfall being just 24 mm over 2 days. This means that the chance of rain is extremely unlikely, and you can still bring light clothing. Environment Hotel NH Central Convenciones includes this congress on its ECO-Meeting programme, which includes efficient use of water and energy, environmentally friendly materials, low fair-trade coffee and voluntary commitment of CO2 emissions. Travel agency The official travel agency is CAC Travel, Calle Virgen de la Victoria 12, 41011 Sevilla, Spain; phone: +34 955 099 142; email: [email protected] The address of the organization is: Department of Microbiology, Faculty of Biology, University of Sevilla, Avenida de la Reina Mercedes s/n, 41012-Sevilla, Spain; phone: +34 954 557 117; fax: +34 954 557 830; e-mail: [email protected], http://congreso.us.es/ibempa vii Meeting program MONDAY, 2 SEPTEMBER 11:00 - 18:00 Registration (Hotel NH Central Convenciones) LECTURE THEATRE OF THE FACULTADES DE CIENCIAS DEL TRABAJO Y DERECHO, UNIVERSITY OF SEVILLE Opening Session. Introduced by Antonio Gil-Serrano (Universidad de Sevilla, Sevilla, España). 18:15 - 18:20 18:20 - 19:15 Welcome address. Keynote Conference: Quorum sensing: a double-edge sword in bacterial-plant interactions. Miguel Cámara. Centre for Biomolecular Sciences, University of Nottingham. Nottingham. Reino Unido. The speaker will be introduced by Ildefonso Bonilla Mangas (Universidad Autónoma de Madrid, Madrid, España). 19:20 - 19:40 19:40 - 20:15 20:15 - 21:30 21:30 - 23:00 Presentation of the Congress picture “Uniendo pueblos”. Musical Performance. Opening Ceremony. Get together Patios of Facultades de Ciencias del Trabajo y Derecho, University of Seville. TUESDAY, 3 SEPTEMBRE LECTURE THEATRE OF THE FACULTADES DE CIENCIAS DEL TRABAJO Y DERECHO. UNIVERSITY OF SEVILLE. Session I Ecology, diversity and evolution of microorganisms beneficial to plants. Chairpersons: Dr. Fábio B. Reis Jr (EMBRAPA Cerrados, Brasil). Dr. María Luisa Izaguirre (IVIC, Venezuela). 09:00 - 09:25 SI-P-1 Diversidad y evolución de las comunidades microbianas en la rizosfera tras un incendio forestal. Fernández-López, M. Estación Experimental del Zaidín, CSIC. Granada. España. 09:25 - 09:55 SI-P-2 Species and symbiovars within Mesorhizobium: diversity and host range. Laranjo, M. Instituto de Ciências Agrárias e Ambientais Mediterránicas, Universidade de Évora, Évora, Portugal. 10:00 - 11:30 Oral Communications SI-CO-1 Diversidad de hongos micorrizógenos arbusculares bajo situaciones contrastantes de fertilización fosfatada. * García, S., Rodríguez Blanco, A. , Pezzani, F. Facultad de Agronomía. Universidad de la República. Av. Garzón 780. Montevideo. Uruguay. viii SI-CO-2 Prospecting metal resistant plant-growth promoting rhizobacteria for rhizoremediation of metal contaminated estuaries using Spartina densiflora. Andrades-Moreno, L., del Castillo, I., Redondo-Gómez, S., Mesa, J., Caviedes M.A., Pajuelo, E., Rodríguez-Llorente, I.D.* Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla. España SI-CO-3 Symbiovar loti-type genes are widely spread across different chromosomal backgrounds, corresponding to up to nine Mesorhizobium genospecies nodulating Cicer canariense. * Pérez-Yépez, J. , Armas-Capote, N., Martínez-Hidalgo, P., Velázquez, E., PérezGaldona, R., Martíne z-Molina, E., León-Barrios, M. Departamento de Microbiología y Biología Celular. Universidad de La Laguna. Tenerife. España. SI-CO-4 Molecular phylogeny and phenotypic characterization of salt tolerant Sinorhizobia nodulating Phaseolus filiformis in Northern Mexico. Rocha, G., Medina, A., Carreño, R., Bustillos, R., Contreras, J.L., Villegas, M.C., Chaintreuil, C., Dreyfus, B., Le Queré, A., Munive, J.A. * Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla, Mexico. 11:30 - 12:00 General discussion. Coffee break. Session II Genetics and genomics of beneficial microorganisms and associated plants. Chairpersons: Dr. Fabricio Cassan (Univ. Nacional de Río Cuarto, Argentina). Dr. Marta Martín (Univ. Autónoma de Madrid, España). 12:00 - 12:25 SII-P-1 Genomics of host specificity in the Rhizobium-legume symbiosis. Imperial, J. ETS de Ingenieros Agrónomos y Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid, Madrid, España. 12:30 - 12:55 SII-P-2 Genomic insights into the rhizosphere lifestyle of rhizobia. Ormeño-Orrillo, E. Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México. 13:00 - 14:30 Oral Communications SII-CO-1 First analyses of the genomic sequence of the soybean symbiont Sinorhizobium fredii HH103. * Vinardell, J.M. , Göttfert, M., Becker, A., Acosta-Jurado, S., Baena, I., Blom, J., Bonilla, I., Buendía, A.M., Crespo-Rivas, J.C., Goesmann, A., Jaenicke, S., Krol, E., Lloret, J., McIntosh, M., Margaret, I., Pérez-Montaño, F., Schneiker-Bekel, S., Serranía, J., Szczepanowski, R., Zehner, S., Pühler, A., Ruiz-Sainz, J.E., Weidner, S. Departamento de Microbiología, Universidad de Sevilla, España. SII-CO-2 Identificación y caracterización del regulador maestro del flagelo lateral de Bradyrhizobium japonicum USDA 110. * Mongiardini, E.J., Quelas, J.I., Lodeiro, A.R. IBBM-Facultad de Ciencias Exactas, UNLP-CONICET, La Plata, Argentina. ix SII-CO-3 Pyrosequencing reveals the presence of diverse bacterial genera which have not previously described to soil and rhizosphere. Lagos, L. *, Jorquera, M., Maruyama, F., Mora, M.L. Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile. SII-CO-4 Temporal profile of nif gene expression in Azotobacter vinelandii: effect of nifA mutation. Navarro-Rodríguez, M.*, Poza-Carrión, C., Jiménez-Vicente, E., Rubio, L.M. Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid. Madrid. España 14:45 - 16:30 General discussion. Lunch at Hotel NH Central Convenciones. HOTEL NH CENTRAL CONVENCIONES 17:00 - 18:00 Meeting of the Latin American Society of Rhizobiology. 18:00 - 19:30 Poster Discussion Sessions I, V and VI. Chairpersons: Dr. Milagros León (Univ. de la Laguna, España). Dr. Maribel Parada (Univ. de la Frontera, Chile). 19:30 - 20:30 Corner business innovation I. Chairpersons: Dr. Alvaro Peix (IRNASA, CSIC, España). Dr. Khalid Alki (AMC Chemical, S.L., España). Participants: Sistemas Genómicos, España. Bruker, España. Corporación Tecnológica de Andalucía, España. 20:30 - 22:00 Posters Session I, V and VI (with snacks and drinks). WEDNESDAY, 4 SEPTEMBER 09:00 - 13:00 13:00 - 15:30 19:00 - 20:30 20:30 Visit to the rice fields of Seville. Tasting of typical products of the Guadalquivir marshes. Visit to the University of Seville. Tapas Tour THURSDAY, 5 SEPTEMBER LECTURE THEATRE OF THE FACULTADES DE CIENCIAS DEL TRABAJO Y DERECHO. UNIVERSITY OF SEVILLE Session III Symbiotic plant/microbe Interactions. Chairpersons: Dr. Carmen Quinto (Instituto de Biotecnología, UNAM, México). Dr. Maria do Carmo Catanho Pereira de Lyra (Instituto Agronômico de PernambucoIP, Brasil). x 09:00 - 09:25 SIII-P-1 Priming plant defences by beneficial soil microorganisms. Pozo-Jiménez, M.J. Estación Experimental del Zaidín, CSIC. Granada. España. 09:30 - 09:55 SIII-P-2 Una visión general del papel de los polisacáridos superficiales de Sinorhizobium fredii en su simbiosis con la soja y otras leguminosas. Ruiz Sainz. J.E. Departamento de Microbiología. Facultad de Biología. Universidad de Sevilla. España 10:00 - 11:30 Oral Communications SIII-CO-1 Boron deficiency affects rhizobia cell surface polysaccharides. Quijada, N.M., Cerda, M.E., Abreu, I.*, Pérez de Nanclares, M., Bonilla, I., Bolaños, L. Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid. Madrid. SIII-CO-2 Threalose is a key carbon regulatory player in the Legume-Rhizobium symbiosis. Sánchez, F.*, Barraza, A., Estrada-Navarrete, G.,Quinto, C., Merino, E. Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México.. SIII-CO-3 RbohB-dependent reactive oxygen species regulates rhizobial infection, bacteroid development and nitrogen fixation in common bean. Arthikala, M.K., Montiel, J., Nava, N., Santana, O., Sánchez-López, R., Cárdenas, L., Quinto, C.* Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México. SIII-CO-4 Quorum Sensing systems and flavonoids via NodD1 coordinate and regulate the biofilm transition in Sinorhizobium fredii SMH12, which is necessary for a successful root colonization of Glycine max cv Osumi. Pérez-Montaño, F.*, Jiménez-Guerrero, I., Del Cerro, P., López-Baena, F.J., Ollero, F.J., Bellogín, R.A., Lloret, J., Espuny, M.R. Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. España 11:30 - 12:00 General discussion. Coffee break. Session IV Associative interaction plant/ beneficial microbe. Chairpersons: Dr. José Miguel Barea (Estación Experimental del Zaidín, CSIC, Spain). Dr. Verónica Reis (EMBRAPA-Agrobiología, Brasil). 12:00 - 12:25 SIV-P-1 Aplicaciones biotecnológicas de la interacción planta-microorganismos: salud, medioambiente y agricultura. Gutiérrez-Mañero, F.J. Facultad de Farmacia. Universidad San Pablo CEU. Madrid. España. xi 12:25 - 12:55 SIV-P-2 Aspectos bioquímicos y moleculares de la interacción Delftia-Rizobio-Alfalfa: un lenguaje de señales químicas. Castro-Sowinski, S. Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay. 13:00 - 14:30 Oral Communications SIV-CO-1 Evaluation of Micromonospora as a potential biocontrol agent. * Martínez-Hidalgo, P. , Martínez-Molina, E., Pozo, M.J. Departamento de Microbiología y Genética, Universidad de Salamanca, España. GIR "Interacciones mutualistas planta-microorganismo", Unidad Asociada al IRNASA. CSIC, Salamanca. España. SIV-CO-2 Movimiento y quimiotáxis en Pseudomonas fluorescens F113, influencia en una colonización competente de la rizosfera de las plantas. Baena, I., Muriel, C., Martín, I., Jalvo, B., Hernanz, A., González de Heredia, E., Barahona, E., Navazo, A., Redondo-Nieto, M., Martínez-Granero, F., Lloret, J., Rivilla, R., Martín, M.* Departamento de Biología. Facultad de Ciencias. Universidad Autónoma de Madrid. SIV-CO-3 Caracterización de la interacción Delftia-Sinorhizobium en alfalfa. Morel, M.A.*, Cagide, C., Dardanelli, M.S., Castro-Sowinski, S. Unidad de Microbiología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Av. Italia 3318, Montevideo, Uruguay. SIV-CO-4 Estrés salino, calidad nutricional y comportamiento postcosecha, en lechuga inoculada con Azospirillum. Fasciglione, G.*, Casanovas, M., Yommi, A., Quillehauqui, V., Roura, S., Barassi, C.A. Unidad Integrada: Facultad de Cs. Agrs. (UNMdP) -EEA Balcarce (INTA). Ruta 226 Km 73,5 CC 276 (7620) Balcarce, Argentina. General discussion. Lunch at Hotel NH Central Convenciones. HOTEL NH CENTRAL CONVENCIONES 17:00 - 18:00 Meeting of the Spanish Society of Nitrogen Fixation 18:00 - 19:30 Poster Discussion Sessions II, III and IV. 14:45 - 16:30 Chairpersons: Dr. Fernando González (Univ. de León, España). Dr. José Antonio Lucas (Univ. CEU San Pablo, España). 19:30 - 20:30 Corner business innovation II. Chairpersons: Dr. Jesús González (Univ. de Granada, España). Dr. Mohamed Dary (ResBioAgro, S.L., España). Participants: Total Biotecnología, Brasil. ResBioAgro, España. ACM Chemical, España. Simborg, España. 20:30 - 22:00 22:00 Posters Session II, III and IV (with snacks and drinks). Removal of posters. xii FRIDAY, 6 SEPTEMBER LECTURE THEATRE OF THE FACULTADES DE CIENCIAS DEL TRABAJO Y DERECHO. UNIVERSITY OF SEVILLE Session V Physiology and biochemistry of beneficial microorganisms and associated plants Chairpersons: Dr. Manuel Sánchez (Univ. de Navarra, Spain). Dr. Carmen Lluch (Univ. de Granada, Spain). 09:00 - 09:25 SV-P-1 Fijación de nitrógeno en leguminosas en entornos variables: el bacteroide, el nódulo y la planta. César Arrese Igor. Universidad Pública de Navarra. Pamplona, España. 09:30 - 09:55 SV-P-2 Molecular physiology of nickel and cobalt homeostasis in Rhizobium leguminosarum. José Manuel Palacios Alberti Escuela Técnica Superior de Ingenieros Agrónomos y Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid, Pozuelo de Alarcón, Madrid, España 10:00 - 11:30 Oral Communications SV-CO-1 Engineering a hydrogen biosensor in the nitrogen-fixing strain Rhodobacter capsulatus. Gónzalez de Heredia, E.M.*, Barahona, E., Jiménez-Vicente, E., Echavarri-Erasun, C., Rubio, L.M. Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón 28223, Madrid. SV-CO-2 MtNramp1 mediates iron import in rhizobia-infected Medicago truncatula cells. Rodríguez-Haas, B., Finney, L., Kryvoruchko, I., González-Melendi, P., Udvardi, M., Imperial, J., González-Guerrero, M.* Centro de Biotecnología y Genómica de Plantas (CBGP) Universidad Politécnica de Madrid. Campus de Montegancedo, Pozuelo de Alarcón, Madrid, España. SV-CO-3 Selección de líneas de garbanzo tolerantes a la salinidad empleando caracteres de producción de biomasa y funcionamiento nodular como indicadores. Gómez, L.A.*, Vadez, V., Vaillhe, H., Pernot, C., Drevon, J.J. Instituto de Suelos. Autopista, Costa-Costa y Antigua Carretera de Vento, Capdevila, Boyeros. La Habana, Cuba. SV-CO-4 Estrés abitotico induce cambios en las señales de comunicación y en la interacción temprana entre maní y rizobacterias. Cesari, A., Paulucci, N., García, M., Dardanelli, M.S. * Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Argentina. General discussion. 11:30 - 12:00 Coffee break. xiii Session VI Inoculants in agriculture and the environment. Chairpersons: Dr. Antonio M. de Ron (Misión Biológica de Galicia, MBG-CSIC). Dr. Yeda Mendes (EMBRAPA-Cerrados, Brasilia, Brasil). 12:00 - 12:25 SVI-P-1 Efficient soil microbial consorcia: a new dimension for sustainable agriculture and environmental sustainability. Cruz, C. Universidade de Lisboa, Lisboa. Portugal. 12:30 - 12:55 SVI-P-2 Oportunidades e ameaças à contribuição da fixação biológica do nitrogênio em leguminosas no Brasil. Nogueira, M.A. Embrapa Soja, Londrina, Paraná, Brazil. 12:00 - 14:30 Oral Communications SVI-CO-1 Selenium biofortification in wheat plants by co-inoculation of selenobacteria strains and arbuscular mycorhizal fungy for obtaining enriched selenium foods. Durán, P.*, Mora, M.M. Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Biotechnological Bioresource Nucleus. Universidad de la Frontera, Temuco-Chile. SVI-CO-2 Evaluación de la degradación de tamo de arroz por microorganismos lignocelulolíticos. Gutiérrez-Rojas, I.*, Pedraza-Zapata, C., Sánchez-Rodríguez, L., Moreno-Sarmiento, N. Instituto de Biotecnología, Ciudad Universitaria, Edificio Manuel Ancizar, Universidad Nacional de Colombia, Bogotá, D.C., Colombia. Departamento de Microbiología, Facultad de Ciencias, Laboratorio de Biotecnología Aplicada, Grupo de Biotecnología Ambiental e Industrial (GBAI), Pontificia Universidad Javeriana, Bogotá, D.C., Colombia. SVI-CO-3 Soil microbial phosphorus, phosphorus availability and crop yield of upland ricecommon bean interc ropping under organic and mineral fertilizer inputs on ferrallitic soil of Malagasy highlands. Henintsoa, M.*, Andriamananjara, A., Razakatiana, A.T.E., Larvy Delarivière, J., Rabeharisoa, L., Becquer, T. Laboratoire des Radioisotopes (LRI), University of Antananarivo, Madagascar. Laboratoire de Microbiologie de l’Environnement (LME), Centre National de Recherches sur l’Environnement (CNRE), Antananarivo, Madagascar. Institut de Recherche pour le Développement (IRD), UMR Eco&Sols, c/o LRI, Madagascar. SVI-CO-4 Sistema uruguayo de fiscalización de inoculantes: complementación interinstitucional y aplicación de tecnologías de la información. Beyhaut, E*, Barlocco, C., Altier, N., Mayans, M. INIA Las Brujas, Ruta 48 Km 10, Canelones, Uruguay. 2 División Control de Insumos, DGSSAA. M.G.A.P, Av. Millán. Montevideo, Uruguay. 14:45 - 16:30 General discussion. Lunch at Hotel NH Central Convenciones. xiv LECTURE THEATRE OF THE FACULTADES DE CIENCIAS DEL TRABAJO Y DERECHO. UNIVERSITY OF SEVILLE Closing Session 18:30 - 19:30 Award “Antonio J. Palomares” Conference. Simplicity turned out complicated: How FixK2, a key regulator of genes for symbiosis, is exquisitely controlled by different means. Mesa, S. Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain. The speaker will be introduced by Eulogio Bedmar (SEFIN President). 19:30 - 19:40 “Bernardo Leicach - BIAGRO” awards ceremony. 19:40 - 20:40 Closing Conference. Uma década de ouro se aproxima para a microbiologia do solo: expectativas da pesquisa, da indústria, dos agricultores e da sociedade. Mariangela Hungría. Embrapa Soja, Londrina, Paraná, Brazil The speaker will be introduced by Tomás Ruíz Argüeso (Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, España). 20:40 21:30 Closing ceremony Buses departure for closing dinner (Hotel NH Central Convenciones) 22:00 Closing dinner. “Hípica Pedro Macías” xv Floor plan xvi Contents Opening Session Quorum sensing: a double-edge sword in bacterial-plant interactions. Miguel Cámara. 2 Centre for Biomolecular Sciences, University of Nottingham. Nottingham NG7 2RD. Reino Unido). Session I. Ecology, diversity and evolution of microorganisms beneficial to plants. SI-P-1 Diversidad y evolución de las comunidades microbianas en la rizosfera tras un incendio forestal. Fernández-López, M. 7 Estación Experimental del Zaidín, CSIC. Granada. España. SI-P-2 Species and symbiovars within Mesorhizobium: diversity and host range. Laranjo, M. 11 Instituto de Ciências Agrárias e Ambientais Mediterránicas, Universidade de Évora, Évora, Portugal. SI-CO-1 Diversidad de hongos micorrizógenos arbusculares bajo situaciones contrastantes de fertilización fosfatada. García, S., Rodríguez Blanco, A.*, Pezzani, F. 15 Facultad de Agronomía. Universidad de la República. Av. Garzón 780. Montevideo. Uruguay. SI-CO-2 Prospecting metal resistant plant-growth promoting rhizobacteria for rhizoremediation of metal contaminated estuaries using Spartina densiflora. Andrades-Moreno, L., del Castillo, I., Redondo-Gómez, S., Mesa, J., Caviedes M.A., Pajuelo, E., Rodríguez-Llorente, I.D.* 17 Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla, c/ Profesor García González 2, 41012 Sevilla, Spain. SI-CO-3 Symbiovar loti-type genes are widely spread across different chromosomal backgrounds, corresponding to up to nine Mesorhizobium genospecies nodulating Cicer canariense. Pérez-Yépez, J., Armas-Capote, N., Martínez-Hidalgo, P., Velázquez, E., PérezGaldona, R., Martínez -Molina, E., León-Barrios, M.* 19 Departamento de Microbiología y Biología Celular. Universidad de La Laguna. Tenerife. España. SI-CO-4 Molecular phylogeny and phenotypic characterization of salt tolerant Sinorhizobia nodulating Phaseolus filiformis in Northern Mexico. Rocha, G., Medina, A., Carreño, R., Bustillos, R., Contreras, J.L., Villegas, M.C., Chaintreuil, C., Dreyfus, B., Le Queré, A., Munive, J.A. * 21 Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla, Mexico. SI-CP-01 Phylogenetic diversity of bradyrhizobia nodulating cowpea (Vigna unguiculata L. Walp) in Extremadura (Spain). Bejarano, A.*, Ramírez-Bahena, M.H., Velázquez, E., Peix, A. 23 SI-CP-02 MALDI-TOFF MS, a tool for diversity analysis and detection of novel rhizobial species nodulating Phaseolus vulgaris. * Flores-Félix, J.D. , García-Fraile, P., Sánchez-Juanes, F., Ramírez-Bahena, M.H., Ferreira, L., Mulas, D., Rivas, R., González-Andrés, F., Peix, A., González-Buitrago, J.M., Velázquez, E. 25 xvii SI-CP-03 Análisis de la biodiversidad de microorganismos en nódulos de Lotus corniculatus. Marcos-García, M., Menéndez, E., Celador-Lera, L., Rivera, L.P., Martínez-Molina, E., Mateos, P.F., Velázquez, E., Rivas, R.* 27 SI-CP-04 Where do PGPR interact with plants? An essential determination to develop effective agricultural inputs based on microorganisms. Mulas, R., Mulas, D.*, Menéndez, E., Rivera, L., Mateos, P., González-Andrés, F. 29 SI-CP-05 Biodiversity of endophytic bacteria and mycorrhizal fungi in roots of pepper(Capsicum annuum L.) in León province (Spain). * Barquero, M., Velázquez, E., Terrón, A. , González-Andrés, F. 31 SI-CP-06 Diversidad de los rizobios que nodulan Medicago marina de regiones del Odiel y San Fernando. Alías-Villegas, C.*, Bellogín, R.A., Camacho, M., Cubo, M.T., Temprano, F., Espuny, M.R. 33 SI-CP-07 Tolerancia a pH y salinidad extremos y propiedades simbióticas de aislamientos de rhizobia de Medicago marina L. Alías-Villegas, C., Espuny, M.R., Bellogín, R.A., Cubo, M.T., Camacho, M., Temprano, * F. 35 SI-CP-08 Estudio de la Biodiversidad microbiana en suelos de las Marismas del Odiel y de las Minas de Rio Tinto. Ruíz-Carnicer, A., Alías-Villegas, C., Bellogín, R.A., Manyani, H., Temprano, F., Espuny, M.R., Camacho, M.* 37 SI-CP-09 Componentes biológicos en el suelo de dos plantaciones similares de ciruelo en producción ecológica y convencional. Daza, A.*, Pérez-Romero, L.F., Arroyo, F.T., García-Galavís, P.A., Camacho, M., Santamaría, C. 39 SI-CP-10 Relative abundance of denitrification genes and diversity of bacterial denitrifiers in sediments with different nitrate concentration. Correa-Galeote, D., Tortosa, G., Bedmar, E.J. * 41 SI-CP-11 Experimental and modelling approach to the legume-Rhizobium interaction: test of plant-host sanctions in co-inoculated plants with fixing and non-fixing strains. Marco, D.E.*, Talbi, C., Bedmar, E.J. 43 SI-CP-12 Identification of rhizobial strains nodulating cultivated grain legumes in Egypt. Zahran, H.H.*, Chahboune, R., Bedmar, E.J., Abdel-Fattah, M., Yasser, M.M., Mahmoud, A.M. 45 SI-CP-13 Genetic diversity and biogeography of rhizobial genospecies nodulating wild chickpea Cicer canariense on La Palma (Canary Is.). Armas-Capote, N., Pérez-Yépez, J., Martínez-Hidalgo, P., Garzón-Machado, V., del Arco-Aguilar, M., Velázquez, E., León-Barrios, M.* 47 SI-CP-14 Identificación de dos bacterias diazótrofas asociadas a una Brassicaceae (Lepidium meyeni Walp.) de suelos altoandinos del Perú. * Chumpitaz, C. , Ogata, K., Santos, R. Zúñiga, D. 49 SI-CP-15 Análisis de la presencia natural de micorrizas en cultivos de algodón inoculados con Bacillus sp. y/o Bradyrhizobium sp. Valencia, C.*, Toro, M., Zúñiga, D. 51 SI-CP-16 Poblaciones microbianas con potencial PGPR en la rizósfera de cultivos de papas nativas amargas del altiplano peruano. Ramos, E.*, Santos, R., Velezmoro, C., Zúñiga, D. 53 xviii SI-CP-17 Capacidad PGPR de cepas de Bacillus y Pseudomonas aisladas de la rizosfera de aguaymanto (Physalis peruviana L.). Cumpa, A.*, Flores, L., Chumpitaz, C., Ogata, K., Zúñiga, D. 55 SI-CP-18 Phenotypic and molecular characterization of Lotus parviflorus nodule bacteria. * Soares, R., Lorite, M.J., Videira e Castro, I., Sanjuán, J. 57 SI-CP-19 Contribution of root nodule bacteria for the sustainability of “montado” (cork oak) ecosystem. Fernández, C., Soares, R., Barrento, M.J., Machado, H., Gomes, A.A., Videira e * Castro, I. 59 SI-CP-20 Estudio del carácter endófito de bacterias del género Pantoea aisladas de los cultivos de arroz de la Marisma del Guadalquivir. Megías, E., Benitez, C., Diaz-Olivares, I., Ollero, F.J., Megías, M. * 61 SI-CP-21 El género Pantoea en los cultivos de arroz de la Marisma del Guadalquivir: diversidad y posible descripción de una nueva especie. Megías, E.*, Fernández, M., Reis Junior, F.B., Márquez, M.C., Ollero, F.J., Megías, M. 63 SI-CP-22 Bacterial biodiversity within the genus Enterobacter in paddies of Guadalquivir marshes. * Fernández, M. , Megías, E., Reis Junior, F.B., Megías, M. 65 SI-CP-23 Isolation and characterization of a novel bacterium isolated from paddies of Guadalquivir marshes. Márquez, M.C.*, Fernández, M., Megías, E., Merchán, F. 67 SI-CP-24 Biodiversidad y performance simbiótica de bacterias noduladoras de soja alóctonas de suelos de Argentina. Covelli, J.M.*, López, M.F., Arrese-Igor, C., Lodeiro, A.R. 69 SI-CP-25 Búsqueda y caracterización de bacterias promotoras del crecimiento vegetal en rizosfera de plantas nativas antárticas. Fernández Garello, P., Braga, L., Senatore, D., Lagurara, P., Yanes, M.L., Vaz, P., Azziz, G., Bajsa, N.* 71 SI-CP-26 Caracterización de las comunidades de hongos micorrícico-arbusculares asociadas a metalofitas en suelos contaminados con Cu. Cornejo, P.*, García, S., Vidal, C., Meier, S., Borie, F. 73 SI-CP-27 Screening for phosphate solubilising rhizobia from faba bean in Marrakech region field cultures. Maghraoui, T.*, Domergue, O., Oufdou, K., Lahrouni, M., Galiana, A., Sanguin, H., Drevon, J.J., de Lajudie, P. 75 SI-CP-28 Estudio de la dinámica poblacional binaria de rizobacterias en Lupinus mediante citometría de flujo. Ruiz Palomino, M., Probanza, A.* 77 SI-CP-29 Utilização da técnica de MLSA em estudos taxonômicos e filogenéticos com Bradyrhizobium e sua importância na descrição de novas espécies. * Delamuta, J.R.M. , Ribeiro, R.A., Hungria, M. 79 SI-CP-30 Mycorrhizal fungal identity as determinant of functional assemblage of plantgrowth promoting bacte ria in the rhizosphere. Meleiro, A.I.*, Carvalho, L., Correia, P., Melo, J., Carolino, M., Cruz, C. 81 SI-CP-31 Diversity and stress tolerance in rhizobia from Parque Chaqueño region of Argentina nodulating Prosopis alba. Chávez Díaz, L., González, P., Rubio, E., Melchiorre, M. * 83 xix SI-CP-32 Condiciones de estrés hídrico o salino modifican la composición y capacidad formadora de biofilm de comunidades bacterianas aisladas de rizósfera de alfalfa. Bogino, P., Abod, A., Nievas, F., Santoro, V., Vicario, J. *, Giordano, W. 85 SI-CP-33 Supervivencia y capacidad formadora de biofilms por rizobacterias. * Abod, A. , Bogino, P., Vicario, J., Giordano, W. 87 SI-CP-34 Caracterización morfológica de rizobios asociados a cultivos de arveja (Pisum sativum L.), chocho (Lupinus mutabilis S.), fréjol (Phaseolus vulgaris L.), haba (Vicia faba L.) y vicia (Vicia atropurpurea) en suelos del Ecuador. * Carpio, M.J., Paucar, B.M. , Alvarado, S.P. 89 SI-CP-35 Bacterias con actividad ACC desaminasa en huertos de aguacate en Michoacán, México. Chávez-Bárcenas, A.T.*, Hernández-Valdés, E.F., Bárcenas-Ortega, A.E., LozunaLópez, F., García-Sau cedo, P.A., Olalde-Portugal, V. 91 SI-CP-36 Caracterização genotípica de bactérias diazotróficas isoladas de plantas de Brachiaria brizantha e B. humidicula no estado de Roraima, Amazônia, Brasil. * Chalita, P.B., Cunha, E.N., Zilli, J.E., Silva, K. 93 Session II. Genetics and genomics of beneficial microorganisms and associated plants. SII-P-1 Genomics of host specificity in the Rhizobium-legume symbiosis. Imperial, J. 96 ETS de Ingenieros Agrónomos y Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid, Madrid, España. SII-P-2 Genomic insights into the rhizosphere lifestyle of rhizobia. Ormeño-Orrillo, E. 100 Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México. SII-CO-1 First analyses of the genomic sequence of the soybean symbiont Sinorhizobium fredii HH103. Vinardell, J.M.*, Göttfert, M., Becker, A., Acosta-Jurado, S., Baena, I., Blom, J., Bonilla, I., Buendía, A.M., Crespo-Rivas, J.C., Goesmann, A., Jaenicke, S., Krol, E., Lloret, J., McIntosh, M., Margaret, I., Pérez-Montaño, F., Schneiker-Bekel, S., Serranía, J., Szczepanowski, R., Zehner, S., Pühler, A., Ruiz-Sainz, J.E., Weidner, S. 103 Departamento de Microbiología, Universidad de Sevilla, Spain. SII-CO-2 Identificación y caracterización del regulador maestro del flagelo lateral de Bradyrhizobium japonicum USDA 110. Mongiardini, E.J.*, Quelas, J.I., Lodeiro, A.R. 105 IBBM-Facultad de Ciencias Exactas, UNLP-CONICET, La Plata, Argentina. SII-CO-3 Pyrosequencing reveals the presence of diverse bacterial genera which have not previously described to soil and rhizosphere. Lagos, L. *, Jorquera, M., Maruyama, F., Mora, M.L. 107 Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile. SII-CO-4 Temporal profile of nif gene expression in Azotobacter vinelandii: effect of nifA mutation. * Navarro-Rodríguez, M. , Poza-Carrión, C., Jiménez-Vicente, E., Rubio, L.M. Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón 28223, Madrid. xx 109 SII-CP-01 Singularidades adaptativas del genoma de Pseudomonas fluorescens F113. Comparación con otras cepas del género Pseudomonas. Redondo-Nieto, M.*, Martínez-Granero, F., Muriel, C., Martín, M., Rivilla, R. 111 SII-CP-02 AmrZ es un regulador transcripcional global en Pseudomonas fluorescens F113. * Martínez-Granero, F., Redondo-Nieto, M., Vesga, P., Martín, M., Rivilla, R. 113 SII-CP-03 The rkp-2 region of Sinorhizobium fredii HH103 is involved in LPS and EPS production. Acosta-Jurado, S.*, Crespo-Rivas, J.C., Murdoch, P.S., Rodríguez-Carvajal, M.A., RuizSainz, J.E., Vinardell, J.M. 115 SII-CP-04 Study of the quorum sensing Sin system of Sinorhizobium fredii HH103. * Crespo-Rivas, J.C. , Pérez-Montaño, F., Acosta-Jurado, S., Payán-Bravo, L., McIntosh, M., Meyer, S., Becker, A., Ruiz-Sainz, J.E., Vinardell, J.M. 117 SII-CP-05 Lotus japonicus Gifu and L. burttii responses to inoculation with a collection of S. fredii HH103 mutants affected in symbiotic signals. Rodríguez-Navarro, D.N., Jin, H., Kawaharada, Y., Sandal, N., Andersen, S.U., Stougaard, J., Ruiz-Sainz, J.E.* 119 SII-CP-06 Analyses of the Sinorhizobium fredii HH103 and USDA257 secretomes in the presence and absence of the flavonoid genistein. Yao, W., Thomas, J.R., Jiménez-Guerrero, I., López-Baena, F.J.*, Ruiz-Sainz, J.E., Vinardell, J.M. 121 SII-CP-07 Ocorrência e caracterização de bactérias isoladas de nódulos de amendoinzeiro (Arachis hypogaea L.) em solos paranaenses, Brasil. * Andrade, D.S. , Cardoso, J.D., Pertinhez, G.N., Saturno, D.F., Lovato, G.M., Nomura, R.B.G., Hungria, M. 123 SII-CP-08 The Type 3 secretion system effector NopP from Ensifer (=Sinorhizobium) fredii HH103 is phosphorylated by a soybean kinase. Calero, B.*, Jiménez-Guerrero, I., Pérez-Montaño, F., Monreal, J.A., Ollero, F.J., López-Baena, F.J. 125 SII-CP-09 A yeast-based array to study the function of Ensifer (=Sinorhizobium) fredii HH103 Type 3 secretion system effectors in symbiosis. Ollero, F.J.*, Jiménez-Guerrero, I., Pérez-Montaño, F., Mesa, B., Medina, C., LópezBaena, F.J. 127 SII-CP-10 Implicación del gen bgvA en la formación de biofilm por Rhizobium tropici CIAT899. Del Cerro, P.*, Ollero, F.J., Megías, M., Bellogín, R.A., Guasch-Vidal, B., PérezMontaño, F., Espu ny, M.R. 129 SII-CP-11 Identification and characterization and of a soybean kinase that phosphorylates the Type 3 secretion system effector NopL from Ensifer (=Sinorhizobium) fredii HH103. * Jiménez-Guerrero, I. , Pérez-Montaño, F., Ollero, F.J., Monreal, J.A., Cubo, M.T., López-Baena, F.J. 131 SII-CP-12 Control de movilidad y producción de EPS I en Sinorhizobium meliloti: nuevas funciones asignadas al sistema NtrY/X. Calatrava*, N., Nogales, J., Ameztoy, K., van Steenbergen, B., Soto, M.J. 133 SII-CP-13 Auxotrophy accounts for nodulation impairment in a Sinorhizobium meliloti mutant defective for meso-diaminopimelate biosynthesis. García-Rodríguez, F.M., Ortigosa, A., Millán, V., Toro, N., Martínez-Abarca, F.* 135 xxi SII-CP-14 Genomic analysis of Azospirillum brasilense Az39 the most extensively used strain for inoculant production in Argentina. Cassán, F.*, Rivera Bottia, D., Molina, R., Revale, S., Vazquez, M., Spaepen, S., Vanderleyden, J., Perticari, A. 137 SII-CP-15 Two-dimensional proteomic reference map of Bradyrhizobium diazoefficiens strain CPAC 7 (=SEMIA 5080). Gomes, D.F.*, Batista, J.S.S., Hungria, M. 139 SII-CP-16 Emprego da metodologia de MLSA em avaliações de filogenia e taxonomia de rizóbios: estudo com Rhizobium spp. microssimbiontes de feijoeiro (Phaseolus vulgaris L.). * Ribeiro, R.A. , Delamuta, J.R.M., Hungria, M. 141 SII-CP-17 Relative expression of hypothetical protein-related genes for Bradyrhizobium diazoefficiens CPAC 7. Gomes, D.F., Rolla-Santos, A.A.P.*, Batista, J.S.S., Hungria, M. 143 SII-CP-18 PoolSeq analysis of the selection of the Rhizobium genotypes by the legume host plant. * Jorrín, B. , Imperial, J. 145 SII-CP-19 Incorporación estable y expresión del gen de la diguanilato ciclasa PleD* en el genoma de bacterias que interaccionan con plantas. Romero-Jiménez, L.*, Rodríguez, D., Prada-Ramírez, H., Gallegos, M.T., Sanjuán, J., Pérez-Mendoza, D. 147 SII-CP-20 Identification of the gene responsible for a non-nodulating phenotype in chickpea. Ali, L., Madrid, E., Sánchez, R., Temprano, F., , Gil, J., Rubio, J., Millan, T.* 149 SII-CP-21 Functional characterization of Ensifer meliloti denitrification genes. Torres, M.J.*, Rubia, M.I., Coba de la Peña, T., Pueyo, J.J., Bedmar, E.J., Delgado, M.J. 151 SII-CP-22 Genome sequence of Herbaspirillum rubrisubalbicans M1, an endophytic diazotroph and mild phytopathogen of sugarcane. Souza, E. M.*, Chubatsu, L., Cardoso, R.A., Raittz, R.T., Weiss, V.A., Monteiro, R.A., Faoro, H., Wassem, R., Baura, V.A., Balsanelli, E., Huergo, L., Muller-Santos, M., Tadra-Sfeir, M., Cruz, L.M., Pedrosa, F.O. 153 SII-CP-23 Sinorhizobium meliloti differentially expressed non-coding RNAs modulating nitrogen fixaion and cell cycle progression. Robledo, M.*, Frage, B., Schlüter, J.P., Becker, A. 155 Session III. Symbiotic plant/microbe Interactions. SIII-P-1 Priming plant defences by beneficial soil microorganisms. Pozo-Jiménez, M.J. 158 Estación Experimental del Zaidín, CSIC. Granada. España. SIII-P-2 Una visión general del papel de los polisacáridos superficiales de Sinorhizobium fredii en su simbiosis con la soja y otras leguminosas. Ruiz Sainz. J.E. 162 Departamento de Microbiología. Facultad de Biología. Universidad de Sevilla. SIII-CO-1 Boron deficiency affects rhizobia cell surface polysaccharides. Quijada, N.M., Cerda, M.E., Abreu, I.*, Pérez de Nanclares, M., Bonilla, I., Bolaños, L. Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid. Madrid. xxii 166 SIII-CO-2 Thehalose is a key carbon regulatory player in the Legume-Rhizobium symbiosis Sánchez, F.*, Barraza, A., Estrada-Navarrete, G., Quinto, C., Merino, E. 168 Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México. Av. Universidad # 2001, Col. Chamilpa. C.P.: 62210, Cuernavaca, Morelos, México. SIII-CO-3 RbohB-dependent reactive oxygen species regulates rhizobial infection, bacteroid development and nitrogen fixation in common bean. Arthikala, M.K., Montiel, J., Nava, N., Santana, O., Sánchez-López, R., Cárdenas, L., Quinto, C.* 170 Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62271, México. SIII-CO-4 Quorum Sensing systems and flavonoids via NodD1 coordinate and regulate the biofilm transition in Sinorhizobium fredii SMH12, which is necessary for a successful root colonization of Glycine max cv Osumi. * Pérez-Montaño, F. , Jiménez-Guerrero, I., Del Cerro, P., López-Baena, F.J., Ollero, F.J., Bellogín, R.A., Lloret, J., Espuny, M.R. 172 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012 - Sevilla, Spain. SIII-CP-01 Proteolytic control of the Bradyrhizobium japonicum transcriptional regulator FixK2. Fernández, N.*, Bonnet, M., Stegmann, M., Maglica, Z., Weber-Ban, E., Hennecke, H., Bedmar, E.J., Mesa, S. 174 SIII-CP-02 Effect of temperature on ectomycorrhizal fungi associated with Pinus sylvestris L. in organic vs mineral soils. Gómez-Gallego, T., García-Rabasa, S., Flores-Rentería, D., Rincón, A.* 176 SIII-CP-03 Ensifer meliloti is the preferred symbiont of Medicago arborea in eastern Morocco soils. Missbah El Idrissi, M., Guerrouj, K., Pérez-Valera, E., Abdelmoumen, H.*, Bedmar, E.J. 178 SIII-CP-04 Interaction between Arbuscular Mycorrhizal Fungi and rhizobia on the growth of subclover under Mn toxicity: The role of Extraradical Mycelium. Alho, L.*, Carvalho, M., Goss, M.J., Brito, I. 180 SIII-CP-05 Autochthonous drought-tolerant arbuscular mycorrhizal fungi and bacteria can increase nutrients acquisition and alleviate drought stress in lavandula plants. Armada, E., Roldán, A., Azcón, R.* 182 SIII-CP-06 Análisis filogenético de la diversidad de hongos formadores de micorrizas arbusculares presentes en los distintos tipos de propágulos asociados a especies de plantas características del Parque Natural Sierra de Baza (Granada, España). Varela-Cervero, S., López-García, A., Berrio, E., Barea, J. M., Azcón-Aguilar, C.* 184 SIII-CP-07 Influencia de la composición de la cubierta vegetal sobre los hongos micorrícicoarbusculares y la estabilización de un suelo degradado en un ecosistema mediterráneo. Cornejo, P., Ferrol, N., Barea, J.M.*, Azcón-Aguilar, C. 186 SIII-CP-08 Salt tolerance evaluation in Casuarina glauca: impact on the photosynthetic apparatus. Batista-Santos, P.*, Graça I., Semedo J., Lidon, F., Alves, P., Scotti, P., Pais, I., Ribeiro, A.I., Ramalho, J.C. 188 xxiii SIII-CP-09 Rhizosphere symbiots valorisation: Common bean-rhizobia symbiosis adaptation to P deficiency in Ain Temouchent agro-ecosystem in Algeria.7 Benadis, Ch.*, Bekki, A., Lazali, M., Drevon, J.J. 190 SIII-CP-10 The genotypic variability of beans affects the growth parameters, the nodulation and the microorganisms which initiate nodulation. Benadis, Ch., Bekki, A.*, Abed, N.H., Ouzane, H., Irekti, H. 192 SIII-CP-11 Phosphoenol pyruvate phosphatase transcript in nodule cortex of Phaseolus vulgaris. * Bargaz, A. , Drevon, J.J., Lazali, M., Ghoulam, C. 194 SIII-CP-12 P-deficiency increases phytase activity and O2 uptake per N2 reduced in Phaseolus vulgaris L. Lazali, M.*, Abadi, J., Ounane, S.M., Bargaz, A., Pernot, C., Drevon, J.J. 196 SIII-CP-13 Effect of inoculation with selected rhizobia on symbiotic nitrogen fixation and Faba bean grains yield in north Tunisia agro-ecosystems. Sifi, B.*, Maazaoui, H., Drevon, J.J. 198 SIII-CP-14 Integrating soil microbial community context in plant response to mycorrhizal symbionts. * Carvalho, L. , Correia, P., Meleiro, A.I., Carolino, M., Dionisio, F., Cruz, C. 200 SIII-CP-15 Identificación y caracterización de sistemas de transporte de hierro en el hongo micorrícico arbuscular Rhizophagus irregularis. Tamayo, E., Ferrol, N.* 202 SIII-CP-16 Structure of the exopolysaccharide isolated from Sinorhizobium fredii HH103. Rodríguez-Carvajal, M.A.*, Acosta-Jurado, S., Rodríguez-Navarro, D.N., Crespo-Rivas, J.C., Margaret, I., Sanjuán, J., Soto, M.J., Vinardell, J.M., Ruiz-Sainz, J.E., Gil-Serrano, A. 204 SIII-CP-17 First evidence for interlinked control of surface motility and biofilm formation in Sinorhizobium meliloti. Amaya-Gómez, C.V., Hirsch, A.H., Soto, M.J.* 206 SIII-CP-18 Symbiotic phenotype of different rhizobial species on Lotus japonicus Gifu and Lotus burttii. Rodríguez-Navarro, D.N.*, Temprano, F., Velázquez, E., Ruiz-Sainz, J.E. 208 SIII-CP-19 Los efectores secretados a través del T3SS suprimen la respuesta de defensa en soja inducida por Ensifer (=Sinorhizobium) fredii HH103. Cubo, M.T.*, Jiménez-Guerrero, I., Pérez-Montaño, F., Ollero, F.J., López-Baena, F.J. 210 SIII-CP-20 Pode Bradyrhizobium japonicum, em combinação com diferentes densidades de plantio de soja alterar componentes do rendimento? * de Luca, M.J. , Nogueira, M.A., Hungria, M. 212 SIII-CP-21 Effects of glyphosate-resistant gene and herbicides on biological nitrogen fixation symbiotic efficiency and grain productivity of soybean in Brazil. * Hungria, M., Mendes, I.C., Nakatami, A.S., Reis-Junior, F.B., Fernandes, M.F. 214 SIII-CP-22 Diversidade genética de bactérias que colonizam nódulos radiculares de Phaseolus vulgaris L. cultivado em campo e em casa de vegetação. * Oliveira-Francesquini, J.P. , Hungria, M., Glienke, C., Kava-Cordeiro, V., GalliTerasawa, L. 216 SIII-CP-23 Symbionts of Mimosa spp. in ultramafic soils in central Brazil. Reis Junior, F.B.*, James, E.K., Hertel Júnior, C.R., Lopes, A.A.C., Alves, M.R.P., Souza, L.M., Baura, V.A., Mendes, I.C., Andrade, L.R.M. 218 xxiv SIII-CP-24 Cloning, Expression and Characterization of a Chitinase class III from Casuarina glauca nodules. Graça, I.*, Liang, J., Guilherme, M., Ferreira-Pinto, M., Ribeiro, A., Pereira A.S, 220 SIII-CP-25 Negative short-term salt effects on the soybean-B. japonicum interaction and partial reversion by calcium addition. Muñoz, N., Rodríguez, M., Robert, G., Lascano, R. * 222 SIII-CP-26 Expresión de supresores de muerte celular en raíces en cabellera de soja: efectos bajo condiciones de estrés y en la interacción con Bradyrhizobium japonicum. * Robert, G. , Muñoz, N., Lascano, R. 224 SIII-CP-27 Efecto del contenido de taninos condensados (TC) sobre la formación de nódulos en especies del género Lotus. Escaray, F.J.*, Estrella, J., Paolocci, F., Damiani, F., Ruiz, O.A. 226 SIII-CP-28 Alteraciones en el proceso de nodulación en ausencia y presencia de fuente de nitrógeno externa de un mutante deficiente en el transporte de nitrato de la leguminosa Lotus japonicus. García-Calderón, M.*, Pal’ove-Balang, P., Pérez, C.M., Betti, M., Marquez, A.J. 228 SIII-CP-29 Phylogenetic diversity and structure of sebacinoid fungi associated with plant communities along an altitudinal gradient. Garnica, S.*, Riess, K., Bauer, R., Oberwinkler, F. 230 SIII-CP-30 Grain yield of cowpea in Ghana responds to Rhizobium inoculation. Atakora, W.K., Guimarães, A.P.*, Fosu, M., Boddey, R.M., Xavier, G.R. 232 SIII-CP-31 Pseudomonas fluorescens enhances photosynthesis and improves bioactive profiles and antioxidant potential in blackberry (Rubus sp. Var. Lochness) in field conditions. García-Seco, D., Bonilla, A., Algar, E., García-Villaraco, A., Gutiérrez Mañero, F.J.*, Ramos Solano, B. 234 SIII-CP-32 Improving opium poppy yield through MAMPs from selected beneficial bacterial strains in field trials. Bonilla, A., Garcia-Seco, D., Muñoz Ledesma, F.J., Lucas, J.A.*, Ramos Solano, B., Gutiérrez Mañero, F.J. 236 SIII-CP-33 Rhizobial cellulase CelC2 and its Ensifer homolog play an important role in plant and nodule development. Menéndez, E.*, Robledo, M., Velázquez, E., Martínez-Molina, E., Rivas, R., Mateos, P.F. 238 SIII-CP-34 Ocorrência de fungos micorrízicos arbusculares na cultura da palma no semiárido de Pernambuco. Mergulhão, A.C.E.S.*, Silva, M.L.R.B., Figueroa, C.S., Lyra, M.C.C.P. 240 SIII-CP-35 Bactérias diazotróficas associadas a cladódios de palma: solubilização de fosfato inorgânico e tolerância à salinidade. Silva, M.L.R.B.*, Figueroa, C.S., Mergulhão, A.C.E.S., Lyra, M.C.C.P. 242 SIII-CP-36 Genotypic characterization of natural rhizobial populations isolated from pea and lentil in two eco-climatic subhumid and semi-arid zones in Algeria. Riah, N.*, Djekoun, A., Heulin, K., Laguerre, G. 244 SIII-CP-37 Native bradyrhizobia isolated from Lupinus mariae-josephae possess an essential T3SS for symbiosis. Durán, D., Pastor, V., Zehner, S., Göttfert, M., Imperial, J., Rey, L., Ruiz-Argüeso, T.* 246 xxv SIII-CP-38 Which phosphatases may contribute to P use efficiency for N2 fixation in the rhizobial symbiosis with legumes? Drevon, J.J.*, Amenc, L. , Pernot, C., Abadie, J., Blair, M., Lazali, M., Bargaz, A., Ghoulam, C., Ounane, S.M., Zaman-Allah, M. 248 SIII-CP-39 nod gene inducers released by Mimosa flocculosa seeds and its effects on common bean growth and yield under Brazilian cerrado soil conditions. Mercante, F.M.*, Otsubo, A.A., Cunha, C.O. 250 SIII-CP-40 Diversidade de fungos micorrízicos arbusculares e colonização do solo em sistema de cultivo consorciado cafeeiro (Coffea arábica L.) e seringueira (Hevea brasiliensis). Colozzi-Filho, A.*, Bugatti, E.P., Garbossi, A., Scaramal, A., Machineski, O., Carrenho, R. 252 SIII-CP-41 Effect of mycorrhizal inoculation on wheat and barley genotypes differing in Altolerance growing a t phytotoxic Al level. Meier, S., Curaqueo, G., Seguel, A., Aguilera, P., Cornejo, P., Borie, F.* 254 SIII-CP-42 Origin of arbuscular mycorrhizal fungi determines plant development and resource distribution between Phaseolus vulgaris L. and Oriza sativa in intercropping. Ramanankierana, H.*, Rasamiarivelo, A.V., Razafimbelo, T., Becker, T., Razakatiana, A.T.E., Manitriniaina, H., Rabeharisoa, L., Randriambanona, H., Drevon, J.J., Duponnois, R. 256 SIII-CP-43 Asociación Española de Leguminosas. De Ron, A.M., De la Cuadra, C., Millán, T* 258 Session IV. Associative interaction plant/beneficial microbe. SIV-P-1 Aplicaciones biotecnológicas de la interacción planta-microorganismos: salud, medioambiente y agricultura. Gutiérrez-Mañero, F.J. 261 Facultad de Farmacia. Universidad San Pablo CEU. Madrid. España. SIV-P-2 Aspectos bioquímicos y moleculares de la interacción Delftia-Rizobio-Alfalfa: un lenguaje de señales químicas. Castro-Sowinski, S. 265 Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay. SIV-CO-1 Evaluation of Micromonospora as a potential biocontrol agent. Martínez-Hidalgo, P.* , Martínez-Molina, E., Pozo, M.J. 269 Departamento de Microbiología y Genética, Universidad de Salamanca, España. GIR "Interacciones mutualistas planta-microorganismo", Unidad Asociada al IRNASA. CSIC, Salamanca. España. SIV-CO-2 Movimiento y quimiotáxis en Pseudomonas fluorescens F113, influencia en una colonización competente de la rizosfera de las plantas. Baena, I., Muriel, C., Martín, I., Jalvo, B., Hernanz, A., González de Heredia, E., Barahona, E., Navazo, A., Redondo-Nieto, M., Martínez-Granero, F., Lloret, J., Rivilla, * R., Martín, M. Departamento de Biología. Facultad de Ciencias. Universidad Autónoma de Madrid. xxvi 271 SIV-CO-3 Caracterización de la interacción Delftia-Sinorhizobium en alfalfa. Morel, M.A.*, Cagide, C., Dardanelli, M.S., Castro-Sowinski, S. 273 Unidad de Microbiología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Av. Italia 3318, Montevideo, Uruguay. SIV-CO-4 Estrés salino, calidad nutricional y comportamiento postcosecha, en lechuga inoculada con Azospirillum. Fasciglione, G.*, Casanovas, M., Yommi, A., Quillehauqui, V., Roura, S., Barassi, C.A. 275 Unidad Integrada: Facultad de Cs. Agrs. (UNMdP) -EEA Balcarce (INTA). Ruta 226 Km 73,5 CC 276 (7620) Balcarce, Argentina. SIV-CP-01 Bioprospecção de Bactérias Isoladas de Milho para Promoção de Crescimento de Plantas. Ikeda, A.C.*, Szilagy-Zecchin, V.J., Hungria, M., Kava-Cordeiro, V., Glienke, C., GalliTerasawa, L .V. 277 SIV-CP-02 Caracterización de una metilobacteria aislada de la superficie del grano de arroz. Gallego Parrilla, J.J., Alías-Villegas, C., Díaz-Olivares, I.M., Gutiérrez Alcántara, R., Madinabeitia-Peiró, N., Bellogín, R.A.*, Espuny, M.R. 279 SIV-CP-03 Resistencia a metales pesados de bacterias aisladas de leguminosas de las Marismas del Odiel y del entorno del Río Tinto. Lara-Dampier, V., Acera-Mateos, P., Alías-Villegas, C., Gómez-Cárdenas, A.J., Temprano, F., Camacho, M., Bellogín, R.A., Espuny, M.R.* 281 SIV-CP-04 Comparación de las características microbiológicas del suelo en huertos de aguacate bajo manejos orgánico y convencional. Bárcenas-Ortega, A.E.*, Chávez-Bárcenas, A.T., García-Saucedo, P.A., OlaldePortugal, V., Tulais-Al varado, C.A., Zavala-Gómez, A. 283 SIV-CP-05 Efecto de la inoculación foliar y radicular de bacterias PGPR en el cultivo de Aguaymanto (Physalis peruviana). Flores, L., Ogata, K.*, Zúñiga, D. 285 SIV-CP-06 Bacillus sp. y hongos micorrícicos para el control biológico del nemátodo Meloidogyne incognita en el cultivo de Aguaymanto (Physalis peruviana). Isla, F.*, Carbonell, E., Ogata, K., Zúñiga, D. 287 SIV-CP-07 Identificación de dos bacterias diazótrofas asociadas a una Brassicaceae (Lepidium meyeni Walp.) de suelos altoandinos del Perú. * Chumpitaz, C., Ogata, K., Santos, R., Zúñiga, D. 289 SIV-CP-08 Selección de cultivos rizobianos aislados de nódulos de leguminosas de diferentes regiones del Perú con capacidad de promover el crecimiento de lechuga, páprika y tomate. Soriano-Bernilla, B.*, Prado-Chávarry, G., Zavaleta-Verde, D., Valdez-Nuñez, R. 291 SIV-CP-09 Promoção de crescimento de tomate (Solanum lycopersicum L.) estimulado por Bacillus amyloliquefaciens FZB42. Szilagyi-Zecchin, V.J.*, Ruaro, L., Mógor, A.F. 293 SIV-CP-10 Efecto de volátiles emitidos por la bacteria bCT34 en la promoción de crecimiento de Arabidopsis thaliana. Parada, M.*, Quiroz, A., Parra, L., Mendoza, D., Fincheira, P. 295 SIV-CP-11 Functionality of phosphate solubilizing bacteria isolated from the rhizosphere of papaya plants under conventional and organic farming systems. * Melo, J. , Azevedo-Junior, R.R. , Eutropio, F.J., Correira, P., Carvalho, L. , Meleiro, A.I., Teixeira, M.S., Carolino, M. , Ramos, A.C., Cruz, C. 297 xxvii SIV-CP-12 Plant-growth promoting Sphingomonas sp. associated with annual ryegrass. Dourado, A.C., Castanheira, N., Cortés Pallero, A., Delgado Rodriguéz, A.I., Alves, P.I.L., Barreto Crespo, M.T., Fareleira, P.* 299 SIV-CP-13 Paenibacillus species isolated from Pisum sativum nodules in Lanzarote present several in vitro plant growth promotion mechanisms. Ramírez-Bahena, M.H., Flores-Félix, J.D., Tejedor, C., León-Barrios, M., Peix, A.*, Velázquez, E. 301 SIV-CP-14 Efecto de rizobacterias en el enraizamiento de miniestacas en dos clones híbridos de Eucalyptus spp. González-Candia, P.*, Sossa, K., Rodríguez, F., Sanfuentes, E. 303 SIV-CP-15 Azoarcus sp. CIB, un nuevo endófito del arroz que degrada anaeróbicamente hidrocarburos aromáticos. Fernández, H., Prandoni, N., Fernández-Pascual, M., Morcillo, C., Fajardo, S., Díaz, E., Carmona, M.* 305 SIV-CP-16 Impacts of rice-bean intercropping on soil microbial activity and the development of rice plants. * Razakatiana, A.T.E. , Henintsoa, M., Becquer, T., Ramanankierana, H., Baohanta, H.R., Raherimandimby, M., Rabeharisoa, L., Duponnois, R. 307 SIV-CP-17 Efeito de bactérias PRPG com indução de ácido salicílico em plântulas de duas cultivares de palma forrageira (Opuntia e Nopalea). Lyra, M.C.C.P.*, Pérez-Montaño, F., Jiménez-Guerrero, I., Madinabeitia, N., DiazOlivares, I.M., Gu tiérrez-Alcántara, R., Ollero, F.J. 309 SIV-CP-18 Análisis cualitativo y cuantitativo de vitamina C en fresas, en presencia de microorganismos endófitos. Aranda-Alonso, C.*, Rodríguez-Carvajal, M.A., Ollero, F.J., Megías, M., Gil-Serrano, A. 311 SIV-CP-19 Occurrence of rhizobacteria with PGPR activities in different ecosystems and agroecosystems of nor thern, central and southern Chile. Barra, P., Jorquera, M., Acuña, J.*, Lagos, L., Marileo, L., Inostroza, N., Mora, M.L. 313 SIV-CP-20 Efecto de plaguicidas sobre la actividad biológica de la cubierta vegetal (L. perenne) de un sistema de biopurificación denominado lecho biológico. Diez, M.C.*, Gallardo, F. 315 Session V. Phisiology and biochemistry of beneficial microorganisms and associated plants. SV-P-1 Fijación de nitrógeno en leguminosas en entornos variables: el bacteroide, el nódulo y la planta. César Arrese Igor. 318 Universidad Pública de Navarra. Pamplona, España. SV-P-2 Molecular physiology of nickel and cobalt homeostasis in Rhizobium leguminosarum. José Manuel Palacios Alberti Escuela Técnica Superior de Ingenieros Agrónomos y Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid, Pozuelo de Alarcón, Madrid, España xxviii 319 SV-CO-1 Engineering a hydrogen biosensor in the nitrogen-fixing strain Rhodobacter capsulatus. Gónzalez de Heredia, E.M.*, Barahona, E., Jiménez-Vicente, E., Echavarri-Erasun, C., Rubio, L.M. 323 Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón 28223, Madrid. SV-CO-2 MtNramp1 mediates iron import in rhizobia-infected Medicago truncatula cells. Rodríguez-Haas, B., Finney, L., Kryvoruchko, I., González-Melendi, P., Udvardi, M., * Imperial, J., González-Guerrero, M. 325 Centro de Biotecnología y Genómica de Plantas (CBGP) Universidad Politécnica de Madrid. Campus de Montegancedo, Pozuelo de Alarcón, 28223 Madrid, España. SV-CO-3 Selección de líneas de garbanzo tolerantes a la salinidad empleando caracteres de producción de biomasa y funcionamiento nodular como indicadores. Gómez, L.A.*, Vadez, V., Vaillhe, H., Pernot, C., Drevon, J.J. 327 Instituto de Suelos. Autopista, Costa-Costa y Antigua Carretera de Vento, Capdevila, Boyeros. La Habana, Cuba. SV-CO-4 Estrés abitotico induce cambios en las señales de comunicación y en la interacción temprana entre maní y rizobacterias. Cesari, A., Paulucci, N., García, M., Dardanelli, M.S. * 329 Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Argentina. SV-CP-01 Movilidad en cepas de rizobios nodulantes de maní. Vicario, J.*, Dardanelli, M.S., Giordano, W.F. 331 SV-CP-02 Effects of phosphorus and nitrogen level fertility on growth parameters of Faba bean inoculated with rhizobia. Maazaoui, H.*, Sifi, B., Drevon, J.J. 333 SV-CP-03 Phytate mineralising bacteria increase in the rhizosphere of nodulated common bean (Phaseolus vulgaris) under P deficiency. Maougal, R.T.*, Brauman, A., Plassard, C., Abadi, J., Djekoun, A., Drevon, J.J. 335 SV-CP-04 Is the contrast between nodulated recombinant imbred lines of common bean (Phaseolus vulgaris L.) linked with available phosphorus in soil ? A multi-local field test in Mediterranean conditions. Alkama, N.*, Jaillard, B. , Ounane, S.M., Ounane, G., Drevon, J.J. 337 SV-CP-05 Phosphoenol pyruvate phosphatase transcript in nodule cortex of Phaseolus vulgaris. * Bargaz, A. , Lazali, M., Ghoulam, C., Drevon, J.J. 339 SV-CP-06 Phytate-mineralizing rhizobia from Vicia faba symbiosis in an agro-ecosystem of south of France. * Domergue, O. , Chouayekh, H., Abadie, J., Amenc, L., Pernot, C., de Lajudie, P., Galiana, A., Drevon, J.J. 341 SV-CP-07 Daily simulation of plant and microbial transfers of carbon between the soil and the atmosphere under wheat-faba bean intercropping. * Ibrahim, H. , Hatira, A., Blavet, D., Drevon, J.J., Pansu, M. 343 SV-CP-08 Effect of phosphorus deficiency on some agrophysiological and biochemical parameters in faba bean (Vicia faba)-rhizobia symbiosis. Makoudi, B., Mouradi, M., Kabbadj, A., Farissi, M., Drevon, J.J., Ghoulam, C.* 345 SV-CP-09 Acúmulo de açúcares e fixação biológica de nitrogênio em genótipos de soja sob restrição hídrica. * Cerezini, P., Pípolo, A.E., Hungria, M., Nogueira, M.A. 347 xxix SV-CP-10 Mannose Rich N-Glycoproteins synthesized during the development of the Rhizobium-pea symbiosis are potential boron ligands. Abreu, I., Valiente, R., Poza, L., Quijada, N.M., Reguera, M., Bonilla, I., Bolaños, L. * 349 SV-CP-11 Regulation of cellulose-like polysaccharide synthesis in Sinorhizobium meliloti. * Baena, I., Valiente, E., Bonilla, I., Martín, M., Rivilla, R., Lloret, J. 351 SV-CP-12 Functional roles of HypC and HupK in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum. Albareda, M.*, Manyani, H., Rey, L., Brito, B., Ruiz-Argüeso, L., Imperial, J., F. Pacios, L., Palacios, J.M. 353 SV-CP-13 Quorum Sensing is essential for an effective symbiosis in R. leguminosarum UPM791. Sánchez-Cañizares, C., Ruiz-Argüeso, T., Imperial, J., Palacios, J.M. * 355 SV-CP-14 DmeRF system is required for nickel and cobalt resistance in Rhizobium leguminosarum bv. viciae. Rubio-Sanz, L.*, Prieto, R.I., Palacios, J.M., Brito, B. 357 SV-CP-15 Efectos de la variación de la transpiración sobre la fijación biológica de nitrógeno y el transporte de metabolitos a larga distancia en guisante. * Aldasoro Galán, J. , Arrese-Igor, C. 359 SV-CP-16 Importancia de la relación fuente/sumidero en la inhibición de la fijación de nitrógeno. García y García, K.*, Arrese-Igor, C. 361 SV-CP-17 Implicación de la trehalosa-6-fosfato en la respuesta a sequía de nódulos de judía. * Hidalgo-García, A. , Argandoña, M., González, E.M., Arrese-Igor, C., Vargas, C., Delgado, M.J. 363 SV-CP-18 Study of sulphur assimilation and sulphur sources used by Rhizobium leguminosarum strain 3841 in free-living and symbiotic conditions. Muñoz-Azcárate, O.*, Karunakaran, R., Arrese-Igor, C., Poole, P.S. 365 SV-CP-19 Towards the optimization of hydrogen production by nitrogenase: In vitro directed evolution of Rhodobacter capsulatus molybdenum nitrogenase. Barahona, E.*, González de Heredia, E.M., Rubio, L.M. 367 SV-CP-20 The FdxN protein is required for the biosynthesis and activity of the NifDK nitrogenase components in Azotobacter vinelandii. Jiménez-Vicente, E.*, Poza-Carrión, C., Navarro-Rodríguez, M., Rubio, L.M. 369 SV-CP-21 Two-Hybrid System in the nitrogen-fixing bacterium Azotobacter vinelandii. Moreno-Urbano, E.*, López-Torrejón, G., Rubio, L.M. 371 SV-CP-22 Boron deficiency in Anabaena PCC7120: potential boron ligands. * Bonilla, I. , Abreu, I., Bolaños, L. 373 SV-CP-23 Metabolismo antioxidante inducido por imazamox en distintos órganos de judía en simbiosis con Rhizobium tropici. * García-Garijo, A., López, M., Palma, F., Tejera, N.A., Lluch, C. 375 SV-CP-24 Implicación de las poliaminas en la respuesta a la salinidad de nódulos de Phaseolus vulgaris. * Cobos, L., López-Gómez, M. , Hidalgo, J., Iribarne, C., Lluch, C. 377 SV-CP-25 Pantoea eucalypti M91 favorece la adquisición de hierro y mejora la performance de Lotus japonicus, ecotipo Gifu, bajo condiciones de estrés alcalino. Campestre, M.P., Castagno, L.N., Estrella, M.J., Ruiz, O.A. * 379 xxx SV-CP-26 Caracterización de aldehído oxidasas en la simbiosis Medicago truncatula-Ensifer meliloti en condiciones de estrés por cadmio. Coba de la Peña, T.*, Pallol, B., Pérez, E., Pueyo, J.J., Lucas, M.M. 381 SV-CP-27 Response of the symbiotic system Medicago truncatula-Ensifer meliloti to cadmium stress: Ionome and ion transporters. Fedorova, E., Coba de la Peña, T., Pueyo, J.J., Lucas, M.M. * 383 SV-CP-28 Nonsymbiotic and truncated hemoglobins of legumes: biochemical characterization and subcellular localization. * Sainz, M., Pérez-Rontomé, C., Ramos, J., Mulet, J.M., James, E.K., Becana, M. 385 SV-CP-29 Producción y actividad nodular de dos variedades de alfalfa (Europa y Aragón) en condiciones de CO2 elevado. Kizildeniz, T., Erice, G., Sanz-Sáez, A., Aguirreolea, J., Sánchez-Díaz, M., Irigoyen, J.J.* 387 SV-CP-30 Análisis de la diversidad de polihidroxialcanoato-sintasas (PhaC) en Bradyrhizobium japonicum USDA 110. Quelas, J.I.*, Pérez-Giménez, J., Mongiardini, J., Carriño, J., Parisi, G., Lodeiro, A.R. 389 SV-CP-31 La mutación de ntrB en Bradyrhizobium japonicum afecta su crecimiento aeróbico en nitrato pero no en amonio. * López, M.F. , Lamelza, F., Lodeiro, A.R., López García, S.L 391 SV-CP-32 Application of calcium, phosphorus and antioxidants do not promote tolerance to high solar irradiance in Rhizobium-nodulated Phaseolus vulgaris L. Izaguirre-Mayoral, M.L.*, Silvera, J., Rodríguez, M. 393 SV-CP-33 Localization and dynamics of the succinoglycan translocon in Sinorhizobium meliloti cells. Rivero, M.R., Medeot, D.B., Contreras-Moreira, B., Liaudat, J.P., Ferrari, W., Rossi, F., Fischer, S.E., Becker, A., Jofré, E.* 395 SV-CP-34 Implication of NRT2.5 and NRT2.6 genes in growth promotion response of Arabidopsis by Phyllobacterium brassicacearum STM196. Kechid, M.*, Desbrosses, G., Varoquaux, F., Djekoun, A., Touraine, B. 397 SV-CP-35 Micorrizas arbusculares: estado actual del conocimiento y perspectivas en pastizales de Uruguay. Pezzani, F.* 399 SV-CP-36 The potential of NIR and MIR spectroscopy to predict the P retention capacity of Malagasy and Brasilan uplands soils. Ramaroson, H.V.*, Marchão, R.L., Vendrame, P.R.S., Razafimahatratra, H., Fanjaniaina, M.L., Andriamalaza, S., Rakotondrazafy, A.F.M., Razafimbelo, T., Rabeharisoa, L., Becquer, T. 401 SV-CP-37 Avaliação da produção de ácido indolacético por bactérias do gênero Azospirillum isoladas de pastagens nativas do Pantanal Sul-Mato-Grossense. Souza, M.S.T., Brasil, M.S.*, Reis Junior, F.B. 403 SV-CP-38 La respuesta de la expresión fitasa nodular a bajo fósforo varía según aislados de rizobios en la simbiosis Phaseolus vulgaris L. Morales Valdés, A.N.*, Gómez, L.A. , Amenc, L., Gregorio, C., Drevon, J.J. 405 SV-CP-39 Oxidases of diterpene phytohormone biosynthesis in Bradyrhizobium japonicum bacteroids. Rojas, M.C.*, Mendez, C., Baginsky, C., Hedden, P., Carú, M. 407 SV-CP-40 Effects of salts levels on phosphorus availability in non calcareous soil. Hamdi, W.*, Seffen, M., Deleporte, P., Drevon, J.J., Blavet, D., Gérard, F. 409 SV-CP-41 Bacterial acid phosphatase activity and localization in Phaseolus vulgaris nodules. Amenc, L.*, Drevon, J.J. 411 xxxi SV-CP-42 Responses of soil catabolic functions in cereal-legume intercrops. Wahbi, S., Baudoin, E., Maghraoui, T., Sanguin, H., Oufdou, K., Hafidi, M., Galiana, A., De Lajudie, P., Prin, Y., Duponnois, R. * 413 SV-CP-43 Managing a pluridisciplinary data base: Integrating knowledge to engineer the complexity of microbe-soil-plant interactions in FabaTropiMed agro-ecosystems Soto, P.*, Blavet, D., Balboa, A., Zanetto, A., Drevon, J.J., Desclaux, D. 415 Session VI. Inoculants in agriculture and the environment. SVI-P-1 Efficient soil microbial consorcia: a new dimension for sustainable agriculture and environmental sustainability. Cruz, C. 418 Universidade de Lisboa, Lisboa. Portugal. SVI-P-2 Oportunidades e ameaças à contribuição da fixação biológica do nitrogênio em leguminosas no Brasil. Nogueira, M.A. 422 Embrapa Soja, Londrina, Paraná, Brazil. SVI-CO-1 Selenium biofortification in wheat plants by co-inoculation of selenobacteria strains and arbuscular mycorhizal fungy for obtaining enriched selenium foods. Durán, P.*, Mora, M.M. 426 Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Biotechnological Bioresource Nucleus. Universidad de la Frontera, Temuco-Chile. SVI-CO-2 Evaluación de la degradación de tamo de arroz por microorganismos lignocelulolíticos. Gutiérrez-Rojas, I.*, Pedraza-Zapata, C., Sánchez-Rodríguez, L., Moreno-Sarmiento, N. 428 Instituto de Biotecnología, Ciudad Universitaria, Edificio Manuel Ancizar, Universidad Nacional de Colombia, Bogotá, D.C., Colombia. Departamento de Microbiología, Facultad de Ciencias, Laboratorio de Biotecnología Aplicada, Grupo de Biotecnología Ambiental e Industrial (GBAI), Pontificia Universidad Javeriana, Bogotá, D.C., Colombia. SVI-CO-3 Soil microbial phosphorus, phosphorus availability and crop yield of upland ricecommon bean interc ropping under organic and mineral fertilizer inputs on ferrallitic soil of Malagasy highlands. Henintsoa, M.*, Andriamananjara, A., Razakatiana, A.T.E., Larvy Delarivière, J., Rabeharisoa, L., Becquer, T. 430 Laboratoire des Radioisotopes (LRI), University of Antananarivo, Madagascar. Laboratoire de Microbiologie de l’Environnement (LME), Centre National de Recherches sur l’Environnement (CNRE), Antananarivo, Madagascar. Institut de Recherche pour le Développement (IRD), UMR Eco&Sols, c/o LRI, Madagascar. SVI-CO-4 Sistema uruguayo de fiscalización de inoculantes: complementación interinstitucional y aplicación de tecnologías de la información. * Beyhaut, E. , Barlocco, C., Altier, N., Mayans, M. 432 INIA Las Brujas, Ruta 48 Km 10, Canelones, Uruguay. 2 División Control de Insumos, DGSSAA. M.G.A.P, Av. Millán 4703, Montevideo, Uruguay. SVI-CP-01 Análise de crescimento e acúmulo de nutrientes pela variedade RB92579 de cana de açúcar plantada em um Argissolo com e sem inoculação e aplicação de Nfertilizante. Reis, V.M.*, de Oliveira, R.P., Schultz, N., de Araújo, A.P., Urquiaga, S. 434 SVI-CP-02 Effect of Maize-common bean intercropping on nitrogen uptake. Latati, M., Pansu, M., Drevon, J.J., Ounane, S.M. * 436 xxxii SVI-CP-03 Leguminous and cereal associations in the Sudano-sahelian agro-systems of Burkina Faso: agronomic and socio-economic determinants. Zongo, K.F.*, Hien, E., Drevon, J.J., Nacro, H.B. 438 SVI-CP-04 Emisión del gas de efecto invernadero óxido nitroso por nódulos de soja. * Tortosa, G., Bedmar, E.J., Mesa, S., Delgado, M.J. 440 SVI-CP-05 Development of biofertilisers and biocontrol agents with autochthonous microorganisms in Spain and Dominican Republic. Mulas, D., Díaz Alcántara, C., Barquero, M., Marcano, I., Urbano, B., Terrón, A., * Velázquez, E., González-Andrés, F. 442 SVI-CP-06 Soybean yield and nodule occupancy as a function of yearly inoculation in the Brazilian Cerrados. Mendes, I.C.*, Reis Junior, F.B., Hungria, M. 444 SVI-CP-07 Recuperação e sobrevivência de Bradyrhizobium em sementes de soja tratadas com fungicidas e inseticidas Ferreira, E.*, Nogueira, M.A., Fukami, J., Gundi, J.S., Terassi, F.S., Conceição, R., Hungria, M. 446 SVI-CP-08 Influence of nitrate fertilization on Hg uptake and oxidative stress parameters in alfalfa plants cultivated in two different Hg-polluted soils. Carrasco-Gil, S., Aguareles, A., Leralta, D., Sobrino-Plata, J.*, Millán, R., Hernández, L.E. 448 SVI-CP-09 Gluconacetobacter diazotrophicus enhances growth in initial stages of Ilex paraguariensis development. Dos Santos, K . C. G . , Lopez, D.S.H., Perez, L.M., Roberti, L., Figueredo, I.E., Schmid, P.G., Peres, C.K., Rojas, C.A.* 450 SVI-CP-10 Nuevas herramientas metodológicas para la evaluación de inoculantes destinados al tratamiento de leguminosas. Penna, C.*, Massa, R., Cabrini, P., Scarabel, D., de Araujo, S.C., Cassan, F. 452 SVI-CP-11 Selección de cepas de rhizobia tolerantes a mercurio por su capacidad de bioacumulación y su efectividad en leguminosas arbustivas. Ruiz-Díez, B., Fajardo, S., Quiñones, M.A., López, M.A., Higueras, P., FernándezPascual, M.* 454 SVI-CP-12 Genetically modified rhizobia (GMR) for phytostabilization of copper in polluted soils. * Pajuelo, E. , Delgadillo, P., Doukkali, J., Pérez-Palacios, B., Caviedes, M.A., RodríguezLlorente, I.D. 456 SVI-CP-13 Utilización de biorrollos vegetales en cultivos de olivar para la reducción de las pérdidas de nitratos por escorrentía. * Pesciaroli, C. , Rodríguez García, E., Rodelas, B., Contreras-Medrano, V., GarcíaMartínez, F.J., G onzález-Martínez, J., González-López, J., Osorio Robles, F. 458 SVI-CP-14 Soybean Biological Nitrogen Fixation in Brazil, inoculant quality 2010- 2011. Beneduzi, A.*, Bangel, E.V., Alvarenga, S.M., Ferreira, S., Bertolo, F., Silva, G., Freire, J.R.J. 460 SVI-CP-15 Ensayo en diferentes cultivos y condiciones de un biofertilizante en base a Pseudomonas spp y Azospirillum spp. Rosas, S.*, Neiderhauser, M., Bettera, C., Bosch, E. 462 SVI-CP-16 Mistura polimérica como veículo na formulação de inoculante rizobiano para feijão-caupi. * Xavier, G.R. , Silva Júnior, E.B., Fernandes Júnior, P.I., Oliveira, P.J., Rumjanek, N.G., Boddey, R.M. 464 xxxiii SVI-CP-17 Substrate-based vs commercial inocula of arbuscular mycorrhizal fungi in lettuce. Garmendia, I., Mangas, V.J.* 466 SVI-CP-18 Algunas experiencias con inoculantes de hongos Glomeromycota y bacterias solubilizadoras de roca fosfórica en sistemas agrícolas venezolanos. * Toro, M. , Mora, E., López-Hernández, D. 468 SVI-CP-19 Plant-growth promotion and biocontrol properties of Colombian Streptomyces spp. isolates to control bacterial rice diseases. Suárez-Moreno, Z.R., Vergara, D.I., Vinchira-Villarraga, D.M., Castellanos, L., Ramos, * F.A., Degrassi, G., Guarnaccia, C., Venturi, V., Moreno-Sarmiento, N. 470 SVI-CP-20 Microbial consortia of mycorrhizal fungi and plant-growth promoting rhizobacteria: a tool to increase maize nutrient use efficiency. Correia, P.*, Carvalho, L., Meleiro, A.I., Reis, A., Costa, F., Delgado, M., Mesquita, L., Dores, J.M., Patanita, M., Castro Pinto, J., Varennes, A., Carolino, M., Cruz, C. 472 SVI-CP-21 Mycelial inoculant production of the ectomycorrhizal fungus Amanita caesarea. Daza, A., Camacho, M., Romero de la Osa, L., Santamaría, C.* 474 SVI-CP-22 Organic selenium forms from selenobacteria strains able to enhance selenium in wheat grain. * Mora, M.L. , Acuña, J.L., Durán, P., Demanet, R. 476 SVI-CP-23 Efecto de metales pesados en el crecimiento de bacterias aisladas del compost de lodos de depuradora. Vela Cano, M.*, Castellano Hinojosa, A., Fernández Vivas, A., Martínez Toledo, M.V. 478 SVI-CP-24 Capacidad de nodulación y fijación de nitrógeno de cepas nativas de Bradyrhizobium sp. en el cultivo de guandul (Cajanus cajan, L.) bajo ambiente controlado, en República Dominicana. Monegro, F., Sosa, R., Vicioso, A.F., Pimentel, A., González-Andrés, F., Díaz Alcántara, C.A.* 480 SVI-CP-25 Mycorrhizal inoculation in organic farming can improve fruit quality. Aguirrebengoa, M., Rivero, J., García, J.M., Azcón-Aguilar, C., López Raez, J.A., Pozo, M.J.* 482 SVI-CP-26 Efecto del herbicida MCPA y flumetsulam en bacterias nitrificantes en suelos volcánicos tratados con urea. Palma, G.*, Jorquera, M., Marileo, G., Briceño, G., Demanet, R., Mora, M.L. 484 SVI-CP-27 Produção de biomassa e fixação biológica de nitrogênio em Flemingia macrophylla. Abboud, A.C.S.*, Salmi, A.P. 486 SVI-CP-28 A construção de uma rede de promoção do benefício da fbn através dos inoculantes: uma proposta metodológica de parceria público privado. Amâncio, C.*, Amâncio, R., Garofolo, A.C., Xavier, G.R., Hungria, M., Cipriano, R., Balbinot Jr, A., Motta, R. 488 SVI-CP-29 FP7 REFERTIL Project: results on Input materials and Compost on Month 24. * * González Granados, I. , Segura Muñoz, I., Estrada de Luis, B.I., García García, R. 490 xxxiv Award “Antonio J. Palomares” Conference Simplicity turned out complicated: How FixK2, a key regulator of genes for symbiosis, is exquisitely controlled by different means. Mesa, S. 493 Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo S uperior de Investigaciones Científicas, Granada, Spain. Closing Conference Uma década de ouro se aproxima para a microbiologia do solo: expectativas da pesquisa, da indústria, dos agricultores e da sociedade. Mariangela Hungría. 497 Embrapa Soja, Londrina, Paraná, Brazil Indexes Author index 506 Participants 519 xxxv Opening Session Opening Session Quorum sensing: a double-edge sword in bacterial-plant interactions. Cámara, M.* Centre for Biomolecular Sciences, School of Life Sciences, University of Nottigham, Nottingham NG7 2RD. * [email protected] Quorum sensing: a universal signaling language Bacteria are highly social organisms capable of sophisticated co-operative behaviours mediated via chemical communication or ‘quorum sensing’ (QS). This is a gene regulatory mechanism usually defined as cell population density dependent regulation and is mediated via self-generated extracellular signal molecules (Figure 1) (Joint et al., 2007; von Bodman et al., 2008; Williams et al., 2007). These low molecular weight compounds or ‘autoinducers’ activate or repress QS target genes once a critical threshold concentration of signal has been reached. The key components of any QS ‘module’ are the QS signal synthase, the signal receptor and the signal molecule (Figure 2) ( Williams et al., 2007). -Butyrolactones N-Acylhomoserine Lactones Many bacteria possess several HO O interacting QS gene regulatory OH O CH modules employing multiple AI-2 2-Alkyl-4signal molecules. Such QS Cyclic Pep des Quinolones HO OH HO O systems are often subject to B O HO DSF O positive feedback CH Furanones CSP-1 (autoinduction) which at high C18-FAME -EMRLSKFFRDFILQRKKpopulation densities results in a marked increase in QS signal Gram-posi ve Gram-nega ve molecule (QSSM) production and consequently enhanced expression of the target gene(s) (Figure 2). Bacterial Farnesol Oxylipids cell-to-cell communication does not however only occur at high cell population Figure 1. Chemical structures of a range of quorum sensing signal molecules in eukaryotes and prokaryotes densities and QS has been adopted as a generic term for the description of any bacterial intercellular communication involving diffusible signal molecules. Bacteria employ a chemically diverse range of molecules as QSSMs some of which interact with receptors at the cell surface while others act following internalization ((Joint et al., 2007; Williams et al., 2007). R1 R2 O H O N H HO O H 3 O O R1 N H O R2 COOH O OH S S H Tyr 3 Ser Thr N H O Phe Me NH Asp Ile 2HN PROKARYOTES O O COOH H3CO EUKARYOTES Until the early 1990’s, N-acylhomoserine lactone (AHL)mediated regulation of gene expression was considered to be interesting but restricted to just a few esoteric bioluminescent marine bacteria related to Vibrio fischeri. However, the Gramnegative plant pathogen Erwinia carotovora was unexpectedly Figure 2: Typical prototype of quorum sensing cascade 2 Opening Session discovered to use N-3-oxohexanoyl-homoserine lactone (3OC6-HSL) to control both the production of a carbapenem antibiotic and virulence. Mutants which could not synthesize 3OC6-HSL were avirulent in a plant model of infection, and virulence could be restored in these mutants by the addition of synthetic 3OC6-HSL. This discovery was followed by a major surge of interest in the role of AHLs in bacterial gene expression. A family of AHL signal molecules has now been identified which varies predominantly in the presence or absence of an acyl chain substituent group at C3 (oxoor hydroxy-) and the length of the N-acyl side chain. In addition, genes encoding homologues of the V. fischeri LuxI and LuxR proteins have identify from many different Gram-negative associated with human, animals and plants. Since this initial discoveries, quorum sensing signal molecules of a novel nature have been identified not only in Gram negative and positive bacteria but also in eukaryotic organisms such as yeast (Figure 1) ( Williams et al., 2007) Quorum sensing and cross-talk QSSMs have been found in different environments from the human host during infection to marine biofilms providing evidence that QS is active in vivo situations. These molecules not only affect the expression the biology of the organisms that make them but can also affect the behaviour of other organisms from different species and kingdom. Figure 3 illustrates some examples of crosscommunication. Cross-talk bacteria-bacteria While most investigations of QS signal transduction pathways have focused on the response of the producer to its own signal, in natural environments and in mixed infections, QS signal molecules are likely impact on the behavior of other microorganisms which ei ther produce similar signals or very different signals. In the latter case, the P. aeruginosa QS signals 3-oxo-C12-HSL has been shown to inhibit the growth of Staphylococcus aureus at concentrations above 30 µM whereas at sub-growth 3 Opening Session inhibitory concentrations inhibited the expression of the staphyloccal agr-mediated quorum sensing system, enhancing the expression of colonisation factors but switching off the production of host-damaging toxins (Qazi et al., 2006). Cross-talk bacteria-yeast Cross-communication has also been found between bacteria and yeast using QSSMs. One example is the interactions observed between P. aeruginosa and Candida albicans which can co-exist in certain environments The QSSM 3-oxo-C12-HSL from P. aeruginosa can suppres filamentation in C. albicans without affecting fungal growth whereas the sesquiterpene farnesol used as a QSSM in C. albicans can interfere with QS in P. aeruginosa (Cugini et al., 2007; Cugini et al., 2010). Current research is looking at how wide spread these mechanisms of cross-talk between eukaryotes and prokaryote using QSSMs are. Cross-talk bacteria eukaryotic cells A significant amount of work has shown that QSSM molecules can have a serious impact in the mammalian host by altering immune responses, producing strong cardiovascular effects resulting in the potential increase in blood flow to the site of infection and disrupting tight cell junction to enable invasion. This multiple impact facilitate the survival of pathogenic bacteria under hostile environment of the host (Williams and Cámara, 2009). Cross-talk between bacteria and plants It has been demonstrated that QS is required for the successful colonisation of plant hosts by bacteria. The best studied QSSMs in this relationship are AHLs. In plantassociated Gram-n egative bacteria, AHLs are used by symbiotic, pathogenic, and biological control strains to regulate a wide range of phenotypes including virulence, rhizosphere competence, conjugation, the secretion of hydrolytic enzymes, and the production of antimicrobial secondary metabolites. Very importantly, AHLs play a key role in bacterial-plant cross-signaling, and some plants have been shown to be able to reprogram gene expression in the presence of these bacterial molecules. In general AHLs with short N-acyl chains can promote plant growth by inducing a change in the hormonal balance between indole acetic acid and cytokinin whereas some AHLs with longer N-acyl chain tend to modulate root development and root hair formation although the mechanisms behind this are still not well understood. Finally some AHLs with long N-acyl chains have been shown to induce resistance against microbial pathogens. Interestingly, plants have develop the ability to interfere with AHL-QS systems by producing small molecules which can interfere with bacterial QS-mediated mechanisms (Gonzalez and Venturi, 2013). The close relationship between QS producers and the plants reach such level that a new class of AHL signals have been found which require the plant precursors for its biosynthesis. These AHLs are pcoumaroyl-AHLs. The precursor molecule p-coumarate is not synthesized by the bacterium but is provided by the plant and it can be found in the plant environment suggesting that bacteria can only synthesise these signals in the proximity of the plant (Schaefer et al., 2008). Cross-talk bacteria-algae The green seaweed Ulva is the most common macro-alga contributing to biofouling of man-made surfaces throughout the world with enormous reproductive potential releasing vast quantities of motile zoospores from each thallus. Zoospores were shown 4 Opening Session to preferentially settle on top of AHL-producing bacterial biofilms and hence using these signal molecules as a cue for the selection of sites for attachment (Joint et al., 2002). The mechanisms responsible for this seems to involve a process of chemokinesis which affects the swimming behaviour of the zoospores through the induction of an increase in intracellular Ca2+ levels. Interestingly, bacteria producing AHL degrading enzyme within microbial biofilms on rocks can interfere with the settlement of zoospores in mixed biofilms showing a direct correlation between the levels of AHLs, degradation activity and spore settlement (Tait et al., 2009). Similarly, reduction of AHL stability by the pH of sea water can affect spore settlement (Tait et al., 2005). Very recently it has been demonstrated that epiphytic bacteria associated with Ulva can interfere with the germination and growth of Ulva zoospores and that this interference is mediated via the production of AHLs by these bacteria, revealing an additional role for AHLs per se in the interactive relationships between marine bacteria and Ulva zoospores (Twigg et al., 2013). In conclussion, quorum sensing is a very versatile bacterial language with an important impact on the relationship between bacterial species and between bacterial communitites and their hosts. Whilst these relationship can sometimes result in very postive symbiosis, in some instances it can have a negative efffect on the host. Understanding better this interactions will be paramount in may areas of research including agriculture. REFERENCES Cugini, C., et al. (2007). Mol. Microbiol. 65: 896-906. Cugini, C., et al. (2010). Microbiology 156: 3096-3107. Gonzalez, J.F., and Venturi, V. (2013). Trends Plant Sci, 18: 167-174. Joint, I., et al. (2007). Philos. T. Roy. Soc. B 362: 1115-1117. Joint, I., et al. (2002). Science 298: 1207. Qazi, S., et al. (2006). Infect. Immun. 74: 910-919. Schaefer, A.L., et al. (2008). Nature 454: 595-599. Tait, K., et al. (2005). Environ. Microbiol. 7: 229-240. Tait, K., et al. (2009). Environ. Microbiol. 11: 1792-1802. Twigg, M., et al. (2013). Environ. Microbiol. (in press). von Bodman, S.B., et al. (2008). J. Bacteriol. 190: 4377-4391. Williams, P., and Cámara, M. (2009). Curr. Opin. Microbiol. 12: 182-191. Williams, P., et al. (2007). Philos. T. Roy. Soc. B 362: 1119-1134. 5 Session I Ecology, diversity and evolution microorganisms beneficial to plants of Session I SI-P-1 Diversidad y evolución de las comunidades microbianas en la rizosfera tras un incendio forestal. Fernández-López, M.1*, Fernández-González, A.J.1, Cobo-Díaz, F.J.1, Villadas, P.J.1, Tringe, S.G.2, Toro, N.1 1 Departamento de Microbiología del Suelo, Estación Experimental del Zaidín, CSIC, calle Profesor Albareda 1, 18008 Granada, España. 2 Joint Genome Institute, Dept. of Energy, Walnut Creek, Califo rnia, USA. * [email protected] Ecología microbiana y nuevas tecnologías de secuenciación masiva. Si como científicos pioneros de los estudios de diversidad microbiana podemos citar a Louis Pasteur y a Robert Koch, en los de ecología microbiana no podemos dejar de mencionar a Vladimir I. Vernadsky, Segei Winogradsky y Martinus Beijerinck. El primero de ellos definió los conceptos de ciclos biogeoquímicos y el de biosfera, el segundo trabajó en los ciclos biogeoquímicos de S, Fe y N, así como desarrolló el concepto de quimioautotrofía, y por su parte Beijerinck ideó los cultivos de enriquecimiento, trabajó en el ciclo del N y aisló los microorganismos responsables de este proceso. A pesar de que sus inicios están en el siglo XIX, el desarrollo real de la ecología microbiana se ha producido gracias a las técnicas de biología molecular. Así no podemos olvidar los trabajos de Carl Woese en la década de los años 70 del siglo XX, a partir de los que se estableció el gen 16S rRNA como herramienta con validez taxonómica y filogenética para identificar microorganismos. La introducción de estas técnicas moleculares ha permitido realizar estimaciones realistas sobre la diversidad de microorganismos presentes en el suelo, constituyendo un tópico citar lo mucho que desconocemos. Así se ha citado ampliamente que los microorganismos constituyen la mayor biodiversidad del planeta (Gans et al., 2005; Rusch et al., 2007) y comprenden la mayor parte de la biomasa de los organismos que lo habitan con 10 3-104 kg de masa microbiana por hectárea de superficie del suelo (Fierer et al., 2007), que sólo hemos sido capaces de cultivar el 1 % del total de microorganismos; o que en 1 gramo de suelo podemos encontrar entre 103 y 104 especies diferentes, representando una abundancia total de 109 procariotas por gramo de suelo (Gans et al., 2005, Torsvik et al., 1990). De lo expuesto anteriormente se deduce que el que un microorganismo pueda ser cultivado es más una excepción que la regla. Todo esto nos lleva a concluir que debemos aplicar técnicas moleculares a los estudios de las comunidades microbianas, si queremos tener una visión realista de lo que está ocurriendo en el medio ambiente. En este contexto, Handelsman y colaboradores acuñaron el término “metagenoma” para definir el conjunto de los genomas de todos los organismos presentes en un determinado hábitat. En su primera acepción, se consideró el metagenoma como el resultado de extraer el ADN de todo un ecosistema para clonarlo en complejas genotecas de ADN recombinante (librerías de ADN ambiental). Posteriormente estas genotecas se sometían a protocolos de búsqueda de compuestos de interés o de identificación de genes que aporten información sobre las rutas metabólicas activas en el ecosistema, el funcionamiento potencial del ecosistema (Hall, 2007). Estas aproximaciones metodológicas también se han aplicado al estudio de la rizosfera tanto de plantas con interés agronómico como forestal (Fernández-López et al., 2013). Hasta ahora, el éxito de estos métodos metagenómicos dependía en gran medida de la capacidad de clonar fragmentos de ADN de suelo suficientemente grandes en los vectores apropiados y de su capacidad para expresar la información genética en hospedadores heterólogos de forma eficiente (Rondon et al., 2000). 7 Session I SI-P-1 Sin embargo el desarrollo de las tecnologías de secuenciación masiva (NGS) a partir del año 2005 nos permite obviar la construcción de estas genotecas para pasar a secuenciar directamente el ADN ambiental extraído del suelo, es decir sin necesidad de clonarlo en ningún tipo de vector. El desarrollo vertiginoso de estas tecnologías de secuenciación masiva es el que nos ha llevado a una nueva era, permitiendo la aparición de aproximaciones de tipo post-genómico (Medini et al., 2008). Al realizar la secuenciación de forma directa se evitan los sesgos motivados por el propio proceso de clonación y obtenemos una visión más realista de los procesos que ocurren en el suelo. Estos nuevos métodos de secuenciación basados en distintas técnicas y puestos a punto por diferentes compañías, tienen en común su bajo coste y su alto rendimiento: el gran número de lecturas que ofrecen en un periodo corto de tiempo. Así la pirosecuenciación 454 Titanium Plus de Roche permite obtener 1 millón de lecturas de 800 bp, la secuenciación por síntesis de Illumina nos ofrece 1.500 millones de lecturas de 2 x 100 bp o la secuenciación por ligación SOLID rinde 171 millones de lecturas de 70 bp (Zhang et al., 2011). Una alternativa es la aplicación de estas técnicas a la secuenciación de amplicones de genes de interés, como puede ser el 16S rRNA, de esta manera se puede conocer la diversidad presente en las comunidades microbianas con una cobertura superior al 90 %. Por otra parte las tecnologías NGS han permitido que se empiece a desarrollar la “metatranscriptómica”. En esencia, esta aproximación consiste en la extracción del ARN total presente en el suelo, para posteriormente sintetizar un cDNA mediante el empleo de oligonucleótidos cebadores al azar y proceder a su secuenciación mediante cualquiera de las técnicas NGS. A diferencia de la metagenómica que nos ofrece una visión del catálogo general de especies y de procesos funcionales que pueden estar presentes y desarrollarse en un ecosistema, la metatranscriptómica, nos ofrece una visión de las especies y procesos metabólicos que están activos. Por tanto este doble abordaje nos permite describir tanto el potencial de cada ecosistema (metagenoma), como los organismos procariotas y procesos que actúan en un momento determinado (metatranscriptoma). Esta aproximación se ha aplicado para el estudio de distintos ecosistemas incluyendo el océano abierto o columnas de agua del mismo (Stewart et al., 2010), suelos, suelos agrícolas, compost, muestras fecales y orales de mamíferos, y aguas residuales (McGrath et al., 2008), así como en suelos forestales (Baldrian et al., 2012). Bosque mediterráneo e incendios forestales. Donde las condiciones ambientales lo permiten, los bosques son la etapa clímax en la sucesión de un ecosistema, presentando al mismo tiempo la menor diversidad de especies y la mayor especialización de las mismas. Esto hace que pequeños cambios ambientales puedan tener un gran impacto sobre estas formaciones. En los ecosistemas de tipo mediterráneo las comunidades vegetales están expuestas a unas condiciones climáticas que pueden considerarse como extremas ya que alternan periodos de heladas invernales, sequía y alta temperatura estival, y ocasionales lluvias torrenciales. Además en un escenario de cambio climático como el que se prevé para la zona mediterránea, con una temperatura mayor y una precipitación menor, catástrofes como los incendios forestales se verán incrementadas. En España, la superficie afectada por incendios forestales alcanzó una media de 127.209 ha al año en el periodo comprendido entre los años 2000-09, incluyendo en esta superficie bosques, matorral y formaciones herbáceas (http://www.magrama.gob.es/es/biodiversidad/temas/defensa -contra-incendiosforestales/estadisticas -de-incendios-forestales/). En la cuenca mediterránea, la intervención humana promoviendo el pastoreo, las talas o las quemas controladas ha 8 Session I SI-P-1 dado lugar al desarrollo de una vegetación específica y adaptada a estas condiciones. Entre esta vegetación podemos destacar a la encina, Quercus ilex subsp. ballota, que tiene capacidad de rebrotar desde la raíz tras un incendio forestal. La encina es la quercínea que ocupa una mayor extensión en España (96.579 Km2), aunque sus bosques son muy escasos ya que en su mayoría han sido manejados hasta la actual fisonomía de dehesas (Felicísimo et al., 2011). Se trata de un árbol o arbusto perenne ampliamente distribuido por toda la región mediterránea, apareciendo desde el nivel del mar hasta los 1.700 m de altitud en exposiciones no umbrías, con climas desde frío semiárido hasta mediterráneo húmedo templado, aunque prevalece en zonas áridas con precipitaciones estivales bajas y máximas elevadas, siendo indiferente al tipo de suelo. En el mes de Septiembre del año 2005 se produjo un incendio forestal en el Parque Nacional de Sierra Nevada, que afectó 3.416,74 has. (Gómez-Zotano et al., 2010). Aunque la cubierta vegetal más afectada en este incendio fue la de matorral, hay que señalar que la quema de vegetación forestal constituyó casi el 21% del total, estando localizada principalmente en los términos municipales de Lanjarón y Lecrín. El encinar afectado supuso el 1,56% del total de la vegetación, con una superficie de 53,41 has; pero si sumamos los distintos tipos de cubiertas vegetales (tomillar, espinal, piornal,…) afectadas que presentaban encinas en su composición, este porcentaje se eleva al 12,06% lo que se traduce en una superficie de 412 has quemadas (Gómez-Zotano et al., 2010). Microbiota rizosferica tras un indendio forestal. Las altas temperaturas alcanzadas en un incendio forestal inducen cambios inmediatos en las propiedades físicas y químicas del suelo, cuya intensidad depende de la gravedad del incendio y del tipo de suelo (Certini 2005). Estos cambios más las condiciones ambientales post-incendio determinarán los cambios de las características biológicas del suelo como la biomasa y la actividad microbiana (Wang et al., 2012). Además se produce una alteración de la estructura de las comunidades microbianas con un incremento de las bacterias formadoras de esporas más resistentes a las altas temperaturas (Yeager et al., 2005; Bárcenas-Moreno et al., 2011). Hay que tener en cuenta que después de un incendio forestal, de forma efímera, hay una ganancia neta de Nitrógeno (N) disuelto. Así se ha concluido que en las zonas mediterráneas hay un incremento del Carbono orgánico del suelo y del N total después del incendio, pero no de la mineralización de N (Wang et al., 2012), por lo que se incrementa la posibilidad de pérdidas de este elemento debido a lixiviación y a una menor actividad. Las bacterias del suelo son componentes esenciales del ciclo biogeoquímico del N, pero han sido poco estudiadas en las condiciones del suelo posteriores a un incendio forestal (Goodale and Aber, 2001). En nuestro grupo estamos trabajando en estas condiciones en la rizosfera de especies forestales, en concreto en encinas. Para estudiar la diversidad y la evolución de las comunidades microbianas hemos optado por la aplicación de técnicas de secuenciación masiva, tanto 454 Titanium como Illumina. Así, la rizosfera de encinas afectadas y no afectas por el incendio forestal del año 2005, la hemos muestreado 3 y 6 años después. El ADN total obtenido del suelo ha sido tratado con 2 aproximaciones: a) Amplificación del gen 16S rRNA y pirosecuenciación para determinar la diversidad microbiana de forma exhaustiva. b) Secuenciación directa del ADN mediante el sistema Illumina para determinar las variaciones del ciclo del N por efecto del incendio. El detalle y las implicaciones de estos resultados se discutirá en la presentación. 9 Session I SI-P-1 AGRADECIMIENTOS Este trabajo ha sido financiado por el Organismo Autónomo Parques Nacionales del Ministerio de Medio Ambiente y Medio Rural y Marino (proyecto OAPN 21/2007), por la Junta de Andalucía (proyecto de excelencia P08-CVI-03549) y por el programa Consolider-Ingenio (CSD 2009-00006). AJFG es beneficiario de una beca FPU del Ministerio de Innovación y Ciencia, y JFCD de una beca predoctoral de la Junta de Andalucía. BIBLIOGRAFÍA Baldrian, P., et al. (2012). ISME J. 6: 248-258. Bárcenas-Moreno, G., et al. (2011). Biol. Fert. Soils 47: 261-272. Certini, G. (2005) Oecologia 143: 1-10. Felicísimo, Á.M., et al. (2011). Impactos, vulnerabilidad y adaptación al cambio climático de la biodiversidad española. 1. Flora y vegetación. Ministerio de Medio Ambiente y Medio Rural y Marino, Madrid. Fernández-López, M., et al. (2013). En: Encyclopedia of Metagenomics. K.E. Nelson, S. Highlander, F. Rodríguez-Valera y B.A. White (eds.). http://www.springerreference.com DOI: 10.1007/SpringerReference_304477. Fierer, N., M. et al. (2007). Appl. Environ. Microbiol. 73: 7059-7066 Gans J, M. et al. (2005). Science 309: 1387-1390. Gómez-Zotano, J., et al. (2005). Cuadernos Geográficos 37: 205-214. Goodale, C.L., and J.D. Aber (2001) Ecol. Appl. 11: 253-267. Hall, N. (2007) J. Exp. Biol. 209: 1518-1525. McGrath, et al. (2008).J. Microb. Meth. 75: 172-176. Medini D, D. et al. (2008). Nature Rev. Microbiol. 6: 419-430 Rondon M.R., P.R. et al. (2000). Appl Environ Microbiol 66:2541-2547. Rusch, D.B., et al. (2007). PLoS Biol. 5: e77. Stewart, F.J., et al. (2010). ISME J 4: 896-907. Torsvik V, et al. (1990). Appl. Environ. Microbiol. 56: 782-787. Wang, Q., et al.. (2012). Forest Ecol. Manag. 271: 91-97. Yeager, C.M., et al. (2005). Appl. Environ. Microbiol. 71: 2713-2722. Zhang, J., et al. (2011). J. Genet. Genomics 38: 95-109. 10 Session I SI-P-2 Species and symbiovars within Mesorhizobium: diversity and host range. Laranjo, M.1, 2*, Oliveira, S.1 1 ICAAM-Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Portugal IIFA-Instituto de Investigação e Formação Avançada, Universidade de Évora, Portugal * [email protected] 2 ABSTRACT The genus Mesorhizobium includes species able to nodulate a wide variety of legumes. Our aim was to investigate the evolutionary relationships among mesorhizobia species that nodulate the same host plant. A multilocus sequence analysis (MLSA) phylogeny of the Mesorhizobium genus showed a higher resolution compared to the 16S rRNA gene phylogeny. The phylogenies of symbiosis genes, such as nodC, are not congruent with the phylogenies based on core genes, reflecting rhizobial host range and legume phylogeny. This suggests that Mesorhizobium species are able to exchange symbiosis genes by lateral gene transfer (LGT), thus acquiring the ability to nodulate a new host. Phylogenetic analyses of the Mesorhizobium genus based on core or accessory genes reveal complex evolutionary relationships and a high genomic plasticity, rendering the Mesorhizobium genus as a good model to investigate rhizobia genome evolution and adaptation to different host plants. INTRODUCTION Rhizobia are the root nodule bacterial symbionts of legumes, which fix atmospheric N2 in a process known as biological nitrogen fixation. All described rhizobial species belong to ten genera of Alpha- and three genera of Betaproteobacteria. Mesorhizobia nodulate a wide variety of legumes, such as chickpea (Sprent, 2009). There are 25 Mesorhizobium species. Additionally, five more species are listed in “IJSEM-Papers in Press”. This genus includes a non-rhizobium-M. thiogangeticum (Ghosh & Roy, 2006). Biovars have been described in bacteria as a group of strains distinguishable from others of the same species on the basis of physiological or biochemical characters. In rhizobia, biovars have been used to distinguish symbiotically distinct groups within a single species (Rogel et al., 2011). These symbiovars may belong to different species due to lateral transfer of symbiosis genes (Laranjo et al., 2008). A Mesorhizobium MLSA phylogeny based on the 16S rRNA gene, the ITS region (16S–23S rRNA) and five core genes: atpD (ATP synthase F1, subunit), dnaJ (DnaJ chaperone), glnA (glutamine synthetase I), gyrB (DNA gyrase subunit) and recA (recombinase A) is presented. Moreover, phylogeny of the symbiosis gene nodC (Nacetylglucosaminylt ransferase) is compared with a legume phylogeny based on the chloroplast maturase K (matK) gene (Wojciechowski et al., 2004). MATERIAL AND METHODS Phylogenetic analyses were conducted with MEGA5 using the maximum likelihood method. The best-fitting evolutionary model of nucleotide substitutions was determined. A bootstrap confidence analysis was performed on 100 replicates. Azorhizobium caulinodans was used as outgroup. RESULTS AND DISCUSSION The 16S rRNA gene based phylogeny including all 25 species is shown in Figure 1. Four species groups (A to D) can be considered and at least two species have an 11 Session I SI-P-2 undefined position, namely M. albiziae and M. chacoense. M. thiogangeticum has a clearly separate position from other mesorhizobia. Mesorhizobium mediterraneum UPM-Ca36 93 Mesorhizobium temperatum SDW 018 Mesorhizobium muleiense CCBAU 83963 47 86 Mesorhizobium robiniae CCNWYC 115 Mesorhizobium caraganae CCBAU 11299 70 Mesorhizobium gobiense CCBAU 83330 A Mesorhizobium metallidurans STM 2683T 59 Mesorhizobium tarimense CCBAU 83306 Mesorhizobium tianshanense A-1BS Mesorhizobium tamadayense Ala-3 41 70 Mesorhizobium huakuii IFO 15243 Mesorhizobium opportunistum WSM2075 Mesorhizobium amorphae ACCC19665 21 61 Mesorhizobium septentrionale SDW 014 B Mesorhizobium plurifarium LMG 11892 95 Mesorhizobium silamurunense CCBAU 01550 Mesorhizobium albiziae CCBAU 61158 95 96 Mesorhizobium australicum WSM2073 Mesorhizobium shangrilense CCBAU 65327 100 90 Mesorhizobium ciceri UPM-Ca7 C Mesorhizobium loti LMG 6125 Mesorhizobium chacoense PR5 Mesorhizobium alhagi CCNWXJ12-2 40 100 Mesorhizobium camelthorni CCNWXJ40-4 D Mesorhizobium thiogangeticum SJT Azorhizobium caulinodans ORS 571 0.01 Figure 1. Mesorhizobium maximum likelihood phylogeny of the 16S rRNA gene (25 species). Tamura 3pa rameter model with a discrete Gamma distribution and invariant sites was used. The 16S rRNA gene phylogeny was compared to a MLSA phylogram of seven loci (16S rRNA, ITS, atpD, dnaJ, glnA, gyrB and recA) (Figure 2). 46 Mesorhizobium tarimense 100 Mesorhizobium tianshanense 100 48 100 73 43 Mesorhizobium gobiense Mesorhizobium mediterraneum Mesorhizobium temperatum Mesorhizobium metallidurans Mesorhizobium amorphae 100 Mesorhizobium septentrionale 74 Mesorhizobium caraganae 100 Mesorhizobium ciceri Mesorhizobium loti 100 56 60 Mesorhizobium australicum Mesorhizobium huakuii Mesorhizobium opportunistum Mesorhizobium plurifarium Mesorhizobium chacoense Azorhizobium caulinodans 0.5 Figure 2. MLSA maximum likelihood phylogeny for the Mesorhizobium genus (15 species). Tamura 3para meter model with a discrete Gamma distribution and invariant sites was used. The resulting MLSA phylogeny with a subgroup of 15 species is highly resolved, with all branches having high bootstrap support (Laranjo et al., 2012). Although most Mesorhizobium species show relatively low sequence divergence at core loci, there are certain groupings of species that are consistently recovered both in the 16S rRNA gene phylogeny and in the MLSA phylogeny, namely M. mediterraneum/M. 12 Session I SI-P-2 temperatum, M. gobiense/M. tarimense/M. tianshanense, M. loti/M. ciceri, M. amorphae/M. septentrionale, as had been observed before between phylogenies of different core loci (Alexandre et al., 2008; Laranjo et al., 2012). This suggests that there is little horizontal gene transfer of core loci between these groupings. However, it is not clear that every species within each group is genetically isolated. On the other hand, there are some species within the Mesorhizobium clade which have a poorly resolved phylogenetic position, such as M. albiziae, M. chacoense and M. thiogangeticum, among others. Contrary to Rhizobium and Sinorhizobium, which carry symbiosis plasmids, Bradyrhizobium and Mesorhizobium species harbour their symbiosis genes clustered in the chromosome. In Mesorhizobium strains symbiosis genes are most commonly located in chromosomal symbiosis islands (Kaneko et al., 2000; Nandasena et al., 2007) and rarely in plasmids (Wang et al., 1999; Zhang et al., 2000). These islands have been shown to be mobile in Mesorhizobium, but not in Bradyrhizobium. 100 100 Mesorhizobium australicum Mesorhizobium opportunistum Mesorhizobium metallidurans 77 Mesorhizobium ciceri 36 99 100 Cicer arietinum Mesorhizobium mediterraneum Mesorhizobium loti 26 Lotus Mesorhizobium tarimense Mesorhizobium amorphae 14 44 Biserrula pelecinus Mesorhizobium chacoense Amorpha fruticosa Prosopis Mesorhizobium temperatum 13 97 Mesorhizobium septentrionale 86 Mesorhizobium gobiense 91 Mesorhizobium tianshanense Glycyrrhiza Astragalus Mesorhizobium caraganae Mesorhizobium huakuii Azorhizobium caulinodans Sesbania 0.1 Figure 3. Mesorhizobium genus maximum likelihood nodC based phylogeny (15 species). Tamura 3parame ter model with invariant sites was used. Legume hosts are indicated on the right-hand side. nifH (data not shown) (Laranjo et al., 2008) and nodC (Figure 3) phylogenies are congruent and evidence a close relationship among rhizobial strains nodulating the same host, as suggested before (Laguerre et al., 2001). LGT of symbiosis genes between the different species in the soil seems to be the most plausible hypothesis to explain incongruence between phylogenies based on symbiosis (e. g. nodC) and core genes (e. g. 16S rRNA). Furthermore, phylogeny of symbiosis genes is compared to host legume phylogeny (Figure 4) based on the chloroplast encoded maturase K gene (matK) (Wojciechowski et al., 2004). 13 Session I SI-P-2 100 100 Astragalus americanus Biserrula pelecinus 41 Oxytropis deflexa Cicer arietinum 68 Alhagi maurorum 100 Caragana arborescens 88 100 Glycyrrhiza lepidota Robinia pseudoacacia 100 Sesbania vesicaria 96 Anthyllis vulneraria 36 100 100 Lotus japonicus Clitoria ternatea Amorpha fruticosa Prosopis glandulosa 100 79 Acacia greggii Albizia julibrissin Oxalis tomentosa 0.02 Figure 4. Maximum likelihood legume phylogeny based on the matK gene (16 legume species). General time reversible (GTR) model with a discrete Gamma distribution was used. In conclusion, rhizobia with different chromosomal backgrounds may carry similar symbiosis genes, explaining how the same legume host is nodulated by several different rhizobium species. However, it is possible that the transfer of symbiosis genes is limited to certain donorrecipient species combinations, which would mean that certain core genome features are needed to support particular symbiovars (Laranjo et al., 2012). ACKNOWLEGEMENTS This work was supported by project (PTDC/BIA/EVF/4158/2012) from Fundação para a Ciência e a Tecnologia (FCT). This work is funded by FEDER Funds through the Operational Programme for Competitiveness Factors - COMPETE and National Funds through FCT under the Strategic Project PEstC /AGR/UI0115/2011. M. Laranjo acknowledges a Post-Doc fellowship (SFRH/BPD/27008/2006) from FCT. REFERENCES Alexandre, A., et al. (2008). Int. J. Syst. Evol. Microbiol. 58: 2839-2849. Ghosh, W., and Roy, P. (2006). Int. J. Syst. Evol. Microbiol. 56: 91-97. Kaneko, T., et al. (2000). DNA Res. 7: 331-338. Laguerre, G., et al. (2001). Microbiology 147: 981-993. Laranjo, M., et al. (2008). FEMS Microbiol. Ecol. 66: 391-400. Laranjo, M., et al. (2012). Syst. Appl. Microbiol. 35: 359-367. Nandasena, K.G., et al. (2007). Int. J. Syst. Evol. Microbiol. 57: 1041-1045. Rogel, M.A., et al. (2011). Syst. Appl. Microbiol. 34: 96-104. Sprent, J.I., (2009). Legume Nodulation: A Global Perspective. Oxford, UK: Wiley-Blackwell. Wang, E.T. et al. (1999). Int. J. Syst. Bacteriol. 49: 51-65. Wojciechowski, M.F., et al. (2004). Am. J. Bot. 91, 1846-1862. Zhang, X. X. et al. (2000). Appl. Environ. Microbiol. 66: 2988-2995. 14 Session I SI-CO-1 Diversidad de hongos micorrizógenos arbusculares bajo situaciones contrastantes de fertilización fosfatada. García, S., Rodríguez Blanco, A*, Pezzani, F. Facultad de Agronomía. Universidad de la República. Av. Garzón 780. Montevideo. Uruguay. * [email protected] RESUMEN Los pastizales de Uruguay presentan bajos niveles de P por lo que son frecuentes los mejoramientos extensivos que implican fertilización fosfatada. Este trabajo tiene como objetivo determinar el efecto de la fertilización fosfatada sobre la diversidad de hongos micorrizógenos arbusculares (HMA) asociados a dos especies de gramíneas de los pastizales de Uruguay. El trabajo se basa en un ensayo con dos niveles de fertilización a largo plazo y parcelas de pastizal natural (PN) sin fertilizar. En base a resultados de composición florística y colonización micorrícica se seleccionaron las especies nativas Paspalum dilatatum y Coelorhachis selloana para el estudio de diversidad de las comunidades de HMA utilizando la técnica Terminal Restriction Fragment Length Polymorphism (T-RFLP). Se presentan resultados correspondientes al primer muestreo (invierno). Se detectaron diferencias significativas en los índices de riqueza y de diversidad entre los niveles de fertilización fosfatada en las comunidades de HMA asociadas a C. selloana. El análisis de cluster demostró una similitud entre todas las comunidades mayor al 55% y formó 2 grupos: uno incluyó las comunidades asociadas a P. dilatatum fertilizadas y sin fertilizar y a C. selloana sin fertilización, mientras que en el otro cluster se separaron las comunidades asociadas a C. selloana con fertilización fosfatada. Estos resultados evidenciarían que los efectos de la fertilización sobre la diversidad de HMA que colonizan las plantas serían dependientes de la especie vegetal. INTRODUCCIÓN Uno de los beneficios más importantes que recibe la planta por los hongos micorrizógenos arbusculares (HMA) es que estos aumentan la absorción de nutrientes, principalmente P que se encuentra poco disponible para las plantas (Smith and Read, 2008). Los pastizales de Uruguay presentan bajos niveles de P por lo que son frecuentes los “mejoramientos extensivos” de los pastizales que implican fertilización fosfatada (Hernández et al., 1995). Según un relevamiento florístico realizado en nuestro sitio de estudio, luego de 10 años de aplicada la fertilización con P, la especie Paspalum dilatatum no modificó su frecuencia relativa en la comunidad en relación al incremento de P, mientras que Coelorhachis selloana disminuyó su frecuencia relativa, viéndose aumentadas las frecuencias de especies invasoras como Cynodon dactylon y Lolium multiflorum (Pezzani et al., 2012) De acuerdo a la bibliografía, la fertilización fosfatada puede tener efectos negativos sobre la colonización micorrícica arbuscular (Covacevich et al., 1995; Aguilar et al., 2004). Para evaluar esto, durante los años 2011 y 2012, se realizaron muestreos estacionales de varias especies de plantas y suelos. Los resultados de colonización radicular por HMA muestran diferencias entre las especies nativas e invasoras. P. dilatatum presentó altos porcentajes de colonización por HMA en todos los tratamientos, aunque los mayores valores fueron observados en el PN y los menores en parcelas con fertilización alta. C. selloana presentó porcentajes menores de colonización por HMA en tratamientos con altos niveles de fertilización con P. El potencial micorrícico (esporas en el suelo) fue afectado negativamente por la fertilización fosfatada (Pezzani et al., 2012). Conocer la diversidad de los HMA en los pastizales, sería un aporte complementario al conocimiento sobre estas interacciones ya que no existen antecedentes sobre este tema en Uruguay. 15 Session I SI-CO-1 El objetivo de este trabajo es determinar el efecto de la fertilización fosfatada sobre la diversidad de hongos micorrizógenos arbusculares (HMA) asociados a dos especies de gramíneas nativas de los pastizales de Uruguay. MATERIAL Y MÉTODOS El trabajo se basa en un ensayo instalado en el año 1996 que consiste en dos niveles de fertilización (media: 30 kg ha -1 de P2O5 anuales y alta: 60 kg ha-1 de P2O5 anuales) y parcelas de pastizal natural (PN) sin fertilizar. Los tratamientos se realizaron en bloques al azar de 2 ha cada uno. Para el estudio de la diversidad de HMA que colonizan las plantas se seleccionaron las especies nativas P. dilatatum y C. selloana. Se realizaron dos muestreos de plantas (invierno y verano). Se extrajeron plantas de ambas especies de cada parcela, se formaron muestras compuestas de raíces y se extrajo el ADN. Para evaluar la diversidad se utilizó la técnica Terminal Restriction Fragment Length Polymorphism (T-RFLP) siguiendo la metodología descripta por Mummey and Rillig (2006). Se utilizaron los primers FLR3-FAM y FLR4, específicos para Glomeromycotas, y la enzima de restricción MboI. Los análisis multivariados se realizaron utilizando el programa PAST y se consideraron los picos cuya abundancia relativa fue >1%. RESULTADOS Y DISCUSIÓN Se presentan resultados correspondientes al primer muestreo. El número de TRFs (biotipos) en las comunidades de HMA varió entre 11 y 19. Las plantas presentaron 7 TRFs (biotipos) en común, mientras que otros TRFs fueron característicos de algunos tratamientos. En las comunidades de HMA asociadas a C. selloana se detectaron diferencias significativas entre los niveles de fertilización fosfatada tanto en el índice de riqueza como en los índices de diversidad, mientras que estas diferencias no se observaron en las comunidades asociadas a P. dilatatum. El análisis de cluster demostró una similitud entre todas las comunidades mayor al 55% y formó 2 grupos. Uno de los clusters incluyó las comunidades asociadas a plantas de P. dilatatum fertilizadas y sin fertilizar y a plantas de C. selloana sin fertilización, mientras que en el otro cluster se separaron las comunidades asociadas a C. selloana con fertilización fosfatada. Estos resultados estarían demostrando que la fertilización fosfatada en las plantas de C. selloana provocó un cambio en la estructura de las comunidades de HMA que las colonizan, mientras que ese cambio no se observó en P. dilatatum. Para profundizar y confirmar estos resultados se está analizando el segundo muestreo (verano). BIBLIOGRAFÍA Aguilar, C., et al. (2004). In: Memorias de la XX Reunión del grupo Técnico Regional del Cono Sur en el Mejoramiento y Utilización de los Recursos Forrajeros del Area Subtropical y Tropical-Grupo Campos. Salto, Uruguay. Pp. 281-282. Covacevich, F., et al. (1995). Ciencia del Suelo. 13: 47-51. Hernández, J., et al. (1995). Boletín de Investigaciones N°43. Facultad de Agronomía. Montevideo. Mummey, D.L., and Rillig, M.C. (2006). Plant Soil 288: 81-90. Pezzani F., et al. (2012). Resumen de la presentación oral en el VII Symposium Nacional y IV Reuni ón Iberoamericana de la Simbiosis Micorrízica. 27-30 mayo 2012. Xalapa. México. p. 115. Smith, S.E., Read, D.J (2008). Arbuscular Mycorrhizal. En: Mycorrhizal Simbiosis. Elsevier. 11-145 . 16 Session I SI-CO-2 Prospecting metal resistant plant-growth promoting rhizobacteria for rhizoremediation of metal contaminated estuaries using Spartina densiflora. Andrades-Moreno, L.1, del Castillo, I.2, Redondo-Gómez, S.1, Mesa, J., Caviedes, M.A.2, Pajuelo, E.2, Rodríguez-Llorente, I.D.2* 1 Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla. Sevilla , Spain. 2 Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla, c/ Profesor García González 2, 41012 Sevilla, Spain. * [email protected] ABSTRACT The total bacterial population in the rhizosphere of Spartina densiflora grown on the Tinto and Piedras river estuaries, with different levels of metal contamination, was analyzed by PCR-DGGE. 22 different cultivable bacterial strains were isolated from the rhizosphere of S. densiflora grown on the Tinto river estuary. 70% of the strains showed one or more PGP properties and high resistance to Cu. A bacterial consortium with PGP properties and high multiresistance to heavy metals was selected for further experiments. This consortium has probed to increase seed germination and to protect for fungus contamination. INTRODUCTION In the salt marshes of the joint estuary of Tinto and Odiel rivers (SW Spain), one of the most polluted areas by heavy metals in the world, S. densiflora grows over sediments with high concentrations of heavy metals (Sáinz et al., 2004). Cambrollé et al. (2008) found that heavy metals accumulated at different rates in S. densiflora tissues and around its roots, concluding that these species could be used for phytoremediaton (either phytoextraction or phytostabilization) of estuarine sediments. Although the importance of the rhizosphere community for plant development in contaminated soils has been recognized, nothing is known about the bacterial population that colonizes the root of S. densiflora grown in contaminated soils. MATERIAL AND METHODS PCR-DGGE analysis. PCR-DGGE analysis was performed as described in del Castillo et al. (2012). Bacterial isolation, identification and PGP properties. Isolation, identification and evaluation of PGP properties of the isolated bacteria were done as described in Andrades-Moreno (2012). RESULTS AND DISCUSSION PCR-DGGE analysis. The biodiversity of bacteria present both in the rhizosphere of S. densiflora (rhizosediment) and in naked soil (sediment) in two estuaries was analysed by DGGE (Figure 1A). Results indicated that the highest similarity corresponded to samples coming from the estuary of the Tinto river, suggesting that soil contamination influences bacterial population in a higher extent than the presence of the plant (Figure 1B). 17 Session I SI-CO-2 Figure 1. PCR-DGGE analysis. (A) Analysis of bacterial populations present in the sediments (S) an d the rhizosediments (Rs) of S. densiflora grown in the estuaries of the Tinto (T) and Piedras (P) river s. (B) Dendrogram showing the similarity between the DGGE profiles corresponding to the four samples base d on the Dice coefficient. Bacterial identification and PGP properties. 22 different cultivable bacterial strains were isolated from the rhizosphere of Spartina densiflora grown on the Tinto river estuary. 16S rDNA was partially sequenced to determine species affiliation. Copper resistance was determined, ranging from 1 to 13 mM. 7 strains were able to fix nitrogen, 10 strains produced siderophores, 10 strains were able to solubilize phosphate and only one strain produced auxins. Based on these characteristics a bacterial consortium comprising 3 strains were selected (Table 1). Table 1. Resistance towards Cu and plant-growth promotion properties of the bacterial consortium selected from the isolated stains. Strain Pseudomonas composti SDT3 Aeromonas aquariorum SDT13 Bacillus thuringiensis SDT14 Cu (mM) 13 7 5 PGP properties Siderophores and phosphate solubilization Siderophores, phosphate and auxins Siderophores, nitrogen fixation, antifungal Increased seed germination induced by the bacterial consortium. Seeds of S. densiflora were germinated in presence or absence of the bacterial consortium. After two weeks, seed germination was observed. Percentage of germination was higher in seeds inoculated with the bacterial consortium, 62% vs 32%. In addition, fungal contamination appeared in 72% of the seeds germinated in absence of the bacteria, while 40% of the inoculated seeds showed fungal contamination. ACKNOWLEGMENTS This work was supported by project RNM07274 of the Junta de Andalucía. J. Mesa was recipient of a FPU grant by the Ministerio de Educación, Cultura y Deporte. REFERENCES Andrades-Moreno, L. (2012). Ph. D. Thesis, Universidad de Sevilla. Cambrollé, J., et al. (2008). Mar. Pollut. Bull. 56: 2037-2042. del Castillo, I., et al. (2012). Water Res. 46: 1723-1734. Sáinz, X., et al. (2004). Environ. Int. 30: 556-557. 18 Session I SI-CO-3 Symbiovar loti-type genes are widely spread across different chromosomal backgrounds, corresponding to up to nine Mesorhizobium genospecies nodulating Cicer canariense. Pérez-Yépez, J.1, Armas-Capote, N.1, Martínez-Hidalgo, P.2, Velázquez, E.2, PérezGaldona, R.1, Mar tínez-Molina, E.2, León-Barrios, M.1* 1 Departamento de Microbiología y Biología Celular. Universidad de La Laguna. Tenerife. Departamento de Microbiología y Genética. Universidad de Salamanca. * [email protected] 2 ABSTRACT The phylogeny based on nodulation genes showed that the rhizobia nodulating Cicer canariense harboured sequences belonging to three different symbiotic lineages. Most isolates showed sequences closely related to symbiovar (sv.) loti which were widely distributed among up to nine Mesorhizobium genospecies. Another two smaller groups of isolates belonged to symbiotypes more restricted to particular species. Differences in N2-fixation capability were noted between strains from different symbiotypes. INTRODUCTION Cicer canariense is a wild chickpea endemic to the Canary Islands. The rhizobia nodulating this legume are found to be highly diverse, belonging or closely related to Mesorhizobium ciceri (the typical symbiont of the domesticated C. arietinum), M. caraganae, M. opportunistum, M. australicum, M. shangrilense, M. amorphae and the M. metallidurans/M. tianshanense/M. tarimense/M. gobiense cluster. The nodC phylogeny has been shown to correlate with the legume host range. Thus, in this work we have sequenced the nodC genes from representative strains of nine mesorhizobium genospecies to build a phylogeny which reflects the symbiovars of the C. canariense symbionts. MATERIAL AND METHODS Sequencing of nodC genes. The nodC genes using the pair of primers nodCF/nodCI as described (Laguerre et al., 2001) or the pair nodCMesoF and nodCMesoR as described (Rivas et al., 2007). The PCR-amplified products were purified (Qiaquick extraction kit, Qiagen) and sequenced in an ABI3730XL (Macrogen, Inc.) or alternatively in a Genetic Analyzer 3500 (Applied Biosystems; Servicio de Genómica, Universidad de La Laguna). Phylogenetic analyses. Sequence alignments (ClustalW) and phylogenetic analyses were conducted using the MEGA 5.0 software package (Tamura et al., 2011). The phylogenetic trees were inferred by the Neighbour-joining method (NJ) using Kimura’s 2-parameter model. Confidence levels of the tree branches were estimated with 1000 bootstrap replications. The sequences of rrs genes were compared with those of bacterial type-strains using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012). Infectivity tests. Sterilized and germinated seeds were inoculated with a high cell density suspension of the corresponding bacteria grown on YEM broth. Inoculated seedlings were grown in vermiculite containing a N-free nutrient solution. Non-inoculated plants were used as negative controls. Nitrogen fixation capacity was deduced from plants and nodules appearance. 19 Session I SI-CO-3 RESULTS AND DISCUSSION The phylogeny based on nodC gene sequences resolved the mesorhizobia nodulating C. canariense into three symbiotic lineages: two symbiotypes correlating with symbiovars ciceri and loti, while the third symbiotype has not been previously described. Surprisingly, most isolates carried nodC sequences highly related (95%) to reference strains from the symbiovar loti, as M. loti NZP 2213T type strain and MAFF303099. The simbiovar loti-type sequences were the most widely distributed among the isolates, since they were detected in nine chromosomal backgrounds of different Mesorhizobium species. A small number of isolates had nodC and nodA sequences identical to those of symbiovar ciceri (such as M. ciceri UPM-CaT type strain) and were only detected in isolates with an M. ciceri-chromosome. Another group of M. caraganae and M. opportunistum isolates presented sequences divergent from other previous known nodC gene sequences. This could be a specific symbiovar for C. canariense, whose closest relatives were a congruent clade of seven Mesorhizobium species (type strains of M. tianshanense, M. gobiense, M. caraganae, M. temperatum, M. septentrionale, M. shangrilense and M. tamadayense) which shared the highest similarities in the range of 84.6-87.0%. These results indicated that C. canariense is a promiscuous legume that can be nodulated by several genomic species and symbiotypes, which make it necessary to determine the combination of core and symbiotic genes producing the most effective symbiosis. Our preliminary results indicate that the isolates with symbiovar loti-type genes produced well developed plants. ACKNOWLEDGEMENTS Supported by Ministerio de Medio Ambiente y Medio Rural y Marino, Organismo Autónomo de Parques Nacionales (Ref. 111/2010). REFERENCES Jarabo-Lorenzo A., et al. (2000). Syst. Appl. Microbiol. 23: 418-425. Laguerre, et al. (2001). Microbiolgy 147: 981-993. Lorite, M.J., et al. (2010). Syst. Appl. Microbiol. 33: 282-290. Rivas, R., et al. (2007). Lett. Appl. Microbiol. 44: 412-418. Tamura, K., et al. (2011). Meth. Mol. Biol. Evol. 28: 2731-2739. 20 Session I SI-CO-4 Molecular phylogeny and phenotypic characterization of salt tolerant Sinorhizobia nodulating Phaseolus filiformis in Northern Mexico. Rocha, G.1, Medina, A.1, Carreño, R.1, Bustillos, R.1, Contreras, J.L.2, Villegas, M.C.3, Chaintreuil, C.4, Dreyfus, B.4, Le Queré, A.5, Munive, J.A.1* 1 Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla, Mexico. 2 Facultad de Arquitectura, Benemérita Universidad Autónoma de Puebla, Benemérita Universidad Autónoma de Puebla, Mexico. 3 Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico. 4 Laboratoire des Symbioses Tropicales et Méditerranéennes, Unité Mi xte de Recherche (113) CIRAD, IRD, Université Montpellier 2, SupAgro, USC INRA, France. 5 Laboratoire de Microbiologie et Biologie Moléculaire, Faculté des Sciences, Université Mohammed V-Agdal, Rabat , Morocco. * [email protected]; [email protected] ABSTRACT The aim of the present work was to establish the phylogenetic relationships of a collection of salt tolerant sinorhizobia, nodulating P. filiformis in northern Mexico, by Multilocus Sequence Analysis (MLSA). This collection was phenotypically and genetically characterized. P.filiformis is nodulated by two groups of sinorhizobia, one of them related to S. meliloti, and the other one related to a new Sinorhizobium species, closely related to S. saheli. It was apparent that there is no strict correlation between the chromosomal genotype of a strain and its salt tolerance. INTRODUCTION The genus Phaseolus comprises around 50 species, all indigenous to the Americas. Among this, P. lunatus, P. vulgaris, P. polyanthus, P. coccineus and P. acitifolius were domesticated by prehispanic civilizations and are widely used for human consumption. Beans, like other legume plants, can establish symbiotic relationships with nitrogen fixing soil bacteria called rhizobia. These bacteria invade root tissues and induce the formation of specialized structures known as nodules, where they fix atmospheric nitrogen. Between a diversity of bean nodulating bacteria, Rhizobium etli is the predominant species in Mexico; although some other rhizobia species have been described as symbionts of Phaseolus vulgaris where bean cultures have been introduced. The number of rhizobia species capable of nodulating bean shows that this legume is a promiscuous host, with a high diversity of interactions RhizobiumPhaseolus. MATERIAL AND METHODS A number of rhizobia strains were isolated from Phaseolus filiformis, a wild bean native to the southwestern United States and northern Mexico. Its common names include slimjim bean, slender-stem bean, and Wright's phaseolus. Strains were identified by 16SrDNA sequence analysis using primers designed by Normand et al. (1991) and Sy et al. (2001). MLSA was performed by sequence analysis of housekeeping genes: recA (modified from Gaunt et al., 2001), dnaK (Stepkowsky et al., 2003), gyrB and rpoB (Martens et al., 2008), atpD and glnII (Vinuesa et al., 2005). Salt tolerance was determined using 8.5 x 10 -4, 0.4, 0.6, 0.8 and 1M of NaCl in YMA medium, and at greenhouse level. This collection was also phenotypically and genetically characterized by carbon source utilization analysis and by restriction patterns produced by ARDRA (using four endonucleases AluI, CfoI, HinfI and NheI) and REP-PCR (Versalovic et al., 1991), using the software FreeTree. 21 Session I SI-CO-4 RESULTS AND DISCUSSION Strains were identified as belonging to genus Sinorhizobium, varying in their tolerance to salt-stress. Seven strains were intolerant to 0.4M NaCl, and only two sinorhizobia strains were tolerant to high levels of salinity (1M); indicating that salt-tolerance is not related to their ecological origin. Greenhouse test showed the salt-tolerance induced in plants inoculated with the most salt-tolerant sinorhizobia strain EMM451. Genotypic characterization showed 4 different genotypes. The phylogenetic relationships of sinorhizobia collection by MLSA, using 6 chromosomal loci, showed that species P. filiformis is nodulated by two groups of sinorhizobia, one of them related to S. meliloti (S. meliloti EMM456), and the other one related to a new Sinorhizobium species, closely related to S. saheli (Figure 1). There is no strict correlation between the chromosomal genotype and the salt tolerance. Salt-tolerance determinants must reside on extrachromosomal elements that may be subject to lateral transfer and recombination. 100 100 S. meliloti S. meliloti EMM456 (P. filiformis) 78 100 S. arboris 100 51 100 60 100 100 100 88 100 87 S. kostiense S. saheli 27 99 S. medicae 100 34 S. meliloti Sinorhizobium sp. (Phaseolus filiformis) S. terangae S. fredii/S. xinjiangense S. adhaerens Rhizobium spp. 47 M loti MAFF303099 0.02 Figure 1. Maximum likelihood species tree inferred from the concatenated alignment recA-dnaK-gyrBr poB-atpD genes, showing the phylogenetic relationships of Sinorhizobium sp. (P. filiormis) strains and Sinorhizobium species. The bar represents the number of expected substitutions per site. Bootstrap values calculated for 1,000 replications are indicated. ACKNOWLEGMENTS This work was granted by ECOS-NORD M08-A02. REFERENCES Gaunt, M., et al. (2001). Int. J. Syst. Evol. Microbiol. 51: 2037-2048. Normand, P., et al. (1991). Int. J. System. Bacteriol. 46: 1-9. Martens, M., et al. (2008). Int. J. Syst. Evol. Microbiol. 58: 200-214. Martens, M., et al. (2007). Int. J. Syst. Evol. Microbiol. 57: 489-503. Stepkowski, T., et al. (2003). Syst. Appl. Microbiol. 26: 483-494. Silva C., et al. (1999). Mol. Ecol. 8: 277-287. Sy A., et al. (2001). J. Bacteriol. 183: 214-220. Versalovic, J., et al. (1991). Nucl. Acids Res. 24: 6823-6831. Vinuesa, P., et al. (2005). Mol. Phyl. Evol. 34: 29-54. 22 Session I SI-CP-01 Phylogenetic diversity of bradyrhizobia nodulating cowpea (Vigna unguiculata L. Walp) in Extremadura (Spain). Bejarano, A.1*, Ramírez-Bahena, M.H.1, 2, Velázquez, E.2, 3, Peix, A.1, 2 1 Instituto de Recursos Naturales y Agrobiología, IRNASA-CSIC, Salamanca, Spain. 2 Unidad Asociada Grupo Interacción Planta-Microorganismo USAL-IRNASA-CSIC. 3 Departamento de Microbiología y Genética, Universidad de Salamanca (USAL), Spain. * [email protected] ABSTRACT Vigna unguiculata, a grain legume worldwide cultivated but poorly cropped in Spain, is a good alternative over other legumes to be included into the mediterranean diet and has a great agronomic interest by its resistance to soil acidity, drought and high temperatures. Cowpea establishes symbiosis mainly with Bradyrhizobium in most of regions, and this rhizobia-legume interaction has been studied in Asia, Africa and America. However up to date there are no data about rhizobia nodulating V. unguiculata in Europe. In this work we analysed the phylogenetic diversity of slow-growing rhizobia isolated from nodules of this legume in Extremadura through the analysis of their 16S rRNA genes and 16S-23S intergenic regions. The results showed that unlike in other continents, V. unguiculata is mainly nodulated in Spain by B. cytisi and B. canariense and by several new phylogenetic lineages within genus Bradyrhizobium. INTRODUCTION Vigna unguiculata (cowpea) is cultivated in concrete regions in Spain where the main production is located in Extremadura region. Cowpea contains high proportions of protein (20-30%), and dietary fiber (20-35%) being of great interest in human and animal nutrition (Fontenele-Urano-Carvalho et al., 2005). Therefore it is a good alternative to be included into the mediterranean diet whose beneficial effects in human health has been widely proven (Sofi et al., 2010). Moreover, this legume is agronomically very interesting since it is very resistant to soil acidity, drought and high temperatures as well as for its ability to establish nitrogen fixing symbiosis with rhizobia mostly belonging to genus Bradyrhizobium. The endosymbionts of Vigna unguiculata have been studied in Africa, Asia and America, but no data about those present in European soils are available. Therefore the main objective of this study was to analyse the phylogenetic diversity of bradyrhizobia nodulating Vigna unguiculata in a soil from Extremadura in which this legume is cultivated and to compare the Spanish cowpea nodulating strains with those isolated from other continents. MATERIAL AND METHODS Vigna unguiculata plants were used as trap plant in an acidic soil from Extremadura and the isolation of rhizobia was performed following the method of Vincent (1970) on YMA plates. RAPD patterns were obtained as was previously described using the primer M13 (Rivas et al., 2006). The mathematical analysis of the patterns was performed using BioNumerics™ software (Applied Maths, Belgium). Amplification and analysis of the 16S rRNA gene and ITS region was performed as previously described (Velázquez et al., 2010). 23 Session I SI-CP-01 RESULTS AND DISCUSSION About fifty slow-growing strains of Bradyrhizobium were isolated from Vigna unguiculata nodules which were grouped by using RAPD fingerprinting. After the mathematical analysis of the obtained patterns a representative strain from groups with similarity values lower than 80% among them was selected for 16S rRNA gene and ITS analyses. These analyses allowed the identification of several Bradyrhizobium species in V. unguiculata nodules as well as the detection of some putative novel species within this genus. The identified species were B. canariense and B. cytisi, which are reported here by the first time in V. unguiculata nodules. Some strains cluster within strain B. japonicum BGA-1, of uncertain taxonomic status since the strains from this group are divergent to that formed by the type strain of B. japonicum USDA 6T. Some other strains formed independent branches in both 16S and ITS phylogenetic trees and then they could constitute new species within genus Bradyrhizobium. A comparison with the strains nodulating Vigna in other continents showed that our Spanish strains belong to different groups than those isolated in other countries with the exception of B. canariense from which a single strain isolated in Japan has been found in nodules of V. unguiculata. Considering that V. unguiculata from tribe Phaseolae is indigenous of South Africa Transvaal region (Paludosi and Ng, 1997), these results suggest an adaptation of this legume to be nodulated by Bradyrhizobium species nodulating Genisteae species in mainland Spain (Velázquez et al., 2010). ACKNOWLEDGMENTS MHRB is recipient of a JAE-Doc researcher contract from CSIC cofinanced by ERDF. REFERENCES Fontenele-Urano-Carvalho, A. et al. (2012). J. Food Compos. Anal. 26: 81-88. Paludosi, S., and Ng, N.Q. (1997). Advances in Cowpea Research. Pp. 1-12. Edited by B.B. Singh, D. R. Mohan Raj, and K.E. Dashiell, L.E.N. Jackai. Nigeria: Copublication of International Insitute of Troplical Agriculture (IITA) and Japan International Research Center of Agricultural Siences (JIRCAS). Rivas, R., et al. (2006). Plant Soil 287: 23-33. Sofi, F., et al. (2010). Am. J. Clin. Nutr. 92: 1189-1196. Velázquez, E., et al. (2010). Antonie Van Leeuwenhoek. 97: 363-376. Vincent J.M. (1970). A Manual for the Practical Study of Root-Nodule. pp. 1-13. Edited by J.M. Vin cent. Blackwell Scientific Publications, Oxford (United Kingdom). 24 Session I SI-CP-02 MALDI-TOFF MS, a tool for diversity analysis and detection of novel rhizobial species nodulating Phaseolus vulgaris. Flores-Félix, J.D.1*, García-Fraile, P.1, Sánchez-Juanes, F.2, 3, 4, Ramírez-Bahena, M.H.5, 6, Ferreira, L.2, 3, 4, Mula, D.7, Rivas, R.1, 6, González-Andrés, F.7, Peix, A.5, 6, González-Buitrago, J.M.2, 3, 4, Velázquez, E.1, 6 1 Departamento de Microbiología y Genética, Universidad de Salamanca (USAL), Spain. 2 Unidad de Investigación. Hospital Universitario de Salamanca, Spain. 3 Departamento de Bioquímica y Biología Molecular. Universidad de Salamanca (USAL), Spain. 4 Instituto de Investigación Biomédica. Salaman ca, Spain. 5 IRNASA-CSIC, Salamanca, Spain. 6 Unidad Asociada Grupo Interacción Planta-Microorganismo USAL-IRNASA-CSIC. 7Instituto de Medio Ambiente y Recursos Naturales, Universidad de León, Spain. * [email protected] ABSTRACT In this work we analysed by MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry) a collection of endosymbionts from P. vulgaris which is nodulated by different genera and species of rhizobia. These endosymbionts were isolated in different regions from the Northwest to the Southest of Spain. The results of MALDI-TOF MS analysis showed that they belonged to Rhizobium and Ensifer but despite a 53% of strains were correctly identified, the remaining 47% of strains was not identified with any of species present in the Rhizobiaceae database. This is a frequent result when the isolates of legume nodules are analysed and grouping techniques are necessary to finish the identification schemes. After the mathematical analysis of spectra from P. vulgaris isolates the dendrogram obtained showed that the unidentified strains presented similarity values lower than 70% with respect to the groups containing the identified ones. The strains belonging to the same species presented similarity internal values higher to 70% allowing the selection of strains for housekeeping gene analysis. The combination of MALDI-TOF MS analysis and gene sequencing allowed the detection of several new species in Phaseolus vulgaris nodules in Spain. INTRODUCTION Phaseolus vulgaris, indigenous of America, is currently the most cultivated legume worldwide after soybean and establishes nitrogen-fixing symbiosis with several fast growing species of genera Rhizobium and Ensifer (Velázquez et al., 2010). Therefore the P. vulgaris-rhizobia symbiosis is a good model to evaluate the reliability of MALDI-TOF MS for the identification of rhizobia at species level as well as for the grouping of unidentified strains in order to select them for identification by gene analyses. MALDI-TOF MS is a technique based on protein analysis that has been recently proposed for differentiation of genera and species from family Rhizobiaceae (Ferreira et al., 2011). It has been previously proven that this technique is useful for identification of legume nodules isolates belonging to already described species, nevertheless frequently nodule isolates that do not belong to described species are isolated. The aim of this work was to analyze the suitability of spectra analysis to group the unidentified rhizobial strains isolated from P. vulgaris at species level in order to select strains for housekeeping gene analysis. MATERIAL AND METHODS The isolates from P. vulgaris nodules growing in different Spanish locations were obtained using the methodology described by Vincent (1970) on YMA plates. For 25 Session I SI-CP-02 MALDI-TOF MS analysis and for DNA isolation the strains were incubated on TY plates (Beringer, 1974) during 24h. The samples preparation, the performing of MALDI-TOF MS analysis and that of recA and atpD genes were performed as was previously described (Ferrerira et al., 2011). RESULTS AND DISCUSSION After comparison of spectra obtained for P. vulgaris isolates with those present in the family Rhizobiaceae database previously build in our laboratory (Ferreira et al., 2011) about 53% of strains were identified as already described species of Rhizobium, such as R. leguminosarum and R. giardinii, and Ensifer, such as E. fredii. The remaining strains although were closely related to other species of these two genera, the score values were lower than 2.0 indicating that they belong to undescribed species. The mathematical analysis of the spectra showed that the identified strains clustered with the type strains of the species in which were placed after MALDI-TOF MS analysis forming groups with internal similarities higher than 70%. The unidentified strains occupied branches or clusters with similarity values lower than 70% with respect to the described species of genera Rhizobium and Ensifer. The analysis of recA and atpD genes showed that the unidentified strains by MALDITOF MS belonged t o groups phylogenetically divergent to those from the already described species from family Rhizobiaceae. These strains were phylogenetically related to R. leguminosarum, R. vallis, R. tibeticum, R. mongolense and Ensifer fredii. Interesting, in León in soils commonly cultivated with P. vulgaris the predominant species was R. leguminosarum. In Barco de Ávila, although this legume is commonly cultivated and R. leguminosarum also was abundant, R. giardinii was frequently found. In Salamanca, in a soil commonly cultivated with Lens culinaris but where P. vulgaris is not cultivated, a new species related to R. leguminosarum was found. In Badajoz in a soil not cultivated with P. vulagris, R. giardinii was found. Finally, in Sevilla, in a soil not cultivated with P. vulgaris, we found two undescribed species related to R. mongolense and E. fredii. ACKNOWLEDGMENTS This work was funded and supported by Junta de Castilla y León project SA183A11-2. MHRB is recipient of a JAE-Doc researcher contract from CSIC, cofinanced by ERDF. REFERENCES Beringer, J.E. (1974). J. Gen. Microbiol. 84: 188-198. Ferreira, et al. (2011). PLoS One. 6: e20223. Velázquez, E., et al. (2010). Microbes for Legume Improvement. Khan, M.S., Zaidi, A., and Musarrat , J. (eds). Dordrecht, The Netherlands: Springer. pp. 1-25. Vincent J.M. (1970). A Manual for the Practical Study of Root-Nodule. pp. 1-13. Edited by J.M. Vin cent. Oxford : Blackwell Scientific Publications. 26 Session I SI-CP-03 Análisis de la biodiversidad de microorganismos en nódulos de Lotus corniculatus. Marcos-García, M.1, Menéndez, E.1, Celador-Lera, L.1, Rivera, L.P.1, Martínez- Molina, E.1, 2, Mateos, P.F.1, 2, Velázquez, E.1, 2, Rivas, R.1, 2* 1 Departamento de Microbiología y Genética. Universidad de Salamanca. Universidad de Salamanca (USAL)-CSIC (IRNASA). * [email protected] 2 Unidad Asociada de I+D RESUMEN Los nódulos de plantas de Lotus corniculatus procedentes de la localidad salmantina de Carbajosa de la Sagrada nos ha proporcionado una elevada biodiversidad de microorganismos, capaces de vivir en el interior de estos nichos seguros junto con el endosimbionte natural de esta planta, Mesorhizobium loti. Las cualidades fenotípicas que presentan varias de estas cepas, algunas de las cuales se encuentran dentro de los géneros Micromonospora y Lysinibacillus, las convierte en potenciales bacterias promotoras del crecimiento vegetal y susceptibles de ser utilizadas como coinoculantes. INTRODUCCIÓN La adaptabilidad a diferentes presiones ambientales que presentan las especies del género Lotus, las convierte en plantas muy importantes desde un punto de vista económico, ya que se utilizan como cultivos altamente productivos en sistemas de pastoreos en una amplia gama de paisajes, incluyendo algunos sometidos con frecuencia a ambientes y condiciones del suelo extremas. En concreto, la especie L. corniculatus se considera una de las leguminosas forrajeras más importantes después de la alfafa (Medicago sativa) y del trébol blanco (Trifolium repens), siendo su valor nutricional similar o incluso superior a la de especies como: L. uliginosus (Schkuhr.), L. tenuis (Waldst et kit.) y L. subbiflorus (Lagasca). Además, es una buena candidata para la restauración y la fitorremediación de ambientes degradados. Esta especie es, con toda probabilidad, la especie de Lotus más comúnmente empleada en restauraciones ecológicas de los suelos afectados por la deficiencia de nutrientes, la salinidad, la sequía o los contaminantes. Conocer la diversidad y la abundancia de los microorganismos que nodulan o que ocupan los nódulos en las especies de Lotus, es un enfoque valioso para una mejor selección y aplicación de las cepas como inoculantes. Estas cepas no sólo deben ser eficientes en la fijación del nitrógeno, como ocurre para las especies de los rhizobia, sino que también deben ser capaces de competir favorablemente con los microorganismos nativos de la planta y la rizosfera circundante. Los estudios realizados hasta la fecha sobre nódulos de L. corniculatus, se han centrado principalmente en las especies del género Mesorhizobium de forma aislada. Conocer la existencia de una mayor diversidad de especies en el interior de estos nichos ecológicos específicos y seguros en cuanto a la presencia de patógenos, abre un abanico de posibilidades en lo que se refiere a la búsqueda de nuevas bacterias promotoras del crecimiento vegetal en plantas no leguminosas. MATERIAL Y MÉTODOS Se obtuvieron 90 aislados que se agruparon utilizando técnicas moleculares como los TP-RAPD. Se secuenció el gen ribosómico 16S de diversos representantes, utilizando para ello los primers 27F y 1522R. El análisis de las secuencias se llevó a cabo con el programa BLASTn. Estas secuencias también se compararon con las cepas tipo utilizando el programa Eztaxon 2.0. Para el análisis filogenético se utilizaron los programas Clustal X y Mega 2.1. Para determinar la potencial capacidad de promoción 27 Session I SI-CP-03 del crecimiento vegetal de cada uno de los aislados se hicieron distintas pruebas de caracterización fenotípica. La solubilización de fosfato fue evaluada en medio YED-P de acuerdo con Peix et al. (2009). La producción de sideróforos se evaluó utilizando el medio de cultivo M9-CAS-AGAR, que es una modificación del medio utilizado por Schwyn y Neilands, donde se observan halos de distintas tonalidades. La determinación cualitativa de la producción de celulosa se llevó a cabo utilizando el medio de cultivo YMA convencional con 25mg/l de rojo congo. La detección de la actividad celulolítica, se determinó utilizando como sustrato carboximetil celulosa sódica, CMC (viscosidad media, Sigma), un compuesto soluble derivado de la celulosa, utilizado para la valoración de la actividad 1,4--glucanásica. La determinación de la formación de biofilms se llevó cabo según Fujishige et al. (2006) con algunas modificaciones. Se realizaron dos ensayos: en placas de microtitulación de poliestireno y en portaobjetos (vidrio). RESULTADOS Y DISCUSIÓN El análisis del gen ribosómico 16S nos mostró una amplia diversidad de especies, siendo las cepas mayoritarias las de la especie Mesorhizbium loti, el endosimbionte natural de L. corniculatus. Dentro del género Mesorhizobium también se aislaron tres especies más: M. australicum, M. amporhae y M. gobiense. Por otra parte, según los datos de secuenciación, aislamos especies no encontradas hasta ahora en nódulos de L. corniculatus y que pertenecen a los géneros: Micromonospora, Dermacoccus, Arthrobacter, Metylobacterium, Acinetobacter, Lysinibacillus y Paenibacillus. En este trabajo, aislamos un total de 90 cepas del interior de nódulos de Lotus corniculatus, obteniendo cepas con capacidad de solubilizar fosfato, promover la síntesis de indol acético, producir sideróforos y celulosa, tener actividad celulolítica y ser capaces de formar biofilms. Esta planta, por tanto, tiene una gran diversidad de microorganismos endofíticos asociados a ella, capaces de mantener una actividad altamente beneficiosa, influyendo de forma directa en el crecimiento de las plantas y sus funciones, no sólo en su actividad cómo planta forrajera sino también como fitorremediadora de suelos en condiciones insalubres. Podemos decir que el potencial que presenta L. corniculatus, hacen de ella una planta con nuevas y amplias posibilidades de estudio y por supuesto, el posible potencial de sus microorganismos endófitos como bacterias promotoras del crecimiento vegetal, nos plantea la posibilidad de aplicarlos a otras especies vegetales, incluidas las no leguminosas. AGRADECIMIENTOS Este trabajo está financiado por el proyecto SA183A11-2 de la Junta de Castilla y León y por el pr oyecto AGL2011-29227 del Ministerio de Ciencia e Innovación. Marta Marcos agradece a la Fundación Miguel Casado San José la financiación de su beca. BIBLIOGRAFÍA Fujishige, N.A., et al. (2006). Bot. J. Linnean Soc. 150: 79-88. Peix, A., et al. (2009). Syst. Appl. Microbiol. 32: 334-341. 28 Session I SI-CP-04 Where do PGPR interact with plants? An essential determination to develop effective agricultural inputs based on microorganisms. Mulas, R.1, Mulas, D.2*, Menéndez, E.3, Rivera, L.3, Mateos, P.3, González-Andrés, F.1 1 Instituto de Medio Ambiente y Recursos Naturales, Universidad de León, León, Spain. 2 Fertiberia S .A., Dpto de I+D+i, Huelva, Spain. 3 Universidad de Salamanca. Dpto de Microbiología y Genética, Salamanca, Spain. * [email protected] ABSTRACT Although PGPR are extensively used in agriculture, they are just becoming popular in Europe now, and this is because the strict regulations (Regulation EC 2003/2003 about fertilisers and Directive 91/414/EEC about plant protection products) and the lack of constancy in their effectiveness in some cases. Studying where they locate at a rhizospheric level can provide valuable information to design agricultural inputs based on microorganisms. This works shows how a phosphate-solubilizing Rhizobium sp strain attaches to barley roots by observing transformed cells (fluorescent) under confocal microscopy. The cells were observed on the root surface, and no endophytic evidence was found for this strain. However, several studies have shown presence of Rhizobium spp. as endophytes in cereal roots, such as rice or maize, indicating that the characteristics of the symbiosis depend on the strain and not on the taxon. INTRODUCTION Bacteria that improve plant nutritional status (PGPR) are gaining acceptance amongst farmers in Europe as an additional input, since a number of studies have shown the feasibility or their use with cereals, legumes, industrial and horticultural crops. However, little is known about the exact location of PGPR in the rhizosphere. Knowing if the bacterium act as an endophyte or stays in the rhizoplane is interesting because it can lead the strategy followed to develop products based on PGPR. Confocal microscopy was first successfully used to view Rhizobium cells in Lucerne by Gage et al. (1996), and since then it is a useful tool to determine the position of the cells in the root. This study aimed at determining the location of a PGPR (Rhizobium sp phosphatesolubilizer) w ith barley (Hordeum vulgare L.) in the rhizosphere, in order to design the best way of delivering the PGPR to this extensive winter crop. MATERIAL AND METHODS A Rhizobium strain belonging to the collection of the University of León, was transformed with the pHC60 plasmid (containing a GFP) harboured by Escherichia coli S17.1 (Simon et al., 1983) by means of biparental mating. The transformed strain was inoculated on barley plants, whose seeds had been previously surface-sterilized (ethanol 70% 1 min, NaClO 7% 7 min). First test: barley plantlets (48 h after seeding) were inoculated with 250 L of the bacterium culture (107 CFU mL-1). Ten days after inoculation (DAI), plant roots were observed by using confocal microscopy to determine the plant root colonization. Second test: barley plants were inoculated by immersion in the bacterium culture for 30 min. Ten DAI, plant roots were observed and the number of bacterial cells present within the roots was determined by the following procedure: surface-sterilized roots (ethanol 70% 1 min, NaClO 7% 5 min) were smashed and the root internal tissue was inoculated on YMA medium plates at the dilutions 10-1, 10-3 and 10-5. A control was used by placing sterile roots over YMA without smashing, which indicated the sterility of the root surface. 29 Session I SI-CP-04 Third test: barley plants were immersed (3 h) in a bacterium suspension in saline solution (108 CFU mL-1). The same observations were done as previously, but the sterilization was less aggressive (NaClO 5% 5 min) to avoid sterilization within the root tissue. With the endophytic isolates from the root that showed a colony growth similar to rhizobia, RAPD profiles were obtained using the PCR primer M-13 as described by Huey and Hall (1989). This method allowed discriminating amongst bacterial strains within the same species, thus indicating whether the isolates were the inoculated strain or not. RESULTS AND DISCUSSION The transformed Rhizobium sp was observed with confocal microscopy, showing an association with the plant roots, but it was not possible to determine whether it happened on the root surface or endophytically (Figure 1). Regarding to the root tissue, no colonies were found in the second test. However, the less aggressive sterilization of the third test, allowed bacterial growth from inside the root, although no external growth was detected. The M13-RAPD profiles indicated that the isolates were different from the inoculated Rhizobium sp, which hints the possibility for the seed to harbor other endophytic bacteria from the early stages, without evidence of endophytic rhizobia. Nonetheless, other studies have shown rhizobium-root endophytic association in nonlegume crops (i. e. Gutiérrez-Zamora and Martínez-Romero, 2001; Yanni et al., 2010; López-López et al., 2010). As a conclusion, the presence of the rhizobia inside the roots is strain-dependent and, therefore it is needed to determine the location of each bacterial strain in the rhizosphere. This information is crucial to design agricultural inputs (such as fertilizers, inoculants and other biostimulants) with efficient performance, and thus, improved nutritional status of the crops. Figure 1. Confocal microscopy images of the first (a), second (b) and third tests (c). Whereas the first test shows presence of bacteria, the second and the third tests show a higher number of bacteria in the barley root. ACKNOWLEDGEMENTS This work has been funded by Fertiberia S.A., Junta de Castilla y León (Research Projects) and the Spanish Ministry of Economy (INNPACTO project IPT-2011-1283-060000, co-funded by the EUFEDER). REFERENCES Gage, D.J., et al. (1996). J. Bacteriol. 178: 71597166. Gutiérrez-Zamora, M.L., and Martínez-Romero, E. (2001). J. Biotechnol. 91: 117-126. Huey, B., and Hall, J. (1989). J. Bacteriol. 171: 2528–2532. López-López, A., et al. (2010). Syst. Appl. Microbiol. 33: 322-327. O'Hara, G.W., et al. (1989). Appl. Environ. Microbiol. 1870-1876. Yanni, Y.G., et al. (2010). Plant Soil 336: 129-142. 30 Session I SI-CP-05 Biodiversity of endophytic bacteria and mycorrhizal fungi in roots of pepper (Capsicum annuum L.) in León province (Spain). Barquero, M.1, Velázquez, E.1, Terrón, A.2*, González-Andrés, F.2 1 Departamento de Microbiología y Genética, Universidad de Salamanca, Salamanca, Spain. 2 Instituto de Medio Ambiente y Recursos Naturales, Universidad de León, León, Spain. * [email protected] ABSTRACT Eigthy one endophytic PGPR strains and 9 AMF isolates were obtained from roots of pepper in the PGI “Fresno-Benavente”, and characterized to be used like biofertilisers. Bacteria were analysed in vitro regarding several plant growth promotion properties, in order to pre-select strains for in field selection. The identification of the bacteria is a key aspect as only safe taxa can be used. In our case an ARDRA test following TP-RAPD were carried out in order to group the strains prior to the sequentation of the gene 16S rRNA for identification. The AMF isolates belonged to the species Glomus mosseae. INTRODUCTION Second generation biofertilisers (Mulas et al. 2013) consist on mixes of different Plant Growth Promoting Rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF), which as a result of a complex mix of mechanisms have one or several of the following effects: improve plant nutrition, stimulates plant growth and overcome plant stresses (Dobbelaere et al., 2003, Singh et al., 2011). The use of biofertilisers in agriculture is an increasing practice, as they improve the efficiency in the use of the mineral nutrients from the soil, reducing nutrient losses an improving the crops yield for reduced rates of chemical fertilisers. By the other hand, rhizobacteria and AMF may play a role like microbial control agents and can be used for the replacement of chemicals according to the EC Regulation 1107/2009. One key point for the success of microorganisms like biofertilisers is the use of native isolates (Mulas et al., 2011, 2013). Castilla y León is a Spanish region with an outstanding agri-food sector. The pepper (Capsicum annuum L.) from Fresno and Benavente, is a product with high added value, labeled with a Protected Geographic Indication (PGI), and for this reason started a research project to develop biofertilisers. Here we present the results of the prospection of native microorganisms and the preliminary characterization of the biodiversity encountered, the first stage to develop an autochthonous biofertiliser. MATERIAL AND METHODS Endophitic bacteria were isolated from surface sterilized roots of pepper crops collected in the in 3 locations and 3 soils per location, inside the PGI region. Crushed roots were plated onto TSA medium and purified. Clones were discarded on the basis of the RAPD (primer M13) profiles. Pure strains were analysed for several activities in vitro: Ca3(PO3)2 solubilization; siderophore production; IAA production; ACC deaminase activity; control of Phitopthora capsicii. The identification of the isolates was based on the sequentiation of the gen 16S rRNA and comparison with the ezTaxon database (ezbiocloud.net). Previously to the sequentiation, isolates were grouped on the basis of and Amplified Ribosomal DNA Restriction Analysis (ARDRA). Each group typically consists on several taxa, so the TP-RAPD (Rivas et al., 2001) profiles within each ARDRA group were obtained in order to group the isolates by recognised taxa and to sequentiate only one strain from each group. AMF were isolated from the rhizosphere of the same plants, using leak (Allium porrum) as trap plant in sterile sand. After this first stage, from each soil 10 spores of the same morphology were used to inoculate a 31 Session I SI-CP-05 leak plant in order to produce inoculum. One spore per soil was used for DNA amplification with the primers of Lee et al. (2008), sequencing the obtained band and comparing it with databases with BLAST-N (Altschut et al., 1990). Figure 1. In vitro test for biocontrol of Phytophtora capsici by bacteria. RESULTS AND DISCUSSION Eighty one different PGPR’s strains were isolated from the locations of Fresno de la Vega, San Cristobal de Entreviñas and Micereces. None of the isolated strains was phosphate solubilizer. Regarding the IAA production, the values ranged from 1.25 µg/ml to 8.93 µg/ml. However only 12 strains from the 81 tested, produce more than 3.0 µg/ml, and half of the 81 produced less than 2.0 µg/ml. The production of IAA was medium-low, compared with the best producers isolated in tropical and subtropical areas. The production of -Ketobutirate showed a broad range from nearly 0 moles mg prot-1 h-1 up to 247, showing a continuous variation within this range. Compared to other works (i.e. Siddikee et al., 2010), the higher values observed in our collection are up to 100 time higher that the higher values of the mentioned work and the lower values are similar in both cases. About siderophores production, more than 50% of the isolates produced them. The isolates belonged to Bacillus pumilus, Bacillus amyloliquefaciens, Pesudomonas geniculata and other species were discarded like Bacillus cereus (human pathogen) and Pseudomonas corrugata (plant pathogen). Five out of the 81 strains controlled Phytophtora capsicii (Figure 1) four belonging to B. pumilus and the last to B. cereus which was discarded. All the AMF isolated belonged to Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe, in contrast to the isolates in tree crops in the same area, which usually belonged to Glomus Intraradices. The selection of the PGPR strains to be tested in microplots in field conditions was based on the level of expression of plant growth promotion properties in vitro, and in the taxonomic identification, as only safe taxa can be used for biofertilisers. AKNOWLEDGEMENTS This work has been funded by Junta de Castilla y León (Project LE029A10-2) and Agencia Española de Cooperación Internacional para el Desarrollo (AECID) (Project A1/035364/11). M. Barquero was granted by AECID. REFERENCES Altschul, S.F., et al. (1990). J. Mol. Biol. 215: 403-410. Lee, J., et al. (2008). FEMS Microbiol. Ecol. 65: 339-349. Mulas, D., et al. (2011). Soil Biol. Biochem. 43: 2283-2293. Mulas, D., et al. (2013). Beneficial plant-microbe interactions: Ecology and applications. CRC Pre ss. Rivas, R., et al. (2001). Electrophoresis 22: 1086-1089. Siddikee, M.A., et al. (2010). J. Microbiol. Biothechnol. 20: 1577-1584. Singh J.S., et al. (2011). Agr. Ecosyst. Environ. 140: 339-353. 32 Session I SI-CP-06 Diversidad de los rizobios que nodulan Medicago marina de regiones del Odiel y San Fernando. Alías-Villegas, C.1*, Bellogín, R.A.1, Camacho, M.2, Cubo, M.T.1, Temprano, F.2, Espuny, M.R.1 1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes 6, 41012-Sevilla. Spain. 2 Centro Las Torres-Tomejil (IFAPA), Apartado Oficial, Alcalá del Río, Sevil la. Spain. * [email protected] RESUMEN La diversidad de los rizobios que nodulan Medicago marina en las Marismas del Odiel y San Fernando, zonas con suelos sometidos a estrés abiótico por pH y salinidad, se ha estimado usando 14 aislamientos de plantas de San Fernando y 18 de las Marismas del Odiel. Tras eliminar redundancias de los perfiles PCR-ERIC se obtuvieron 12 aislamientos diferentes, 7 de las Marismas del Odiel y 5 de San Fernando. La secuenciación del gen ARNr 16s mostró que todas las cepas pertenecen al género Sinorhizobium, estando estrechamente relacionadas con S. meliloti LMG 6133T. Para estudiar si son distintas cepas de S. meliloti se han realizado RFLP del gen nod C y de la región ITS. Según el perfil RFLP de nod C, se han obtenido dos grupos diferenciados (I y II), con las enzimas MspI y RsaI, pero éstos no coinciden exactamente con las áreas geográficas donde se han recogido las muestras y no corrobora que todos los aislamientos sean S. meliloti, puesto que al digerir con MspI el perfil de S. meliloti coincide con el grupo II y al digerir con RsaI en perfil de S. meliloti coincide con el grupo I. Por otra parte, según el perfil RFLP de la región ITS, todos los aislamientos mostraron el mismo perfil que S. meliloti, con excepción del aislamiento ORT17 que mostró un perfil distinto con la enzima MspI. INTRODUCCIÓN La agricultura actual sufre la sobreexplotación de los recursos y el empleo masivo de fertilizantes, lo que hace preciso la recuperación de suelos que presentan algún tipo de estrés abiótico por pH o salinidad extremos que imposibilitan el cultivo. Así, se planteó la búsqueda de bacterias simbióticas fijadoras de nitrógeno atmosférico en ambientes con estos tipos de estrés, con el objetivo de introducir las parejas simbióticas rizobioleguminosa más adecuadas a los parámetros físico-químicos de los suelos a recuperar. Con este fin se ha iniciado el estudio de los simbiontes de M. marina aislada de dos zonas, las Marismas del Odiel (Huelva) y la zona dunar de San Fernando (Cádiz) Además, dada la relativa abundancia de esta leguminosa en las playas de San Fernando una vez determinados los mejores simbiontes, podría ser utilizada para la estabilización de sistemas dunares. MATERIAL Y MÉTODOS Para el aislamiento de bacterias, los extractos de los nódulos previamente desinfectados (hipoclorito de sodio al 5% durante 4 minutos) fueron sembrados sobre placas de YMA con Rojo Congo (25 mg/L). La PCR-ERIC se hizo utilizando los cebadores consenso ERIC 1R y ERIC 2, siguiendo el protocolo descrito por Versalovic et al. (1991). La amplificación del gen ARNr 16S se realizó con los cebadores 616F y 1522R. Los fragmentos del gen ARNr 16s amplificados fueron secuenciados por Stab-Vida (Portugal) usando los cebadores 519F, 907R, 616F y 1522R. La identificación de las secuencias se realizó empleando la base de datos EzTaxon-e. Para el análisis de datos, se realizaron alineamientos con el programa Clustal X (Thomson et al., 1997). Los 33 Session I SI-CP-06 análisis filogenéticos y de evolución molecular se hicieron usando el programa MEGA versión 4 (Tamura et al., 2007); con tres algoritmos diferentes para realizar el árbol, máximum-likelihood, (Felsenstein, 1981), maximum-parsimony (Kluge y Farris, 1969) y neighbour-joining (Saitou y Nei, 1987). El gen nod C se amplificó con los cebadores nodCF2 y nodCI (Laguerre et al., 2001) y el método de Laguerre et al. (1994). El producto de PCR se digirió independientemente con los enzimas MspI y RsaI. La región ITS se amplificó con FGPS1490 y FGPS132 (Laguerre et al., 1996) y según el método de Vinuesa et al. (1998). El producto de PCR se digirió independientemente con MspI y AluI. RESULTADOS Y DISCUSIÓN Del aislamiento de los nódulos de las plantas de M. marina se obtuvieron 32 clones que, tras el empleo de la PCR-ERIC, se agruparon en 12 perfiles distintos, 7 de las Marismas del Odiel y 5 de las Dunas de San Fernando. La secuencia del gen del ARNr 16S ha identificado todos los aislamientos como S. meliloti; la mayor parte de ellos con porcentajes de identidad que oscilan entre el 99,3 y 100%. Tan solo uno de los aislamientos de San Fernando (SF 1.2) muestra un porcentaje de identidad del 98%. Los árboles filogenéticos construidos muestran la relación de todos los aislamientos con S. meliloti y la proximidad de los diferentes aislamientos entre sí. Los perfiles RFLP con MspI y RsaI del gen nodC agrupan a los aislamientos en dos tipos de perfil no coincidentes entre ellos. Los perfiles RFLP de la región intergénica ITS muestran que todos los aislamientos presentan el mismo perfil que S. meliloti, excepto el aislamiento ORT17 al ser digerido con MspI. Los resultados obtenidos muestran que los aislamientos de M. marina, pertenecen al género Sinorhizobium y están estrechamente emparentados con S. meliloti, según nos indican los porcentajes de similitud del gen ARNr 16S y los árboles filogenéticos, pero que existen diferentes grupos de cepas que difieren de la cepa tipo de S. meliloti en el patrón de los fragmentos de restricción del gen nodC. Además, los dos perfiles obtenidos por RFLP no se corresponden con la diferente localización geográfica de los aislamientos. AGRADECIMIENTOS Este trabajo ha sido financiado por el proyecto de Excelencia P10-AGR5821 de La Junta de Andalucía . Cynthia Alías Villegas disfruta una beca predoctoral del mismo programa. BIBLIOGRAFÍA Laguerre, G., et al. (1994). Appl. Environ. Microbiol. 60: 56-63. Laguerre, G., et al. (1996). Appl. Environ. Microbiol. 62: 2029-2036. Laguerre, G., et al. (2001). Microbiology 147: 981-993. Versalovic, et al. (1991). Nucl. Acids Res. 19: 6923-6831. Vinuesa, P., et al. (1998). Appl. Environ. Microbiol. 64: 2096-2104. 34 Session I SI-CP-07 Tolerancia a pH y salinidad extremos y propiedades simbióticas de aislamientos de rhizobia de Medicago marina L. Alías-Villegas, C.1, Espuny, M.R.1, Bellogín, R.A.1, Cubo, M.T.1, Camacho, M.2, Temprano, F.2* 1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes 6, 41012-Sevilla. España. 2 IFAPA-Centro Las Torres-Tomejil. Carretera Sevilla-Alcalá del Río km. 12, 2. 41200 Alcalá del Río (Sevilla), España. * [email protected] INTRODUCCIÓN Medicago marina L. es una leguminosa que crece en suelos arenosos o gravosos en numerosos lugares del litoral de la Península Ibérica y de la cuenca mediterránea (Castroviejo et al., 2000). Esta planta podría tener interés para fijar las dunas costeras o servir de planta pionera para facilitar la colonización posterior de los suelos por otras plantas. Es capaz, como muchas leguminosas, de formar nódulos y establecer simbiosis con bacterias del suelo para fijar nitrógeno molecular. Poco se sabe de las características de los rhizobia capaces de nodular M. marina y sería de interés conocer sus propiedades simbióticas con otras especies de Medicago o géneros afines y fisiológicas como tolerancia a pH y salinidad extremos. MATERIAL Y MÉTODOS Se aislaron rhizobia de nódulos de dos poblaciones de plantas de M. marina, una de las marismas costeras de los ríos Tinto y Odiel (37º 10´ 28,5´´ N, 6º 55´51,5 W), en la provincia de Huelva y otra de la playa de San Fernando (36º 28´52,5´´ N, 6º 15´49,4´´ W) en Cádiz. Los nódulos se esterilizaron en superficie y su contenido se sembró en placas con medio sólido de extracto de levadura manitol (ELM), según la técnica habitual (Vincent, 1970). Se realizaron mediante PCR y desarrollo en geles de agarosa perfiles ERIC de ADN para diferenciar los aislamientos (de Bruijn, 1992) tomando de cada grupo similar un aislamiento representativo. Los aislamientos ORT11, 12, 13, 15, 16, 17, 18 y 19 proceden de Huelva y ORT51, 52, 53, 54 y 55 son originarios de Cádiz. Para estudiar el crecimiento a distintos pH se utilizó medio líquido de ELM estabilizando el pH en valores entre 5 y 8,5 con tampones adecuados. Para la tolerancia a salinidad se usó el mismo medio líquido con concentraciones crecientes de cloruro sódico desde 300 mM hasta 900 mM. Los aislamientos se crecieron en tubos con 3 mL de medio, inoculando cada tubo con 50 µL de un cultivo crecido hasta fase estacionaria. Hubo tres repeticiones por cepa bacteriana. Después de 4 días de crecimiento en agitación (180 rpm, 28 ºC) se evaluó el crecimiento midiendo la D.O. de los cultivos a 600 nm. Para la comprobación de las propiedades simbióticas de los aislamientos se utilizaron semillas de M. marina, M. sativa (cv. Aragón), M. polymorpha (cv. Santiago), M. truncatula, M. murex, M. arabiga, M. minima, M. orbicularis y Melilotus indicus. Las semillas se esterilizaron previamente en superficie con etanol al 70% (30 s.) e hipoclorito sódico al 5% (6 min.) y posterior lavado (6 veces) con agua estéril. Las semillas, germinadas en agar agua (10%), se sembraron en tubos de 20 x 200 mm, con una tira de papel de filtro como soporte y 15 mL de medio de cultivo sin nitrógeno (Rigaud y Puppo, 1975). Las plantas se inocularon, a las 24 h de la siembra, con 0,1 mL de una suspensión concentrada de 108 células/mL de cada cepa bacteriana (2 repeticiones/cepa y planta). Las plantas se crecieron en invernadero entre 16 ºC (noche) y 25 ºC (día) y con un fotoperíodo de 14 h de luz. Después de 2 meses se observó la nodulación (presencia y tamaño de nódulos) y la efectividad de la fijación por comparación con los controles de las plantas no inoculadas. Como referencia se utilizaron las cepas LMG6133T de Sinorhizobium meliloti, LMG19920T o M19-1 de S. medicae e ISLU16 de Bradyrhizobium sp. (Lupinus). 35 Session I SI-CP-07 RESULTADOS Y DISCUSIÓN El crecimiento de los aislamientos de M. marina a distintos pH en el rango 5-8,5 mostró un patrón similar, entre ellos y con las cepas de referencia de S. meliloti y S. medicae. Ninguna de las cepas creció a pH 5, mientras que a pH 5,5 mostraron algo de crecimiento y a pH 7, 8 y 8,5 crecieron satisfactoriamente. La cepa ISLU16 de Bradyrhizobium sp., sin embargo, creció bien a pH 5 y también a 5,5, 7 y 8, viéndose disminuido el crecimiento a pH 8,5. La tolerancia a salinidad de los distintos aislamientos fue muy parecida y semejante a la de S. meliloti: todas las cepas crecieron bien hasta con 700 mM de NaCl, poco a 800 mM y nada a 900 mM. La cepa tipo de S. medicae no creció en medios con 800 mM de NaCl. ISLU16 fue incapaz de crecer en medios con más de 300 mM de NaCl. En la Tabla 1 se muestran los resultados de nodulación y fijación de los aislamientos con las distintas especies de medicagos y con Melilotus indicus. Su comportamiento simbiótico fue muy semejante entre ellos y muy parecido al de la especie tipo de S. meliloti y muy diferente al de S. medicae (Garau et al., 2005). Con las especies M. truncatula, M. minima y M. orbicularis la efectividad fijadora de nitrógeno de los aislamientos fue variable. Tabla 1. Nodulación y fijación de nitrógeno de aislamientos de Medicago marina con algunas especie s de Medicago y Melilotus indicus1. Bacteria 1 M. marina M.. sativa M. polymorpha M. truncatula M. murex M. arabiga M. minima M. orbicularis M. indicus ORT11 nod+fix+ nod+fix+ Ab. nod+fix+ Ab. Ab. nod+fix+ nod+fixnodORT12 nod+fix+ nod+fix+ Ab. nod+fixAb. Ab. nod+fix+ nod+fixAb. ORT13 nod+fix+ nod+fix+ Ab. nod+fixAb. Ab. nod+fix+ nod+fix+ nodORT15 nod+fix+ nod+fix+ Ab. nod+fixAb. Ab. nod+fixnod+fix+ Ab. ORT16 nod+fix+ nod+fix+ Ab. nod+fixnodAb. nod+fixnod+fix+ Ab. ORT17 nod+fix+ nod+fix+ Ab. nod+fixAb. Ab. nod+fix+ nod+fix+ ORT18 nod+fix+ nod+fix+ Ab. nod+fixAb. Ab. nod+fixnod+fixAb. ORT19 nod+fix+ nod+fix+ Ab. nod+fix+ Ab. Ab. nod+fixnod+fixnodORT51 nod+fix+ nod+fix+ Ab. nod+fixAb. Ab. nod+fix+ nod+fixnodORT52 nod+fix+ nod+fix+ Ab. nod+fixAb. Ab. nod+fix+ nod+fixAb. ORT53 nod+fix+ nod+fix+ Ab. nod+fixAb. Ab. nod+fix+ nod+fixAb. ORT54 nod+fix+ nod+fix+ Ab. nod+fixAb. Ab. nod+fix+ nod+fixAb. ORT55 nod+fix+ nod+fix+ Ab. nod+fixnodAb. nod+fix+ nod+fixnodS. meliloti2 nod+fix+ nod+fix+ Ab. nod+fixAb. Ab. nod+fix+ nod+fixAb. S. medicae3 nod+fixnod+fix+ nod+fix+ nod+fix+ nod+fixnod+fix nod+fix+ nod+fix+ nod+fix+ nod+fix+ + T Ab.: Abultamientos blancos en las raíces. 2 Cepa LMG19920 . 3 Cepa M19-1. AGRADECIMIENTOS Este trabajo se ha realizado gracias a la financiación de los proyectos P10-AGR5821 de La Junta de Andalucía y RM2010-00014-00-00 del INIA. BIBLIOGRAFÍA Castroviejo, S., et al.(Eds.) (2000). Flora Iberica. VII(II). Real Jardín Botánico. Madrid. p. 755 . de Bruijn, F. (1992). Appl. Environ. Microbiol. 58: 2180-2187. Garau, G., et al. (2005). Plant Soil.176: 263-277. Rigaud, J., Puppo, A. (1975). J. Gen. Microbiol. 88: 223-228. Vincent, J.M. (1970). A manual for the practical study of root nodule bacteria. IBP Handbook No. 5 . Balckwell Sci. Pub. Oxford (Reino Unido) 36 Session I SI-CP-08 Estudio de la biodiversidad microbiana en suelos de las Marismas del Odiel y de las Minas de Rio Tinto. Ruíz-Carnicer, A.1, Alías-Villegas, C.2, Bellogín, R.A.2, Manyani, H.1, Temprano, F.3, Espuny, M.R.2, Camacho, M.3* 1 ResBioagro. 3ª planta Edificio Citius. Reina Mercedes, 4. Sevilla. 2 Departamento de Microbiología , Facultad de Biología, Universidad de Sevilla. Sevilla. 3 IFAPA Centro Las Torres-Tomejil. Sevilla. * [email protected] RESUMEN Se ha hecho un estudio de la diversidad microbiana presente en suelos altamente contaminados de las Marismas del Odiel y de las Minas de Río Tinto para determinar cómo afectan distintos factores (acidez y salinidad) a las poblaciones bacterianas e aislar aquellas especies mayoritarias en cada uno de ellos. De 300 aislamientos realizados, se han seleccionado aquellos que poseen alguna característica promotora del crecimiento de la planta (PGP) o alguna actividad enzimática útil en la industria. Los resultados muestran la gran biodiversidad presente en las distintas zonas, así como la selección de 28 bacterias altamente prometedoras. INTRODUCCIÓN El impacto de la actividad humana influye de forma notable en la degradación de los estuarios. Entre estos, el estuario de Huelva está considerado como uno de los más contaminados de Europa (Sainz et al., 2004; Ruiz et al., 2008) debido, por una parte al aporte altamente ácido que recibe de los Ríos Tinto y Odiel tras recorrer gran parte de la Faja Pirítica Ibérica y por otra, a los vertidos provenientes del polo industrial onubense. Hasta ahora se han hecho numerosos estudios sobre la influencia de estos vertidos sobre la flora y fauna de la zona (Mateos-Naranjo et al., 2011; Sánchez, 2006), especialmente tras los intentos de recuperación llevados a cabo en parte del estuario (Rubio et al., 2001), pero los estudios acerca de las comunidades microbianas son escasos o nulos. En este trabajo se ha comparado, mediante la técnica de la electroforesis en gel con gradiente de desnaturalización (DGGE), las poblaciones bacterianas presentes en tres zonas diferenciadas del estuario: zona del Dique Juan Carlos (suelo básico, no salino), zona recuperada (suelo básico, salino) y zona del Rio Tinto (suelo ácido, no salino). Además, se han aislados una serie de bacterias que pueden ser en utilizadas tanto en los procesos de restauración (aplicadas como inoculantes) como en distintos procesos industriales. MATERIAL Y MÉTODOS Toma de muestras: se tomaron muestras de suelo rizosférico de varias sub-parcelas de cada zona de muestreo. Se mantuvieron en frio hasta su procesamiento. Aislamiento de bacterias y pruebas bioquímicas. Se llevaron a cabo utilizando los métodos descritos por Tsavkelova et al. (2007), con ligeras modificaciones. Extracción de ADN de suelo. Se realizó siguiendo el protocolo del kit PowerSoilTM DNA Isolation Kit. Se comprobó el resultado de la extracción en un gel de agarosa al 1%. Electroforesis en gradiente desnaturalizante (DGGE). Se hizo siguiendo el protocolo descrito por Heuer et al. (1997) con un gradiente de agentes desnaturalizantes comprendido entre el 55% y el 70%. El posterior análisis de las bandas se realizó con el programa Quantity One. 37 Session I SI-CP-08 RESULTADOS Y DISCUSIÓN De las 300 cepas aisladas se han seleccionados 28 en base a la determinación de las siguientes propiedades: Actividades enzimáticas: lipasa, proteasa, celulasa y amilasa Características como PGP: producción de ácido indol-acético, de sideróforos, solubilización de fosfatos y actividad ACC (1-aminociclopropano carboxilato) desaminasa Características fisiológicas: tolerancia a salinidad y a pH De las 28 cepas seleccionadas, 15 proceden de Rio Tinto, 12 del Dique Juan Carlos y tan solo 1 procede de la zona de recuperación, lo que parece indicar que las zonas más contaminadas son lugares más apropiados para el aislamiento de bacterias con alguna característica deseable. La mayoría de los aislamientos son capaces de crecer a concentraciones de 600 mM de NaCl, a un amplio rango de pH (de 4.5 a 9) y poseen una o dos actividades enzimáticas. Sólo uno de ellos (RBA-OR120) resultó positivo en las cuatro actividades. Respecto a las propiedades como PGP, la mayoría de las cepas exhibió al menos dos de ellas, presentando 5 de los aislamientos todas las estudiadas. En cuanto a la diversidad bacteriana de las distintas zonas, en cada punto de muestreo aparecieron entre 25-30 especies distintas, existiendo una mayor heterogeneidad entre los distintos puntos en la zona del Rio Tinto (Figura 1). Por otra parte, en términos globales, la zona que presentó una mayor diversidad bacteriana fue la zona recuperada, donde se observaron más de 70 especies distintas. Figura 1. DGGE correspondiente a la zona de Rio Tinto, con 13 puntos de muestreo. AGRADECIMIENTOS Este trabajo ha sido financiado por el Proyecto de Excelencia P10-AGR5831 de la Junta de Andalucía y la beca del mismo programa de la Junta de Andalucía de Cynthia Alias Villegas BIBLIOGRAFÍA Heuer, H., et al. (1997). Appl. Environ. Microbiol. 63: 3233-3241. Mateos-Naranjo, E., et al. (2011). Ecotox. Environ. Safe. 74: 2040-2049. Rubio, J.C., et al. (2001). III Congreso Forestal Español. Tomo: 3CFE04-061-T4: pág. 392-395. Ruiz, F., et al. (2008). Mar. Pollut. Bull. 56: 1258-1264. Sainz, A., et al. (2004). Environ. Int. 30: 557-566. Sánchez, M., et al. (2006). Arch. Fur Hydrobiologie 166: 199-223. Tsavkelova, E.A., et al. (2007). Microbiol. Res. 162: 69-76. 38 Session I SI-CP-09 Componentes biológicos en el suelo de dos plantaciones similares de ciruelo en producción ecológica y convencional. Daza, A. *, Pérez-Romero, L.F., Arroyo, F.T., García-Galavís, P.A., Camacho, M., Santamaría, C. IFAPA Centro “Las Torres-Tomejil”, 41200-Alcalá del Río (Sevilla), España. * [email protected] RESUMEN Se han analizado la composición biológica del suelo en dos plantaciones de ciruelo japonés (Prunus salicina Lindl.), en un suelo franco limoso, una manejada en producción ecológica y la otra en producción convencional. Las poblaciones de bacterias y hongos cultivables han sido el doble en el suelo ecológico, que contuvo también poblaciones superiores de rizobios. El grado de micorrización de las raíces de ciruelo y de la herbácea Conyza bonariensis, común en ambos suelos, ha sido elevada y no ha mostrado diferencias significativas. Las poblaciones de lombrices fueron alrededor de tres veces más abundantes en el suelo ecológico. INTRODUCCIÓN La Agricultura Ecológica ha crecido exponencialmente en todo el mundo en las dos últimas décadas. Su defensa se basa tanto en aspectos relacionados con la calidad y sanidad de los productos como en los beneficios medioambientales que dicha práctica agraria conlleva. En este estudio se ofrecen datos que indican que determinados componentes vivos del suelo, claves para su sostenibilidad y fertilidad, se ven favorecidos por el manejo ecológico MATERIAL Y MÉTODOS El estudio se ha desarrollado en dos parcelas experimentales de 5500 m2 cada una de la finca experimental del IFAPA Centro “Las Torres-Tomejil” en Alcalá del Río (Sevilla). Desde su plantación, en enero de 2005, una parcela está en producción ecológica y la otra en producción convencional. La fertilización en la parcela ecológica ha sido a base de estiércol de origen animal (3-4 kg m-2) y la siembra de cubiertas vegetales conteniendo leguminosas. Las poblaciones bacterianas y fúngicas cultivables se han cuantificado en los medios sintéticos Nutrient Agar y PDA, respectivamente. Las poblaciones de rizobios se determinaron por el método del número más probable. El grado de micorrización en raíces de ciruelo (Prunus salicina Lindl.) y la especie herbácea Conyza bonariensis L. se determinó según Smith y Read (1997). Las lombrices se calcularon tomando muestras de suelo de 0,5 m2 y 40 cm de profundidad. RESULTADOS Y DISCUSIÓN Se ha constatado que las cubiertas vegetales de leguminosas aportaron cantidades importantes de los principales macronutrientes (Tabla 1). 39 Session I SI-CP-09 Tabla 1. Aportación bruta en materia seca y principales macroelementos de las diferentes cubiertas vegetales utilizadas en años sucesivos en la plantación ecológica de ciruelos Tipo de cubierta kg/ha Materia seca N P K Glycine max (pre-plantación) 10.060 320 34 206 Vicia faba 6.320 180 14 185 Brassica napus + Vicia sativa 2.500 70 8 75 Vegetación espontánea 2.913 56 9 75 Vicia sativa + Avena sativa 8.320 180 19 220 Vicia faba 10.353 225 24 274 Con relación a las poblaciones de bacterias y hongos, a partir del año 2008 se viene observando de forma consistente que éstas son aproximadamente dos o tres veces más abundantes en el suelo ecológico. En la Figura 1 se representa el periodo 2011-2013. Número de hongos por gramo de suelo seco Número de bacterias por gramo de suelo seco 400000 350000 120000000 100000000 300000 250000 80000000 Ecológico 60000000 Convencional 40000000 20000000 Ecológico 200000 150000 100000 Convencional 10/02/2013 10/01/2013 10/12/2012 10/11/2012 10/10/2012 10/09/2012 10/08/2012 10/07/2012 10/06/2012 10/05/2012 10/04/2012 10/03/2012 10/02/2012 10/01/2012 10/11/2011 10/02/2013 10/01/2013 10/12/2012 10/11/2012 10/10/2012 10/09/2012 10/08/2012 10/07/2012 10/06/2012 10/05/2012 10/04/2012 10/03/2012 10/02/2012 10/01/2012 10/12/2011 10/11/2011 10/12/2011 50000 0 0 Figura 1. Poblaciones de bacterias y hongos en los suelos ecológico y convencional en el periodo 2 0112013. En ocasiones se sembró también una muestra de la cubierta vegetal en la parcela convencional, para comparar la nodulación y las poblaciones de rizobios. Los datos obtenidos con Vicia faba y Vicia sativa se presentan en la Tabla 2. Tabla 2. Número y peso seco de nódulos correspondientes a 12 plantas en los suelos ecológico y convencional Especie Tratamiento Nº nódulos Peso nódulos NMP (g) (Nº rizobios/g suelo) Convencional 105 0,403 499 Vicia faba Ecológico 1094 1,888 10.429 Vicia sativa Convencional Ecológico 92 143 0,157 0,254 nd nd Se ha observado un elevado grado de micorrización (>70%) en las raíces de ciruelo y de la herbácea Conyza bonariensis, pero sin diferencia significativa entre tratamientos. Sin embargo, sí existieron claras diferencias en las poblaciones de lombrices, tres o cuatro veces más abundantes en el suelo ecológico. AGRADECIMIENTOS Los autores agradecen la financiación recibida del INIA (Proyecto RTA2010-00046-00-00) BIBLIOGRAFÍA Smith S.E., and Read, D.J. (1997). Mycorrhizal Symbiosis. Academic Press, San Diego. 40 Session I SI-CP-10 Relative abundance of denitrification genes and diversity of bacterial denitrifiers in sediments with different nitrate concentration. Correa-Galeote, D., Tortosa, G., Bedmar, E.J.* Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Granada, España. Apartado Postal 419, 18080-Granada. * [email protected] INTRODUCTION Denitrification is the biological process by which nitrate is sequentially reduced to dinitrogen gas (N2) via the intermediate compounds nitrite (NO2-), nitric oxide (NO) and nitrous oxide (N2O). Respiratory nitrate reduction is catalysed by a membrane-bound (Nar) or periplasmic (Nap) nitrate reductase encoded by the narGHIJ or the napABC operon, respectively. The reduction of nitrite to nitric oxide is carried out by two structurally different, but functionally similar, enzymes, the Cu-containing and the cd1containing nitrite reductase encoded by the nirK and nirS genes, respectively. The last step of denitrification is the reduction of nitrous oxide and is catalysed by nitrous oxide reductase encoded by the nosZ gene. Denitrifiers include more than 60 genera of Bacteria, and some Archaea and Fungi, and can represent up to 5% of the total soil microbial community (Henry et al., 2006). Denitrification is one of the main branches of the global N cycle, and is responsible for the return of fixed nitrogen to the atmosphere. It also accounts for significant losses of fertilizer nitrogen from agricultural soils (Philippot et al., 2006) and contributes to modify the global atmospheric chemistry, essentially through the greenhouse effect and destruction of the Earth’s ozone layer through stratospheric nitric oxide production. Here we describe differences in the abundance and in the biodiversity of two sampling sites with contrasting nitrate content. MATERIAL AND METHODS Sampling sites. Two sites, the lagoon of el Acebrón (S1) and la Cañada creek (S2), representing the sites with the lowest (0.27 ± 0.05 mg NO3−/kg sediment) and highest (32.77 ± 1.95 mg NO3−/kg sediment) nitrate concentration along la Rocina stream, which feeds Marisma del Rocio in Doñana National Park (South West, Spain), were selected for sampling. Sediment samples were taken as indicated earlier (Tortosa et al., 2011) in April and October years 2008, 2009 and 2010. Samples were placed on ice while returned to the laboratory and then stored at -80 ºC until use. DNA extraction. DNA was extracted from 250 mg of each subsample according to Correa-Galeote et al. (2013a). Quantification of the denitrification-associated microbial community. The size of the denitrifier community was estimated by quantitative, real-time PCR (qPCR) of narG, napA, nirK, nirS and nosZ gene fragments using reaction mixtures, primers and thermal cycling conditions described previously (Correa-Galeote et al., 2013a). The total bacterial community was quantified using 16S rRNA gene as molecular marker as described by Correa-Galeote et al. (2013b). Clone library construction and DNA sequencing. nosZ amplicons from samples taken in April and October 2009 and 2010 from sites S1 and S2 were purified using the QIAquick PCR purification kit (Quiagen) and cloned in Escherichia coli JM109 using the pGEM-T Easy cloning kit. Cells were screened by PCR using the vector primers Sp6 and T7 (Invitrogen). Nucleotide sequences of clones 41 Session I SI-CP-10 containing inserts of the expected size were determined at the DNA Sequencing Service of Estación Experimental del Zaidín, CSIC, Granada, Spain. Nucleotide sequences were compared to entries in GenBank using BlastN (Altschul et al., 1990). RESULTS AND DISCUSSION Relative abundance of the denitrification genes. For the 3 years under study, regardless of the sampling dates, the relative abundance of the narG and napA genes in sediments taken at S2 was higher than that in sediments sampled at S1. Whereas the relative contributions of narG to the total eubacterial community in April and October were similar at S1, there was a significant difference between April and October at S2. However, there were no significant differences in relative abundance of the nirK gene between S1 and S2 for the three years under study. Whereas values of relative contribution of nirK were similar at S1, the contribution was higher in October than in April at S2. The percentage of the nirS gene was also higher in S2 than in S1. Despite differences in the relative contribution of the nirS gene both for years and sampling dates, the contribution of nirS to the total eubacterial community was higher in October than in April at S1 and also at S2. For the 3 years under study, the relative abundance of nosZ was higher in S2 than in S1, regardless of the sampling. Whereas no differences in the relative contribution of nosZ to the total eubacterial community were found between the months of April and October at S1, there was a statistically significant difference between April and October at S2. Taken together, our data show that relative abundance of the narG, napA, nirS, nirK and nosZ genes was 15.54%, 8.96%, 6.86%, 5.72% and 0.26%, respectively. Biodiversity of bacterial denitrifiers. For the 2 years under study, when sequences of the nosZ gene from the eight clone libraries were compared, the number of operational taxonomic units (OTUs) found in sediments from S1 were 29 for samples taken in both April 2009 and 2010, and 27 and 29 for samples taken in October 2009 and 2010, respectively. Similarly, the number of OTUs in sediments from S2 collected in April 2009 and 2010 were 25 and 26, respectively. However, the number of OTUs in sediments taken at S2 in October 2009 and 2010 were 35 and 34, respectively. Nitrate concentration in sediments from S2 was always higher in October (47 and 34 mg NO3 -/kg in years 2009 and 2010, respectively) than in April (31 and 26 mg NO3-/kg in years 2009 and 2010, respectively). Thus, it is possible that increase in the number of OTUs could be due to the higher nitrate concentration. The most abundant genera in sediments were Acidovorax, Leptothrix, Azospirillum, Paracoccus and Bradyrhizobium. ACKNOWLEDGEMENTS This work was supported by ERDF-cofinanced grants P09-RNM-4746 from Consejería de Economía, Innovación y Ciencia (Junta de Andalucía, Spain). REFERENCES Correa-Galeote, D., et al. (2013a). FEMS Microbiol. Ecol. 83: 340-351. Correa-Galeote, D., et al. (2013b). J. Metagenomics doi:10.4303/mg/235702 Henry, D., et al. (2006). Appl. Environ. Microbiol. 72: 5181-5189. Philippot, L., et al. (2006). In: Molecular Approaches to Soil, Rhizosphere and Plant Microorganis ms Analysis. Cooper JE and Rao JR, eds. CABI International, Cambridge. Pp. 146-164. Tortosa, G., et al. (2011). Ecol. Eng. 37: 539-548. 42 Session I SI-CP-11 Experimental and modelling approach to the legume-Rhizobium interaction: test of plant-host sanctions in co-inoculated plants with fixing and non-fixing strains. Marco, D.E.1*, Talbi, C.2, Bedmar, E.J.2 1 Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba and CONICET, Argentina. 2 Departmento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Agencia CSIC, Granada, Spain. * [email protected] ABSTRACT We tested the plant host sanction hypothesis using soybean plants co-inoculated with two rhizobial strains, a normally N2 fixing strain and a mutant derivative that lacks nitrogenase activity but has the same nodulation abilities. We found no evidence of functioning plant host sanctions to cheater rhizobia based on nodular rhizobia viability in co-inoculated plants. INTRODUCTION The origin and persistence in nature of symbiotic interactions is difficult to explain since the existence of exploitative, cheating' partners that could erode the interaction is common. Host sanctions against non N2 fixing, cheating symbionts have been proposed as a force stabilizing mutualism in legume-Rhizobium symbiosis (Denison, 2000). Penalizations would include decreased nodular rhizobial viability and/or early nodule senescence in nodules occupied by cheating rhizobia. We analyze the ecological and evolutionary stability of Rhizobium-legume symbiosis when "cheating" strains are present, using a combination of experiments and modelling. Co-occupation of the same nodule by strains with different fixation abilities is an important source of concern in cultivated legumes (Rolfe and Gresshoff, 1980). Effects of co-occupation of nodules by non-fixing rhizobia would be diluted by fixing rhizobia occupying the same nodule (Denison, 2000). We performed experiments with soybean plants co-inoculated with two rhizobial strains, a normally N2 fixing strain and an isogenic non-fixing, cheating mutant derivative that lacks nitrogenase activity but has the same nodulation abilities. MATERIAL AND METHODS Bacterial strains and inoculum preparation. Mutant derivatives BJD321 (nopB-lacZuidA, Zehner et al ., 2008) and A3 (nifH::Tn5, Nod+ Fix-; Hahn et al., 1984) strains derived from Bradyrhizobium japonicum USDA110 were grown in PSY medium (Regensburger and Hennecke, 1983) and used for inoculations (10 9 cells/ml). Coinocula were prepar ed by mixing bacterial solutions containing similar number of colony forming units (CFUs). Strain A3 lacks nitrogenase activity but shows similar infection and nodule formation levels respect to USDA110 and BJD321. Plant experimental setting. Seeds of soybean (Glycine max) cv. Williams were surfacesterilized, ge rminated and sowed in Leonard jar assemblies. Seedlings (2/jar) were inoculated independently with 1 ml of a bacterial suspension made from cultures of each BJD321, A3 and a mixture of each strain. Jars were periodically supplied with a sterile N2-free nutrient solution. Plants were placed in a growth chamber under 600 µEm-2 s-1 photosynthetically active radiation, at 25/18 °C day/night temperature, and 16/8 h photoperiod. Four weeks after inoculation nodules of each plant were collected. Determination of nodule occupancy and viable rhizobial counts. Collected nodules were individually surface sterilized using Cl2Hg (2.5%), manually crushed, homogenized and 43 Session I SI-CP-11 resuspended in Tris-HCL mannitol buffer containing. Each crushed nodule was smeared on PSY plates supplemented with selective antibiotics depending on the strain to determine if the nodule was occupied by BJD321, A3 or both strains. To determine rhizobial viability, appropriate serial dilutions from each nodule of another set of homogenized nodules were plated (three replicates per dilution) in PSY supplemented with selective antibiotics depending on the strain. Plates were incubated at 28 °C for a week or until no further growth was detected, and CFUs were counted. Plant dry weight and N content were determined in plants that had been heated at 60 ºC for 48 h. RESULTS AND DISCUSSION Plants with all nodules occupied by non-fixing rhizobia were not able of maintaining good vegetative conditions as plants with co-occupied or exclusively occupied nodules with fixing rhizobia, and ultimately they died due to N starvation about 5 weeks after inoculation. Thus, comparisons were performed using plants co-inoculated and plants inoculated only with strain BJD321. In co-inoculated plants nodule co-occupation did not differ (36.35 % BJD321, 33.32 % BJD321 and 27.28 % A3, 2 = 6.00, p = 0.199, n = 66). Co-inoculated plants and plants only inoculated with strain BJD321 did not differ in dry weight (Kolmogorov-Smirnov Z = 0.707, p = 0.699, n = 6). Total plant N did also not differ between treatments (Kolmogorov-Smirnov Z = 1.299, p = 0.068, n = 6). Nodule mass did not differ between co-inoculated plants and plants inoculated with BDJ321 only (2 = 1.66, p = 0.56, n = 66). Number of CFUs did not differ between cooccupied nodules and BDJ321 or A3 single-occupied nodules in co-inoculated plants (Figure 1). Figure 1. Number of CFUs in cooccupied nodules or singleoccupied nodules in co-inoculated plants. No evidence of functioning plant host sanctions to cheater rhizobia based on nodular rhizobia viability in co-inoculated plants was found. These experimental results will be incorporated to the mathematical model (Marco et al., 2009) to check for plant population persistence in presence of cheating rhizobia. ACKNOWLEDGEMENTS We thank Michael Goffert for B. japonicum BJD321 and Hans-Martin Fischer for A3 strains. DM was granted with a Sabbatical leave from the Ministry of Education of Spain. REFERENCES Denison, R.F. (2000). Amer. Nat. 156: 567-576. Hahn, M., et al. (1984). Plant Mol. Biol. 3:159-168. Marco, D.E., et al. (2009). J. Theor. Biol. 259: 423-433. Regensburger, B., and Hennecke, H. (1983). Arch. Microbiol. 135: 103-109. Rolfe, B.G., and Gresshoff, P.M., 1980. Austr. J. Biol. Sci. 33, 491-504. Zehner, S., et al. (2008). Mol. Plant Microbe Interact. 21: 1087-1093. 44 Session I SI-CP-12 Identification of rhizobial strains nodulating cultivated grain legumes in Egypt. Zahran, H.H.1*, Chahboune, R.2, Bedmar, E.J.2, Abdel-Fattah, M.1, Yasser, M.M.1, Mahmoud, A.M1. 1 Department of Botany and Microbiology, Faculty of Science, University of Beni-Suef, Beni-Suef 6251 1, Egypt. 2 Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Agencia Estatal Consejo Superior de Investigaciones Científicas (EEZ-CSIC). Apartado Postal 419, 18080-Granada, Spain. * [email protected] INTRODUCTION Members of the Leguminosae (Fabaceae), comprising 17,000 to 19,000 species, play an important ecological role, with representatives in nearly every terrestrial biome on Earth. These plants are best characterized by their ability to establish N2-fixing symbiotic associations with Alphaproteobacteria of the genus Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Ensifer (for reviews see Rivas et al., 2009; Velázquez et al., 2010) collectively referred to as rhizobia. Other non-rhizobial genera have been shown to nodulate legumes (for a review see Velázquez et al., 2010), and they can also be nodulated by Betaproteobacteria of the genus Burkholderia (for reviews see Angus et al., 2010; Bontemps et al., 2010; Gyaneshwar et al., 2011). During the infection process, an exchange of molecular signals occurs between the two partners, leading to the formation of root nodules, where nitrogen fixation takes place. In Egypt, the grain legumes chickpea (Cicer arietinum), lentil (Lentils esculentus), common bean (Phaseolus vulgaris), pea (Pisum sativum) and broad bean (Vicia faba) are widely used for people consumption. Cultivated all along the River Nile, no data exist on the bacterial diversity within nodules formed on those legume crops. In this regard, a culture collection of more than 50 rhizobial-like strains isolated from root nodules of those legumes were obtained whose phenotypic characteristics and nodulation capacity have been published (Zahran et al., 2012). Our research had the aim of identifying the strains isolated from nodules formed on agriculturally-grown chickpeas, lentils, common beans, peas and broad beans, and to identify them on the basis of their 16S rRNA gene phylogenies. MATERIAL AND METHODS Isolation of bacteria from nodules and culture conditions Nodules (10-12/plant) were collected from roots of healthy C. arietinum, L. esculentus, P. vulgaris, P. sativum and V. faba plants agriculturally-grown in the Beni-Suef Governorate (Egypt). They were surface-sterilized and crushed in a drop of sterile water. The resulting suspension was streaked onto Petri dishes containing yeast extractmannitol (Y EM) medium supplemented with 0.025 g/l Congo Red. Colonies forming units were selected as indicated earlier (Chahboune et al., 2011). DNA extraction and PCR amplifications For DNA extraction and PCR amplifications, genomic DNA was isolated as described previously (Chahboune et al., 2011). Repetitive extragenic palindromic (REP)polymerase chain react ions (PCR) were performed using primers REPIR-I and REP2-I according to de Bruijn (1992). PCR amplifications of 16S rRNA gene fragments were carried out using the two opposing primers 41f and 1488r as previously reported (Chahboune et al., 2011). Amplification products were purified and subjected to cycle sequencing using the same primers as for PCR amplification. The obtained sequences 45 Session I SI-CP-12 were compared to the GenBank database using the BLAST program and also with the sequences held in EzTaxon-e server (Kim et al., 2012). Phylogenetic trees were prepared as indicated earlier (Chahboune et al., 2011). Nitrogenase activity was determined as acetylene-dependent ethylene production as described previously (Zahran et al., 2012). RESULTS AND DISCUSSION A total of 54 bacterial strains, 12 from P. sativum, 11 from each C. arietinum and V. faba, and 10 from each L. esculentum and P. vulgaris, respectively, were isolated from extracts of nodules from healthy, agriculturally-grown plants in Beni-Suef Governorate (Egypt). According to the results, the 54 isolates showed 15 different REP-PCR patterns. The nearly complete sequence of the 16S rRNA gene from a representative strain of each REP pattern revealed they all were members of the family Rhizobiaceae of the Alphaproteobacteria. Of all the 15 strains, 12 were classified into the genus Rhizobium and 3 into the Mesorhizobium group. The maximum likelihood phylogenetic tree and EzTaxon-e analysis inferred from the 16S rRNA genes sequences indicated that strains BSPV2, BSPV7, BSPS4, BSVF2, BSVF5, BSVF9, BSLE4 and BSLE10 clustered with R. leguminosarum USDA 2370T with identity values higher than 99.6%, and that strains BSPS7, BSPS10 and BSPV9 grouped with R. pisi DSM 30132T, R. etli CFN 42T and R. mesosinicum CCBAU 25010T, respectively, each of them with identity values higher than 99.8%. Also, the strain BSCA1 clustered with M. amorphae ACCC 19665T, and strains BSCA8 and BSCA9 did with M. robiniae CCNWYC 115T, the all three strains with 100% identity for the 16S rRNA gene sequences of their corresponding type strains. The 15 rhizobial strains identified in this study nodulated their original host plants. Nodules fixed N2, with values of nitrogenase activity, determined as acetylene-dependent ethylene production, varying from 51 nmol C2H4 plant-1 h-1 in C. arietinum nodulated by strain BSCA8 to 480 nmol C2H4 plant-1 h-1 in P. vulgaris inoculated with strain BSPV2. ACKNOWLEDGEMENTS This study was supported by ERDF-cofinanced grant RNM4746 from Consejería de Economía, Innovación y Ciencia (Junta de Andalucía, Spain). A. M. Mahmoud thanks the Egyptian Government for a Partnership and Ownership Initiative (PAROWN) grant to support his stay at Department of Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Agencia Estatal CSIC, Granad a, Spain. REFERENCES Angus, A.A., et al. (2010). Mol. Ecol. 19: 28-23 Bontemps, C., et al., (2010). Mol. Ecol. 19: 44-52. Chahboune, R., et al. (2011). Syst. Appl. Microbiol. 34: 440-445 De Bruijn, F.J. (1992). Appl. Environ. Microbiol. 58: 2180-2187. Gyaneshwar, P., et al. (2011). Mol. Plant Microbe Interact. 24: 1276-1288. Kim, O.S., et al. (2012). Int. J. Syst. Evol. Microbiol. 62: 716-721. Rivas, R., et al. (2009). Rev. Microbiol. Insight, 2: 251-269. Velázquez, E., et al. (2010). In: Proteobacteria: Phylogeny, metabolic diversity and ecological ef fects. Sezenna, M.L. (ed). Nova Science Publishers Inc. New York, USA, pp37-56. Zahran, H.H., et al. (2012). Aust. J. Basic Appl. Sci., 6: 571-583. 46 Session I SI-CP-13 Genetic diversity and biogeography of rhizobial genospecies nodulating wild chickpea Cicer canariense on La Palma (Canary Is.). Armas-Capote, N.1, Pérez-Yepez, J.1, Martínez-Hidalgo, P.2, Garzón-Machado, V.3, del Arco-Aguilar, M.3, Velázquez, E.2, León-Barrios, M.1* 1 Departamento de Microbiología y Biología Celular. 2 Departamento de Microbiología y Genética, Universidad de Salamanca. 3 Departamento de Biología Vegetal (Botánica). Universidad de La Laguna. Tenerife. Spain. * [email protected] ABSTRACT We have characterized a collection of one hundred and thirteen rhizobial strains that nodulate Cicer canariense and show the distribution of the bacterial genotypes at ten locations on the island of La Palma (six inside Caldera de Taburiente National Park and four in areas to the northwest and south of the Park). Analysis of the 16S rRNA gene classified these isolates within the genus Mesorhizobium and distinguished nine genotypes. The five predominant genotypes belonged or were close to reference strains of M. caraganae (32 isolates), M. tianshanense/M. metallidurans/M. gobiense (29 isolates), M. opportunistum (14 isolates) M. ciceri (12 isolates) and M. tamadayense (11 isolates). M. tamadayense isolates were exclusive at one location outside the Park. The M. tianshanense and M. ciceri groups predominated in planted plots and showed scarce genetic diversity at infraspecific level. The M. caraganae and M. opportunistum groups were widely distributed inside and outside the Park, though they predominated in natural populations, and were genetically diverse. INTRODUCTION Cicer canariense is a perennial wild chickpea endemic to the Canary Islands, the only native Cicer species in the archipelago. It is found almost exclusively on La Palma, in the dry mesocanarian bioclimatic belt characteristic of pine woodland (Pinus canariensis). Natural populations of C. canariense are fragmented and contain a low number of individuals, and currently it is catalogued as “endangered” (EN) in the Red List of the Spanish Vascular Flora (Moreno, 2008), according to the IUCN (2001) criteria. Herbivores have probably been the most important threat for C. canariense (mouflons and rabbits on Tenerife, and Barbary sheep, goats and rabbits on La Palma) (Garzón-Machado et al., 2010). In this study we investigated for the first time the genetic diversity of the rhizobia nodulating C. canariense in natural and reinforced populations on La Palma (Canary Islands). MATERIAL AND METHODS Random Amplified Polymorphic DNA (RAPD) fingerprints were obtained as previously described (Ramírez-Bahena et al., 2012). Restriction Fragment Length Polymorphism of PCR-amplified 16S rRNA (rrs) genes (16S-RFPL). The rrs genes were amplified, restricted and analysed as described (Jarabo-Lorenzo et al., 2000). Sequencing of rrs genes. The rrs genes were amplified as above described and the purified products (Qiaquick extraction kit, Qiagen) were sequenced in an ABI3730XL (Macrogen, Inc.) or in a Genetic Analyzer 3500 (Servicio de Genómica, ULL). Phylogenetic analyses were conducted as described (Lorite et al., 2011). The sequences of rrs genes were compared with those of bacterial type strains using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012). 47 Session I SI-CP-13 RESULTS AND DISCUSSION A collection of one hundred and thirteen rhizobia that nodulate C. canariense were characterized. Isolations were done from populations at ten sampling sites on La Palma, which included six locations with natural and planted populations within the Caldera de Taburiente National Park and another four localities with natural populations outside the Park. The genetic diversity and phylogeny were estimated by RAPD profiles, 16S-RFLP analysis and sequencing of the 16S rRNA genes. 16S-RFLP classified the isolates within the genus Mesorhizobium and distinguished nine different ribotypes. Four branches were minority genotypes (3-5 isolates), whereas another five contained the predominant genotypes that clustered with reference strains M. tianshanense/M. metallidurans/M. gobiense (M. tianshanense-group) M. caraganae, M. opportunistum, M. ciceri and M. tamadayense. The 16S sequences confirmed homogeneity for M. ciceri, M. opportunistum and M. tamadayense RFLP-groupings. They also resolved further internal divergence within the two larger groups (M. caraganae and M. metallidurans) outlining two novel Mesorhizobium species. RAPD fingerprints were used to estimate the genetic diversity at infraspecies level, showing that highly similar isolates belonged to the same RFLP group and were in general recovered from the same location. The geographical distribution of these genotypes varied among the different locations. Some genotypes predominated or were restricted to a single location, whereas others were widely distributed. Isolates from the M. tianshanense-group were detected at six locations, although 23 out of 29 isolates were recovered from the same single location. The M. ciceri-genotype isolates were detected at four sampling sites but predominated at two neighbouring locations. The M. tamadayense genotype showed infraspecific diversity, despite being exclusive to a single location outside the Park. Isolates from the M. caraganae and M. opportunistum groups were widely distributed inside and outside the Park, although they predominated in natural populations and were genetically diverse. ACKNOWLEGMENTS Supported by the Ministerio de Medio Ambiente y Medio Rural y Marino, Organismo Autónomo de Parques Nacionales (Ref. 111/2010). REFERENCES Garzón-Machado, V., et al. (2010). Biol. Conserv. 143: 2685-2694. Jarabo-Lorenzo, A., et al. (2000). Syst. Appl. Microbiol. 23: 418-425. Lorite, M.J., et al. (2010). Syst. Appl. Microbiol. 33: 282-290. Moreno, J.C. coord. (2008). Lista Roja 2008 de la flora vascular española. Ministerio de Medio Amb iente y Medio Rural y Marino, y Sociedad Española de Biología de la Conservación de Plantas, Madrid. 86 pp. Ramírez-Bahena, M.H., et al. (2012). Syst. Appl. Microbiol. 35: 334-341. 48 Session I SI-CP-14 Identificación de dos bacterias diazótrofas asociadas a una Brassicaceae (Lepidium meyeni Walp.) de suelos altoandinos del Perú. Chumpitaz, C. *, Ogata, K., Santos, R., Zúñiga, D. Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso, Dpto. Biología, Universidad Nacional Agraria La Molina. Lima12, Perú. www.lamolina.edu.pe/lmt * [email protected] RESUMEN Se amplificó el gen nifH de la dinitrogenasa reductasa en dos cepas, LMTZ064-109 y LMTZ064-119, asociadas a la rizosfera de Lepidium meyenii Walp. Los aislados pertenecen a los géneros Stenotrophomonas y Rahnella, respectivamente, siendo ambas Gram negativas, cocobacilos y oxidasa negativa. La aparición de un producto de aproximadamente 370 pb perteneciente al gen nifH confirmó la capacidad fijadora de nitrógeno molecular de ambas cepas. INTRODUCCIÓN La maca (L. meyenii Walp.) es una Brassicaceae que habita en regiones altoandinas del Perú entre los 3700-4500 msnm. Es muy apreciada por sus cualidades nutricionales y medicinales (Tovar, 2001). Al ser un cultivo muy extractivo genera un empobrecimiento de los suelos, disminuyendo los rendimientos (Zúñiga, 2009). Los diazótrofos, por otro lado, son un grupo de microorganismos capaces de fijar N 2 a formas más asimilables en diferentes ambientes. El gen nifH de la Fe-proteína de la nitrogenasa (enzima responsable del proceso de fijación) es usado como un indicador determinante de esta capacidad (Zehr et al., 2003). El objetivo de este estudio fue identificar la capacidad fijadora de N2 de los aislados bacterianos asociados al cultivo de maca. MATERIAL Y MÉTODOS Reactivación y caracterización morfológica y bioquímica de bacterias conservados. Amplificación BOX PCR de acuerdo a Versalovic et al. (1991). Amplificación del gen nifH de acuerdo a Barua et al. (2011). Análisis filogenético por medio del gen ARN ribosomal 16S según Weisburg et al. (1991), usando la base de datos del Genbank (NCBI). RESULTADOS Y DISCUSION Un total de siete bacterias (LMTZ064-21, LMTZ064-30, LMTZ064-94, LMTZ064-95, LMTZ064-109, LMTZ064-119 y LMTZ064-120) fueron seleccionadas de la colección de bacterias promotoras de crecimiento vegetal (186 cepas) aisladas de suelos de cultivo L. meyenni (Garcia, 2011). Estas cepas fueron caracterizadas morfológicamente de acuerdo a su crecimiento en medio mineral sin nitrógeno (MMN-) por 48 horas a 28 °C. Las colonias tuvieron formas convexas, incoloras, de borde liso, mucosas y diámetro entre 0.5-2 mm. Todas resultaron ser Gram negativas, cocobacilos y oxidasa negativa. De acuerdo al análisis BOX PCR se obtuvieron cinco perfiles distintos (Figura 1A), a los cuales se les evaluó su capacidad fijadora de nitrógeno por medio de la amplificación del gen nifH de la dinitrogenasa reductasa (Fe-proteína). Solo dos aislados (LMTZ064-109 y LMTZ064-119) resultaron ser nifH positivos (Figura 1B). 49 Session I SI-CP-14 A B A B C C D E E A B C D E nifH 370pb Figura 1. A) Perfiles BOX A1R, A (21), B (30), C (94, 95), D (109) y E (119, 120). M: marcador 1 kb (fermentas). B) PCR nifH de perfiles BOX diferentes. Cepas nifH+: LMTZ064-109 y LMTZ064-119. CIAT899 Rizhobium tropici (control+). M: marcador 100 pb (Axygen). Figura 2. Árbol filogenético en base al gen del ARNr 16S mostrando la localización de los aislados LMTZ064-109 y LMTZ064-119. Se realizó un análisis Neighbor-joining con un valor bootstrap calculado para 1.000 réplicas. Escala: 2% de secuencias divergentes. De acuerdo al análisis filogenético realizado (Figura 2), se encontró que la cepa LMTZ064-109 está relacionada a S. maltophila ATCC 13637T con 98%. Stenotrophomonas spp. es un habitante común de suelos y plantas (Ryan et al., 2009), especies de este género pueden actuar como promotores de crecimiento vegetal mediante diferentes mecanismos como la fijación de nitrógeno y producción de fitohormonas (Liba et al., 2006). Por otro lado, la cepa LMTZ064-119 tuvo un 100% de similitud con Rahnella aquatilis HX2. Esta especie se reportó por primera vez como una enterobacteria fijadora de nitrógeno presente en rizosferas de cultivos de maíz y trigo (Berge et al., 1991) y su capacidad como PGPR en rizosfera de cultivos de soja y papa (Kim et al., 1997; Kohashikawa, 2010) al producir fitohormonas y solubilizar sales fosfato. En este trabajo se pudo determinar que las dos bacterias aisladas del cultivo de maca fueron capaces de fijar nitrógeno molecular. Este parámetro es importante a tener en cuenta para la producción de inoculantes bacterianos, ya que además de reducir el uso de fertilizantes químicos, promueve el crecimiento de cultivos de importancia económica dentro de una agricultura sustentable; mejorando a su vez la calidad del suelo. AGRADECIMIENTOS Perú biodiverso GTZ-CONCYTEC, 2009. Procyt 309-2009-CONCYTEC. FDA Biol.111-UNALM. BIBLIOGRAFÍA Barua, M.S., et al. (2012). Microbiol. Res. 167: 95-102. Berge, O., et al. (1991). Can. J. Microbiol. 37: 195-203. Garcia, F. (2011). Tesis Doctoral. Universidad Nacional Agraria La Molina. Lima, Perú. Kim, K,Y., et al. (1997). FEMS Microbiol. Lett. 153: 273-277. Kohashikawa, N. (2010). Tesis Doctoral. Universidad Nacional Agraria La Molina. Lima, Perú. Liba, C.M., et al. (2006). J. Appl. Microbiol. 101: 1076-1086. Ryan, R.P., et al. (2009). Nature Rev. Microbiol. 7: 514-525. Tovar, O. (2001). Publicación CONCYTEC. Lima 144 pp. Versalovic, J., et al. (1991). Nucl. Acids Res. 19: 6823-6831. Weisburg, W.G., et al. (1991). J. Bacteriol. 173: 697-703. Zehr, J.P., et al. (2003). Environ. Microbiol. 5: 539-554. Zúñiga, D. (2009). Perú biodiverso GTZ-Concytec. Lima, Perú. 50 Session I SI-CP-15 Análisis de la presencia natural de micorrizas en cultivos de algodón inoculados con Bacillus sp. y/o Bradyrhizobium sp. Valencia, C.1*, Toro, M.2, Zúñiga, D.1 1 Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso. Dpto. de Biología, Facultad de Ciencias, Universidad Nacional Agraria La Molina, La Molina, Lima, Perú. Web: www.lamolina.edu.pe/lmt. 2 Laboratorio de Estudios Ambientales, Instituto de Zoología y Ecología Tropical, Facultad de Ciencias, Universidad Central de Venezuela. * [email protected] RESUMEN Para el presente estudio, se extrajeron las raíces de cultivos de algodón (Gossypium barbadense L.) inoculados con Bacillus sp. (B) y/o Bradyrhizobium sp. (Br), B + Br (I), y los controles N+ (Nitrato de Potasio) y N- (Sin inocular). Para la tinción se realizó una modificación optimizada del protocolo propuesto por Phillips & Hayman. Una vez teñidas las raíces se calculó el porcentaje de Longitud de Raíz Colonizada (%LRC) para determinar la influencia de las diferentes bacterias sobre la presencia natural de micorrizas arbusculares (MA). Se observó que el %LRC (28.77%) disminuye cuando el cultivo es fertilizado con nitrato de potasio, mientras que la colonización micorrícica se promueve cuando las plantas son inoculadas con Bacillus sp., alcanzando en promedio un 70.98%. INTRODUCCIÓN Las bacterias promotoras de crecimiento (PGPRs) están asociadas con la rizósfera de las plantas y son capaces de ejercer un efecto benéfico en el crecimiento de plantas. Bajo ciertas condiciones, estas bacterias son capaces de promover la germinación de esporas de hongos y la elongación del tubo germinativo o incluso aumentar la densidad de la colonización micorricica (Jaizme et al., 2006) Las MA son asociaciones ecológicamente mutualistas que se establecen entre un selecto grupo de hongos (Glomeromycota) y la gran mayoría de las plantas. Aproximadamente un 80% de las familias de plantas existentes tienen la potencialidad de formar este tipo de asociación (Cuenca et al., 2007). La distribución de micorrizas en el suelo puede ser afectada directamente por el pH obteniéndose una buena germinación de esporas de MA en un intervalo amplio de pH que va de 5 a 8 (Alvarado et al., 2004). MATERIAL Y MÉTODOS Las muestras fueron tomadas de un cultivo de algodón cosechado a los 180 días, el cual había sido inoculado con B, Br y la interacción de ambos (I) en diferentes momentos: a la siembra (M1), en la aparición del hipocotilo (M2) y en ambos momentos (M12) (Zúñiga, 2011). El ensayo fue a nivel de invernadero y el sustrato utilizado para las plantas fue una mezcla de suelo agrícola y arena (2:1). Las raíces fueron extraídas y lavadas; para la tinción de raíces se utilizó el protocolo de Phillips y Hayman (1970) con las modificaciones propuestas por Chávez et al. (2013). Las observaciones se realizaron con un lente objetivo de 4x y se contaron los campos colonizados (Sieverding, 1983). Los datos del %LRC se analizaron con el software estadístico Statgraphics centurión. RESULTADOS Y DISCUSIÓN El protocolo optimizado previamente para la tinción (Datos no mostrados) permitió visualizar las características morfológicas de las MA (vesículas y arbúsculos) en raíces de algodón (Figura 1). El análisis de varianza multifactorial (ANOVA) mostró que el 51 Session I SI-CP-15 factor “tipo de inóculo” tuvo un efecto altamente significativo sobre el %LRC (p = 0.0000), mas no el factor “momento de inoculación”. Mediante la prueba de rangos múltiples LSD, el tratamiento N+ afectó negativa y significativamente (28.77%) la infección natural de los hongos micorrícicos, reportados por primera vez en el cultivo de algodón en nuestro país (Figura 2). De acuerdo a lo indicado por Cornejo (2008), ello podría deberse a la reducción del pH del suelo causada por la producción de H+ generado en el proceso de nitrificación. A su vez el control sin inocular N- presentó un %LRC significativamente menor (53.81%) que las raíces inoculadas con B (70.98%), se resalta que el porcentaje de infección en condiciones naturales es un indicador de que el algodón es sensible a la infección por micorrizas. Sin embargo, cuando es inoculado con la cepa de Bacillus sp. el %LRC se incrementa, esto es posible gracias a que las PGPRs y las MA comparten la misma zona de infección y pueden crear una interacción que favorece tanto el crecimiento de la planta como el aumento de la biomasa del hongo, en contraste, otros autores señalan que no existe una interacción positiva entre las PGPRs y las MA (Jaizme et al. 2006). B A A Porcentaje de colonización Figura 1. Infección micorrícica en raíces de algodón inoculadas con Bradyrhizobium sp. A: Vesícula s, B: Hifas. Aumento 10X (Izquierda) y 4X (Derecha). 100 80 60 M1 40 M2 M12 20 0 B Br I Tratamientos N+ NFigura 2. Porcentaje de colonización para los tratamientos usados en la inoculación (B: Bacillus s p., Br: Bradyrhizobium sp., I: Bacillus sp., + Bradyrhizobium sp. y dos controles N+: Con Nitrógeno y N-: Sin Nitrógeno). Las series representan los momentos en los que se realizó la inoculación: a la siembra (M1), aparición del hipocotilo (M2) y en ambos momentos (M12). Las barras señaladas con las mismas letra s no difieren significativamente (p ≤ 0.05). AGRADECIMIENTOS Protec-CONCYTEC-OAI N° 278-2009. FDA Biol. 111-UNALM. BIBLIOGRAFÍA Alvarado, A., et al. (2004). Agronomía Costarricense, 28: 89-100. Chávez-Bárcenas, A.T., et al. (2013). African J. Microbiology Res. En prensa. Cornejo, P., et al. (2008). Chilean J. Agric. Res. 68: 119-127. Cuenca, G., et al. (2007). Interciencia, 32: 23-29. Jaizme Vega, M., et al. (2006). Fruits, 61: 1-7. Sieverding, E. (1983). Manual CIAT, Cali, 123 pp. Zuñiga, D. (2011). Informe final Proyecto Protec-CONCYTEC, 2009. 52 Session I SI-CP-16 Poblaciones microbianas con potencial PGPR en la rizósfera de cultivos de papas nativas amargas del altiplano peruano. Ramos, E.1*, Santos, R.1, Velezmoro, C.2, Zúñiga, D.1 1 Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso. Facultad de Ciencias. Universidad Nacional Agraria La Molina. Lima 12, Perú. 2 Facultad de Industrias Alimentarias. Universidad Nacional Agraria La Molina. Lima 12, Perú. * [email protected] RESUMEN Se evaluaron las poblaciones microbianas de la rizosfera de papas nativas amargas de las variedades locka (Solanum juzepczukii) y occucuri blanco (S. curtilobum) cultivadas en Ilave - Puno (Perú), entre las coordenadas geográficas 16º06´00” y 69º38´00” de latitud sur y oeste respectivamente y 3860 m.s.n.m. INTRODUCCIÓN Alrededor del 65% de la población andina económicamente activa se dedica a la agricultura y la papa es el principal alimento básico, no gramíneo, de la dieta de sus pobladores. Las papas nativas amargas (Solanum juzepczukii y S. curtilobum) pueden crecer a altitudes de hasta 4200 m.s.n.m., donde los riesgos de pérdida de cosechas debido a heladas son mucho mayores para papas de variedad “dulce”. De acuerdo a la Dirección de Información Agraria de Puno, en la campaña 2010-2011, este departamento cultivó alrededor de 46 966 ha de papa, con rendimientos promedio de 9 641 kg/ha. Las estadísticas oficiales no diferencian áreas específicas para papas dulces y amargas, sin embargo puede inferirse que alrededor del 26% son papas amargas, obtenidas principalmente por producción orgánica. Algunas poblaciones microbianas de papas han sido caracterizadas, sin embargo, a pesar de la importancia de los cultivos de papas amargas en el Altiplano, pocos estudios se han realizado al respecto. Así, el presente estudio pretende evaluar las poblaciones microbianas de papas nativas amargas cultivadas en Ilave (Puno, Perú). MATERIAL Y MÉTODOS La zona de estudio se situó en la cuenca del río Ilave, de la provincia de El Collao, al sur del departamento de Puno, con temperaturas que oscilan entre 1-15 °C y con precipitación media anual de 620 mm. Se eligieron las comunidades andinas de Jallamilla y Concahui. Se tomaron muestras de suelo rizosférico (franco arenoso, pH 5.1-5.5) de papas amargas de las variedades locka y occucuri blanco en 3 periodos de evaluación: floración, tuberización y cosecha, en los meses de enero, abril y mayo del 2009 de manera respectiva. También se tomaron muestras de suelo no rizosférico. Poblaciones microbianas de la rizosfera de papas amargas. Se llevó a cabo la cuantificación de bacterias esporulantes mesófilas aerobias y anaerobias (EMA y EMANA) y psicrófilas aerobias y anaerobias (EPA y EPANA), mediante el método de APHA (1998). Los aerobios mesófilos viables (AMV), Pseudomonas (PS), mohos y levaduras (MYL) fueron analizados según la ICMSF (2000). Los diazotrofos (DIAZO) se ensayaron de acuerdo a Zapater (1975). Los datos fueron sometidos al análisis comparativo de medias (prueba de Duncan, p≤0.05) utilizando el programa R. Caracterización molecular de los microorganismos aislados. Se realizó mediante perfiles de amplificación BOX-PCR y secuenciamiento del gen ARNr 16S, según las técnicas descritas por Versalovic et al. (1991). 53 Session I SI-CP-16 RESULTADOS Y DISCUSIÓN Se observaron diferencias significativas en los grupos microbianos de las poblaciones de EMA, EMANA, EPA, EPANA y DIAZO, según la época de muestreo, no encontrándose diferencias según la comunidad ni variedad cultivada. La influencia rizosférica solo se evidenció en las poblaciones de DIAZO de vida libre y PS. Así, durante la floración, en los suelos cultivados, se registraron poblaciones de 10 5 NMP/g PS, 107 NMP/g DIAZO y 105 UFC/g EMA (Figura 1). Tabla 1.Clasificación filogenética de las cepas aisladas de la rizosfera de papas amargas. Figura 1. Poblaciones microbianas de la rizosfera de papas amargas en tres estados fenológicos del cultivo Se observaron 25 diferentes perfiles de amplificación BOX-PCR entre los aislados provenientes de la rizosfera de papas nativas amargas. Se identificó B. simplex NBRC 15720T, reportado en la literatura como secretor de auxinas y B. safensis FO-036bT, de esporas altamente resistentes (Tabla 1). Otras cepas estuvieron relacionadas con B. pumilus ATCC 7061T, conocido promotor de crecimiento vegetal, Brevibacillus laterosporus DSM 25T, de uso potencial como controlador biológico y Lysinibacillus parviboronicapiens BAM-582T. Se encontró además la presencia de cepas del género Pseudomonas relacionadas a P. fragi ATCC 4973T que promueve la movilización de fósforo, P. gessardii CIP 105469T que produce enzimas de gran resistencia al frío y P. azotoformans IAM1603T de demostrada capacidad de fijación de nitrógeno. Las cepas anteriormente mencionadas vienen siendo ensayadas para la evaluación de sus propiedades PGPR. AGRADECIMIENTOS Proyecto PROCOM 255-2008-CONCYTEC-OAJ, Consorcio de productores de tunta “Los Aymaras”, FDA Biol.111- UNALM. BIBLIOGRAFÍA APHA, AWWA, WPCF (1998). Standard Methods for Examination of Water and Waste Water. 20 ed. ICMSF (2000). Microorganismos de los alimentos. Técnicas de análisis microbiológicos. 2 a ed. Versalovic, J., et al. (1991). Nucl. Acids Res. 19: 6823-6831. Zapater, J. (1975). Anales científicos de la UNALM 13: 45-57. 54 Session I SI-CP-17 Capacidad PGPR de cepas de Bacillus y Pseudomonas aisladas de la rizosfera de aguaymanto (Physalis peruviana L.). Cumpa, A. *, Flores, L., Chumpitaz, C., Ogata, K., Zúñiga, D. Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso, Dpto. Biología, Universidad Nacional Agraria La Molina. Lima-Perú. www.lamolina.edu.pe/lmt. * [email protected] RESUMEN 176 cepas, tanto de Bacillus como de Pseudomonas fueron aisladas del suelo y la rizosfera del cultivo de aguaymanto (Physalis peruviana L.) proveniente de las regiones de Lima y Junín en el Perú. Se realizaron dos pruebas de caracterización PGPR: solubilización de fosfatos y antagonismos contra hongos. Además se realizó una prueba de emergencia en semillas de aguaymanto. Las cepas Ba60 y Pa15 promovieron de forma significativa la emergencia de las semillas de aguaymanto y la cepa Ba53 demostró una alto porcentaje de inhibición (59%) del hongo Fusarium sp. INTRODUCCIÓN Las bacterias promotoras de crecimiento vegetal o PGPR (por sus siglas en inglés) otorgan múltiples beneficios a las plantas con las que interactúan, tales como el incremento de la germinación, colonización de raíces, estimulación del crecimiento de las plantas, control biológico, inducción de la resistencia a patógenos y mejoramiento en la asimilación de agua y nutrientes (Barka et al., 2000). El objetivo de este trabajo fue evaluar la capacidad PGPR de las bacterias aisladas y determinar su efecto en la emergencia de semillas de aguaymanto. MATERIAL Y MÉTODOS Producción de ácido indol acético (Gordon y Webber, 1951) Solubilización de fosfatos bicálcico y tricálcico (Nautiyal, 1999) Pruebas de antagonismo (Ahmed et al., 2007 modificado). Prueba de emergencia (Zúñiga, 2012) RESULTADOS Y DISCUSIÓN Producción de ácido indol acético Se evaluaron en total 176 cepas: 71 cepas de Bacillus sp. y 105 cepas de Pseudomonas spp. aisladas de la rizosfera de aguaymanto. De todas las cepas, 86 (81.9%) Pseudomonas spp. y 58 (81%) Bacillus sp. produjeron AIA. La producción más alta la registraron las cepas Pa87 y Ba64, con valores de 54.31 y 44.56 ppm de ácido indol acético respectivamente. 55 Session I SI-CP-17 Solubilización de fosfatos bicálcico y tricálcico. Tabla 1. Porcentaje de cepas analizadas, distribuidas por género y según su capacidad para solubil izar fosfato Solubilización de fosfatos Medio Cepas No solubiliza Bajaa Mediab Altac Muy altad Bicálcico Tricálcico Pseudomonas 40% 27.62% 12.38% 20% 0% Bacillus 92.96% 7.04% 0% 0% 0% Pseudomonas 70.48% 24.77% 2.86% 0% 0% Bacillus 74.65% 23.94% 1.41% 0% 0% a b c d h: halo de solubilización medido en cm; h<0.5, 0.51.5 Emergencia(%) Los mejores halos de solubilización de fosfato bicálcico se obtuvieron con las cepas Pa15 y Pa85 (1.45cm y 1.5 respectivamente) y de tricálcico con las cepas Ba60 y (0.7cm y 0.43cm respectivamente). Prueba de antagonismo. De entre las 40 cepas evaluadas la mejor fue la cepa Ba53 con un porcentaje de inhibición del 59% (Figura 1). Es posible que el mecanismo empleado por estas bacterias sea el de antibiosis con producción de metabolitos secundarios volátiles o difusibles que pueden inhibir o restringir el crecimiento del patógeno (Rodríguez et al., 2010). Prueba de emergencia en semillas de aguaymanto (Physalis peruviana). Se hallaron diferencias significativas en la emergencia de semillas de aguaymanto al inocular la cepa Ba18 (T2) y la cepa Pa15 (T4) en comparación con el control sin inóculo (T1) (Figura 2) Cepas Figura 2. Efecto de las cepas PGPR aisladas en la emergencia de plántulas de aguaymanto a nivel de laboratorio. Figura 1. Cultivo dual de cepas Ba53, Ba54 y Ba56. Entre ellas, la cepa Ba53 presenta un porcentaje de inhibición de 59% AGRADECIMIENTOS Este trabajo fue financiado por el proyecto Procyt-325-011-CONCYTEC-OAJ y la cuenta FDA-Biología, Lima-Perú. BIBLIOGRAFÍA Ahmed, H., et al. (2007). Biological Control 40: 97-106. Barka, E., et al. (2000). FEMS Microbiol. Lett. 186: 91-95. Gordon, S., and Weber, R. (1951). Plant Physiol. 26: 192-195. Nautiyal, C. (1999). FEMS Microbiol. Lett. 170: 65-70. Rodríguez, J. (2010). Revista de la Facultad Nacional Agraria de Medellín. 63: 5499-5509. Zúñiga, D. (2012). Manual de Microbiología Agrícola. Universidad Nacional Agraria La Molina. 56 Session I SI-CP-18 Phenotypic and molecular characterization of Lotus parviflorus nodule bacteria. Soares, R.1, 2, Lorite, M.J.2, Videira e Castro, I.1, Sanjuán, J.2* 1 2 Laboratory of Soil Microbiology, INIAV, Oeiras, Portugal. Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, CSIC, Granada, Spain. * [email protected] ABSTRACT A total of thirteen root nodule bacterial isolates from Lotus parviflorus plants were isolated from Estarreja, Portugal. Five isolates were slow growing strains and eight fast growers. A high diversity was revealed by ERIC-PCR, grouping them in ten clusters at 75% similarity. The isolates were identified as bacteria belonging to the genera Rhizobium, Bradyrhizobium, Sphingobacterium, Lysobacter and Pseudomonas. All isolates nodulated at least one of the various Lotus hosts tested. INTRODUCTION The legume nodulating bacteria collectively known as Rhizobia, are able to establish interactions with compatible plant species that culminate into the formation of nitrogen fixing root nodules. It has been thought for long time that Lotus spp. would only form specific symbioses with either slow-growing Bradyrhizobia or with intermediategrowing Mesorhizobia (Jarvis et al., 1982; Jordan, 1982), but recent studies have shown that actually various genus of Rhizobia, such as Rhizobium, Mesorhizobium, Bradyrhizobium, Sinorhizobium, Agrobacterim and Aminobacter can establish specific interactions with Lotus spp. (Estrella et al., 2009; Lorite et al., 2010a, 2010b). In fact, most Lotus species root nitrogen-fixing symbioses remain unexplored (Lorite et al., 2010a, b). Our goal was to characterize L. parviflorus root nodule symbionts, which have never been described previously. MATERIAL AND METHODS Bacteria were isolated from root nodules of Lotus parviflorus wild plants growing in a field of Estarreja, Portugal. The bacterial isolates were grown on Yeast Manitol Agar (YMA) (Vincent, 1970), on YMA with congo red and YMA with Bromothymol Blue (Somasegaran and Hoben, 1994).Total DNA was extracted following Estrella et al. (2009). ERIC-PCR was performed following the procedure of Caetano-Anollés and Gresshoff (1997) with De Brujin (1992) primers. 16S rRNA and glnII gene amplifications were performed as described in Herrera-Cervera et al. (1999) and Stepkowski et al. (2005), respectively. recA and atpD gene were amplified as described in Gaunt et al. (2001). nodC and nifH genes were amplified as reported Laguerre et al. (2001) and nodA following Moulin et al. (2004). The amplified DNAs were extracted with the QIAEXII kit and sequenced with an ABI373 automated sequencer. Plant nodulation assays were performed using L. parviflorus PI415815, L. parviflorus PI283615, L. tenuis cv. INTA PAMPA, L. tenuis cv. Esmeralda, L. corniculatus cv. São Grabiel, L. pedunculatus cv. Sunrise, grown on a modified Agar Jensen medium. Three individual plants were inoculated with each isolate; non-inoculated or KNO3 fertilised control plants were also included. RESULTS AND DISCUSSION ERIC-PCR fingerprinting of the thirteen isolates showed the presence of ten different clusters at 75% similarity, indicating a very high genetic diversity. Indeed, phylogenetic analysis of the 16S rRNA gene sequences suggests that the isolates belong to either of 5 57 Session I SI-CP-18 different genera: Rhizobium, Bradyrhizobium, Sphingobacterium, Lysobacter, and Pseudomonas. A majority of the isolates belong to the Rhizobium/Agrobacterium genera that are not typical Lotus symbionts and only one isolate was placed into the Bradyrhizobium genus. Phylogenetic analyses of glnII, recA and atpD genes are in progress to verify genus and species assignment. Since original L. parviflorus host plants were wild, non-cultivated ones, seeds were not available and nodulation tests had to be done on different Lotus spp. and genotypes. All isolates were able to nodulate at least one of the Lotus plant hosts tested, and only the Bradyrhizobium Lp11 isolate displayed a Fix+ phenotype in L. pedunculatus and L. parviflorus PI283615, although it was Fix- in the other L. parviflorus genotype PI415815. All other bacteria-Lotus combinations were Fix-, suggesting a very narrow host range of the L. parviflorus isolates. This will need to be confirmed with additional host range tests together with sequence analysis of symbiotic genes. ACKNOWLEDGEMENTS This work was supported by a BIOFAG network grant and by an ERASMUS mobility grant to R. Soares. REFERENCES Caetano-Anollés, G., and Gresshoff, P.M. (1997). DNA Markers Protocols, Applications, and Overview s. Wiley-Liss, Inc. De Bruijn, F.J. (1992). Appl. Environ. Microbiol. 58: 2180-2187. Estrella, M.J., et al. (2009). Appl. Environ. Microbiol. 75: 1088-1098. Gaunt, M.W., et al. (2001). Int. J. Syst. Evol. Microbiol. 51: 2037-2048. Herrera-Cervera, J.A., et al. (1999). FEMS Microbiol. Ecol. 30: 87-97. Jarvis, B.D.W., et al. (1982). Int. J. Syst. Bacteriol. 32: 378-380. Jordan, D.C. (1982). Int. J. Syst. Bacteriol. 32: 136-139. Laguerre, G., et al. (2001). Microbiology 147: 981-993. Lorite, M.J., et al. (2010a). Appl. Environ. Microbiol. 76: 4019-4026. Lorite, M.J., et al. (2010b). Syst. Appl. Microbiol. 33: 282-290. Moulin, L., et al. (2004). Mol. Phylogenet. Evol. 30: 720-732. Somasegaran, P., and Hoben, H.J. (1994). Methods in legume-Rhizobium Technology. Paia: Univ. Hawaii. Stepkowski, T., et al. (2005). Appl. Environ. Microbiol. 71: 7041-7052. Vincent, J.M. (1970). A Manual for the Practical Study of Root Nodule Bacteria. Oxford: Blackwell Scientific. 58 Session I SI-CP-19 Contribution of root nodule bacteria for the sustainability of “montado” (cork oak) ecosystem. Fernández, C.1, Soares, R.1, Barrento, M.J.2, Machado, H.2, Gomes, A.A.1, Videira e Castro, I.1* 1 Laboratório de Microbiologia do Solo, UEISAFSV, INIAV,I.P., Oeiras, Portugal. Sanidade Vegetal, UEISAFSV, INIAV,I.P., Oeiras, Portugal. * [email protected] 2 Laboratório de ABSTRACT A total of fifty-three root nodule bacterial strains from Trifolium subterraneum plants were isolated from soils of the Southern of Portugal and were subsequently characterized. Besides nitrogen fixation, other important functions were also searched such as solubilization of mineral phosphate, hydrolysis of plant polymers and production of siderophores. Antagonistic activity against pathogenic disease caused by Phytophtora cinnamomi was also investigated. INTRODUCTION Nitrogen is the primary nutrient limiting plant production in most natural ecosystems. Legumes are important components of the strategy for increasing productivity and sustainability of disturbed ecosystems, using symbiotic nitrogen fixation as a major process of providing nitrogen to the soils. Therefore, legumes and their root nodule bacteria, are considered as a powerful management tool for improving pastures yield in the “montado” ecosystem (“dehesas” in Spain), which is an agroforestry system associated with the exploitation of cork oaks. In addition to nitrogen fixation, rhizobia can also promote the growth of plants either directly through the solubilization of minerals, such as phosphorus, or indirectly as a biocontrol agent by inhibiting the growth of pathogens. In Portugal, the decline caused by Phytophthora cinnamomi is the main disease that in the last decades has led to the widespread mortality of Quercus suber (Moreira and Martins, 2005). The aim of the present work was to characterize and select root nodule bacteria from Trifolium subterraneum plants grown in soils from Q. suber “montado” in different sanitary and vegetative states in the Southern of Portugal (Grândola hill), in order to evaluate their potential as plant growth promoters. So, besides nitrogen fixation, other important functions were also searched, such as solubilization of mineral phosphate, hydrolysis of plant polymers and production of siderophores, as well as antagonistic activity against the P. cinnamomi. MATERIAL AND METHODS For each soil sample, rhizobial natural population was estimated by the most probable number (MPN) plant infection method using T. subterraneum cv. Clare as trap host. For evaluating the effectiveness of rhizobial population, plants of T. subterraneum inoculated with each soil were grown during 8 weeks in controlled environmental conditions and the effectiveness was determined by the shoots dry weight. A total of 53 root nodule bacterial strains were isolated on yeast manitol agar from nodules of T. subterraneum (cv. Clare) grown in three different soils and subsequently characterized. Biodiversity analysis was performed by ERIC-PCR fingerprinting (De Bruijn, 1992). In vitro tests for determination phosphate-solubilization (Peix et al., 2001), siderophore production (Perez-Miranda et al., 2007) and cellulase and pectinase activities (Verma et al., 2001) were conducted qualitatively. Tests of antifungal activity were performed in PDA plates inoculated first with fungus mycelium and two days later 59 Session I SI-CP-19 with 3µl of each bacterial culture (grown for 48 h in TY liquid medium). Tests with clovers inoculated separately with two strains of P. cinnamomi and two strains of selected bacteria were performed in controlled environmental conditions, during 8 weeks. Two strains of P. cinnamomi isolated from two different sites of Grândola Hill, were used. RESULTS AND DISCUSSION The size of the rhizobial population revealed great differences among the soil samples tested, with values ranging from over 104 bact.g-1 of soil to less than10 bact.g-1 in soils with low fertility. The results for nitrogen fixing capacity were variable. Values similar to controls with nitrogen were observed only in one of the soil samples analyzed. In soils with low fertility, the rhizobial population was ineffective (similar to controls without nitrogen). Analyzing the patterns obtained by ERIC-PCR, it was verified the existence of several clusters, at 80% similarity, indicating a high genetic diversity among the population studied. However, isolates from soils with low fertility were maintained almost together. Results obtained from plant growth promoting in vitro activities showed that within the set of isolates, approximately more than half (60%) presented cellulase activity, 18% could hydrolyse pectin, 12% were capable to solubilize mineral phosphate, but only 6% were able to produce siderophores. These last isolates have also an antagonistic activity against P. cinnamomi. This relationship may be important, since siderophore production in iron stress conditions provides bacteria an added advantage, resulting in exclusion of pathogens due to iron starvation. The antagonistic activity of bacterial strains against P. cinnamomi was demonstrated by the differences observed between the development of plants inoculated simultaneously with the mycelium and rhizobia isolates and plants inoculated only with P. cinnamomi which showed root damage and yellow leaves. These results indicate that root nodule bacteria could have a potential as biological control agent against this disease. ACKNOWLEDGEMENTS This work was supported by Fundo Florestal Permanente. C. Fernandez is granted by Quercus Vprogram a Leonardo da Vinci (FUNDECYT, España). REFERENCES De Bruijn, F.J. (1992). Appl. Environ. Microbiol. 58: 2180-2187. Moreira, A.C., and Martins, J.M.S. (2005). For. Path. 35: 145-162. Peix, A., et al. (2001). Soil Biol. Biochem. 24: 389-395. Perez-Miranda, et al. (2007). J. Microbiol. Meth. 70: 2420-2428. Verma, S.C., et al. (2001). J. Biotech. 91: 127-141. 60 Session I SI-CP-20 Estudio del carácter endófito de bacterias del género Pantoea aisladas de los cultivos de arroz de la Marisma del Guadalquivir. Megías, E.1, Benitez, C.1, Diaz-Olivares, I.2, Ollero, F.J.2, Megías, M.1* 1 Universidad de Sevilla. Departamento de Microbiología y Parasitología. Departamento de Microbiología. * [email protected] 2 Universidad de Sevilla, RESUMEN Muchas de las especies del género Pantoea se han descrito como bacterias endófitas en diferentes plantas, incluidas las plantas de arroz. En este estudio se describe el carácter endófito de aislados seleccionados de plantas de arroz y su relación con la movilidad de la cepa y la producción de moléculas de homoseril-lactona. Se exponen los resultados de cuatro cepas del género Pantoea respecto a la movilidad estudiada en medio sólido. Todas presentan movilidad tipo swimming y producen AHL. Las cuatro cepas se comportan como cepas endófitas, según los resultados observados en los ensayos de reinoculación. Asimismo estudiamos la influencia de la movilidad y la producción de AHLs en el carácter endófito. INTRODUCCIÓN El género Pantoea pertenece a la familia Enterobacteriaceae, y comprende actualmente 19 especies. Presentan una distribución ubicua y se han descrito como endófitos de plantas de arroz (Mano et al., 2007; Okunishi et al., 2005; Sun et al., 2007; Verma et al., 2001). La definición de endófito aceptada en la actualidad es la propuesta por Hallman et al. (1997), que describe a los endófitos como bacterias que pueden ser aisladas del interior de una planta o tejido vegetal esterilizado superficialmente, y no provocan daños aparentes en la planta. Para acceder al interior de la planta los endófitos utilizan como vía de entrada preferencial las zonas de aparición de las raíces secundarias (Dong et al., 2003), aunque también pueden entrar a través de los estomas, las lenticelas y heridas de la planta. Una vez en el interior de la planta los endófitos pueden permanecer en un tejido específico, como el córtex radicular, o colonizar la planta sistémicamente moviéndose a través de los vasos conductores o el apoplasto (Hurek et al., 1994). A día de hoy se desconocen los mecanismos moleculares que intervienen en el proceso de colonización y movimiento de la bacteria en el interior de la planta. De este modo, el objetivo de este trabajo es determinar si la movilidad de las bacterias y su capacidad de producir moléculas de comunicación del tipo AHLs, inciden sobre la capacidad de colonización y movimiento de los potenciales endófitos. MATERIAL Y MÉTODOS Como material biológico se utilizaron 4 cepas previamente identificadas y clasificadas por análisis filogenético como miembros del género Pantoea, se trata de las cepas AMG-153 (P. dispersa), AMG-461 (P. deleyi), AMG-501 (P. ananatis) y AMG-521 (Pantoea sp.). Se siguió el protocolo descrito por Kearn and Losick (2003) para determinar la movilidad de tipo swimming y de tipo swarming a las 48 horas. La producción de AHLs de cada una de las cepas se calculó siguiendo el método descrito por McClean et al. (1997). Para describir el carácter endófito se llevaron a cabo ensayos de reinoculación en condiciones controladas en el laboratorio, permitiendo de esta forma observar si las bacterias se encontraban o no en el interior de la parte aérea de la planta de arroz. La presencia de la cepa se confirmó mediante ensayos de BOX-PCR (Johnson and Clabots, 2000) frente al inóculo utilizado. 61 Session I SI-CP-20 Se obtuvieron mutantes espontáneos inmóviles de las cepas AMG-501 y AMG-521, mediante el uso de resistencias naturales a antibióticos como la rifampicina y la espectinomicina. Para la obtención de mutantes no productores de AHLs se insertó el plásmido pME6863 (Reimmann et al., 2002 ) en el interior de las cepas AMG-501 y AMG-521, inhibiéndose la producción de moléculas de señalización poblacional. Se comprobó la capacidad de colonización de los mutantes frente al silvestre, mediante ensayos de reinoculación. RESULTADOS Y DISCUSIÓN Todas las cepas analizadas presentaban movilidad de tipo swimming, cubriendo prácticamente la totalidad de la placa a las 48 horas. En ninguna de las cuatro cepas se observó movilidad de tipo swarming. Los resultados de producción de AHLs nos muestran que todas las cepas producen AHLs en mayor o menor medida. En los ensayos de reinoculación todas las cepas estaban presentes en el homogeneizado de la parte aérea de la planta. En todos los casos se confirmó por BOX-PCR que la cepa inoculada coincidía con las colonias recuperadas del interior de la planta. Se han obtenido resultados preliminares en los ensayos de reinoculación destinados a determinar la capacidad de colonización de la planta por parte de las cepas mutantes (en movilidad y en producción de AHLs), frente a las cepas silvestres. Actualmente se están llevando a cabo nuevos ensayos que confirmen los resultados observados. En este trabajo hemos confirmado por ensayos microbiológicos la presencia de las bacterias en el interior de los tejidos aéreos de la planta de arroz, pero no hemos llevado a cabo ningún ensayo confirmativo por microscopía confocal o electrónica, uno de los requisitos mencionados en la literatura (Reinhold-Hurek and Hurek, 1998) para poder afirmar que una cepa es endófita. El objetivo futuro pasa por realizar este tipo de ensayos de microscopía confocal en plantas de arroz inoculadas con cepas que potencialmente actúan como endófitos. Desde el punto de vista biotecnológico, y a la luz de las evidencias mencionadas en la bibliografía, los endófitos pueden resultar interesantes al participar en diferentes procesos relacionados con una posible mejora de las condiciones de la planta, tales como el control de determinados patógenos (Pusey et al., 2011), la interacción con la producción de hormonas por parte de la planta (Feng et al., 2006) o la capacidad de fijar nitrógeno atmosférico (Loiret et al., 2004). Es por ello interesante determinar qué factores intervienen en el proceso de colonización de las plantas y en el movimiento bacteriano en el interior de las mismas. BIBLIOGRAFÍA Dong, Y., et al. (2003). Plant Soil 257: 49-59. Feng, Y,. et al. (2006). J. Appl. Microbiol. 100: 938-945. Hallman, J., et al. (1997). Can. J. Microbiol. 43: 895-914. Hurek, T., et al. (1994). J. Bacteriology. 176: 1913-1923. Johnson, J.R., and Clabots, C. (2000). Clin. Diagn. Lab. Immunol. 7: 258-264. Loiret, F.G., et al. (2004). J. Appl. Microbiol. 97: 504-511. Mano, H., et al. (2007). Microbes Environ. 22: 175-185. McClean, K.H., et al. (1997). Microbiology 143: 3703-3711. Okunishi, S., et al. (2005). Microbes Environ. 20: 168-177. Pusey, P.L., et al. (2011). Phytopathology 101: 1234-1241. Reimmann, C., et al. (2002). Microbiology 148: 923-932. Reinhold-Hurek, B., and Hurek, T. (1998). Nuc. A. Sym. Series, 41: 95-98. Sun, L., et al. (2007). Microbial Ecol. 55: 415-424. Verma, S.C., et al. (2001). J. Biotech. 91: 127-141. 62 Session I SI-CP-21 El género Pantoea en los cultivos de arroz de la Marisma del Guadalquivir: diversidad y posible descripción de una nueva especie. Megías, E.1*, Fernández, M.1, Reis Junior, F.B.2, Márquez, M.C.1, Ollero, F.J.3, Megías, M.1 1 Universidad de Sevilla. Departamento de Microbiología y Parasitología. Universidad de Sevilla. Departamento de Microbiología. * [email protected] 2 Embrapa Cerrados. 3 RESUMEN El género Pantoea se engloba dentro de la familia Enterobacteriaceae y presenta una distribución ubicua, se han descrito algunas especies como endófitos de plantas de arroz. De las cepas aisladas como endófitos de plantas de arroz de la Marisma del Guadalquivir, 29 fueron clasificadas mediante la secuenciación del ARNr 16S como pertenecientes al género Pantoea. Se realizo el análisis por MLSA basado en cuatro genes housekeeping, atpD, gyrB, infB y rpoB, para clasificar las cepas dentro del género. En este análisis se observó que 18 de los 29 aislados se encontraban estructurados en un clado diferente próximo filogenéticamente a P. ananatis, P. allii, y P. stewartii. Se postula que estos aislados podrían ser una especie nueva en el género, a pesar de que la distancia filogenética con el grupo más cercano (P. ananatis), es pequeña. Actualmente se están realizando ensayos de DDH (DNA-DNA hibridization) con el fin de determinar si las 18 cepas dentro del clado problema pueden ser consideradas como una nueva especie. INTRODUCCIÓN El género Pantoea se engloba dentro de la familia Enterobacteriaceae, y comprende actualmente 19 especies. Presentan una distribución ubicua y se han descrito como endófitos de plantas de arroz (Mano et al., 2007; Okunishi et al. 2005; Sun, et al., 2007; Verma et al., 2001). Las evidencias encontradas en la literatura ponen de manifiesto la capacidad de algunas cepas de este género para actuar en beneficio de la planta, bien actuando como bacterias PGPR (Feng et al., 2006), bien como agentes de biocontrol (Pusey et al., 2011). Esto las hace interesantes desde el punto de vista biotecnológico ya que pueden tener diversas aplicaciones sobre diferentes cultivos, y en particular en los cultivos de arroz. El objetivo de este trabajo es determinar la diversidad de bacterias endófitas del género Pantoea asociada a las plantas de arroz en los arrozales de la Marisma del Guadalquivir, y encontrar cepas con posibles aplicaciones biotecnológicas. MATERIAL Y MÉTODOS A las cepas aisladas como endófitos se les extrajo el ADN (NucleoSpin® Tissue de MN) y se amplificó el gen del ARNr 16s mediante los cebadores F27 y R1488 (Edwards et al., 1989). Para la amplificación de los genes atpD, gyrB, infB y rpoB, se utilizaron cebadores diseñados en nuestro laboratorio a partir de los descritos por Brady et al. (2008). Los productos de PCR obtenidos se enviaron a secuenciar a la empresa Sistemas Genómicos S.L. El tratamiento de las secuencias fue análogo para cada uno de los genes trabajados y también se siguió la misma metodología para el concatenado de los 4 genes housekeeping, base del análisis por MLSA. Cada paquete de genes se alineó mediante el programa ClustalX 2.0 (Larkin et al.,2007) y los alineamientos fueron debidamente editados mediante los programas Dambe (Xia et al., 2001) y BioEdit (Hall, 1999). Para los análisis de las secuencias por Maximum-likelihood (ML), se eligió el modelo de sustitución adecuado para cada set de datos mediante el programa jModelTest 2.1.1 63 Session I SI-CP-21 (Darriba et al., 2012). Los árboles filogenéticos se construyeron utilizando el programa PhyML 3.0 (Guindon, 2010) partiendo de 200 árboles iniciales construidos en PAUP* y un árbol construido por BioNJ (método habitual de inicio del programa PhyML). Se eligieron los árboles con mejor lnL y se analizaron y editaron utilizando MEGA 5.0. Para las pruebas bioquímicas se siguieron las indicaciones descritas por el fabricante en el protocolo de las galerías Api 50CH (Biomerieux). Los resultados se analizaron utilizando el programa Bionumerics. RESULTADOS Y DISCUSIÓN El árbol filogenético obtenido del concatenado de genes housekeeping permite ubicar 9 de las 29 cepas analizadas en diferentes especies del género Pantoea. Dos de las cepas analizadas se encontraban asociadas filogenéticamente al grupo externo, usado para enraizar el árbol, por lo que estas dos, presuntamente asociadas al género Enterobacter, se desecharon en posteriores análisis. El resto de las cepas, un total de 18, se agrupaban en un único clado próximo a las especies P. ananatis, P. stewartii y P. allii. Este grupo se repetía, con pequeños cambios en la filogenia, en los análisis gen a gen y en el análisis del ARNr 16s. El análisis de los datos de consumo de azucares (pruebas Api 50CH) expresados en forma de matriz de distancias y dendrograma construido por UPGMA, soporta los resultados obtenidos en el análisis filogenético, apareciendo el clado formado por las 18 especies con la misma estructura que en los árboles filogenéticos. Las cepas agrupadas en el clado problema presentan una distancia filogenética pequeña en relación a la especie P. ananatis, siendo esto insuficiente para considerar a este clado como una nueva especie. Brady et al (2008, 2009, 2010a, 2010b, 2011, 2012) estructura el género Pantoea, describe nuevas especies y reubica especies anteriormente consideradas Pantoea en otros géneros, apoyándose en la hibridación ADN-ADN para confirmar la descripción de las nuevas especies. Por otro lado, es interesante mencionar la solidez de nuestros resultados filogenéticos en relación a aquellos aportados por Brady et al en los trabajos arriba mencionados. En ellos no mencionan los modelos de sustitución empleados y aplican rearreglos de rama basados en Bootstrap, método mucho más lento y menos informativo que los rearreglos mediados por algoritmos SH-like. En conclusión, los resultados de filogenia mostrados en este trabajo están mejor adaptados a las nuevas corrientes en filogenia computacional. BIBLIOGRAFÍA Brady, C.L., et al. (2008). Syst, Appl. Microbiol. 31: 447-460. Brady, C.L., et al. (2009). Int. J. Syst. Evol. Microbiol. 59: 2339-2345. Brady, C.L., et al. (2010a). Int. J. Syst. Evol. Microbiol. 60: 2430-2440. Brady, C.L., et al. (2010b). Int. J. Syst. Evol. Microbiol. 60: 484-494. Brady, C.L., et al. (2011). Int. J. Syst. Evol. Microbiol. 61: 932-937. Brady, C.L., et al. (2012). Int. J. Syst. Evol. Microbiol. 62: 1457-1464. Darriba, D., et al. (2012). Nature Methods 9: 772. Edwards, U., et al. (1989). Nucl. Acid. Res. 17: 7843-7853. Feng, Y., et al. (2006). J. Appl. Microbiol. 100: 938-945. Guindon, S. (2010). Syst. Biol. 59: 307-321. Hall, T.A. (1999). Nucleic Acid Symposium Series 41: 95-98. Larkin, M.A., et al. (2007). Bioinformatics 23: 2947-2948. Mano, H., et al. (2007). Microbes Environ. 22: 175-185. Okunishi, S., et al. (2005). Microbes Environ. 20: 168-177. Pusey, P.L., et al. (2011). Phytopathol. 101: 1234-1241. Sun, L., et al. (2007). Microbial Ecol. 55: 415-424. Verma, S.C., et al. (2001). J. Biotech. 91: 127-141. Xia, X., et al. (2001). J. Heredity 92: 371-373. 64 Session I SI-CP-22 Bacterial biodiversity within the genus Enterobacter in paddies of Guadalquivir marshes. Fernández, M.1*, Megías, E.1, Reis Junior, F.B.2, Megías, M.1 1 Universidad de Sevilla, Dpto. de Microbiología y Parasitología. España 2 Embrapa Cerrados, Brasil. * [email protected] ABSTRACT Associated with paddies there are many microbial communities that could be used as biological agents in order to improve rice crops yields. Bacteria associated with rice plants from different areas of paddies of Guadalquivir marshes were isolated and studied focusing on Enterobacter. Using cultural and biochemical tests and 16S rDNA gene sequencing we obtained 13 strains belonging to 5 different species of the genus Enterobacter, and we observed that they were more common in areas irrigated with river water than those irrigated with pit water. Now we are working on the remaining strains isolated to identify them, studying by rpoB gene the strains already identified by 16S rDNA gene sequencing to complete our results and testing the plant growthpromoting capacities of our strains. INTRODUCTION The Guadalquivir marshes constitute an area placed in western Andalusia (Spain). This area has approximately the 40% of Spain rice production. Associated with paddies there are many microbial communities that could be used as biological agents in order to improve rice crops yields, since these microorganisms are able to carry out fundamental processes for rice plants. The aim of this study is to isolate and identify bacteria present in rice plants grown in the marshes of the Guadalquivir river, focusing on genus Enterobacter, which has been described as one of the most commonly isolated genus of bacteria from rice plants (Cottyn et al., 2001). MATERIAL AND METHODS Rice plants of Puntal variety were collected in July 2011 from different areas in Guadalquivir marshes, each of these areas with a different kind of irrigation: “Hato ratón” area with pit water irrigation, “Los Alemanes” area with river water irrigation and “La compañía” area with river water irrigation of better quality since this area is closer to the river and water has not been recycled. Plants from each area were divided in different zones: rhizosphere soil, roots and aerial part. Roots and aerial part were surface sterilized with ethanol 70% and NaClO 5%. To obtain the extract of each part, sterilized roots and sterilized aerial part along with NaCl 0.9% were triturated with Polytron PT 2100. From each different sample 1 ml was pre-cultivated in TSB medium. Secondly, the preculture was cultivated in Mossel enrichment medium for Enterobacteriaceae and finally, that culture was seeded in MacConkey agar plates. Pink and red colonies were selected and pure cultures were done form each of them. These strains were subjected to IMViC (indole, methyl-red, Voges-Proskauer and citrate tests) in order to identify if they belonged to the genus Enterobacter, and DNA from those strains that were identified as Enterobacter was extracted and 16S rDNA gene amplified by PCR with primers F27 and R1488 (Edwards et al., 1989). Then PCR products were sequenced and analyzed with online servers Blast and EzTaxon (Chun et al., 2007) to identify isolates. 65 Session I SI-CP-22 RESULTS AND DISCUSSION We obtained a total of 61 isolates from the different parts of plants and different zones. From “La Compañía” area we obtained: 18 isolates from soil samples, 5 isolates from roots samples and 3 isolates from aereal part samples. From “Los Alemanes” area we obtained: 15 isolates from soil samples, 2 isolates from roots samples and 3 isolates from aereal part samples. From “Hato Ratón” area, we obtained 12 isolates from soil samples and 3 isolates from roots samples. 32 isolates were positive for Enterobacter by the IMViC tests, and 21 of them have been already identified by the 16S rDNA gene amplification as bacteria belonging to 5 different species of genus Enterobacter (Enterobacter ludwigii, E. aerogenes, E. absuriae, E. hormaechei and E. cancerogenus) and other related genera.We will complete these results by rpoB gene sequencing to make phylogenetic analysis. Our results show that rice plants from paddies of Guadalquivir marshes constitute a source of bacteria from several species of genus Enterobacter, and it can be observed that this endophytic community of rice plants was influenced by paddy irrigation type, being more abundant in areas irrrigated with river water than those irrigated with pit water. The presence of Enterobacter strains in the endosphere of rice plants also demonstrates the ability of these bacteria to colonize rice plants, even the aereal part, suggesting that they act as endophytes that could have several plant growth-promoting capacities that we are now testing. REFERENCES Cottyn, B., et al. (2001). Phytopathol. 91: 282-292. Edwards, U., et al. (1989). Nucl. Acid. Res. 17: 7843-7853. Chun, J., et al. (2007). Int. J. Syst. Evol. Microbiol. 57: 2259-2261. 66 Session I SI-CP-23 Isolation and characterization of a novel bacterium isolated from paddies of Guadalquivir marshes. Márquez, M.C.*, Fernández, M., Megías, E., Merchán, F. Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla. * [email protected] ABSTRACT During an investigation of the presence of bacteria associated with rice plant, we isolated 32 strains from different areas of paddies of Guadalquivir marshes. The phylogenetic relationships of these isolates were investigated performing a comparative analysis of the sequences of the 16S rRNA. This study showed that one of these isolates, designated as strain S.EP-T3-1, represents a novel sub line within the family Enterobacteriaceae. Currently a polyphasic taxonomic approach of this strain is performing to determine its exact taxonomic position. INTRODUCTION The family Enterobacteriaceae comprises a heterogeneous group of bacterial species currently distributed among almost fifty genera. Many representatives are known to exhibit plant-growth-promoting effects that include production of phytohormones, fixation of atmospheric nitrogen and biocontrol activity (Götz et al., 2006; Ruppel and Merbach, 1997; Ruppel et al., 1992;). In this work, the phylogenetic relationships of 32 strains with potential plant growth promoting capacities, has been investigated by comparative 16S rRNA analysis. Besides, further studies based on a polyphasic characterization are carrying out with one of these isolates, strain S.EP-T3-1, to confirm its precise taxonomic position as a novel taxon within the family Enterobacteriaceae. MATERIAL AND METHODS Bacterial strains. A total of 32 strains were isolated from rice plant of Puntal variety in July 2011. Klebsiella oxytoca CECT 860T was obtained from the Spanish Type Culture Collection and used as reference for comparative phenotypic studies. Sequence analysis. The phylogenetic position of all the isolates was determined by almost-complete 16S rRNA gene sequences analysis. Sequencing of the 16S rRNA genes was performed according to Márquez et al. (2011). Phylogenetic analyses were carried out with the ARB software package (Ludwig et al., 2004). Phenotypic study. Phenotypic characteristics of strain S.EP-T3-1 were observed on TSA after 24 h of incubation at 37 ºC. Gram staining, motility, microscopic observations, and biochemical analysis were performed according to standard laboratory procedure (Barrow and Feltham, 2004). The API20E and API50CH (bioMerieux, Inc. Durham, NC) were used to obtain the biochemical profile according to the manufacturers’ instructions. The G+C content of DNA was determined by the thermal denaturation method described by Marmur and Doty (1962). RESULTS AND DISCUSSION Comparative 16S rRNA gene sequence analysis revealed that the 32 isolates allocate into different genera of the family Enterobacteriaceae: Enterobacter, Klebsiella, Citrobacter and Leclercia. From the results obtained in this study we observed that one 67 Session I SI-CP-23 of these strains, designated as strain S.EP-T3-1, was loosely clustered to members of this family, at the highest 16S rRNA similarity of 94% to Klebsiella oxytoca. In general, organisms sharing less than 97.0% 16S rRNA gene sequence similarity do not have DNA-DNA reassociation values greater than 70%, so DNA-DNA hybridization has not been performed. The DNA G+C content of strain S.EP-T3-1 is 55.4 ± 0.9 mol%. Well-isolated colonies of S.EP-T3-1 appear within 24 h of incubation at 37 ºC on TSA medium. The colonies are cream, circular with entire margin and convex elevation. They have a glistening texture due to capsule production. The size of a single colony of an 18–24 h culture is about 0.15-0.2 cm in diameter. The isolate is capable of growing over a temperature range of 10–43 ºC with an optimum of 37 ºC. The cells are Gramnegative straight rods (0.7-1 µm wide and 2-3 µm long) occurring singly and sometimes in pairs. Biochemical tests have been performed on the isolate to confirm its taxonomic position and to specifically differentiate it from K. oxytoca. In contrast to the type strain of K. oxytoca, our isolate is motile and negative for indol test, nitrite reduction and lactose fermentation. The API 20E and API50CH profiles are also clearly different between both strains. The data obtained up to now in the phenotypic and phylogenetic characterization suggest that strain S.EP-T3-1 may constitute a novel taxon of the family Enterobacteriaceae. Besides, we are carrying out the study of its chemotaxonomic features in order to complete its characterization and confirm its precise taxonomic position. REFERENCES Barrow, G.L., and Feltham, R.K.A. (2004). Cowan and steel’s manual for the identification of medic al bacteria, 3rd edn. Cambridge University Press, Cambridge. Götz, M., et al. (2006). FEMS Microbiol. Ecol. 56: 207-18. Ludwig, W., et al. (2004). Nucleic Acids Res. 32: 1363-71. Marmur J., and Doty, P. (1962). J. Mol. Biol. 5: 109-18. Márquez, M., et al. (2011). Syst. Appl. Microbiol. 34: 424-8. Ruppel, S., et al. (1992). Plant Soil 145: 261-73. Ruppel, S., and Merbach, W. (1997). Microbiol. Res. 152: 377-83. 68 Session I SI-CP-24 Biodiversidad y performance simbiótica de bacterias noduladoras de soja alóctonas de suelos de Argentina. Covelli, J.M.1*, López, M.F.1, Arrese-Igor, C.2, Lodeiro, A.R.1 1 IBBM-Facultad de Ciencias Exactas, UNLP-CONICET, La Plata, Argentina. Ciencias del Medio Natural, Universidad Pública de Navarra, Navarra, España. * [email protected] 2 Departamento de RESUMEN Aislamientos de bacterias noduladoras de soja tomados de cinco suelos de Argentina mostraron una considerable biodiversidad genotípica agrupada por la acidez del suelo de origen, e incluyeron rizobios de crecimiento rápido y bradirrizobios de crecimiento lento. Los rizobios tendieron a ser malos fijadores de N 2, mientras que los bradirrizobios tuvieron una performance similar o en ocasiones superior a las de Bradyrhizobium japonicum E109 o USDA 110, usadas como referencia. La competitividad para nodular frente a una E109 SmR/SpR fue en general baja, más particularmente la de los rizobios. INTRODUCCIÓN El cultivo de leguminosas más importante de la Argentina es la soja, cuya superficie sembrada ha superado en 2012 los 19 millones de hectáreas. Gran parte de esta superficie se inocula con B. japonicum para aprovechar la nodulación y fijación biológica de N2 (FBN), pero en las zonas con historia previa de cultivo de soja a menudo la inoculación no tiene efecto sobre el rendimiento. Esta falta de respuesta se debe, en gran medida, a que los suelos donde se ha cultivado soja ya recibieron inoculaciones en años anteriores y por lo tanto, cuentan con una población de rizobios noduladores de soja derivados de dichos inoculantes (población alóctona). Frecuentemente esta población alóctona ocupa la mayor parte de los nódulos en desmedro del inoculante, lo cual restringe su impacto en la FBN global del cultivo (López-García et al., 2009). Un problema que surge en esta situación es si la calidad simbiótica de la población alóctona se asemeja a la del inoculante. Esto no tiene por qué ser así, ya que la alta performance de la FBN no es un carácter sujeto a selección positiva en el ambiente edáfico. Por lo tanto, una vez que la población alóctona se establece en el suelo, puede derivar genéticamente y/o intercambiar material genético con la población bacteriana autóctona de forma tal de dar origen, en sucesivas generaciones, a una población cuya calidad fijadora de N2 sea altamente heterogénea. Sin embargo, éste no parece ser el caso de la performance para la nodulación: ya que el nódulo es un ambiente protector para los rizobios, la capacidad de nodular sí puede estar sujeta a selección positiva en el ambiente y por lo tanto, la población alóctona puede estar formada por genotipos altamente competitivos para nodular. En este trabajo hemos puesto a prueba las tres hipótesis, a saber: 1) la población alóctona posee una significativa biodiversidad; 2) la población alóctona es variable en su FBN, 3) la población alóctona es altamente competitiva para nodular. MATERIAL Y MÉTODOS El análisis de biodiversidad se llevó a cabo a través de la huella digital de ADN por PCR con primers Box A1R (Versalovic et al., 1994), y posterior análisis con GelCompare II.4.0 La FBN se estimó en plantas de soja DonMario 3010 cultivadas en solución mineral libre de N (Lodeiro et al., 2000) a través del peso seco de nódulos (PSN), el peso seco de la parte aérea (PSA) y la concentración de ureidos en hojas 69 Session I SI-CP-24 (CUH) (Trijbels y Vogels, 1966) registrados al inicio de la floración. La competición para la nodulación se midió coinoculando cada cepa en una relación 1:1 con LP 3018 (E109 SmR/SpR) a plantas de soja cultivadas como se describió, y registrando la ocupación de nódulos por cada cepa alóctona (SmS o SpS) o LP 3018, a los 25 días postinoculación, en placas réplica con antibióticos selectivos (Althabegoiti et al., 2011). RESULTADOS Y DISCUSIÓN Biodiversidad de la población alóctona: Hemos estudiado la distribución de genotipos de 100 aislamientos obtenidos de cinco zonas del cinturón sojero de Argentina. La mitad de los aislamientos provenían de nódulos obtenidos en el campo y la otra mitad fueron obtenidos en laboratorio a partir de muestras de suelo. De este modo, estaban representados, por un lado, los genotipos capaces de nodular en las condiciones específicas de suelo y clima al momento de muestrear, y por otro lado, los genotipos potencialmente capaces de nodular bajo condiciones ideales. Observamos una considerable biodiversidad, con al menos 44 cepas diferentes en la colección de aislamientos. Los genotipos se agruparon en ocho clados, pero el tipo de muestreo no influyó en la distribución de genotipos entre los clados. Sin embargo, dicha distribución estuvo significativamente sesgada por la zona de origen, y en particular, por la acidez del suelo de origen, cuyos valores de pH iban de 6,1 a 4,8. Por lo tanto, confirmamos que la población alóctona sufrió procesos de diversificación genética en el suelo. Heterogeneidad en la FBN: Dado que la colección de aislamientos contenía tanto rizobios de crecimiento rápido como bradirrizobios de crecimiento lento, elegimos cinco cepas de rizobios y cinco de bradirrizobios para continuar con los estudios. Como cepas de referencia utilizamos B. japonicum E109 y USDA 110. El PSN, el PSA y la CUH mostraron correlación positiva estadísticamente significativa, con lo cual se tomaron como representativos de la capacidad FBN. Esta capacidad divergió entre los rizobios y los bradirrizobios. Mientras solo una de las cinco cepas de rizobios mostró una performance FBN similar a las cepas de referencia, cuatro de las cinco cepas de bradirrizobios se comportaron igual o mejor que las cepas de referencia. Por lo tanto, confirmamos que la performance FBN de la población alóctona es heterogénea y observamos que diverge entre rizobios y bradirrizobios. Competitividad para la nodulación: En este caso, solo una de las cepas de rizobios y dos de las de bradirrizobios compitieron igual que LP 3018, pero ninguna fue más competitiva que dicha cepa de referencia. Dado que las 10 cepas elegidas al azar representan aproximadamente el 25 % del total de cepas cuyos genotipos fueron establecidos, no podemos asegurar que las cepas alóctonas son más competitivas que la cepa E109, utilizada mayoritariamente en los inoculantes comerciales. En este trabajo se evaluó la competitividad intrínseca de cada cepa, pero no la que podría ejercer en su ambiente edáfico original. Por lo tanto, estos resultados indican que las condiciones del ambiente original de la población alóctona tendrían una importante gravitación para determinar la alta competitividad para nodular observada a campo. AGRADECIMIENTOS Este trabajo fue financiado por la Agencia Nacional de Promoción de la Investigación CientíficoTec nológica, la Universidad Nacional de La Plata y Barenbrug-Palaversich SA (todas de Argentina) y la Dirección General de Investigación Científica y Técnica de España, proyecto AGL2011-30386-C02-01. BIBLIOGRAFÍA López-García, S.L., et al. (2009). Agron. J. 101: 357-363. Versalovic, M., et al. (1994). Methods Mol. Cell Biol. 5: 25-40. Lodeiro, A.R., et al. (2000). Plant Sci. 154: 31-41. Trijbels, F., and Vogels, G.D. (1966). Biochim. Biophys. Acta. 113: 292-301. Althabegoiti, M.J., et al. (2011). FEMS Microbiol Lett 319: 133-139. 70 Session I SI-CP-25 Búsqueda y caracterización de bacterias promotoras del crecimiento vegetal en rizosfera de plantas nativas antárticas. Fernández Garello, P.1, Braga, L.1, Senatore, D.1, Lagurara, P.1, Yanes, M.L.1, Vaz, P.1, Azziz, G.2 y Bajsa, N.1* 1 Laboratorio de Ecología Microbiana-Instituto de Investigaciones Biológicas Clemente Estable. Urugu ay. Laboratorio de Microbiología, Facultad de Agronomía - UDELAR. Uruguay. * [email protected] 2 RESUMEN En este trabajo se aislaron y caracterizaron bacterias rizosféricas a partir de las plantas nativas antárticas Colobanthus quitensis y Deschampsia antarctica. Se estudiaron las características promotoras del crecimiento vegetal de los aislamientos, encontrándose producción de proteasas, sideróforos, celulasas y capacidad para solubilizar fosfato. Algunos de los aislamientos presentaron varias actividades simultáneamente. INTRODUCCIÓN La Antártida posee uno de los ambientes terrestres más severos, caracterizado por bajas temperaturas y falta de agua líquida, que limita la abundancia y actividad de organismos terrestres. La vegetación terrestre nativa de la Antártida marítima incluye dos especies de plantas vasculares: Colobanthus quitensis (clavel antártico) y Deschampsia antarctica (pasto antártico). Dadas las condiciones ambientales adversas, es probable que estas plantas presenten asociaciones con microorganismos que favorezcan su crecimiento. El objetivo de este trabajo fue aislar y caracterizar bacterias promotoras del crecimiento vegetal a partir de rizósfera de clavel y pasto antárticos. MATERIAL Y MÉTODOS Se colectaron plantas en varios sitios de la Isla Rey Jorge (Archipiélago Shetland del Sur) en el verano de 2012 y 2013. A partir de suspensiones de raíces, utilizando medios semiselectivos e incubando a 4 °C y 25 °C, se aislaron Pseudomonas fluorescentes (King’s B), actinobacterias (almidón-caseína), bacterias heterótrofas (tripticasa-soja) y hongos filamentosos (agar malta). Se evaluó su capacidad celulolítica (carboximetil celulosa), proteolítica (medio con leche descremada), de producción de sideróforos (CAS), solubilización de fosfato (NBRIP) y producción de biosurfactantes (ensayo de la gota). RESULTADOS Y DISCUSIÓN De 16 muestras de pasto y 17 de clavel se obtuvieron 667 aislamientos de heterótrofos, 546 de Pseudomonas fluorescentes, 125 de actinobacterias y 16 de hongos filamentosos (Tabla 1). El 63% de los heterótrofos evaluados presentaron actividad proteolítica, el 61% mostró capacidad para solubilizar fosfato, el 41% produjo sideróforos, mientras que sólo el 1% mostró actividad celulolítica. De las actinobacterias evaluadas, el 67% presentó actividad celulolítica, el 3% mostró capacidad para solubilizar fosfato y el 15% produjo sideróforos. No se obtuvieron actinobacterias capaces de producir proteasas extracelulares ni biosurfactantes en las condiciones de cultivo ensayadas. Todas las Pseudomonas fluorescentes evaluadas mostraron actividad proteolítica, en tanto que el 70% fue capaz de solubilizar fosfato (Figura 1). Considerando los aislamientos evaluados hasta el momento, no se observaron diferencias en la proporciones con actividad a ambas temperaturas. Se encontró un mayor porcentaje de actinobacterias y heterótrofos productores de sideróforos aislados de clavel que de pasto, mientras que los heterótrofos productores de proteasas fueron más abundantes en pasto que en clavel. 71 Session I SI-CP-25 Tabla 1. Colección de bacterias rizosféricas. Número de aislamientos de bacterias heterótrofas, Pseudomonas fluorescentes y actinobacterias obtenidos a partir de pasto, clavel o suelo a las dos temperaturas de incubación utilizadas. nº aislamientos 4ºC Muestra nº aislamientos 25ºC Pseudomonas Actinobacterias Heterótrofos Pseudomonas Actinobacterias Pastos 153 172 23 157 203 42 Claveles 85 37 13 165 90 24 Suelo 57 6 9 50 53 14 TOTAL 295 215 45 372 346 80 Porcentaje de aislamientos que presentaron actividad Heterótrofos 100 80 60 40 20 0 actividad proteolítica Actinobacterias actividad celulolítica producción de sideróforos Bacterias heterótrofas solubilización de fosfato Pseudomonas fluorescentes Figura 1. Actividades encontradas en los diferentes grupos bacterianos. Porcentaje de aislamientos de actinobacterias, bacterias heterótrofas y Pseudomonas fluorescentes que presentaron las actividade s ensayadas (la actividad celulolítica en Pseudomonas no fue determinada hasta el momento). Estos estudios se continuarán para analizar todos los aislamientos de la colección, además de determinar otras propiedades tales como producción de fitohormonas, fijación de nitrógeno, y capacidad de antagonismo in vitro. Los aislamientos que resulten de interés serán identificados filogenéticamente. AGRADECIMIENTOS Financiación: Instituto Antártico Uruguayo y ANII (Beca de Iniciación a la Investigación a P.F.G.) . 72 Session I SI-CP-26 Caracterización de las comunidades de hongos micorrícicoarbusculares asociadas a metalofitas en su elos contaminados con Cu. Cornejo, P.*, García, S., Vidal, C., Meier, S., Borie, F. Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile. * [email protected] RESUMEN En un ecosistema contaminado con Cu en la Zona Central de Chile (Valle de Puchuncaví) se analizaron las comunidades de hongos formadores de micorrizas arbusculares (HMA) y las características del suelo rizosférico de tres especies metalofitas. Se observó la presencia de un ecotipo de Acaulospora presente en las tres rizosferas, que representaba la práctica totalidad de esporas asociada a Oenothera picensis, especie endémica que acumula importantes cantidades de Cu en su raíz y parte aérea, y que puede representar una alternativa para realizar procesos de biorremediación. INTRODUCCIÓN La minería del Cu representa en Chile la principal actividad económica, viéndose también seriamente afectados los ecosistemas que se encuentran aledaños a los sitios de producción, principalmente por la depositación de material enriquecido en metales y lluvia ácida (Meier et al., 2012a). Bajo estas condiciones solamente unas pocas especies de plantas pueden establecerse y crecer, conocidas como metalofitas, algunas de las cuales pueden establecer simbiosis con HMA. Estos hongos favorecen la captación de agua y nutrientes de estas plantas, y las protegen frente al estrés abiótico generado por la presencia de éstos contaminantes en el ambiente, disminuyendo el estrés oxidativo de la planta (Meier et al., 2011), o inmovilizando éstos contaminantes en sus estructuras de resistencia, hifas o la glomalina que liberan en el suelo (Cornejo et al., 2008, 2013). En este estudio se analizan las comunidades de HMA asociadas a la rizosfera de tres metalofitas endémicas de la Zona Central de Chile, en el Valle de Puchuncaví, una zona que ha recibido por varias décadas el impacto del funcionamiento de una fundición de Cu. MATERIAL Y MÉTODOS Se recolectó suelo rizosférico de las metalofitas Baccharis linearis (Asteraceae), Imperata condensata (Poaceae) y Oenothera picensis (Onagraceae), las que crecen de forma natural en suelos contaminados (hasta 830 mg Cu kg -1) en el Valle de Puchuncaví. Las esporas se extrajeron mediante tamizado húmedo y posterior decantación en gradientes de sacarosa, recuperadas desde la suspensión, cuantificadas y analizadas morfológica y molecularmente. Con las abundancias por cada ecotipo de HMA encontrado, se caracterizó la diversidad de las comunidades con los índices de riqueza (S`), equidad de Shannon-Wienner (H`) y Dominancia de Simpson (). En el suelo obtenido se determinó igualmente el pH, el P disponible, el porcentaje de agregados estables al agua (AEA), las actividades microbianas dehidrogenasa (DH) y fosfatasa ácida (P-asa), y la longitud del micelio micorrícico. RESULTADOS Y DISCUSIÓN Se observó en todos los casos una fuerte acidificación del suelo, varias unidades por debajo de los valores normales para la zona y suelo (alrededor de 7). Cornejo et al. (2008) reportan para esta zona valores extremos de pH y de disponibilidad de Cu (más 73 Session I SI-CP-26 de 330 mg Cu kg-1) en el suelo rizosférico de I. condensata, que a pesar de lo anterior, en este estudio presentó las comunidades de HMA más diversas, así como también los mayores valores de micelio fúngico y AEA. Esto puede deberse a que también en estos suelos es donde se han encontrado los mayores valores de glomalina, que permitirían una modificación del medio ambiente en el cual se están desarrollando los HMA y otros microorganismos. Por otra parte, estudios en esta especie han demostrado una gran capacidad de exudación de ácidos orgánicos, especialmente cítrico, que posee una alta capacidad para detoxificar el Cu libre en el medio (Meier et al. 2012b), por lo que las plantas de I. condensata pueden hacer frente por varias vías al estrés ambiental. Tabla 1. Principales resultados químicos, bioquímicos y de descripción de la comunidad de hongos arbusculares asociados a plantas de especial interés ecológico del valle de Puchuncaví, Región de Valparaíso, Chile. Especie pH1 P2 AEA3 PDH5 Mic.6 Esp.7 S`8 H`9 10 4 asa B. linearis 4.7 30.1 57.1 2.0 7.3 1.2 207 5.7 1.2 0.4 I. 4.5 49.4 89.4 2.6 6.3 4.6 148 5.3 1.4 0.2 condensata O. picensis 5.0 24.1 66.2 2.2 11.1 2.5 582 2.3 0.1 0.9 1 pH suelo/agua en relación 2/5; 2 P según método Olsen (ppm); 3 Agregados estables al agua (%); 4 A ctividad Fosfatasa ácida (mg de PNP g-1 h-1); 5 Actividad dehidrogenasa (mg INTF g-1 h-1); 6 Longitud de mi celio (m g-1); 7 Densidad de esporas (esporas en 100 g de suelo); 8 Riqueza de especies; 9 Índice de equidad de Sha nnon-Wienner; 10 Índice de dominancia de Simpson. La alta dominancia en O. affinis se debe a la presencia mayoritaria (más de 90%) de un ecotipo de Aculospora en la comunidad de HMA asociada a su rizosfera. Este ecotipo también compone las comunidades de las otras plantas estudiadas, y se ha detectado por medios moleculares usando PCR-TTGE (Cornejo et al., 2004) colonizando las raíces, por lo que puede ser un candidato idóneo para obtener inoculantes que puedan mejorar el establecimiento y crecimiento de especies micorrizables en suelos contaminados con Cu. Sin embargo, los aspectos funcionales de la simbiosis necesitan un mayor grado de comprensión, sobre todo por la capacidad que pudieran tener algunos HMA en particular de producir sustancias como la glomalina, que sería un componente idóneo para mejorar los procesos de fitoestabilización. En este sentido, existen indicios que la glomalina se produce en mayor cantidad a altos niveles de estrés, por lo que ecotipos de HMA no adaptados a estas condiciones igualmente pudieran ser una herramienta, si genética y funcionalmente pudieran tener la capacidad de generar elevadas cantidades de glomalina (Meier et al., 2012c) AGRADECIMIENTOS Estudio financiado por el Proyecto FONDECYT 1120890, CONICYT-Chile. BIBLIOGRAFÍA Cornejo, P., et al. (2004). FEMS Microbiol. Lett. 241: 265-270. Cornejo, P., et al. (2008). Sci. Total Environ. 406:154-160. Cornejo, P., et al. (2013). Soil Biol. Biochem. 57: 925-928. Meier, S., et al. (2011). Appl. Soil Ecol. 48:117-124. Meier, S., et al. (2012a). Crit. Rev. Environ. Sci. Technol. 42: 741-775. Meier, S., et al. (2012b). Ecotox. Environ. Safe. 75: 8-15. Meier, S., et al. (2012c). Appl. Soil Ecol. 61: 280-287. 74 Session I SI-CP-27 Screening for phosphate solubilising rhizobia from faba bean in Marrakech region field cultures. Maghraoui, T.1, 2*, Domergue, O.2, 3, Oufdou, K.1, Lahrouni, M.1, Galiana, A.2, Sanguin, H.2, Drevon, J.J.3, de Lajudie, P.2 1 Laboratory of Biology and Biotechnology of Microorganisms, Faculty of Sciences Semlalia, Cadi Ayyad University, PO Box 2390, Marrakech, Morocco. 2 INRA, IRD, CIRAD, LSTM, Campus de Baillarguet TA A82/J 34398 Montpellier. 3 INRA, Eco&Sols, 1 Place Viala, 34060 Montpellier, France . * [email protected] ABSTRACT Rhizobia and phosphorus (P)-solubilising bacteria are important to plant nutrition. These microbes also play a significant role as plant growth-promoting rhizobacteria (PGPR) in the biofertilization of crops. The present study was conducted in order to select rhizobial strains adapted to low P, isolated from faba bean nodules in Morocco and to investigate their ability to solubilise the mineral P. Our results revealed that among 100 isolates, 15 are able to solubilising the mineral P using TCP test. 40 strains were evaluated for their effect on the growth of faba plant and were genotypically characterized. Preliminary results show contrasting effect of strains on nodulation rate and plant growth. INTRODUCTION Low soil phosphorus availability is a major constraint for crops specially when they are depending on symbiotic nitrogen fixation. Phosphorus is often present in soil as insoluble complexes thus unavailable to plant. Phosphorus complexes with aluminum, iron, silicium, and other metallic ions in acidic soil (Whitelaw, 2000) or with calcium carbonate in alkaline soil (Gyaneshwar et al., 2002). Some rhizobial strains, beneficial N2-fixing symbiotic partners of legumes, were reported to solubilize both organic and inorganic phosphate complexes (Richardson, 2001; Alikhani et al., 2006; Jisha and Joseph, 2008). Using selecting rhizobia, as phosphate-solubilizing microorganisms (PSM) as inoculums would have thus a dual beneficial nutritional effect resulting both from P mobilization and N 2-fixation (Peix et al., 2001). The current study was designed to select phosphate solubilising rhizobia in order to circumvent phosphorus deficiency in soil in an environmentally-friendly, and sustainable manner. MATERIAL AND METHODS The rhizobial strains were isolated from nodules of faba bean plants in Marrakech region according to Vincent (1970). We evaluated their P solubilising capacity using four solid media and two phosphorus forms: NBRIY with CaHPO 4, NBRIY Ca3(PO4)2, TCP NH4Cl and TCP KNO3 with Ca3(PO4)2. The characterization of Rhizobium strains was performed by PCR amplification and sequencing of 16s rRNA coding gene, recA, of nodD and pqqC gene encoding the pyrroloquinoline quinone synthase C. Infectivity and efficiency of rhizobial strains was studied on plants in hydroaeroponic conditions. RESULTS AND DISCUSSION We screened a collection of 100 rhizobial strains isolated from P deficient and P sufficient soils of field cultivated with faba bean in the Houz region. 15 strains were able to solubilise the two types of complex phosphorus on the four mediums used. The 75 Session I SI-CP-27 diameter of peripheral halo zone formed by these strains on NBRIY CaHPO 4 was larger than the others media. Using the second source of phosphorus complex Ca 3(PO4)2, the largest diameter were obtained on TCP NH4Cl medium (Figure 1). Figure 1. Rhizobial isolates forming peripheral halo zone on TCP NH4 medium. Figure 2. Faba bean plants inoculated with rhizobial strains selected, in hydroaeroponic conditions. The phosphate solubilising rhizobia strains were tested for their efficiency to grow with faba bean plants under phosphorus deficiency in hydroaeroponic cultures (Figure 2). The preliminary results indicate that inoculation with selected strains contribute to a difference in root and shoot weight, plants size, and in nodulation. P and N content determination in the different compartments of the plant is in progress. 16s rRNA and rec A gene sequences of twenty strains shows that they all belong to Rhizobium leguminosarum. nodD and pqqC gene analysis are in progress ACKNOWLEDGEMENTS This work was supported by the Great Federative Project FABATROPIMED of Agropolis Foundation under the reference ID 1001-009 and AVERROES scholarship programme provided by the EU for the stay of Tasnime MAGHRAOUI in Montpellier. REFERENCES Alikhani, H.A., et al. (2006). Plant Soil 287: 35-41. Arcand, M.M., Schneider, K.D. (2006). Ann. Acad. Bras. Ciênc. 78: 791-807. Gyaneshwar, P., et al. (2002). Plant Soil 245: 83-93. Hamdali, H. et al. (2008). Appl. Soil Ecol. 38: 12-19. Valverde A., et al. (2007). First international Meeting on Microbial Phosphate solubiliszation. 27 3-276. Vincent J.M. (1970). A manual for the practical study of root-nodule. Blackwell Scientific Publica tions, Oxford. Whitelaw, M.A. (2000). Adv. Agron. 69: 99-104. Peix, A., et al. (2001). Soil Biol. Biochem. 33: 103-110. 76 Session I SI-CP-28 Estudio de la dinámica poblacional binaria de rizobacterias en Lupinus mediante citometría de flujo. Ruiz Palomino, M.1, 2, Probanza, A.1* 1 Universidad San Pablo CEU. Facultad de Farmacia. Departamento de Biología. Madrid. Departamento de Biología. * [email protected] 2 C. San Agustín RESUMEN El presente estudio analiza mediante Citometría de Flujo (CF) la dinámica de dos poblaciones bacterianas (Bacillus P1C11 y Pseudomonas P1C10) en Lupinus albus L., creciendo en hidroponía. Se evalúa el crecimiento de ambas poblaciones solas o en interacción doble a 3 diferentes proporciones. El estudio se completa sin el efecto de la planta, en medios ricos (caldo nutritivo) y restrictivos (solución de Hoaglands) respecto a fuentes de carbono. INTRODUCCIÓN El conocimiento de la dinámica e interacción entre poblaciones bacterianas rizosféricas no sólo tiene interés básico, sino que también es imprescindible optimizar biotecnologías aplicadas, como puede ser la introducción de PGPRs para biorremediación, fitoprotección o la mejora de la producción. En este sentido hay algunos estudios de interacción poblacional binaria (del Gallo et al., 1986) pero sin el efecto planta. La CF es una técnica con buen rendimiento analítico en estudios de ecología microbiana realizados en varios ambientes (Bergquist et al., 2009; Wang et al., 2010) pero su aplicación es infrecuente en el caso de muestras de suelo/planta, aunque rinde interesantes resultados (Müller y Nebe von Caron, 2010) singularmente en estudios de rizosfera (Gamalero et al., 2004; Rochat et al., 2010). MATERIAL Y MÉTODOS La Figura 1 esquematiza del diseño de los experimentos y protocolo seguidos. La CF (en concreto FC/FACS, Flow Cytometry/Fluorescence-Activated Cell Sorting) se realizó con un equipo FACS CALIBUR (Becton Dickinson) usándose tampón PBS como líquido envolvente, con una velocidad de fujo para contar 200 eventos s -1. Las bacterias fueron enumeradas usando SSC (side angle-scattered light) y la emisión de fluorescencia en verde (FL1, Fluorocrome light), tras tratarlas con el flourocromo SYTO 13. La adquisición y análisis de los datos se realizó con el programa CellQuestn. RESULTADOS Y DISCUSIÓN Dinámicas poblacionales en caldo nutritivo. En los ensayos de Bacillus/Pseudomonas 100:0 ó 0:100, se observa que a lo largo del tiempo analizado descienden los recuentos de microorganismos, debido probablemente a que ha superado la fase estacionaria, y la disminución de nutrientes impide el mantenimiento de dicha fase. En el caso de aquellos cultivos mixtos de Bacillus/Pseudomonas 75:25 y 50:50, las tendencias de las curvas de crecimiento son similares. La población de Bacillus en cualquier caso es dominante frente a las Gram negativas, pero ambas poblaciones mantienen aproximadamente los mismos valores iniciales. Sin embargo, en el caso de los cultivos mixtos con un porcentaje inicial de Pseudomonas del 75%, se muestra claramente un gran crecimiento de Bacillus, superando a término las primeras. En este caso (en minoría), la población de Pseudomonas es menos competitiva que la población de Bacillus. A término los niveles de cada población son análogos a los otros dos mixtos. 77 Session I SI-CP-28 Dinámicas poblacionales en solución de Hoagland. En al cultivo puro de Bacillus, se mantiene la población, con oscilaciones menores. La CF muestra que una gran parte de estas bacterias están esporuladas, por lo que mantiene un contingente poblacional estacionario (e inactivo). En el cultivo puro de Pseudomonas, observamos el descenso poblacional más dramático que en el del caso del caldo nutritivo. Las muestras pertenecientes a cualquiera de las tres combinaciones ensayadas, tienen perfiles similares a los del ensayo en caldo nutritivo: se mantienen estables, aunque atendiendo a la localización de las partículas en las representaciones gráficas obtenidas en los análisis (SSC y FL1) habría diferenciaciones en cuanto a la morfología y estado del ciclo vegetativo de las dos cepas utilizadas. Figura 1. Esquema del diseño de los experimentos y protocolo seguidos. Figura 2. Análisis de recue ntos bacterianos por citometría de flujo a los 4 días de cultivo, en cultivo hidropónico de Lupinus, en las muestras con inoculación inicial Bacillus: Pseudomonas, 100:0, 75:25, 50:50, 25:75 y 0:100, respectivamente. Eje X: dispersión de luz por tamaño de la partícula (SSC); eje Y: luz absorbida p or el fluorocromo SYTO 13 (FL1) en escala logarítmica. Los ejes internos separan las regiones de recuent os: superior derecha (Bacillus) y superior izquierda (Pseudomonas) (100.000 eventos analizados) Dinámicas poblacionales en cultivo hidropónico de Lupinus. En el cultivo de Bacillus 100% se mantiene durante los primeros 4 días una curva de población similar a la obtenida en los estudios sin influencia de la planta. Pese a algunas oscilaciones observamos aquí claramente la influencia de los exudados radicales en el medio de cultivo, ya que se mantiene durante más tiempo y más activas (no esporuladas) el contingente poblacional. En el caso del cultivo puro de Pseudomonas, se detectan fluctuaciones, tal vez relacionables con pulsos cíclicos (circadiano) de exudación radical. Las muestras pertenecientes a cualquiera de las tres combinaciones ensayadas resultan en un dominio de Pseudomonas a término, con cierta densodependencia inicial (Figura 2) y que relacionamos (entre otras razones) con la mayor capacidad colonizadora de Pseudomonas. BIBLIOGRAFÍA Bergquist, P., et al. (2009). Extremophiles 13: 389-401. del Gallo, M., et al. (1986). Plant Soil 90: 107-116. Gamalero, E., et al. (2004). FEMS Microbiol. Ecol. 48: 79-87. Müller, S., and Nebe Von Caron, G. (2010). FEMS Microbiol. Rev. 34: 554-587. Rochat, L., et al. (2010). Mol. Plant Microbe Interact. 23: 949-961. Wang, Y., et al. (2010). Trends Biotechnol. 28: 416-424. 78 Session I SI-CP-29 Utilização da técnica de MLSA em estudos taxonômicos e filogenéticos com Bradyrhizobium e sua importância na descrição de novas espécies. Delamuta, J.R.M.1*, Ribeiro, R.A.2, Hungria, M.2 1 Programa de Pós-Graduação em Microbiologia; Universidade Estadual de Londrina, Bolsista CAPES. Laboratório de Biotecnologia do Solo, Embrapa Soja, Londrina. Bolsistas CNPq. * [email protected] 2 RESUMO A metodologia de MLSA (Multilocus Sequence Analysis) tem sido cada vez mais utilizada como ferramenta para se inferir a taxonomia, a filogenia e a evolução de bactérias simbióticas fixadoras de nitrogênio, conhecidas como rizóbios. Como exemplo, o emprego do MLSA foi decisivo para a recente descrição da nova espécie Bradyrhizobium diazoefficiens por nosso grupo de pesquisa (Delamuta et al., 2013). Neste estudo, utilizando-se a sequência concatenada dos genes atpD, glnII e recA, as relações filogenéticas de 13 estirpes de Bradyrhizobium foram determinadas e os resultados indicam a existência de possíveis novas espécies dentro do gênero. INTRODUÇÃO Estudos sobre taxonomia, diversidade e evolução de procariotos, incluindo rizóbios, têm como base técnicas polifásicas, incluindo dados fenotípicos e genotípicos (Zhang et al., 2012). Na última década, a análise de genes housekeeping utilizando a técnica de MLSA (Multilocus Sequence Analysis) tem sido empregada com sucesso em estudos sobre as relações taxonômicas e filogenéticas entre rizóbios, mostrando-se como uma ferramenta poderosa para a descrição de novas espécies (Delamuta et al., 2013). MATERIAL E MÉTODOS Treze estirpes de Bradyrhizobium foram escolhidas de estudos anteriores (Delamuta et al., 2012; Menna et al., 2009). Além da sequência do gene ribossomal 16S, três genes housekeeping (atpD, glnII, e recA) foram utilizados na análise filogenética por MLSA, usando-se parâmetros pré-definidos, com o modelo de distância Tamura-Nei e o algoritmo de Neighbor-Joining. O suporte estatístico foi avaliado pela análise de bootstrap, com 1.000 repetições. RESULTADOS A árvore construída com o gene 16S RNAr dividiu as estirpes de Bradyrhizobium em dois grandes grupos (Figura 1A), com o primeiro (G-I) reunindo oito estirpes SEMIAs e as estirpes tipo do grupo B. japonicum e o segundo grupo (G-II) incluindo as SEMIAs 6154, 6028, 6053, 6148 e 6145 e a estirpe tipo de B. elkani. Na Fig. 1A também fica evidenciado que a posição taxonômica das estirpes não ficou claramente definida com base apenas no gene 16S RNAr. Para uma análise taxonômica mais refinada das estirpes, as sequências dos genes atpD, glnII e recA foram concatenadas (Figura 1B). Com base no MLSA, a SEMIA 656 foi agrupada com B. huanghuaihaiense CCBAU 23303T, o par de estirpes 6002-6144 agrupou com B. arachidis CCBAU 051107T e as SEMIAs 6028, 6053 e 6145 mostraram uma maior similaridade com B. pachyrhizi PAC 48T. Interessantemente, sete estirpes ocuparam posições isoladas na árvore. 79 Session I SI-CP-29 DISCUSSÃO O gênero Bradyrhizobium é considerado ancestral de todos os rizóbios e tem sido isolado de várias leguminosas (Lloret et al., 2005). Contudo, apesar de sua posição evolutiva, poucas espécies estão atualmente descritas no gênero. Uma possível explicação para isso é que a taxonomia moderna é baseada no gene 16S RNAr, cuja sequência é extremamente conservada nesse gênero. Nesse contexto, a técnica de MLSA está se mostrando cada vez mais útil nos estudos filogenéticos e uma prova disto é a sua utilização, nos últimos anos, na descrição e/ou reclassificação de novas espécies de Bradyrhizobium (Guerrouj et al., 2013; Wang et al., 2013), inclusive com a recente descrição de B. diazoefficiens (Delamuta et al., 2013). Os resultados deste estudo indicam que as sete estirpes que ocuparam posições isoladas na árvore concatenada provavelmente representam novas espécies e confirmam que esta técnica é importante e eficaz para revelar a diversidade ainda pouco explorada dentro do gênero Bradyrhizobium e, certamente, de outras espécies fixadoras de nitrogênio de importância econômica e ambiental. B) A) Figura 1. Relações filogenéticas entre as estirpes de Bradyrhizobium deste estudo e as estirpes ti po com base no (A) gene 16S RNAr e (B) nos genes housekeeping concatenados (atpD, glnII, e recA). REFERÊNCIAS Delamuta, J.R.M., et al. (2012). Braz. J. Microbiol. 43: 698-710. Delamuta, J.R.M., et al. (2013). Int. J. Syst. Evol. Microbiol. (online). Guerrouj, K., et al. (2013). Syst. Appl. Microbiol. 36: 218-223. Lloret, L., et al. (2005). Rev. Latinoam. Microbiol. 47: 43-60. Menna, P., et al. (2009). Int. J. Syst. Evol. Microbiol. 59: 2934-2950. Wang, J.Y., et al. (2013). Int. J. Syst. Evol. Microbiol. 63: 616-624. Zhang, Y.M., et al. (2012). PLoS ONE 7(9): e44936. doi:10.1371. 80 Session I SI-CP-30 Mycorrhizal fungal identity as determinant of functional assemblage of plant-growth promoting bacteria in the rhizosphere. Meleiro, A.I.1*, Carvalho, L.1, Correia, P.1, Melo, J.1, 2, Carolino, M.1, Cruz, C.1 1 Centre for Environmental Biology, Faculty of Sciences, University of Lisbon. Campo Grande, Bloco 2 C2, 1749-016 Lisboa, Portugal. Enviromental Microbiology and Biotecnology Lab, University of Vila Velha (UVV). Street Comissário José Dantas de Melo 21, 29102-770, Vila Velha, Espirito Santo State , Brazil. * [email protected] ABSTRACT We found that rhizospheric abundance and functionality of groups of plant-growth promoting bacteria related with phosphorus and nitrogen nutrition can vary with arbuscular mycorrhizal fungal taxa colonizing maize roots, which indicates that mycorrhizal fungi can modulate bacterial functional diversity in the plant rhizosphere. INTRODUCTION The rhizosphere microbiome can modulate plant health and productivity, and understanding how microbes are assembled in the rhizosphere can offer us new strategies to maximize the sustainable production of food (Bisseling et al., 2009). Arbuscular mycorrhizal fungi (AMF) are root symbionts enhancing plant nutrition, and whilst recent evidences indicate that AM fungi can potentially shape total soil microbial community composition by acting as large suppliers of carbon to the rhizosphere (Singh et al., 2008; Drigo et al., 2010), little is known about their influence on the functional diversity of microbial communities. In this work, we addressed whether there is a specific mycorrhizal fungal selection for particular soil bacterial functional groups with emphasis on phosphorus (P) solubilizing and nitrogen (N) fixing bacterial communities. MATERIAL AND METHODS Rhizospheric soil samples were collected from greenhouse experiments where maize plants were inoculated with one of five AMF species (belonging to Glomeraceae and Gigasporaceae families), and with (1) a known soil community of six species of plantgrowth promoti ng rhizobacteria involved in P and N nutrition, or with (2) a whole soil biotic community. P-solubilizing and N-fixing bacterial cell densities for each AMF treatment in both soil communities were determined by culturing methods. The most promising P-solubilizing bacteria were isolated from each AMF treatment, and their functional P-solubilizing capacities were determined. Bacterial isolates identification was evaluated by 16S rRNA Gene Sequencing. RESULTS AND DISCUSSION AMF taxa significantly affect the rhizospheric densities of P-solubilizing and N-fixing bacteria. When a known community consisting of P-solubilizer and N-fixing bacterial species were added, two distinct groups of AMF species were found regarding Psolubilizing bacteria , one (AMF from Glomeraceae family) with highest bacterial densities, while other (AMF from Gigasporaceae family) greatly decreasing bacterial densities (Figure 1). N-fixing bacteria densities also varied according with AMF identity; we found highest densities with one AMF species from the Glomeraceae family, whereas lowest densities were observed in one AMF species from the Gigasporaceae family (Figure 1). When AMF were inoculated into a whole soil community, similar results were found (data not shown). 81 Session I SI-CP-30 Figure 1. Phosphorus-solubilizing and nitrogen-fixing bacterial densities in rhizospheric soils of maize plants inoculated with an AMF species (species 1, 2 and 3 belong to Glomeraceae family, and specie s 4 and 5 to Gigasporaceae family) and with a known community of six PGPR consisting of P-solubilizing and N-fixing bacteria (means ± sd). Overall, the functional characterization of bacterial isolates varied with AMF taxa (data not shown). Our results indicate that functional assemblage of soil bacterial community is influenced by AMF identity. This suggests an ongoing mycorrhizal fungal-bacterial interaction in soil influencing plant productivity. ACKNOWLEDGEMENTS This work was supported by the project PTDC/AGR-PRO/115888/2009 and SFRH/BPD/33633/2009 (to LC) of the Fundação para a Ciência e a Tecnologia of Portugal. REFERENCES Bisseling, T., et al. (2009). Science 324: 691. Drigo, B., et al. (2010). Proc Natl. Acad. Sci. USA 107: 10938-10942. Singh, B.K., et al. (2008). Environ. Microbiol. 10: 534-541. 82 grant Session I SI-CP-31 Diversity and stress tolerance in rhizobia from Parque Chaqueño region of Argentina nodulating Prosopis alba. Chávez Díaz, L.1, González, P.1, Rubio, E.2, Melchiorre, M.1, 3* 1 Instituto de Fisiología y Recursos Genéticos Vegetales (IFRGV), Centro de Investigaciones Agropecuarias-Instituto Nacional de Tecnología Agropecuaria (CIAP-INTA). 2 Instituto de Zoología y Microbiología Agrícola (IMYZA), Instituto Nacional de Tecnología Agropecuaria (INTA). 3 Facultad d e Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba. * [email protected] ABSTRACT The genetic diversity, symbiotic effectiveness, drought tolerance and Indol Acetic Acid (IAA) production of indigenous rhizobial populations in the Parque Chaqueño of Argentina able to nodulate Prosopis alba were assayed. The populations were sampled at five locations from the Arid, Semi-arid and Humid Chaco in the Parque Chaqueño region. A set of rhizobial strains able to nodulate P. alba was obtained and selected based on their molecular diversity by BOX-PCR. The highest molecular variability was observed in rhizobial isolates from Semi-arid Chaco. High level of indolic compound production and tolerance to osmotic treatment were significantly correlated with water restrictions of the environments where the strains belonged. The strains were identified by 16S rDNA sequencing as belonging to the genera Mesorhizobium, Bradyrhizobium and Sinorhizobium. To our knowledge, this is the first report of P. alba nodulation by strains other than Mesorhizobium chacoense, which was already described for the Parque Chaqueño. INTRODUCTION The Parque Chaqueño in Argentina is a very extensive region, subjected to abiotic stresses commonly modifying biodiversity of soil microorganisms and their capacity to establish symbiotic relationships. To our knowledge, there is no information about rhizobium diversity in areas of different moisture in the Parque Chaqueño region, or about the physiological characteristics of rhizobia that might contribute to improve Prosopis nodulation, growth and survival. We hypothesize that rhizobia from Arid, Semi-arid and Humid Chaco of the Parque Chaqueño region of Argentina, able to nodulate P. alba, have genetic polymorphism and the strain behavior under osmotic stress and IAA production is correlated with the hydric characteristics of the environment where the strains come from. MATHERIAL AND METHODS Soil samples were collected from five locations in Parque Chaqueño region, Argentina: 1) San Miguel 31°45’59’’ S, 65°25’00’’ W, Córdoba province; Arid Chaco; 2) Colonia Benítez 27°20’00” S, 58°55’60’’ W, Chaco province; Humid Chaco; 3) Padre Lozano 23°12’51’’ S, 63°50’39’’ W, NE of Salta province; Semi-arid Chaco; 4) Isla Cuba 24°17’31’’ S, 61°51’10’’ W, Formosa province; Semi-arid Chaco; 5) Bolsa Palomo 24° 13’15’’S, 61°57’42’’W, Formosa province; Semi-arid Chaco. Rhizobial isolates were obtained from nodules developed in P. alba trap plants 120 days after sowing. DNA of rhizobial isolates and reference strains was used to perform RepPCR fingerpri nting with BOX-A1R primer. PCR electrophoretic pattern were analyzed, for each location with (UPGMA) clustering method and Dice’s coefficient as similarity index. These data were used to describe molecular variability by the Principal Coordinate Analysis (PCO) (Balzarini et al., 2011). The selected rhizobial isolates were assayed for their tolerance to water deficit treatment (0.6 Mpa, -2 Mpa) and indolic compound production (Glickman and Dessaux, 1995) in free-living state. 16S rRNA of 83 Session I SI-CP-31 rhizobial isolates representing the main BOX-REP-PCR clusters was sequenced and published in data base. RESULTS AND DISCUSION PCO analysis showed the highest molecular variability in rhizobial isolates from Semiarid Chaco (F igure 1). Tolerance to water deficit treatment as well as higher levels of indolic compound production showed that strains from San Miguel (Arid Chaco) and Padre Lozano (Semiarid Chaco), bot h locations with higher soil water shortage conditions, showed the highest survival under water deficit and higher levels of indolic compound production. 16S rDNA from rhizobial strains that showed higher contribution to P alba growth were analyzed. Three of the five strain from Bolsa Palomo (2 KC 759691, 3 KC 759692 and 54 KC 759694) were classified as Mesorhizobium genus; and the other two (12 KC 759693 and 63 KC 759695) as Sinorhizobium. Isolates from Padre Lozano (53 KC 759699 and 40 KC 759698) were species of Bradyrhizobium and Sinorhizobium genera respectively. The isolates obtained from Colonia Benitez (66 KC 759696) and Isla Cuba (36 KC 759697) were species of Mesorhizobium and Rhizobium genera, respectively (Figure 2). To our knowledge, this is the first report of P. alba nodulation by strains other than Mesorhizobium chacoense, which was already described for the Parque Chaqueño. 0,40 PCO 2 (8.2%) 0,20 B. japonicum USDA138 0,00 R. etli SC15 M. loti M. chacoense -0,20 E. meliloti1021 ACKNOWLEGMENTS -0,40 -0,40 -0,20 REFERENCES Bolsa Palomo Isla Cuba 0,00 0,20 0,40 PCO 1 (11%) Padre Lozano Colonia Benitez San Miguel Figure 1. PCO based on UPGMA similarity matrix and Dice’s coefficient of the rep-PCR (primer BOX-A1R) products of DNA from rhizobial isolates from locations in Parque Chaqueño in Argentina Figure 2. 16S rDNA gene phylogeny of Parque Chaqueño rhizobial isolates from P. alba and other. The tree was constructed from 1163 bp aligned nucleotide sequence data by using the UPGMA algorithm. The numbers at branch points are the significant bootstrap values (1000 replicates). Accession nº are written in parentheses after the species name. ACKNOWLEDGEMENTS This work was supported by INTA-PNFOR-044341 and Secyt-UNC 162/12 projects. REFERENCES Balzarini, M.G., et al. (2011) In: Phytopathology in the Omics Era. (Rodriguez Herrera, R. Aguilar , C.N., Simpson-Williamson, J.K., and Gutierrez Sanchez, G., eds). Research Signpost, Kerala, India, pp 12 0. Glickman, E., and Dessaux, Y. (1995). Appl. Environ. Microbiol. 61: 793-796. 84 Session I SI-CP-32 Condiciones de estrés hídrico o salino modifican la composición y capacidad formadora de biofilm de comunidades bacterianas aisladas de rizósfera de alfalfa. Bogino, P., Abod, A., Nievas, F., Santoro, V., Vicario, J. *, Giordano, W. Departamento de Biología Molecular, Facultad de Cs Exactas, Físico-Químicas y Naturales, Universid ad Nacional de Río Cuarto. Río Cuarto, Argentina. * [email protected] RESUMEN Se aislaron comunidades bacterianas procedentes de suelo rizosférico de alfalfa sometido a distintas condiciones: a) control, b) estrés hídrico y c) estrés salino. Las mismas se caracterizaron en función de la capacidad formadora de biofilm total (CFBT), la cual fue mayor en la comunidad procedente de la condición de estrés hídrico. La composición fue estudiada mediante métodos moleculares y se determinó mayor variabilidad para las comunidades obtenidas bajo condiciones de estrés. INTRODUCCIÓN En los suelos el potencial agua () incluye el potencial osmótico (por solutos disueltos) y el potencial mátrico (fuerza de retención del agua por el suelo) (Papendick y Campbell, 1980). Aunque tales potenciales son termodinámicamente equivalentes, representan formas de deprivación del agua que posiblemente afecten a la fisiología bacteriana de maneras distintas. Ha sido estimado que más del 99% de toda la actividad bacteriana en los ecosistemas naturales está asociada con bacterias organizadas en biofilms (Potera, 1996). Aunque la vida en biofilm presenta varias ventajas para las bacterias del suelo, entre las que se pueden mencionar la protección contra la desecación (Flemming, 1993), es escaso el conocimiento de los mecanismos que utilizan las bacterias para desarrollar estructuras del tipo biofilm, como así también de su composición y funcionalidad en los distintos micronichos edáficos (Chang et al., 2007). El objetivo del presente trabajo fue estudiar las comunidades multibacterianas establecidas en estructuras tipo biofilm asociadas a rizósfera de M. sativa bajo distintas condiciones de disponibilidad de agua. MATERIAL Y MÉTODOS Suelo y tratamientos. Se tomaron porciones de 0-20 cm de diferentes sitios de un suelo ubicado en la localidad de Bulnes, Córdoba, Argentina. Se homogeneizó, se dispuso en macetas, se sembraron semillas de alfalfa variedad Pampeana Córdoba y se establecieron tres condiciones: Control (regado regularmente), Estrés Hídrico (escaso riego) y Estrés Salino (NaCl 2,5 g por Kg de suelo). Se mantuvieron 40 días en cámara de crecimiento. Aislamiento de las comunidades rizosféricas. Se obtuvieron los suelos rizosféricos (SR) para cada condición (SRC: Control; SRM: Mátrico; SRS: Salino) y se realizaron recuentos en medio rico (AN). De las placas de mayor dilución, se aislaron 95 cepas por tratamiento. Formación de biofilm. Se siguió la metodología descrita por O’Toole y Kolter (1998). Caracterización molecular. Se realizó un análisis de restricción del gen ARNr 16S (ARDRA) con la enzima HaeIII. La identidad se obtuvo mediante secuenciación del gen ARNr 16S. 85 Session I SI-CP-32 RESULTADOS Y DISCUSIÓN Tamaño y CFBT de las comunidades aisladas de suelos rizosféricos de alfalfa. Los recuentos obtenidos para los suelos rizosféricos (SRC, SRS y SRM) fueron superiores al recuento obtenido en el suelo entero (SE), esto es debido a que la rizosfera representa un micronicho nutricionalmente más rico. Las ufc ml-1 en la rizosfera de alfalfa bajo condiciones de estrés (SRM y SRS) fueron menores en comparación con SRC (sin estrés). Interesantemente, la CFBT fue mayor para la comunidad obtenida del suelo rizosférico sometido a estrés por desecación (SRM), mientras que la comunidad recuperada del suelo rizosférico salino (SRS) fue la que presento la CFBT más reducida. Podría especularse que la condición de estrés afecta la composición de los exudados radicales y por lo tanto la comunidad bacteriana que se establece en las proximidades de las raíces. Considerando la fisiología bacteriana, es probable que condiciones de desecación conduzcan a la selección de cepas con mayor CFB para colonizar la superficie de las raíces otorgando ventajas de supervivencia, mientras que otros mecanismos no necesariamente relacionados a la CFB tendrían mayor relevancia en la supervivencia bacteriana en condiciones de estrés salino. Caracterización de las comunidades rizosféricas. Las cepas aisladas de cada tratamiento fueron agrupadas en subpoblaciones de alta y baja CFB (ACFB y BCFB respectivamente). La mayoría de los perfiles de ARDRA obtenidos en los subgrupos de ACFB fueron diferentes a los de BCFB, demostrando que los ribotipos que constituyen cada subpoblación son diferentes y la existencia de cierta correspondencia entre fisiología (CFB) y caracterización genética. En general, las subpoblaciones de ACFB y BCFB procedentes de suelos rizosféricos sometidos a condiciones de estrés (SRM y SRS) presentaron mayores perfiles de restricción respecto de la condición no sometida a estrés (SRC), indicando la posible existencia de procesos de diversificación en tales situaciones. Podría especularse que ante la presencia de estrés, un grupo más variado de bacterias se establece como consorcio mixto en las zonas cercanas a las raíces de alfalfa, probablemente en respuesta a la búsqueda de micronichos menos hostiles. Por el contario, en condiciones no estresantes, la comunidad mixta se establecería con predominio de los grupos más efectivos para colonizar la zona próxima a las raíces y a que en tal condición otros grupos no necesitan del microambiente rizosférico para sobrevivir. En función de su pertenencia a perfiles de ARDRA mayoritarios, se seleccionaron cepas para su identificación mediante secuenciación del gen ARNr 16S. Se destaca la identidad de las cepas aisladas con cepas típicas encontradas en los suelos, en particular resulta interesante la presencia de cepas filogenéticamente vinculadas a los rizobios como parte del biofilm mixto que constituye la comunidad asociada a rizósfera de alfalfa. AGRADECIMIENTOS Este trabajo ha sido financiado por medio del Programa PPI SECYT-UNRC 18/C408 y de los Proyectos PICT FONCYT 2007-02228, 2011-0965 y PIP CONICET 112-201101-00086. BIBLIOGRAFÍA Flemming, H.C. (1993). Water Sci. Technol. 27: 1-10. Chang, W.S., et al. (2007). J. Bacteriol. 189: 8290-8299. Papendick, R.I., and Campbell, G.S. (1980). Water potential relations in soil microbiology. p. 1-2 2 Potera, C. (1996). 273: 1795-1797. O'Toole, G.A., and Kolter, R. (1998). Mol. Microbiol. 28: 449-461. 86 Session I SI-CP-33 Supervivencia y capacidad formadora de biofilms por rizobacterias. Abod, A.*, Bogino, P., Vicario, J., Giordano, W. Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto. Río Cuarto, Argentina. * [email protected] RESUMEN Las rizobacterias constituyen un amplio grupo de bacterias las cuales han desarrollado estrategias como la agregación microbiana y la formación de biofilms para adaptarse a diversas condiciones edafo-ambientales. En el presente trabajo se estudió la supervivencia y capacidad de formación de biofilm de distintas rizobacterias bajo condiciones de estrés hídrico y salino. También se evaluó el desarrollo de biofilms mixtos formado entre rizobios y Pseudomonas. En general, las rizobacterias que presentaron elevada capacidad para formar biofilms en condiciones de desecación también mostraron mayores niveles de supervivencia en esa condición de estrés. INTRODUCCIÓN La rizósfera es definida como la zona del suelo inmediata al sistema radical de los vegetales, donde tiene lugar una interacción dinámica con los microorganismos. En la rizósfera existe un variado grupo de bacterias conocidas como PGPR (Plant Growth Promoting Rhizobacteria) (Spaepen et al., 2009). La capacidad para desarrollar estructuras del tipo biofilm representa una estrategia clave para responder a variaciones en la disponibilidad nutricional, sobrevivir en el suelo y mejorar sus posibilidades para colonizar la planta hospedadora. La mayoría de los biofilms en la naturaleza consisten de consorcios microbianos de múltiples especies (Kawarai et al., 2007), cuya funcionalidad y arquitectura es influida por diversas situaciones estresantes (Chang et al., 2007). MATERIAL Y MÉTODOS Cepas bacterianas: se utilizaron las cepas silvestres de Sinorhizobium meliloti (Rm1021 y Rm8530) y la mutante no productora de EPS II, Rm8530 expA. Además se usó la cepa WCS417r de P. fluorescens. Medio de cultivo: Se utilizó el medio LB reducido (LB/2) para generar un potencial osmótico de -0,25 MPa, al cual se le agregaron cantidades crecientes de NaCl o de PEG 8000 para generar condiciones de reducción del potencial agua (estrés salino o hídrico respectivamente) del orden de -0,5 y -1,0 MPa. Cuantificación de la formación de biofilm: La capacidad de las cepas de formar biofilm creciendo individualmente y en co-cultivo fue examinada por el método basado en la tinción y medición de la absorbancia del colorante cristal violeta (O'Toole y Kolter, 1998), adaptada en nuestro laboratorio para ensayos en rizobios (Rinaudi et al., 2006). Ensayos de viabilidad bacteriana en condiciones de estrés hídrico y salino: Se utilizó arena estéril como sustrato, la misma se dispuso en tubos de vidrio y se humedeció a capacidad de campo con suspensiones conteniendo 106 UFC de cada cepa de S. meliloti. Los tubos fueron incubados durante distintos períodos de tiempo a 30°C. Se incluyeron controles sin deshidratar. Para el estrés salino, se procedió de la misma manera, pero la arena fue humedecida con soluciones de 430 mM y 250 mM de NaCl conteniendo 10 6 UFC de cada cepa de S. meliloti. Los controles fueron humedecidos con solución fisiológica (NaCl 155 mM). Se realizaron recuentos bacterianos a diferentes tiempos. 87 Session I SI-CP-33 RESULTADOS Y DISCUSIÓN Ensayos de viabilidad bacteriana en arena. Estrés hídrico. La cepa Rm8530 presentó mayor capacidad de supervivencia que las cepas Rm1021 y Rm8530 expA. Interesantemente, se observó que Rm8530 incrementó el número de ufc por gramo de arena en condiciones de desecación. Probablemente la producción de EPSII por esta cepa y la vinculación del mismo con la capacidad de formar biofilms (Rinaudi y Giordano, 2010) permitan explicar estos resultados. Estrés salino. La cepa Rm1021 tuvo mejor capacidad de supervivencia cuando la concentración de NaCl fue de 155 mM mientras que la exposición a altas concentraciones de NaCl (250 y 430 mM) determinó una marcada disminución de la viabilidad celular, con reducciones de alrededor de 5 órdenes de magnitud. En los casos de Rm8530 y Rm8530 expA la exposición al estrés salino no determinó grandes cambios de la viabilidad celular. Especulamos que la presencia del elemento regulador ExpR (Marketon y González, 2002) en Rm8530 y Rm8530 expA estaría vinculado a la supervivencia de estas cepas en condiciones salinas. Cuantificación de la formación de biofilm por consorcios mono y multiespecies. Se determinó que frente a condiciones de estrés hídrico y salino, a medida que el potencial agua del medio se redujo, la cepa WCS417r de P. fluorescens disminuyó el crecimiento y aumentó la formación de biofilm, este efecto fue más marcado en presencia del soluto PEG respecto de NaCl. En el caso de la cepa Rm1021, la formación de biofilm fue reducida en todas las variables estudiadas excepto en el tratamiento con PEG -1,0 MPa, en cuyo caso se registró un aumento de dicho parámetro. La cepa Rm8530 no fue afectada frente a estrés salino en su capacidad para formar biofilm, por el contrario, condiciones de estrés hídrico ocasionaron un gran aumento en la formación de biofilm. Cuando se examinó el biofilm mixto entre Pseudomonas y Rm8530 se vio que la presencia de NaCl (-0,5 y -1,0 MPa) produjo incrementos en el crecimiento y formación de biofilm respecto de LB/2 (-0,25 MPa), mientras que la adición de PEG (1,0 MPa) determ inó un crecimiento despreciable y una marcada formación de biofilm por el co-cultivo. Llamativamente, los recuentos diferenciales indicaron que el biofilm formado en medio conteniendo PEG -1,0 MPa estuvo formado casi exclusivamente por la cepa de rizobio, mientras que en condiciones de estrés salino (NaCl -1,0 MPa) el biofilm mixto estuvo constituído por ambas cepas en proporciones similares. Se puede concluir que ante un estrés salino, la bacteria usa diferentes mecanismos (como la acumulación de solutos compatibles) para afrontar y persistir en esta condición. Por el contrario, en condiciones de estrés hídrico la estrategia de supervivencia más conveniente sería el desarrollo de estructuras tipo biofilms. AGRADECIMIENTOS Este trabajo ha sido financiado por medio del Programa PPI SECYT-UNRC 18/C408 y de los Proyectos PICT FONCYT 2007-02228, 2011-0965 y PIP CONICET 112-201101-00086. BIBLIOGRAFÍA Chang, W.S., et al. (2007). J. Bacteriol. 189: 8290-8299. Kawarai T., et al. (2007). Appl. Environ. Microbiol. 73: 4673-4676. Marketon M.M., and González J.E. (2002). J. Bacteriol. 184: 3466-3475. O'Toole G.A., and Kolter R. (1998). Mol. Microbiol. 28: 449-461. Rinaudi L., et al. (2006). Res. Microbiol. 157: 867-875. Rinaudi L., and Giordano W. (2010). FEMS Microbiol. Lett. 304: 1-11. Spaepen S., et al. (2009) Adv. Bot. Res. 51: 283-320. 88 Session I SI-CP-34 Caracterización morfológica de rizobios asociados a cultivos de arveja (Pisum sativum L.), chocho (Lupinus mutabilis S.), fréjol (Phaseolus vulgaris L.), haba (Vicia faba L.) y vicia (Vicia atropurpurea) en suelos del Ecuador. Carpio, M.J., Paucar, B.M.*, Alvarado, S.P. Departamento de Manejo de Suelos y Aguas (DMSA), Estación Experimental Santa Catalina (EESC), Instituto Nacional Autónomo de Investigaciones Agropecuarias (INIAP). Quito, Ecuador. * [email protected]; [email protected] RESUMEN Una de las alternativas para cubrir la deficiencia de nitrógeno (N) en los suelos es el uso de microorganismos fijadores de este elemento. Rizobios asociados con arveja, chocho, fréjol, haba y vicia en suelos ecuatorianos fueron aislados y caracterizados. Se identificaron diferencias en los parámetros de elevación y producción de goma entre aislados del mismo cultivo y entre cultivos. INTRODUCCIÓN Uno de los principales elementos que limitan la producción agrícola en suelos de la sierra ecuatoriana es N, y para suplir este requerimiento se aplican fertilizantes nitrogenados con una baja eficiencia; y consecuentemente, con problemas de tipo económico y ambiental (Beman et al., 2005; Galloway et al., 2008; Schlesinger, 2009). Existen varias alternativas para manejar adecuadamente la fertilidad de los suelos y la nutrición de los cultivos, entre estas se encuentran el uso de abonos verdes dentro de un sistema de rotación de cultivos y el uso de microorganismos eficientes. Los microorganismos fijadores de N contribuyen con un aporte de N al suelo de forma natural (Revelo et al., 2010; Bernal, 2012), sin dañar el medio ambiente y a la vez favorecen la economía del agricultor (Campaña, 2003; González, 2001; Subía, 2001). En el Ecuador se ha realizado investigación sobre rizobios en algunos cultivos (fréjol, arveja, alfalfa, trébol, soya y maní), pero sólo se cuenta con caracterización fenotípica y genotípica del rizobio de fréjol y maní, habiéndose evidenciado una alta heterogeneidad en los suelos ecuatorianos (Bernal, 2004; Aguilar et al., 2004). Con respecto a los rizobios asociados al cultivo de haba, chocho y vicia, no se han realizado estudios y en lo referente a arveja hay poca información. En el presente estudio se realizó el aislamiento, purificación y caracterización morfológica de los rizobios asociados a los cultivos de arveja, chocho, fréjol (voluble), haba y vicia como parte inicial de la evaluación de los mismos en cultivos con potencial uso como abonos verdes. MATERIAL Y MÉTODOS Las muestras de rizobios colectadas en cultivos de arveja, chocho, fréjol, haba y vicia en la provincia Imbabura-Ecuador, se tomaron en la época seca (octubre) y época lluviosa (enero), con cultivos en estado de floración. El aislamiento se realizó utilizando medio Levadura manitol agar (LMA) con rojo congo, y luego se purificó para realizar la caracterización mediante las siguientes pruebas: Tiempo de crecimiento, acidificación o alcalinización del medio LMA (Levadura manitol agar) con ABT (Azul de bromotimol) y características morfológicas (textura, cantidad de goma, aspecto, forma, elevación y borde) (CIAT, 1988; Jerez, 2004; Marquina et al., 2011). 89 Session I SI-CP-34 RESULTADOS Y DISCUSIÓN Los aislamientos de rizobios asociados a arveja, fréjol, haba y vicia, tienen características similares en textura, aspecto y borde, diferenciándose entre ellas por la cantidad de goma que producen. En tanto, los rizobios asociados a chocho fueron muy diferentes con respecto a los antes mencionados por la abundante cantidad de goma y por la elevación convexa. Esto indicaría que son diferentes tipos de géneros concordante con lo manifestado por Marquina et al. (2011). En cuanto a los aislamientos de rizobios asociados a arveja se observaron diferencias en la elevación y en la cantidad de goma producida; mientras los aislados de haba se diferenciaron por la cantidad de goma producida. Estos resultados posiblemente se deben a que sean rizobios de diferente especie o biovar en cada caso, pues las muestras efectivamente fueron recolectadas en diferentes localidades. Los aislamientos de rizobio de fréjol tienen características distintas en elevación a los reportados por Marquina et al. (2011), lo cual indicaría que podría tratarse de otra especie y además estas fueron recolectadas en diferentes localidades. En cuanto al tiempo de crecimiento, todos los rizobios aislados son de crecimiento rápido y modifican el pH del medio a ácido, lo cual es consistente con lo manifestado por el CIAT (1988). En general, estos resultados corroboran con investigaciones previas, donde se menciona la gran variabilidad de rizobios en suelos ecuatorianos (Bernal, 2004; Aguilar et al., 2004). AGRADECIMIENTOS Esta investigación es parte del Proyecto PIC 12 INIAP 009 “Manejo adecuado de abonos verdes y microorganismos fijadores de nitrógeno dentro de sistemas de producción agroecológicos” financiado por el Gobierno Nacional del Ecuador a través de SENESCYT y ejecutado por el Departamento de Manejo de Suelos y Aguas de la EESC del INIAP. BIBLIOGRAFÍA Aguilar, M., et al. (2004). Proc. Natl. Acad. Sci. USA 101: 13548-13553. Beman, M.J., et al. (2005). Nature 434: 211-214. Bernal, G. (2004). INIAP. Resumen Ejecutivo. 80 p. Bernal, G. (2012). Memorias SECS. 9 p. Campaña, D. (2003). Tesis de Ing. Agr. UCE. 117 p. CIAT (1988). Simbiosis leguminosa rizobio -Man. met. eval., sel. manejo agr. 178 p. Galloway, J., et al. (2008). Curr. Sci. 94: 1375-1381. Jerez, M. (2004). Tesis Ing. Agr. UCE. 127 p. González, P. (2001). Tesis Ing. Agr. UCE. 102 p. Marquina, M., et al. (2011). Biol. Trop. 59: 1017-1036. Revelo, J., et al. (2010). Informe Proyecto SENESCYT. 12 p. Schlesinger, W.H. (2009). Proc. Natl. Acad. Sci. USA 106: 203-208. Subía, C. (2001). Tesis Ing. Agrp. ESPE. 151 p. 90 Session I SI-CP-35 Bacterias con actividad ACC desaminasa en huertos de aguacate en Michoacán, México. Chávez-Bárcenas, A.T.1*, Hernández-Valdés, E.F.1, Bárcenas-Ortega, A.E.1, LozunaLópez, F.1, García -Saucedo, P.A.1, Olalde-Portugal, V.2 1 Facultad de Agrobiología “Presidente Juárez” de la Universidad Michoacana de San Nicolás de Hidalgo, México. 2 CINVESTAV Unidad Irapuato, México. * [email protected]; [email protected] RESUMEN Se determinó la cuenta total de bacterias viables con actividad ACC desaminasa en suelo de huertos de aguacate ‘Hass’ bajo dos tipos de manejo del cultivo, orgánico y convencional, en el municipio de Uruapan, Michoacán, México. En suelos bajo manejo orgánico se encontró 40 % más UFC de bacterias que en los de manejo convencional. Adicionalmente se seleccionaron 19 aislados con morfología colonial distinta que mostraron un efecto positivo en el desarrollo radicular de sorgo. INTRODUCCIÓN El aguacate presenta raíces poco profundas y carentes de tricoblastos (Salazar, 2002), por lo que las relaciones simbióticas con microorganismos de la rizosfera seguramente juegan un papel muy importante en su desarrollo. El cultivo del aguacate en Michoacán se lleva a cabo bajo dos esquemas de manejo contrastantes, el convencional con el uso de fertilizantes y plaguicidas químicos sintéticos y el orgánico en el que se utilizan insumos de origen orgánico como estiércol animal, compostas y control biológico de plagas. Un grupo de bacterias promotoras del crecimiento vegetal (BPCV) disminuye los niveles de etileno, que inhiben la elongación radical, por acción de la enzima 1amino-ciclopro pano-1-carboxilato (ACC) desaminasa (Caballero, 2006). En este trabajo se reporta la presencia de BPCV con actividad ACC desaminasa en suelo adyacente a las raíces de aguacate con mayor abundancia en huertos bajo manejo orgánico respecto al manejo convencional. MATERIAL Y MÉTODOS En cuatro huertos de aguacate ‘Hass’ establecidos por pares, uno de manejo orgánico frente a otro de manejo convencional, se tomaron muestras compuestas de 200 g de suelo cercano a raíces jóvenes de árboles de aguacate (cuatro árboles por huerto). Se determinó la cuenta total microbiana en medio mínimo de sales con ACC como única fuente de nitrógeno (Penrose y Glick, 2003). Colonias aisladas de rizosfera de aguacate por cultivo de raíces en el mismo medio selectivo fueron clasificadas por su morfología colonial y su efecto en el desarrollo de raíces se evaluó en co-cultivo in vitro con sorgo. RESULTADOS Y DISCUSIÓN El número de bacterias con actividad ACC desaminasa es mayor en suelos de huertos bajo manejo orgánico que en aquellos bajo manejo convencional. Todos los huertos presentaron bacterias viables con actividad ACC desaminasa, sin embargo en los huertos bajo manejo orgánico se encontró mayor número de bacterias que en los de manejo convencional (Figura 1). Estos resultados sugieren que bajo las condiciones de manejo orgánico en huertos de aguacatero se promueve un mayor desarrollo de poblaciones de bacterias con capacidad para disminuir las concentraciones de etileno en raíces de plantas. Estos resultados coinciden con los datos de biomasa y actividad microbiana total, así como porcentaje de materia orgánica en los mismos huertos (datos no mostrados) y con Sánchez et al. (2006), al comparar biomasa y 91 Session I SI-CP-35 actividad microbiana edáfica en manejo agroecológico y convencional de maracuyá. Se sugiere que el esquema de manejo orgánico del aguacate en Uruapan, Michoacán, promueve mejores condiciones ambientales para el desarrollo de bacterias benéficas, respecto al manejo convencional. . Figura 1. Efecto del tipo de manejo del cultivo del aguacate en Uruapan, Michoacán sobre la abundancia de bacterais con actividad ACC desaminasa. Letras distintas indican diferencias estadísticas significativas. Figura 2. Co-cultivo in vitro de semillas de sorgo y aislado bacteriano. Semillas desinfectadas se sembraron en medio MS al 10 % (flecha blanca), al mismo tiempo se aplicó una azada de uno de los aislados bacterianos (a) o agua estéril (b) en el otro extremo de la caja de Petri (flecha negra). Las bacterias con actividad ACC desaminasa aisladas de huertos de aguacatero promueven el desarrollo radicular. El desarrollo de raíces de sorgo co-cultivadas in vitro con 19 de los 20 morfotipos de aislados de bacterias con actividad ACC desaminasa obtenidos, mostraron un mayor crecimiento que el testigo 72 h después de la siembra, sobresaliendo el crecimiento de las raíces de plantas con los aislados C18 y C22 (Figura 2). Adicionalmente se observó que en las raíces co-inoculadas con los aislados C10 y C12 se promovió el desarrollo de tricoblastos. Estos resultados muestran que la bacterias aisladas pueden considerarse promotoras del crecimiento vegetal, lo cual se ha reportado para diversas especies de bacterias de la rizobiota con estas propiedades bioquímicas en distintas especies vegetales (Yim et al., 2013). AGRADECIMIENTOS A CONACYT y CIC-UMSNH, México, por el financiamiento del proyecto BIBLIOGRAFÍA Caballero, M.J. (2006). Rev. Lat Amer. Microbiol. 48: 154-161. Penrose, D.M, and Glick, B.R. (2003). Physiol. Plant. 118: 10-15. Salazar, G.S. (2002). Nutrición del aguacate, principios y aplicaciones. Primera Edición INIFAP, INPOFOS. Querétaro, Qro. pp:165. Sánchez, M., et al. (2006). Acta Agron. 55: 7-12. Yim, W., et al. (2013). Plant Physiol. Biochem. 14: 95-104. 92 Session I SI-CP-36 Caracterização genotípica de bactérias diazotróficas isoladas de plantas de Brachiaria brizantha e B. humidicula no estado de Roraima, Amazônia, Brasil. Chalita, P.B.1, Cunha, E.N.1, Zilli, J.E.2, Silva, K.1* 1 Laboratório Microbiologia do Solo. Embrapa Roraima, Rodovia BR-174, Km 8, Distrito Industrial, CEP 69301-970, Boa Vista-RR, Brasil. 2 Embrapa Agrobiologia, Rodovia BR-465 Km 7, CEP 23890-000, Seropédica-RJ, Brasil. * [email protected] RESUMO Através do sequenciamento parcial do gene 16S rDNA de 29 bactérias diazotróficas isoladas de Brachiaraia brizantha e B. humidicula em Roraima na região Amazônica, Brasil, foi verificada que a maioria das bactérias pertencem ao gênero Bacillus. INTRODUÇÃO Estudos com bactérias diazotróficas associadas a espécies de Brachiaria são importantes para o fornecimento deste nutriente que é um dos mais limitantes nas pastagens brasileiras. No Brasil, a bovinocultura de corte é fortemente sustentada por diferentes espécies do gênero Brachiaria, tornando-as principal fonte de nutrientes para animais em pastejo. Portanto o objetivo deste trabalho foi caracterizar genotipicamente bactérias isoladas de plantas de Brachiaria no Estado de Roraima, Amazônia, Brasil. MATERIAL E MÉTODOS Vinte e nove bactérias isoladas de plantas de braquiária (Tabela 1) no Estado de Roraima foram caracterizadas genotipicamente através da amplificação do gene 16S rDNA utilizando os iniciadores Y1 (5’ TGG CTC AGA ACG AAC GCT GGC GGC 3’) e B3 (TAC CTT GTT ACG ACT TCA CCC CAG TC). O sequencimaneto parcial foi realizado utilizando o inciador Y1 no sequenciador 3730xl. Tabela 1. Local de coleta e planta de isolamento das 29 bactérias caracterizadas genotipicamente. Planta de isolamento Brachiaria humidicula Local de coleta B. brizanta Parte aérea Raiz Parte aérea Raiz Fazenda São Paulo – Roxinho* ERR 535, ERR 536 ERR 513, ERR 514, ERR 515, ERR 516, ERR 517 ERR 537, ERR 538 ERR 518, ERR 519, ERR 520, ERR 521, ERR 522, ERR 523, ERR 524 Campo Experimental Água Boa – Boa Vista** ERR 525, ERR 526, ERR 527, ERR 528, ERR 529, ERR 530 ERR 539, ERR 540, ERR 541 ERR 531, ERR 532, ERR 533, ERR 534 * Área de transição cerrado-mata: B. brizanta (N 02º17’34,4”, W 61º15’09,8”); B. humidicula (N 02º 18’05,5”, W 61º15’32,9”). ** Área de cerrado: B. brizanta (N 02º39’34,7”, W 60º50’00,8”); B. humidicula (N02o3 9’51,7”, W60o50’60,5”). RESULTADOS E DISCUSSÃO Através do sequenciamento do gene 16S rDNA (Figura 1) foi possível observar que a maioria dos isolados pertence aos gênero Bacillus. Além do gênero Bacillus, foram encontrados isolados próximos a Brevibacillus e Lysinibacillus. Também foi encontrado um isolado próximo de Agrobacterium tumefaciens e dois isolados próximos a Klebsiella. Estes resultados demonstram há existência de outros gêneros diferentes dos 93 Session I SI-CP-36 encontrados em outros trabalhos no Brasil, como Azospirillum (Magalhães and Döbereiner, 1984; Reis Júnior et al., 2004), Burkholderia (Boddey, 2003) e Herbaspirillum (Baldani et al., 1992). 54 (AJ831844) Bacillus aerophilus partial 28K ERR520 ERR532 ERR541 (AJ831842) Bacillus altitudinis 41KF2b ERR524 44 ERR526 ERR537 ERR521 ERR525 27 ERR523 ERR531 ERR516 26 (D16273) Bacillus megaterium IAM 13418 85 ERR518 (AB021192) Bacillus mycoides (D16281) Bacillus thuringiensis IAM 12077 57 ERR535 59 ERR529 ERR538 28 ERR540 ERR513 ERR514 (AF234854) Bacillus safensis FO-036b 87 34 (AY876289) Bacillus pumilus ATCC 7061 ERR527 ERR539 ERR536 69 (FJ233848) Bacillus rhizospherae SC-N012 (AB112715) Brevibacillus reuszeri DSM 9887T 100 ERR515 100 (X60608) B.azotofixans 37 (D16276) Paenibacillus polymyxa IAM 13419 100 (AY677116) Lysinibacillus massiliensis 74 ERR528 (D14500) Agrobacterium tumefaciens 100 ERR530 (EF488759) Enterobacter oryzae Ola 51 (AF129440) Klebsiella oxytoca ERR533 84 ERR534 0.005 Figura 1. Árvore filogenética do sequenciamento parcial do gene 16S rDNA das bactérias isoladas de braquiária em Roraima. Árvore inferida através do método de Neighbor-joining, utilizando o modelo de Jukes-Cantor, com 10 00 repetições. AGRADECIMENTOS A Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) pelo financiamento, Projeto FBNPlus: 02.09.01.011.00.00. REFERÊNCIAS Baldani, V.L.D., et al. (1992). Symbiosis, 13: 65-73. Boddey, L.H. (2003). Tese (Doutorado em agronomia). UFRRJ, Seropédica, Rio de Janeiro. Magalhães, F.M.M., and Döbereiner, J. (1984). R. Microbiologia 15: 246-252. Reis Júnior, F.B., et al. (2004). R. Bras. Ci. Solo, 28: 103-113. 94 Session II Genetics and genomics of beneficial microorganisms and associated plants Session II SII-P-1 Genomics of host specificity in the Rhizobium-legume symbiosis. Imperial, J.1, 2*, Laguerre, G.3, Brito, B.1, Jorrín, B.1 1 Centro de Biotecnología y Genómica de Plantas (CBGP) Universidad Politécnica de Madrid. Campus de Montegancedo, Pozuelo de Alarcón, 28223 Madrid, España. 2 Consejo Superior de Investigaciones Científicas (CSIC). Madrid, España. 3 LSTM, CIRAD-IRD, Université de Montpellier 2, Supagro, USC INRA, Montpellier. Deceased, 17/1/2013. * [email protected] ABSTRACT Most Rhizobium leguminosarum bv. viciae isolates are able to specifically nodulate plants of any of four different legume genera: Pisum, Lens, Vicia, and Lathyrus. However, previous evidence suggests that some genotypes are more adapted to a given plant host than others, and that the plant host can select specific genotypes among those present in a given soil population. We have used a population genomics approach to confirm that this is indeed the case, and to analyze the specific genotypic characteristics that each plant host selects. One of the key aspects of the Rhizobium-legume symbiosis is its well-known specificity: specific rhizobia nodulate and fix nitrogen in specific legume hosts. However, this specificity is not absolute. Some tropical legumes (such as Phaseolus or siratro) are quite broad in their specificity requirements and are promiscuously nodulated by a large number of different rhizobial species and genera, whereas some rhizobia (such as Sinorhizobium sp. NGR234) are able to establish symbioses with very different plants. Some rhizobia, such as Sinorhizobium meliloti (Ballard et al., 2005) or Bradyrhizobium japonicum (Koch et al., 2010) can nodulate different hosts depending on a specific genetic complement, often uncovered after mutant screening or isolation of specific strains that are symbiotically active with just some of the hosts. However, in some cases very subtle mechanisms of adaptation to a specific plant host might be in operation. This, for example, seems to be the case of rhizobial species were all (or most) of the isolates can effectively establish a diazotrophic symbiosis with plants of several different genera, often from different habitats and with different lifestyles, and is well exemplified by Rhizobium leguminosarum bv. viciae. Isolates belonging to this biovar establish effective symbioses with legumes belonging to four genera: Pisum, Lens, Lathyrus and Vicia. The last genus, in particular, includes species as diverse as vetch (V. sativa) and broad bean (V. faba). One set of nodulation and nitrogen fixation genes, harbored on a symbiotic plasmid, allows successful establishment and development of symbiosis with the different hosts (Surin and Downie, 1989) and, in cross-inoculation experiments, when challenged with any one of the above legume hosts, any R. leguminosarum bv. viciae strain is able to establish an efficient symbiosis. However, it has long been hypothesized that different rhizobial strains may be more adapted to a specific plant host than others, which may result in selection and enrichment of a specific strain or set of strains by the legume host from those present in a particular soil. Molecular evidence for plant-mediated selection of specific rhizobial genotypes from soil populations has been obtained by the research groups of Gisèle Laguerre (Depret et al., 2004; Laguerre et al., 2003; Louvrier et al., 1995) and J. Peter W. Young (Mutch and Young, 2004; Palmer and Young, 2000). They used molecular markers and specific PCR amplification to obtain evidence that different plant hosts enrich specific genotypic marker variants of R. leguminosarumbv. viciae from those available in the soil. 96 Session II SII-P-1 These molecular studies were limited by the number and nature of the markers selected, and did not clarify the bases for enrichment of a given specific genotype. With the development and availability of new generation sequencing technologies this problem has been reappraised using genomic and population genomic approaches. Genomics of Rhizobium. After the original reports on genome sequencing of model rhizobia (Mesorhizobium loti 2000, Sinorhizobium meliloti 2001, Bradyrhizobium japonicum 2002, R. etli 2006 and R. leguminosarum 2006, a very large number of rhizobial genomes have been sequenced or are in the process of being sequenced, and among them about 100 Rhizobium isolates (as many as 96 complete or ongoing genome sequence projects in GOLD, the Genomes OnLine Database –http://www.genomesonline.org, as of June 30, 2013). Although data are still quite recent, several general conclusions on the genomic structure and organization of Rhizobium rhizobia emerge. In general, the Rhizobium contain large genomes, of ca. 7 Mb, and even larger in the case of members of the genus Bradyrhizobium (ca. 9 Mb).The occurrence of very large genomes in soil bacteria has been interpreted as an adaptation to this habitat, a complex, hostile and changing environment that demands the large metabolic and behavioral plasticity that can be provided by a large gene-encoding capacity. Contrary to members of the genus Bradyrhizobium, members of the genus Rhizobium (and both Sinorhizobium) present a multi-partite genome, harboring several large plasmids, some of which resemble chromosomes (“chromids”, Harrison et al., 2010). On average, 30-40% of the genome in these bacteria is present in the form of plasmids (Galardini et al., 2013; Harrison et al., 2010; Mazur et al., 2011). This characteristic is shared with the Roseobacter clade (Petersen et al., 2013), and affords a large genomic plasticity, especially since many of these plasmids incorporate conjugative systems (Crossman et al., 2008). This plasticity liberates these bacteria from the constraints of long replication times associated to a single, very large chromosome, the situation found with the bradyrhizobia. Genomics and the rhizobia in the soil. Soil microbial communities have the highest level of prokaryotic diversity (up to 109 microorganisms per gram, Knietsch et al., 2003). Metagenomic approaches would appear to be the ideal approximation to such a complex system, allowing the study of the nature, composition and function of microbial communities in soil. However, even metagenomics is limitedby this complexity, in view of: a) the very large size of these datasets limits our ability to analyze them; b) soil changes rapidly not only temporally but also spatially, even at the micro level, and its physicochemical properties affect microbial distribution within the soil matrix, imposing important technical and methodological problems. Despite these caveats metagenomics constitutes a powerful approach to obtain information about the nature, composition and function of microbial communities in soil. The specificity of the Rhizobium-legume symbiosis has classically allowed the use of most probable number (MPN) techniques to enumerate soil Rhizobia that are able to nodulate trap plants. For R. leguminosarum bv. viciae, representative abundances are on the order of 104-105 viable cells per gram of soil). Louvrier and collaborators developed a semi-selective medium to isolate R. leguminosarum directly from soils (Louvrier et al., 1995). The numbers they obtained in soils from Eastern France were ca. 104 per gram of soil. Overall, it can be concluded that, although cultivation of the plant host results in an increase in rhizobial soil counts (modest in the case of R. leguminosarum), established soil populations of R. leguminosarum are, at most, on the order of 104 to 105 per gram of soil. If typical soils contain ca. 10 9 bacteria per gram of soil, R. 97 Session II SII-P-1 leguminosarum would amount to less than 0.01% of the total soil microbiota. In practical terms, this implies that even in one of the largest metagenomic datasets (ca. 1 Tb), at most 100 Mb would be R. leguminosarum DNA. This, barring the crucial problem of how to specifically identify these sequences, would represent at most a 15x coverage of a single R. leguminosarum genome and would barely be representative of the population diversity. Thus, the low natural abundance of rhizobia in soils precludes the use of purely metagenomic methods to study their diversity and demands an alternative approach. In view of these difficulties, we decided to adopt a Pool-Seq approach to the study of genotype selection by the host plant. Kofler and collaborators, working with Drosophila melanogaster populations, proposed for the first time the Pool-Seq term in 2011 for the next-generation sequencing and analysis of pooled DNA samples from natural populations. It constitutes a feasible (and affordable) genome-wide approach for comparison of population samples, thus allowing an easy scaling from the limitations of single markers to population genomics (Kofler et al., 2011 and references therein). We reasoned that sequencing pooled DNA samples from R.leguminosarum bv. viciae nodule isolates obtained from different legume plant hosts used as rhizobial traps would allow an experimental test of the hypothesis that different plant hosts select specific subpopulations of rhizobia from the available population present in a given soil. We compared four populations (P. sativum, L. culinaris, V, sativa and V. faba) originating from the same agricultural soil and consisting each of one hundred nodulae isolates that were grown independently and then pooled; the genomic DNA of the pools was extracted and sequenced (Illumina Hi-Seq 2000, 180 bp PE libraries, 100 bp reads, 12 Mreads) at BGI (Hong Kong and Shenzhen, China). For analysis of the rhizobial Pool-Seq data, two specific considerations were taken into account.First, plant-specific subpopulations derive from the same unselected, resident soil population, whose genomic composition is, by definition, unknown, since their low numbers preclude any unselected genomic analysis. It is likely that this resident population contains both major and minor genomic types, resulting both from the soil’s edapho-climatic properties and from its agricultural history. Thus, specific selection by the legume host will operate –if it does– on this original composition which, although distorted by the plant effect, will still be present in the plant specific isolates. Second, the large size and the multipartite composition of the R. leguminosarum genome favor both an open pan-genome and a large non-conserved genome. With R. leguminosarum bv. viciae we have estimated that 20-30% of the genes are strain specific. This suggests that plant host selection of specific rhizobial genotypes may implicate specific genes or groups of genes (eg. transport and metabolism of substrates). However, identification of these genes from Pool-Seq data is technically complex, since any DNA assembly will result, necessarily, in the formation of chimaeras with no biological meaning. With these limitations in mind, we decided to restrict the Pool-Seq comparative data analysis to conserved genes, and reads for those genes were identified following recruitment by a reference genome, which in our case was that of R. leguminosarum bv. viciae 3841. A data analysis pipeline was designed and implemented, where reads are aligned to the reference genome, and both coverage and single nucleotide polymorphism (SNP) analysis are performed and compared between subpopulations. These analyses were carried out both for the complete genome and for relevant markers (16S rDNA, nod genes, nif genes, recA and glnII housekeeping markers, etc.). The data clearly show, both at the genome-wide and at the specific marker levels, that specific genotypes are indeed selected by the plant host, thus confirming previous indications. 98 Session II SII-P-1 A major outcome of this study is one of methodology for the study of natural rhizobial populations in the soil. Given unlimited resources for sequencing and data analysis, it is clear that individual genome sequencing of isolates, followed by assembly and multiple genome comparisons represents a more powerful tool than the Pool-Seq approach. However, such a situation is unlikely to occur, and the advantages and disadvantages of Pool-Seq must be evaluated for each project. When this project was designed (2010), it was not feasible to individually sequence and assemble 200 rhizobial strains. Even with today’s higher capacity and lower costs, the pooled DNA approach allows for higher sequencing depths, and thus for potentially better descriptions of the populations, and for more facile analysis with our optimized pipeline. However, the Pool-Seq approach suffers from important drawbacks for this type of analysis in rhizobia. First, since the plant specific genotype enrichment will necessarily reflect the original structure of the rhizobial population in the soil, and this can vary from soil to soil, the analysis should be repeated with different types of soil. More importantly, the impossibility to assemble reads without the generation of chimeras makes it very difficult to identify specific genes that are not present in the reference genome but that may be specifically enriched in plant-selected subpopulations. These genes are important because they can provide not only specific host-linked markers but also evidence for the structural or functional nature of the phenotypes selected by the plant. We are addressing these limitations by means of two complementary approaches. First, the complexity of the plant-enriched subpopulations can be reduced by any number of typing methods, for instance RAPD analysis, making this reduced number of isolates more amenable to direct genome sequencing and assembly. Second, the Pool-Seq pipeline for coverage and SNP data analysis can be repeated with different R. leguminosarum reference genomes in order to incorporate coverage and SNP analyses for genes that were absent from the original reference genome. Results from both these strategies strengthen the power of the PoolSeq approach with a mínimum investment in sequencing and data analysis. ACKNOWLEDGMENTS This work was supported by the Spanish Consolider-Ingenio Program (Microgen Project, CSD200900006) to J.I. We thank Rosabel Prieto for help with isolation of strains from root nodules and DNA preparations. We also thank Gonzalo Martín for IT. REFERENCES Ballard, R.A., et al. (2005). Aust. J. Exp. Agr. 45: 209-216. Crossman, L.C., et al. (2008). PloS ONE 3: e2567. Depret, G., et al. (2004). FEMS Microbiol. Ecol. 51: 87-97. Galardini, M., et al. (2013). Genome Biol. Evol. 5: 542-558. Harrison, P.W., et al. (2010). Trends Microbiol. 18: 141-148. Knietsch, A., et al. (2003). J. Mol. Microbiol. Biotechnol. 5: 46-56. Koch, M., et al. (2010). Mol. Plant-Microbe Interact. 23: 784-790. Kofler, R., et al. (2011). Bioinformatics 27: 3435-3436. Laguerre, G., et al. (2003). Appl. Environ. Microbiol. 69: 2276-2283. Louvrier, P., et al. (1995). Soil Biol. Biochem. 27: 919-924. Mazur, A., et al. (2011). BMC Microbiol. 11: 123. Mutch, L.A., and Young, J.P.W. (2004). Mol. Ecol. 13: 2435-2444. Palmer, K.M., and Young, J.P.W. (2000). Appl. Environ. Microbiol. 66: 2445-2450. Petersen, J., et al. (2013). Appl. Microbiol. Biotechnol. 97: 2805-2815. Surin, B.P., and Downie, J.A. (1989). Plant Mol. Biol. 12: 19-29. 99 Session II SII-P-2 Genomic insights into the rhizosphere lifestyle of rhizobia. Ormeño-Orrillo, E.*, Martínez-Romero, E. Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apdo. Postal 565-A, CP 62251, Cuernavaca, Morelos, Mexico. * [email protected] Rhizobia is a common name used to refer to members of several genera of - and Proteobacteria which are able to induce the formation of root or stem nodules in legume plants, invade the nodules and differentiate into nitrogen-fixing intracellular bacteroids. These bacteria are used in agriculture as biofertilizers to reduce or replace the use of nitrogen chemical fertilizers (Hungria et al., 2000). The term rhizosphere refers to the root surface and adjacent soil that is influenced by root exudates. Despite being best known as symbiotic partners of legumes, rhizobia are able to colonize the rhizospheres of non-legume plants (Rosenblueth and Martínez-Romero, 2004) and act as plant growth promoting bacteria (PGPB) in several crops (García-Fraile et al., 2012 and references therein). Rhizobia have been safely used in agriculture for over a hundred years, an advantage over other PGPB known to be potential human pathogens. Extensive studies of many rhizobia-legume models have produced a detailed inventory of molecular determinants and mechanisms used by rhizobia during nodulation and nitrogen fixation with legumes. In contrast, little is known about the genetic repertoire used by rhizobia during rhizosphere colonization. Here we report on genes that may promote rhizosphere colonization in the genomes of two rhizobial strains used as models in our research group, Rhizobium tropici CIAT 899 and Rhizobium phaseoli Ch24-10, and report on the transcriptome of the latter strain during rhizosphere colonization. Rhizosphere colonization-related traits encoded in selected rhizobial genomes. Strain Ch24-10 was isolated from the stem of a maize plant (Zea mays cv. Criollo Cholula) growing intercropped with common bean (Phaseolus vulgaris) (Rosenblueth and Martínez-Romero, 2004). Originally ascribed to Rhizobium etli, Ch24-10 has been recently reclassified as R. phaseoli (López-Guerrero et al., 2012). This strain is both a maize endophyte and common bean rhizobia as it possesses a symbiotic plasmid. R. tropici CIAT 899 was isolated from a P. vulgaris nodule (Martínez-Romero et al., 1991) but is also able to colonize the rhizosphere and interior of maize roots (Rosenblueth and Martínez-Romero, 2004). Both strains are competitive for rhizosphere colonization and behave as PGPB. Recently we obtained the genome sequences of Ch24-10 (López-Guerrero et al., 2012) and CIAT 899 (Ormeño-Orrillo et al., 2012). Analysis of both genomes revealed a wide array of genes that may promote colonization and support a rhizospheric lifestyle. Like other rhizosphere bacteria, both strains encode hundreds of transporters in their genomes that may allow them to take up numerous nutrients available in root exudates. Genes coding for enzymes like peptidades/proteases, glycosyl hydrolases, oxireductases and oxygenases are also abundant in both genomes and many of them could be involved in the catabolism of rhizosphere nutrients. Traits known to promote rhizosphere colonization in other bacteria were found encoded in both genomes. These included siderophore production, bacteriocin synthesis, antimicrobial efflux pumps, vitamin prototrophy (except for biotin in CIAT 899 although it possesses redundant biotin uptake transporters), surface polysaccharides, type IV pili, motility and chemotaxis. Both strains have genes involved in biosynthesis 100 Session II SII-P-2 of the plant hormones auxins and gibberellins supporting their role as PGPB. In CIAT 899, and acdS gene coding for a 1-aminocyclopropane-1-carboxylate deaminase may promote plant growth by reducing ethylene levels. Genes for biosynthesis of cellulose microfibrils and adhesin-like proteins which may contribute to root surface attachment were also found in both genomes. In CIAT 899, filamentous hemagglutinin-like and YadA-like adhesins may be exported by several type V secretion systems. Both strain genomes encoded type IV secretion systems although they are more related to conjugation machineries than to effector-exporting systems. In Ch24-10, two type VI secretion systems were found that may be related to its superior competitive ability (Records, 2011). Rhizobial genes expressed at the rhizosphere of maize and common bean. We investigated the transcriptome of R. phaseoli Ch24-10 during its early interaction with maize or common bean roots. Around 1250 genes were highly expressed in either plant rhizosphere. Transporter genes comprised 15% of these transcripts. Genes coding for uptake transporters of oligopeptides, sugars and amino acids were the most abundant, but uptake transporters of other molecules like iron/siderophore, sulphate, nitrate, and some metals were also expressed suggesting that Ch24-10 is able to use a wide range of the nutrients present in root exudates. We have reported that rhizobia can simultaneously utilize different carbon sources (Romanov and Martínez-Romero, 1994) a feature that seems advantageous for rhizosphere colonization given the diversity of nutrients present in root exudates (López-Guerrero et al., 2013; Ormeño-Orrillo and Martínez-Romero, 2013). Several expressed genes like putA, hut, asn and rha for proline, histidine, asparagine and rhamnose catabolism, respectively, as well as some glycosidase and peptidase genes are surely involved in utilization of substrates imported by the uptake transporters. In rhizospheres, bacteria are exposed to plant antimicrobials and expression of genes involved in resistance to these compounds seems to be advantageous. Several drug efflux transporters were found among the Ch24-10 highly expressed genes, including the Rmr extrusion pump involved in common bean nodulation proficiency (GonzalezPasayo and Martínez Romero, 2000) that was found expressed in both rhizospheres. The lps2 gene, required for biosynthesis of the O antigen moiety of lipopolysaccharide (LPS) (Garcia-de los Santos and Brom, 1997), was highly expressed. This observation is in agreement with our report that O antigen-defective mutants of R. tropici CIAT 899 are impaired in maize root colonization as LPS seems to protect rhizobia from plant antimicrobials (Ormeño-Orrillo et al., 2008). Besides plant antimicrobials, bacteria seem to face other adverse conditions in the rhizosphere (Matilla et al., 2007; Ramachandran et al., 2011). Various genes involved in osmotic stress response were highly expressed in our system including ndv for perisplasmic glucan biosynthesis, kup and kdp for potassium transport, several genes for utilization of external osmoprotectants like betaine, and treS and treY for biosynthesis of trehalose (another osmoprotectant). Other stress-related genes included relA required for biosynthesis of the alarmone ppGpp, katG coding for a catalase/peroxidase, and a cyclopropane fattyacyl-phospholip id synthase gene. Synthesis of vitamins seems to be required for successful rhizosphere colonization (Karunakaran et al., 2006). Accordingly, we found that thiamin biosynthesis genes were highly expressed. Genes for the flagellum and type IV pilus, surface appendages that are important in other bacteria for rhizosphere colonization, were expressed by R. phaseoli 101 Session II SII-P-2 Ch24-10. Other expressed genes included those involved in the synthesis of some known, poorly characterized or putative novel colonization factors like siderophores, cellulose microfibrils (Ausmees et al., 1999), trifolitoxin (Robleto et al., 1998), two different type VI secretion systems (Records, 2011) and an alpha-2-macroglobulin (Budd et al., 2004). Interestingly, genes putatively involved in biosynthesis of the plant hormone gibberellin were also expressed in t he rhizosphere by Ch24 -10. Several orthologues to previously reported R. leguminosarum genes expressed in plant rhizospheres (Ramachandran et al. 2011) were highly expressed in maize and common bean roots by R. phaseoli Ch24-10. As in R. leguminosarum, many R. phaseoli Ch24-10 highly expressed genes were hypothetical. Expanding our understanding of rhizobial genes expressed at the rhizosphere will surely aid in selecting or designing competitive and proficient PGPB rhizobial strains. REFERENCES Ausmees, N., et al. (1999). Microbiology 145: 1253-1262. Budd, A., et al. (2004). Genome Biol. 5: R38. García-de los Santos. A., and Brom, S. (1997). Mol. Plant Microbe Interact. 10: 891-902. García-Fraile, P., et al. (2012). PLoS ONE 7: e38122. González-Pasayo, R., and Martínez-Romero, E. (2000). Mol. Plant Microbe Interact. 13: 572-577. Hungria, M., et al. (2000). In: Nitrogen Fixation: From Molecules to Crop Productivity (Current Pl ant Science and Biotechnology in agriculture Volume 38). Pedrosa, F.O., Hungria, M., Yates, G., and Newton, W.E. (eds.). ISBN: 978-0-7923-6233-3. Springer. pp. 515-518. Karunakaran, R. et al. (2006). J. Bacteriol. 188: 6661-6668. López-Guerrero, M.G. et al. (2012). Syst. Appl. Microbiol. 35: 353-358. López-Guerrero, M.G., et al. (2013). Front. Plant Sci. 4: 188. Martínez-Romero, E., et al. (1991). Int. J. Syst. Bacteriol. 41: 417-426. Matilla, M.A., et al. (2007). Genome Biol. 8: R179. Ormeño-Orrillo, E., et al. (2008). Environ. Microbiol. 10: 1271-1284. Ormeño-Orrillo, E., and Martínez-Romero, E. (2013). Syst. Appl. Microbiol. 36: 145-147. Ormeño-Orrillo, E., et al. (2012). BMC Genomics 13: 735. Ramachandran, V.K, et al. (2011). Genome Biol. 12: R106. Records, A.R. (2011). Mol. Plant Microbe Interact. 24: 751-757. Robleto, E.A., et al. (1998). Appl. Environ. Microbiol. 64: 2630-2633. Romanov, V.I., and Martínez-Romero, E. (1994). Plant Soil 161: 91-96. Rosenblueth, M., and Martínez-Romero, E. (2004). Arch. Microbiol. 181: 337-344. 102 Session II SII-CO-1 First analyses of the genomic sequence of the soybean symbiont Sinorhizobium fredii HH103. Vinardell, J.M.1*, Göttfert, M.2, Becker, A.3, Acosta-Jurado, S.1, Baena, I.4, Blom, J.5, Bonilla, I.4, Buendía, A.M.1, Crespo-Rivas, J.C.1, Goesmann, A.5, Jaenicke, S.5, Krol, E.3, Lloret, J.4, McIntosh, M.3, Margaret, I.1, Pérez-Montaño, F.1, Schneiker-Bekel, S.5, Serranía, J.3, Szczepanowski, R.5, Zehner, S.2, Pühler, A.5, Ruiz-Sainz, J.E.1, Weidner, S.5 1 Departamento de Microbiología, Universidad de Sevilla, Spain. 2 Institut für Genetik, Technische Universität Dresden, Dresden, Germany. 3 LOEWE-Zentrum für Synthetische Mikrobiologie, Marburg, Germany. 4 Departamento de Biología, Universidad Autónoma de Madrid, Madrid, Spain; 5 Centrum für Biotechnologie, Universität Bielefeld, Bielefeld, Germany. * [email protected] ABSTRACT Sinorhizobium fredii HH103 is a fast-growing rhizobial strain with a broad host-range including soybean. Here we present its genome, which consists of one chromosome and six plasmids with a total size of 7239684bp. INTRODUCTION Sinorhizobium fredii shows an extremely broad host-range of nodulation, which includes determinate (such as Glycine max and Vigna unguiculata) and indeterminate (such as Cajanus cajan and Glycyrrhiza uralensis) nodule forming legumes. Among the different S. fredii strains, HH103 is particularly interesting because it can effectively nodulate both Asiatic and American (i.e., commercial) soybeans. S. fredii is closely related to S. meliloti and Sinorhizobium sp. NGR234, although these bacteria do not nodulate soybeans. S. fredii HH103 genes involved in the production of Nod factors, the symbiotic T3SS, and surface polysaccharides have been already reported (for a review see Margaret et al., 2011). The chemical structure of LCOs, cyclic glucans, capsular polysaccharides, and exopolysaccharides has been determined. Field trials using strain HH103 as soybean inoculant have also been carried out. This makes S. fredii HH103 to one of the best studied rhizobial strains. MATERIAL AND METHODS The complete genomic sequence of S. fredii HH103 has been established as described by Weidner et al. (2012), leading to a circa 35-fold coverage. Manual annotation of the genome and comparative analyses are being carried out by using the GenDB and EDGAR software respectively (CeBiTec, University of Bielefeld, Germany). Molecular biology techniques have been carried out as described by Vinardell et al. (2004). RESULTS AND DISCUSSION Our first attempt to assemble the complete genome of S. fredii HH103 led to a genome structure composed of 6 replicons: the chromosome and five plasmids (Margaret et al., 2011, Weidner et al., 2012). New experiments indicate the presence of a putative sixth plasmid (pSfHH103a2) that comigrates with the smallest plasmid in agarose gels. The existence of this new plasmid has been confirmed by purification of the small plasmids of HH103 and hybridisation experiments. The manual annotation of the genome (about 6919 genes) is close to completion. Table 1 summarizes the main characteristics of the different replicons constituting the S. fredii HH103 genome. Figure 1 shows the number of genes shared by S. fredii strains HH103 and USDA257 and by Sinorhizobium sp. NGR234 (panel A). It also depicts a comparison of HH103, S.meliloti 1021, and the 103 Session II SII-CO-1 slow-growing soybean symbiont Bradyrhizobium japonicum USDA110 (panel B). As expected, HH103 shares a higher number of genes with strains USDA257 and NGR234 than with S. meliloti 1021. Not surprisingly, the number of genes shared by HH103 and B. japonicum USDA110 is very low. The analysis of the set of symbiotic genes shared by S. fredii strains and B. japonicum might help to identify the essential bacterial traits for nodulation of soybeans. Table 1. Main characteristics of the seven replicons constituting the S. fredii HH103 genome. GC Gene Other interesting Replicon Size (bp) Symbiotic genes a (%) number characteristics b sinIsinR (QS) CG, KPS, LPS, Putative secretion systems: Sec4014 nolR, exoR, exoS, Chromosome 4305723 62.61 independent, T1SS, T2SS, mucR1, mucR2 T2/T4SS, 2096125 62.38 1973 EPS, KPS, LPS minCDE;T2SS, T3SS, T4SS pSfHH103e Met tRNA (anticodon: CAT) nod, noe, nol, nif, 582757 59.57 651 T3SS (sym), T4SS, pSfHH103d fix, nop, tts trb (T4SS)- traItraR (QS) T4SS 144082 58.68 167 nopM2, nopP2 Not essential for growth or pSfHH103c symbiosis c Several unsuccessful attempts 61880 58.47 58 pSfHH103b have been carried out to cure it Not essential for growth or 24036 58.21 18 pSfHH103a1 symbiosis c Several genes encoding 25081 58.02 38 pSfHH103a2 chaperonines 7239684 62.14 6919 Total a CG, cyclic glucans. KPS, K-antigen capsular polysaccharide. LPS, lipopolysaccharide. EPS, exopolysaccharide; b QS, quorum sensing. TXSS, type X secretion system; c Elimination of this plas mid neither affected growth in TY or YMA media nor symbiotic ability with soybean, cowpea, pigeonpea. Figure 1. Number of shared genes between HH103 and different rhizobial strains (EDGAR software). ACKNOWLEDGEMENTS This work was supported by grants BIO2008-05736-C02-01/02 and BIO2011-30229-C02-01 from the Spanish Ministry of Science and Innovation and grant 0313805A from the Bundesministerium für Forschung und Technologie, Germany REFERENCES Margaret, I., et al. (2011). J. Biotechnol. 155: 11-19. Vinardell, J.M., et al. (2004). Mol. Plant-Microbe Interact. 17: 676-685. Weidner, S., et al. (2012). J. Bacteriol. 194: 1617-1618. 104 Session II SII-CO-2 Identificación y caracterización del regulador maestro del flagelo lateral de Bradyrhizobium japonicum USDA 110. Mongiardini, E.J. *, Quelas, J.I., Lodeiro, A.R. IBBM-Facultad de Ciencias Exactas, UNLP-CONICET, La Plata, Argentina * [email protected] RESUMEN En este trabajo se identificó el regulador maestro del flagelo lateral de Bradyrhizobium japonicum USDA 110 mediante mutagénesis insercional. Además se muestran resultados preliminares relacionados con el mecanismo de acción de dicha proteína mediante mutagénesis puntual de un residuo clave relacionado con su funcionalidad. INTRODUCCIÓN Bradyrhizobium japonicum es una bacteria Gram negativa que se utiliza en la industria agrícola como fertilizante biológico para soja, dada su capacidad para nodular raíces de soja y una vez allí, fijar nitrógeno atmosférico en simbiosis con esta leguminosa. En los suelos donde se cultiva soja a menudo existen poblaciones de rizobios capaces de nodular sus raíces compitiendo de esta manera con el fertilizante biológico. En este sentido, trabajos previos de nuestro laboratorio demostraron que la movilidad bacteriana puede jugar un papel en la competitividad del fertilizante biológico para nodular. Así, cuando en el campo se inocularon bacterias seleccionadas en el laboratorio por su mayor movilidad, éstas fueron capaces de competir mejor que las bacterias establecidas en el suelo y concomitantemente, condujeron a aumentos de rendimiento en grano (Althabegoiti et al., 2008; López-García et al., 2009). La capacidad de movimiento de estas bacterias está dada por una estructura denominada flagelo. B. japonicum es el único rizobio que cuenta con dos sistemas flagelares, uno lateral e inducible y otro subpolar y constitutivo. Remarcablemente, encontramos que las bacterias seleccionadas por mayor movilidad tienen desreprimida la expresión del flagelo lateral (Althabegoiti et al., 2008). Dada la importancia de la regulación de este flagelo en la competitividad para nodular y la escasez de información al respecto, decidimos profundizar nuestro conocimiento sobre este tema. En general, la regulación de la expresión de los flagelos bacterianos se da en diferentes etapas y está controlada a modo de cascada por varios reguladores transcripcionales. El regulador clave que desencadena la síntesis de los flagelos se denomina regulador maestro. (Smith and Hoover, 2009). En este trabajo se identificó y se comenzó con la caracterización del regulador maestro que controla la expresión del flagelo lateral en B. japonicum. MATERIAL Y MÉTODOS Se construyó un mutante insercional en el ORF blr6846 introduciendo un cassette de resistencia a kanamicina entre los nucleótidos 7542881 y 7543631, y en paralelo se construyeron plásmidos replicativos llevando el gen completo, para todo lo cual se emplearon las metodologías ya descriptas (Quelas et al., 2010). Con una modificación de estos métodos se realizaron los reemplazos de aminoácidos en la secuencia codificada. La extracción de flagelina de cultivos líquidos y su visualización en SDSPAGE se reali zaron como se describió (Althabegoiti et al., 2008). 105 Session II SII-CO-2 RESULTADOS Y DISCUSIÓN Mediante estudios bioinformáticos se identificó el probable regulador maestro que controla la expresión del flagelo inducible, cuyo gen estaría codificado en el locus blr6846. Esta proteína fue previamente caracterizada en Ensifer meliloti y Brucella melitensis (Léonard et al., 2007, Rotter et al., 2006). Este regulador pertenece a la familia de proteínas de reguladores de respuesta de sistemas de dos componentes presentando un dominio sensor capaz de ser fosforilado y otro de unión a ADN que le permite reconocer las regiones donde ejerce su acción. En E. meliloti y B. melitensis se probó que este regulador de respuesta presenta una mutación puntual en el residuo que debería sufrir la fosforilación, de manera que su funcionalidad no dependería de esta modificación post-traduccional y por lo tanto sería independiente de su par quinasa. Sin embargo, un análisis de la secuencia aminoacídica de esta proteína en B. japonicum muestra que este residuo no presenta dicha mutación sino que se encuentra conservado como en los típicos reguladores de respuesta de sistemas de dos componentes. Así, se esperaría que para ejercer su función dependa de una fosforilación previa. El mutante insercional construído en blr6846 resultó incapaz de expresar el flagelo lateral en las condiciones de inducción. La mutación se corroboró complementando en trans con el gen salvaje completo en un vector replicativo, lo cual restituyó el fenotipo salvaje. Para probar si el regulador de respuesta de B. japonicum depende de una modificación por fosforilación para ejercer su función, se construyó un mutante puntual en el residuo Asp58 por Gly58, residuo que no puede sufrir este tipo de modificaciones. De esta manera se determinó que el mutante puntual con el reemplazo Asp58Gly sigue siendo capaz de expresar el flagelo inducible, lo cual indica que la presencia del residuo Asp58, y por lo tanto la fosforilación, no sería necesaria para cumplir su función. Este hecho particular está siendo investigado con mayor detalle en el laboratorio. AGRADECIMIENTOS Este trabajo fue financiado por la ANPCyT de Argentina. EJM, JIQ y ARL son miembros de la carrera de investigador científico de CONICET, Argentina. BIBLIOGRAFÍA Althabegoiti, M.J., et al. (2008). FEMS Microbiol. Lett. 282: 115-123. Léonard, S., et al. (2007). J. Bacteriol. 189: 131-141. López-García, S.L., et al. (2009). Agronomy J. 101: 357-363. Quelas, J.I., et al. (2010). Mol. Plant-Microbe Interact. 23: 1592-1604. Rotter, C., et al. (2006). J. Bacteriol. 188:6932-6942. Smith, T.G., and Hoover, T.R. (2009). Adv. Appl. Microbiol. 67: 257-295. 106 Session II SII-CO-3 Pyrosequencing reveals the presence of diverse bacterial genera which have not previously described to soil and rhizosphere. Lagos, L.1*, Jorquera, M.1, Maruyama, F.2, Mora, M.L.1 1 Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile. Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo, Japan. * [email protected] 2 ABSTRACT Rhizosphere is a complex environment that harbors diverse microorganisms which carry out essential functions for plants. Multitude of biotic and abiotic factors are assumed to influence the structure and activity of microbial communities in the rhizosphere. In this work the diversity of bacterial communities in ryegrass (Lollium perenne var. Nui) rhizosphere grown in two Andisols was analyzed by pyrosequencing technique. Our results reveal the presence of 65 bacterial genera which have no records in the GenBank as groups present in plant rhizospheres and soils. However, the distribution and functions of these 65 bacterial groups in soil or rhizosphere remain unknown. INTRODUCTION The rhizosphere, defined as soil zone affected by plant roots, is a complex environment. The diversity of microbial communities in the rhizosphere differs in their composition, activity and abundance according to diverse biotic and abiotic factors. Bacterial diversity in these environments is much greater than previous estimated by using conventional molecular techniques (T-RFLP and DGGE). In traditional molecular studies, dominant populations have masked the detection of low abundance OTUs, genetic diversity, and their individual distribution in soil environments. The study of soil metagenome (all the genetic material present in soil), particularly of the rhizosphere, could be crucial to a better understanding how structure, diversity and activity are distributed along the roots of plants. In this context, the objective of this study was to explore by using pyrosequencing the diversity of bacterial communities in rhizosphere of ryegrass grown in two Andisols under greenhouse conditions. MATERIAL AND METHODS Sterile seeds of Lollium perenne var. Nui were sown in rhizoboxes containing unsterile volcanic soils (Andisols) and maintained for 30 days under greenhouse conditions. Rhizosphere soil samples were aseptically collected from adhering soil to roots and immediately processed in laboratory. Total bacterial DNA was extracted from rhizosphere samples by using Power Soil® DNA Isolation Kit (MoBio Inc., USA) according to manufacturer instructions. Then, the DNA extracts were analyzed by pyrosequencing at Macrogen, Inc. (Seoul, Korea). Pyrosequencing was performed by using Genome Sequencer-FLX system (Roche). The genera identified were compared with those present in the National Center for Biotechnology Information’s GenBank database (NCBI; www.ncbi.nlm.nih.gov/). RESULTS AND DISCUSSION The sequencing data revealed a high diversity of low-abundance bacterial groups and the occurrence of 65 genera which have not been previously described for soil or rhizosphere according to GenBank database. The genera included: Dethiosulfovibrio (Synergistetes), Asteroleplasma (Tenericutes), Aspromonas, Desulfopila, Sulfurimonas, Albidiferax (Proteobacteria), Vitellibacter (Bacteroidetes), Anaerofustis (Firmicutes), 107 Session II SII-CO-3 Bryobacter (Acidobacteria) isolated from diverse habitats, such as: sediments and waters from sea and lakes, gastrointestinal tract and gut of human and animals, slurry and industrial sludges, sedimentary rock, cave, glacial, etc. Other genera present have been related with functions such as biopolymer-degrader (Proteobacteria; Rugamonas), sulfate-reducer (Proteobacteria; Desulfopila), thermophilic alkane-degrader (Firmicutes; Coprothermobacter), succinate-utilizing bacteria (Firmicutes; Succiniclasticum) oil-degrader, (Actinobacteria; Aestuariimicrobium), glycogenaccumulator (Actinob acteria; Micropruina). In traditional molecular studies, dominant populations have masked the detection of low-abundance OTUs, genetic diversity, and their individual distribution in soil environments. Pyrosequencing studies, has recently uncovered relatively “rare” (Dini-Andreote and Elsas, 2013) species in soil communities. The distribution of these 65 bacterial groups and their role in rhizosphere in nutrient cycling and beneficial effects on plant growth remain unknown. ACKNOWLEGMENTS This work was supported by the CONICYT Doctoral Scholarships 21120698 and FONDECYT Project no. 1120505. REFERENCES Dini-Andreote, F., and Elsas, J.D. (2013). Plant and Soil DOI 10.1007/s11104-013-1687-z. 108 Session II SII-CO-4 Temporal profile of nif gene expression in Azotobacter vinelandii: effect of nifA mutation. Navarro-Rodríguez, M.*, Poza-Carrión, C., Jiménez-Vicente, E., Rubio, L.M. Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón 28223, Madrid. * [email protected] ABSTRACT Azotobacter vinelandii, a free-living bacterium, is considered a model organism for the study of biological nitrogen fixation. Expression of nitrogen fixation (nif) genes is regulated in response to environmental conditions by the NifA activator and the NifL anti-activator. An A. vinelandii mutant strain, UW356, carrying a chromosomal inframe deletion of nifA was generated and its nif gene expression profile compared to that of the wild-type strain. Quantitative real-time PCR was performed normalizing absolute mRNA values. In addition, quantitative immunoblot analysis of Nif protein accumulation was carried out at different derepression times. Our results show that nifU, nifS and nifV gene expression is not exclusively dependent on nifA. INTRODUCTION Azotobacter vinelandii is one of the model organisms to study the biological process of nitrogen fixation. This complex process carried out by the nitrogenase enzyme(s) plays a key role in the biogeochemical nitrogen cycle. The majority of biological nitrogen fixation is carried out by the molybdenum nitrogenase, although alternative vanadium and iron-only nitrogenases exists. Molybdenum-dependent nitrogen fixation depends on nif genes, which are grouped into operons and expressed from NifA-dependent promoters (Setubal J.C. et al., 2009). NifA transcriptional activating ability is modulated by association/dissociation with the nifL protein that depends on the nitrogen and environmental conditions such as the presence of oxygen or the C/N balance of the cell (Dixon R., 1998). Therefore, when nifA does not interact with nifL, it activates nif gene transcription. MATERIAL AND METHODS A mutant strain of Azotobacter vinelandii that carried a chromosomal in-frame deletion of the nifA gene was generated by replacing it with an spectinomicin resistant cassette, UW356 (ΔnifA::Spc). Cultures of wild-type and UW356 strains were grown in modified Burk media supplemented with ammonium at 30ºC and 200 rpm. Cells were collected by centrifugation and resuspended in media with or without ammonia. Twenty three-ml aliquots were collected at different times after resuspension (0', 10', 30', 60', 120', 180', 240' where time 0' indicates 0 minutes after ammonium removal from the culture medium) and divided into three samples: 20 ml for RNA extraction and RT-qPCR analysis, 1ml for immunoblot assays, 1ml for acetylene reduction assays and 1ml for OD measure. Total RNA was isolated using RiboPureTM-Bacteria Kit (Ambion, life technologies), treated with Turbo DNA-free™ (Ambion, life technologies) and the cDNA synthesis was carried out using High Capacity cDNA Reverse Transcription Kit (Ambion, life technologies). Each reaction used 2 µg of total RNA and an aliquot of 1 µl of a 1:10 dilution (1:10,000 for reference gene 16SrDNA) of the cDNA was used as template for RT-qPCR amplification. Immunoblot analysis (Brandner et al., 1989) has been described. Protein samples were prepared by mixing pelleted cells with Laemmli sample buffer 2X supplemented with DTT and 2-mercaptoethanol to a final OD600 of 109 Session II SII-CO-4 4. In vivo nitrogenase activity was determined by the acetylene reduction assay at 30ºC for 15 minutes as described (Stewart et al., 1967). RESULTS AND DISCUSSION Our results show a clear and fast pulse of nifA expression peaking at 10-30 minutes after ammonium removal and descending rapidly afterwards. This peak is followed by expression of structural and biosynthetic nif genes, which peaks have variable times between 30 min and 120 min and exhibit smoother profiles. In all cases, nif genes within each of the operons followed similar expression patterns. Surprisingly, the nifA mutant showed significant expression on nifU, nifS and nifV genes, indicating that they were not strickly dependent on nifA. t= 4h 0m t= 30 m t= 1h t= 2h t= 0 t= 1 t= 4h t= 3h t= 2h m m t= 1h t= 30 t= 0 t= 10 t= 3h ΔnifA DJ nifH nifD nifK 0 50 0 50 0 5 0 5 0 0.5 0 0.5 nifY nifE nifN nifX nifU nifS nifV nifA nifB fdxN nifQ nafY nifL Figure 1. Different responses of nif gene expression under diazotrophic conditions in the mutant U W356 and. wild-type strains of A. vinelandii. Gene expression was quantified by q-RTPCR. mRNA expressio n data is visualized with the Multi Experimental Viewer program (http:://www.tm4.org/). Color bar sh ows normalized absolute signal. Each data point represents the mean of three biological replicates. REFERENCES Dixon, R. (1998). Arch. Microbiol. 169: 371-380. Brandner, J.P., et al. (1989). J. Bacteriol. 171: 360-368. Stewart W.D.P., et al. (1967). Proc. Natl. Acad. Sci. USA. 58: 2071-2078. Setubal J.C., et al. (2009). J. Bacteriol. 191: 4534-4545. Figure 3 110 Session II SII-CP-01 Singularidades adaptativas del genoma de Pseudomonas fluorescens F113. Comparación con otras cepas del género Pseudomonas. Redondo-Nieto, M.*, Martínez-Granero, F., Muriel, C., Martín, M., Rivilla, R. Departamento de Biología. Facultad de Ciencias. Universidad Autónoma de Madrid. c/ Darwin, 2. 2802 9. Madrid. * [email protected] RESUMEN Pseudomonas fluorescens F113 es una rizobacteria promotora del crecimiento vegetal (PGPR) que presenta actividad protectora ante patógenos fúngicos y además sirve de modelo para la colonización de la rizosfera. La secuenciación completa de su genoma y su posterior comparación con otras Pseudomonas ha puesto de manifiesto una alta conservación del genoma central. A pesar del alto grado de conservación, P. fluorescens F113 muestra un gran número de rutas propias que le conceden una alta capacidad competitiva y adaptativa en la rizosfera. INTRODUCCIÓN Pseudomonas fluorescens F113 es una PGPR aislada de la rizosfera de remolacha que posee un elevado potencial como cepa biocontroladora dada su capacidad para la producción de compuestos antibióticos y antifúngicos. Además, su capacidad adaptativa y competitiva en rizosfera conferida por la plasticidad genómica debida a fenómenos de variación de fase hace de F113 un interesante candidato para aplicaciones biotecnológicas. Se ha completado la secuenciación y el borrador de la anotación de esta cepa y se ha comparado con los genomas de otras Pseudomonas con el fin de establecer por un lado las relaciones filogenómicas y por otro los genes compartidos y genes propios de F113 que le confieren ventajas adaptativas. MATERIAL Y MÉTODOS El genoma de F113 fue secuenciado por las plataformas 454 FLX Titanium (Roche) y Solexa (actualmente Illumina). Para el ensamblaje se recurrió a varios algoritmos, Velvet, MIRA y NewBler. Los contigs obtenidos con cada software fueron comparados mediante Blast y analizados con scripts propios con el fin de fusionarlos. Las regiones que no pudieron ser calculadas se cerraron amplificándolas por PCR y secuenciándolas por Sanger. La anotación se llevo a cabo utilizando el protocolo RAST y Blast2GO. La búsqueda de ortólogos se realizó con el paquete de software OrthoMCL. Para el estudio filogenómico, se descargaron todos los genomas completos y borradores del género Pseudomonas presentes en la base de datos PATRIC y para calcular las distancias se utilizó una aproximación del tipo “Composition Vector” usando el algoritmo CVtree con una ventana de 6 aminoácidos por péptido. Los árboles se construyeron mediante el método de Neighbor-Joining. RESULTADOS Y DISCUSIÓN El genoma de P. fluorescens F113 está compuesto por un cromosoma circular con un tamaño de 6,8 Mpb, un contenido G+C medio del 60,8% y una densidad codificante del 86,7%. La anotación actual muestra que F113 presenta 5862 secuencias codificantes, nueve ncRNAs, cinco operones de rRNA y 66 loci de tRNA (Redondo-Nieto et al., 2012). El genoma de F113 presenta una variedad de genes codificantes que le confieren ventaja en el ambiente rizosférico. Estos incluyen adaptaciones metabólicas inusuales dentro de la especie P. fluorescens, como puede ser la capacidad denitrificadora y 111 Session II SII-CP-01 metabolismo de sustancias terpénicas cíclicas; nuevos sistemas de movilidad como son un segundo aparato flagelar no descrito hasta la fecha en esta especie y sistemas quimiotácticos adicionales; genes codificantes de posibles toxinas para distintos organismos y un vasto número de genes implicados en el ensamblaje de distintos sistemas de secreción de tipo II, III y VI. Las características fenotípicas de F113 en el momento de ser aislada hicieron que fuera incluida en el grupo de las P. fluorescens, pero como ya ha sido indicado por Silby et al. (2009), Loper et al. (2012), y más recientemente por Redondo-Nieto et al. (2013), la clasificación de este grupo requiere un estudio en mayor profundidad. El análisis filogenómico del genoma y/o borradores de 162 cepas del género Pseudomonas disponibles en las bases de datos, han mostrado una distribución prácticamente concordante con la obtenida por métodos filogenéticos como MLSA. El denominado grupo P. fluorescens está compuesto por 50 cepas de las especies P. fluorescens, P. brassicacearum, P. protegens, P. mandelii, P. chlororaphis, P. tolaasii, P. extremaustralis y Pseudomonas spp. Según el árbol filogenómico, este grupo se forma a una profundidad mayor que otros grupos más compactos como P. aeruginosa o P. syringae. Por ello se realizó un análisis más detallado de esas 50 cepas y se concluyó que el grupo P. fluorescens puede clasificarse en 5 subgrupos, estando F113 en el subgrupo I con otras dos cepas de P. fluorescens y dos de P. brassicacearum. El análisis de ortólogos muestra que F113 comparte un 35% con todas las cepas asignadas al grupo de P. fluorescens pero ese valor se eleva al 76% al compararlo con las cepas de su subgrupo. Estos datos junto con que las cepas del subgrupo I comparten habilidades inusuales como la denitrificación, variación de fase durante la colonización de la rizosfera y sistemas propios de quimiotaxis altamente conservados, conducen a la conclusión de que estas cepas corresponden a la misma especie. Adicionalmente F113 presenta 344 genes que no presentan el resto de las cepas del subgrupo I entre los que podemos destacar los dirigidos al ensamblaje de un segundo aparato flagelar y a la producción de un posible antibiótico de tipo poliquétido. Estos grupos de genes podrían explicar la excelente capacidad competitiva de F113 de rizosfera y están actualmente en estudio. AGRADECIMIENTOS Esta investigación ha sido financiada por los proyectos BIO2009 08254, BIO2012 316034, Microambiente CM. BIBLIOGRAFÍA Loper, J.E., et al. (2012). Plos Genet. 8(7):e1002784. Redondo-Nieto, M., et al. (2012). J. Bacteriol. 194: 1273-1274. Redondo-Nieto, M., et al. (2013). BMC Genomics 14: 54. Silby, M., et al. (2009). Genome Biol. 10: R51. 112 Session II SII-CP-02 AmrZ es un regulador transcripcional global en Pseudomonas fluorescens F113. Martínez-Granero, F., Redondo-Nieto, M., Vesga, P., Martín, M., Rivilla, R. * Departamento de Biología. Facultad de Ciencias. Universidad Autónoma de Madrid. * [email protected] RESUMEN Mediante análisis de ChIP-seq se ha determinado que la proteína AmrZ es un regulador transcripcional de múltiples genes implicados en movimiento, producción de exopolisacáridos y homeostasis de hierro, entre otros. Se ha determinado un motivo conservado para la unión de AmrZ al ADN. INTRODUCCIÓN AmrZ (originalmente denominada AlgZ) es una proteína reguladora de la familia ArcC que contiene un dominio de unión a ADN Ribbon-Helix-Helix (RHH). Esta proteína fue descrita originalmente en Pseudomonas aeruginosa como un activador transcripcional que regulaba positivamente la producción del exopolisacárido alginato. A su vez, AmrZ se auto-regula de forma negativa y reprime la expresión de genes implicados en la síntesis de otro exopolisacárido. AmrZ también regula el movimiento a nivel transcripcional mediante el control de la producción de pili tipo IV y del flagelo. AmrZ requiere para su transcripción el factor sigma alternativo AlgU y está conservada en los genomas de todas las pseudomonas secuenciadas hasta la fecha. Pseudomonas fluorescens F113 fue aislada de la rizosfera de remolacha y su capacidad para producir compuestos con actividad antibiótica y antifúngica la convierte en una estirpe de interés en biocontrol. Los usos de F113 en sistemas integrados planta/microorganismo dependen de la eficacia de la colonización de la rizosfera. Uno de los caracteres de interés en la colonización competitiva de la rizosfera es la capacidad de movimiento (Capdevila et al., 2004; Martínez-Granero et al., 2006). En esta cepa, AmrZ regula la movilidad a través de la represión de fleQ, que a su vez codifica el principal regulador de la síntesis del flagelo (Martínez-Granero et al., 2012). Debido al importante papel que AmrZ puede tener en la ecología de las pseudomonas y aprovechando la secuenciación completa del genoma de F113 (Redondo-Nieto et al., 2013), hemos realizado un análisis genómico por inmunoprecipitación de cromatina (ChIP-seq) para determinar los sitios de unión de AmrZ al ADN y analizar la importancia de esta proteína en la adaptación ambiental de las pseudomonas. MATERIAL Y MÉTODOS Se inmunoprecipitó ADN de un derivado de F113 que contenía una fusión traduccional HA-AmrZ, utilizando un anticuerpo anti-HA. Se construyó una librería que se secuenció a través de Illumina. Las lecturas obtenidas se alinearon frente al genoma de F113 usando el programa BowTie 2. Los picos de lecturas acumuladas fueron detectados mediante el programa MACS 1.4. Los picos observados se validaron por PCR cuantitativa de un experimento de ChIP independiente. Se estudió la expresión génica de genes seleccionados mediante PCR cuantitativa en fondos genéticos wt y amrZ-. Se utilizó el programa MEME para determinar los motivos de unión de AmrZ al ADN. 113 Session II SII-CP-02 RESULTADOS Y DISCUSIÓN El análisis del ChIP-seq mostró la presencia de 154 picos que podrían corresponder a sitios de unión de AmrZ. El 76% de estos picos se localizaban en regiones intergénicas indicando que AmrZ se une fundamentalmente a regiones promotoras. Se validaron 20 de estos picos mediante PCR en tiempo real de un experimento independiente de ChIP. Los picos se asignaron a genes implicados en múltiples funciones, destacando genes relacionados con homeostasis de hierro, movilidad, quimiotaxis y producción de polisacáridos. Algunos de los picos se asociaron a genes que codifican proteínas reguladoras y de transducción de señal, incluidas proteínas implicadas en la síntesis y/o degradación del c-di-GMP. El análisis de las secuencias contenidas en los picos, permitió determinar un motivo de unión de AmrZ, que contenía la secuencia identificada previamente por cristalografía en los promotores de algD y amrZ en P. aeruginosa. Se analizó la expresión de 20 de estos genes, pertenecientes a distintas clases funcionales en la estirpe silvestre y en un mutante amrZ, mostrando que en las condiciones estudiadas, AmrZ participaba en la expresión de algunos de ellos. Además, se seleccionaron 12 genes implicados en la homeostasis de hierro y que estaban precedidos por un pico. El análisis de expresión mostró que AmrZ reprimía a todos estos genes en condiciones de deficiencia de hierro. El alto número de sitios de unión de AmrZ al ADN, así como la identidad de los genes regulados por esta proteína, muestran que AmrZ es un importante regulador global de la transcripción en P. fluorescens. Además, al regular a otras proteínas reguladoras y señalizadoras, es de esperar que el regulón de AmrZ sea muy amplio, convirtiendo a AmrZ en uno de los reguladores más importantes para la adaptación de las pseudomonas a condiciones ambientales cambiantes. AGRADECIMIENTOS Esta investigación ha sido financiada por los proyectos BIO2009 08254, BIO2012 316034, Microambiente CM. BIBLIOGRAFÍA Capdevila, S., et al. (2004). Microbiology SGM 150: 3889-3897 Martínez-Granero, F., et al. (2006). Appl. Environ. Microbiol. 72: 3429-3434 Martínez-Granero, F., et al. (2012). PLoS ONE Vol 7: e31765 Redondo-Nieto, M., et al. (2013). BMC Genomics 14:54 114 Session II SII-CP-03 The rkp-2 region of Sinorhizobium fredii HH103 is involved in LPS and EPS production. Acosta-Jurado, S.1*, Crespo-Rivas, J.C.1, Murdoch, P.S.2, Rodríguez-Carvajal, M.A.3, Ruiz-Sainz, J.E.1, Vinardell, J.M.1 1 Departamento de Microbiología and 2 Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012-Sevilla, Spain; 3 Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla. C/ Profesor García González 1, 41012-Sevilla, Spain * [email protected] ABSTRACT Bacterial surface polysaccharides play a crucial role in the rhizobia-legume symbiotic interaction. In this work we characterize the Sinorhizobium fredii HH103 rkp-2 genetic region and demonstrate its involvement in exopolysaccharide (EPS) and lipopolysaccharide (LPS) production. This region is composed of two genes, lpsL and rkpK. Inactivation of any of these genes results in mucus reduction, LPS electrophoretic profile alterations, reduced capacity to form biofilms, and enhanced autoaggregation in liquid cultures. In addition, rkp-2 mutants, in contrast to other S. fredii HH103 mutants affected in surface polysaccharide production, bind Congo red and calcofluor. INTRODUCTION In the rhizobia-legume symbiotic interaction, bacterial surface polysaccharides appear to play a crucial role either acting as signals required for the progression of the interaction and/or preventing host defence mechanisms (Fraysse et al., 2003). We have previously described the rkp-1 and rkp-3 regions, which are required for KPS (Kantigen polysacchar ide) production (Margaret et al., 2012a,b) in S. fredii HH103. Here we present our first studies of the HH103 rkp-2 region, which comprises two genes: lpsL, that encodes a UDP-glucuronate 4-epimerase, and rkpK, that encodes a UDPglucose 6-dehydrogen ase. Here we report that, in contrast to that described for S. meliloti, the HH103 rkp-2 region is not involved in KPS synthesis. However, HH103 rkp-2 mutants are affected in LPS and EPS production. MATERIAL AND METHODS Mutagenesis of the lpsL and rkpK genes, LPS extraction, separation on SDS polyacrylamide gel electrophoresis, silver staining and immunostaining procedures were performed as described by Margaret et al. (2012a,b). To observe EPS production on solid YM medium, rhizobial strains were grown for 120 h at 28°C and then incubated 48 h at room temperature. Congo red adsorption was observed on plates of YM medium supplemented with 25mg/L of this dye. Calcofluor binding was tested on TY plates containing calcofluor 0.02% m/v. Biofilm formation on plastic surfaces was studied by using the method of O'Toole and Kolter (1998), with some modifications. The autoaggregation assays was carried out as described by Sorroche et al. (2012). RESULTS AND DISCUSSION S. fredii HH103 mutant derivatives SVQ701 (lpsL::lacZ-GmR), SVQ703 (rkpK::lacZGmR), and SVQ704 (rk pK::lacZ-GmR) were unable to produce EPS as observed on YMA plates (Figure 1 Panel A) and after precipitation with 3 volumes of cold ethanol. This defect could be due to the fact that glucuronic acid (whose production is dependent on RkpK) is present in the HH103 EPS structure. All the mutants tested were able to 115 Session II SII-CP-03 absorb Congo red and calcofluor (Figure 1, panels B and C). Moreover, HH103 rkp-2 mutants produced a LPS which showed an altered profile in SDS-PAGE experiments (Figure 1, panel D) and was not recognized by the monoclonal antibody NB6-228.22 (It recognizes the wild type LPS) (Figure 1, panel E). All S. fredii HH103 rkp-2 mutants were unable to form biofilms and showed increased autoaggregation ability in MGM liquid medium (Figure 1, panel F and G) when compared with the parental strain HH103. The fact that HH103 does not contain glucuronic acid in its KPS justifies that HH103 rkp-2 mutants were not affected in KPS production (data not shown) in contrast to Sinorhizobium meliloti Rm41 (whose KPS contains glucuronic acid). We have also obtained a mutant in the rkpZ gene, SVQ702 (rkpZ::lacZ-GmR). Strain SVQ702 did not show differences with regard to HH103 in any of the characteristics studied above. A B C SVQ269 SVQ701 SVQ701 SVQ269 SVQ269 SVQ702 SVQ703 SVQ701 SVQ703 SVQ702 bluB SVQ704 SVQ704 D 1 2 SVQ703 SVQ704 E 3 1 2 3 G F Figure 1. (A,B,C) Growth of HH103 RifR (= SVQ269) and different rkp derivatives on YMA (A), and YMA supplemented with calcofluor (B) or Congo red (C). S.meliloti bluB was used as a positive cont rol for calcofluor binding. (D) SDS-PAGE and silver-staining or (E) immuno-staining (monoclonal antibody N B6228.22) of lipopolysaccharides (LPS) crude extracts from HH103 and its rkp-2 derivatives. (F) Bi ofilm and (G) autoaggregation of HH103 and its rkp-2 derivatives. 1. SVQ269, 2. SVQ701, and 3. SVQ703. ACKNOWLEDGEMENTS This work was supported by grants from the Spanish Ministry of Science and Innovation (BIO20113022 9-C02-01) and from the Andalusian Consejería de Innovación, Ciencia y Empresa (Proyecto de Excelencia CVI2506). Authors also thank Modesto Carballo (CITIUS, Biology Service, University of Sevilla) for helping with biofilm measurement experiments. REFERENCES Fraysse, A.L., et al. (2003). Eur. J. Biochem. 270: 1365-1380. Margaret, I., et al. (2012a). Arch. Microbiol 194: 87-102. Margaret, I., et al. (2012b). Mol. Plant-Microbe Interact. 25: 825-838. O'Toole, G.A., and Kolter, R. (1998). Mol Microbiol 28: 449-461. Sorroche, F.G., et al. (2012). Appl Environ Microbiol.78: 4092-101. 116 Session II SII-CP-04 Study of the quorum sensing Sin system of Sinorhizobium fredii HH103. Crespo-Rivas, J.C.1*, Pérez-Montaño, F.1, Acosta-Jurado, S.1, Payán-Bravo, L.1, McIntosh, M.2, Meyer, S.2, Becker, A.2, Ruiz-Sainz, J.E.1, Vinardell, J.M.1 1 Departamento de Microbiología Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012, Sevilla, Spain. 2 LOEWE Center for Synthetic Microbiology (SYNMIKRO) and Department of Biology, Philipps-Universität Marburg, Marburg, Germany. * [email protected] ABSTRACT Quorum sensing (QS) is a genetic regulation system depending on population density. In rhizobia the small autoinducer molecules involved in QS regulation are N-AcylHomoserine-Lactone s (AHLs). Two kinds of AHLs produced by HH103 were detected in chromatoplate assays using the biosensor strain Agrobacterium tumefaciens NT1 (pZRL4). The kinetic productions of both AHLs are different and vary according to the different points of the growing curve. An in silico search in the S. fredii HH103 genomic sequence revealed the presence of two genes putatively responsible for AHL synthesis: sinI and traI. Inactivation of these genes removed the production of AHLs. Chromatoplate assays and expression experiments revealed that the production of these AHLs is repressed by genistein, a nod gene inducer flavonoid. INTRODUCTION QS is a regulatory process by which bacteria perceive their population density (Fuqua et al., 1994). Bacteria synthesize small molecules called autoinducers that are able to move across the membrane. The concentration of these molecules in the extracellular medium increases according to the number of bacteria. When the concentration of these molecules reaches a threshold level, they can activate or suppress the expression of genes involved in a collective behavior. In rhizobia, as in other Gram negative bacteria, the QS system autoinducer molecules known to date are AHLs. Some recognised features of QS responses observed in rhizobia are the strain-specificity and the high diversity of genes involved in this mechanism (Sánchez-Contreras et al., 2011). In this work we have verified that S. fredii HH103 produces at least two different AHLs in a population density-depending manner, and we have initiated the study of the complex genetic regulation by AHLs in this strain. MATERIAL AND METHODS The biosensor strain Agrobaterium tumefaciens NT1 (pZRL4) was used to detect AHLs produced by HH103 in culture supernatants by Thin Layer Chromatography (TLC) assay experiments (Holden et al., 1999). Surface polysaccharide analyses and rt-RTPCR experiments were done according to Crespo-Rivas et al. (2009). Fluorescence measurement experiments were done according to McIntosh et al. (2009). RESULTS AND DISCUSSION In order to find genes involved in AHL synthesis in S. fredii HH103, we analysed its genomic sequence to find orthologues of Vibrio fischeri luxI. Thus, the sinI and traI genes were identified. In order to verify whether these genes are responsible for AHL synthesis, two HH103 mutants were generated: SVQ693 (sinI::lacZ-GmR) and SVQ697 (traI:: SpcR). By using the TLC approach described above, two different AHLs were found to be produced by HH103: a long acyl chain AHL and a short acyl chain AHL. The analysis of mutants SVQ693 and SVQ697 demonstrated that these AHLs are synthesized by SinI and TraI respectively (Figure 1, panels A, B, and C). It was also 117 Session II SII-CP-04 observed that the rate of production of each of these AHLs is not dependent on the presence of the other AHL. The absence of long acyl chain AHL did apparently not affect the production of LPS, EPS, KPS neither its swimming mobility (data not shown), which could be due to the absence of a functional expR gene. We have also studied whether AHL production in HH103 could be affected by the presence of flavonoids, as it has been described for S. fredii SMH12 (Perez-Montaño et al., 2011). TLC assays (Figure 1D) revealed that the production of the two types of AHLs decreased when HH103 was grown in the presence of genistein, although this reduction was clearly more evident for the long acyl chain AHL (dependent on sinI). rtRT-PCR and f luorescence measurement experiments (using a transcriptional fusion between the sinI promoter region and the gfp gene) showed that the presence of genistein led to a reduction of sinI expression (Figure 1 E and F), which is in agreement with the results obtained in TLC assays. E A B C D F Figure 1. (A, B, C, D) TLCs from supernatant culture extracts at different O.D. of Sinorhizobium f redii HH103 (A) and its sinI (B) and traI (C) derivatives; D: HH103 cultured in the presence or absence of genistein. E and F: rt-RT-PCR relative expression data of nodA and sinI genes in HH103 (= 269) in the presence (+) or absence of genistein. St= AHL standards, Gen= genistein. CONCLUSIONS S. fredii HH103 produces at least two kinds of AHLs whose synthases are encoded by sinI and traI. The production of these AHLs is somehow repressed by genistein, which clearly suggests a connection between QS and the nod regulon in S. fredii HH103. ACKNOWLEDGEMENTS This work was supported by grants from the Spanish Ministry of Science and Innovation (BIO20113022 9-C02-01) and from the Andalusian Consejería de Innovación, Ciencia y Empresa (Proyecto de Excelencia CVI2506). Authors also thank Modesto Carballo (CITIUS, Biology Service, University of Sevilla) for helping with fluorescence measurement experiments. REFERENCES Crespo-Rivas, J.C., et al. (2009). Mol. Plant-Microbe Interact. 22: 575-588. Fuqua, W.C., et al., (1994). J. Bact. 176: 269–275. Holden, M.T., et al. (1999). Mol. Microbiol. 33, 1254-1266. McIntosh, M., et al. (2009). Mol. Microbiol. 74: 1238–1256. Pérez-Montaño, F., et al. (2011). Res. Microbiol. 162: 715-23. Sánchez-Contreras, M., et al. (2011). Fundamentos y aplicaciones agroambientales de las interaccio nes beneficiosas plantas-microorganismos. Edita: Sociedad Española de Fijación de Nitrógeno (SEFIN), España pp.227-240 ISBN: 978-84-614-7364-9. 118 Session II SII-CP-05 Lotus japonicus Gifu and L. burttii responses to inoculation with a collection of S. fredii HH103 mutants affected in symbiotic signals. Rodríguez-Navarro, D.N.1 Jin, H.2, Kawaharada, Y.2, Sandal, N.2, Andersen, S.U.2, Stougaard, J.2, Ruiz-Sainz, J.E.3* 1 IFAPA, Centro Las Torres-Tomejil. Apartado Oficial 41200, Alcalá del Río, Sevilla, Spain. 2 Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark. 3 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes 6, C.P. 41012. Sevilla, Spain. * [email protected] ABSTRACT A collection of S. fredii HH103 mutants affected in genes that are known to be symbiotically relevant has been tested with the model legume Lotus japonicus Gifu and also with L. burttii. S. fredii HH103 and most of its mutant derivatives analysed did not form nitrogen fixing (Fix+) nodules with L. japonicus. However, S. fredii HH103 nodD2 or nolR mutants showed improved nodulation capacity with L. japonicus. Most of the S. fredii HH103 mutants were still able to form Fix+ nodules with L. burttii. Mutations affecting S. fredii HH103 surface polysaccharide (SP) production [exopolysaccharides (EPS), lipopolysaccharides (LPS), capsular polysaccharides (KPS) or cyclic glucans (CG)] did not prevent the formation of Fix+ nodules with L. burttii. Fix+ nodules formed by inoculation of L. burttii with S. fredii HH103 appear to be the result of crack entry. These results indicate that when S. f. HH103 invades L. burttii roots through cracks, the bacterial SP are not strictly required for successful nodulation. INTRODUCTION Sinorhizobium fredii HH103 is a fast-growing strain able to nodulate American and Asiatic soybean (Glycine max) cultivars as well as many other legumes. The S. fredii HH103 genome has been sequenced (Margaret et al., 2011; Weidner et al., 2012). For details about the HH103 genome see the abstract/poster Vinardell et al. S. fredii HH103 effectively nodulates with L. burttii but it only induces the formation of ineffective pseudonodules on L. japonicus roots (Sandal et al. 2012). L. japonicus is being used as a model in genetic studies of determinate nodule forming legumes. L. burttii is becoming attractive for genetic studies because it represents a valid alternative to L. japonicus MG-20 as a L. japonicus Gifu mapping partner. In fact, we have demonstrated the utility of the L. japonicus Gifu x L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for S. fredii nodulation (Sandal et al. 2012). Numerous S. fredii HH103 mutants have been tested for their symbiotic capacity with soybean. We have now investigated the symbiotic capacity of these mutants with Lotus burttii and L. japonicus. Plant tests on L. burttii should reveal which mutations impair the symbiosis of S. fredii HH103 with L. burttii. Plant tests on L. japonicus might lead to the identification of HH103 genes that block nodulation on this plant. The infection of L. japonicus by Mesorhizobium loti is through the formation of infection threads but in the background of particular symbiotic mutants, examples of crack entry were also found (Madsen et al., 2010). Here we are investigating the mode of infection in the symbiosis between L. burttii and S. fredii HH103. 119 Session II SII-CP-05 MATERIAL AND METHODS Surface sterilization of L. japonicus Gifu and L. burttii seeds and nodulation tests in square plastic boxes were carried our as described by Sandal et al. (2012). In other cases, miniaturized Leonard jars were used to grow the plants. RESULTS AND DISCUSSION Mutants affected in genes that are transcriptionally activated by flavonoids. 1. Mutants affected in genes that are transcriptionally activated by flavonoids. Mutated in nod genes (nodA and noeI) or in genes involved in the secretion of Nops (nodulation outer proteins) through the Type Three Secretion System (T3SS). Only the nodA mutant, unable to produce Nod factors, failed to nodulate with L. burttii. None of the mutants gained the capacity to form Fix+ nodules with L. japonicus. 2. Mutants affected in surface polysaccharide production. None of the HH103 mutants affected in EPS biosynthesis, in KPS production, in LPS biosynthesis, or in CG production (cgs, also called ndvB) totally lost the capacity to induce pink (Fix+) nodules on L. burttii roots. To our knowledge, this is the first report in which a rhizobial mutant unable to produce CG is still capable of inducing pink nodules on any of its host legumes. Three pink nodules were also found in L. japonicus plants inoculated with an HH103 lpsB mutant. 3. Mutants affected in regulatory genes. Mutated in transcriptional regulators of nodulation genes (nodD2 and nolR), in a regulator of the T3SS (ttsI), or in a transcription elongation factor (greA). All these mutants formed pink nodules with L. burttii. L. japonicus roots inoculated with mutants in nodD2 or nolR formed pseudonodules and pink nodules, indicating that these mutations enhance the symbiotic capacity of S. fredii HH103 with L. japonicus. The symbiosis of L. burttii with S. fredii HH103 is established through crack entry. The symbiosis between L. burttii and Mesorhizobium loti is established through infection threads. In contrast to this microscopic analysis of L. burttii plants inoculated with S. fredii HH103 DsRED, including microtome and vibratome sections, revealed microcolonies at the root hair tips but infection threads formed very rarely and they did not reach any further than the middle of the root hair. Infection threads were not detected in the nitrogen fixing nodules. Therefore the infection most likely is formed through crack entry. The rate of crack entry infections is clearly higher in symbiosis between S. fredii HH103 and L. burttii than in L. burttii and L. japonicus symbioses with M. loti where it is only noticed as a rare event in particular plant mutant backgrounds. The analysis of S. fredii HH103 mutants on L. burttii can therefore show which bacterial genes are needed for crack entry. For instance, it seems that cyclic glucans are not needed for crack entry in L. burttii but Nod factors are needed. ACNOWLEDGEMENTS This work was supported by grants from the Andalusia Government P07-CVI-02506 and P11-CVI-7500. REFERENCES Madsen, L.H., et al. (2010). Nat. Commun. 1: 1-12. Margaret, I., et al. (2011). J. Biotechnol. 155: 11-19. Sandal, N., et al. (2012). DNA Res. 19: 317-323. Weidner, S., et al. (2012). J. Bacteriol. 194: 1617-1618. 120 Session II SII-CP-06 Analyses of the Sinorhizobium fredii HH103 and USDA257 secretomes in the presence and absence of the flavonoid genistein. Yao, W.1, Thomas, J.R.1, Jiménez-Guerrero, I.2, López-Baena, F.J.2*, Ruiz-Sainz, J.E.2, Vinardell, J.M.2 1 Department of Biology, University of York. Y010 5DD, York, United Kingdom. 2 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012 - Sevi lla, España. * [email protected] ABSTRACT The genome of the fast-growing soybean-rhizobia S. fredii HH103 has recently been sequenced. This bacterium possesses many protein secretion systems to interact with the environment and also with its eukaryotic counterparts. The identification of proteins secreted by HH103 could provide valuable information for identifying particular genes that could play relevant roles in the symbiosis. INTRODUCTION Plant rhizosphere bacteria must overcome different environmental and biotic stresses to establish a successful relationship with their host plants. These bacteria use different protein secretion systems (Type 1 to Type 7) to deliver effectors and toxins that modify the host physiology to promote colonization. S. fredii HH103 is a broad host-range bacterium that possesses twelve putative protein secretion systems (Margaret et al., 2011; Weidner et al., 2012). Interestingly, cultivar specificity, which is the impairment to nodulate agronomically improved American soybean varieties shown by some S. fredii strains (such as S. fredii USDA257), is mediated by proteins secreted through the Type 3 secretion system (Yang et al., 2010). In this work, we have identified the extracellular proteins (the secretome) that are present in S. fredii HH103 and USDA257 liquid cultures grown with and without genistein, a flavonoid that is present in the soybean rhizosfere and activates the transcription of rhizobial nodulation genes. MATERIAL AND METHODS Protein samples were briefly subjected to SDS-PAGE such that proteins entered only 1 cm into the gel. In-gel tryptic digestion was followed by LC-MS/MS using a Waters nanoAcquity coupled to a Bruker maXis. Tandem mass spectral data were matched to proteins in the NCBInr database using Mascot (Matrix Science). Mascot-calculated emPAI values and direct comparison of Mascot ions scores were used to asses protein relative abundance in different samples. The S. fredii HH103 genome sequence was analyzed by using the GenDB software (CeBiTec, University of Bielefeld, Germany). RESULTS AND DISCUSSION The analyses of the S. fredii HH103 and USDA257 secretomes have shown that these bacteria secrete a high number of proteins to the extracellular milieu. Thus, whereas 119 proteins were detected in USDA257 uninduced cultures, 126 were found in the presence of genistein. In the case of HH103 cultures, 37 proteins were identified in the absence of genistein and 119 in the presence of the flavonoid. The analyses of these data showed some differences between S. fredii strains USDA257 and HH103. For instance, five effector proteins (NopA,B,L,P,X) secreted through the USDA257 T3SS were detected in t he presence of genist ein. In contrast, seven HH103 putat ive effectors (NopA,B,L,P,X plus NopC and NopM) were identified. These results are in agreement 121 Session II SII-CP-06 with previous reports suggesting that S. fredii USDA257 might not be particularly efficient in Nop secretion (Krishnan et al., 2011). The secretome analyses of strains HH103 and USDA257 showed other interesting differences. For instance, 8 putative components of the flagellum were identified in the secretome of USDA257 but only three of them were detected in HH103 cultures. This difference in the number of putative flagella components identified in USDA257 and HH103 cultures occurs in the presence and absence of genistein. S. fredii strains USDA257 and USDA191 also differ in the number of putative flagella proteins (Krishnan et al., 2011). Five proteins that might play symbiotic roles were chosen for further work (Table 1). Table 1. Proteins of the S. fredii HH103 and USDA257 secretomes that were chosen for further analy ses. Predicted functions of some HH103 and USDA257 orthologues. Putative metal binding protein Phosphoserine aminotransferase Peptidyl-prolyl cistrans isomerase, cyclophilin type Peptidyl-prolyl-cistrans isomerise, cyclophilin type Peptidase M29, aminopeptidase II USDA257 genes Presence of protein in Gen Gen + HH103 genes Presence of protein Gen - Gen + c53490 + 02943 + serC_c51910 + serC_02741 + ppi2_c40150 + ppiA_01477 + ppi1_c40140 D ppiB_01476 + c61510 03876 + Gen-, absence of genistein; Gen+, presence of genistein; USDA257 and HH103 genes occupying the sal e line are orthologues. Cyclophilins proteins belong to a group of proteins that have peptidyl-prolyl cis-trans isomerase activity and are found in all prokaryotes and eukaryotes investigated. Recent studies have implicated cyclophilins in a diverse array of cellular functions, including roles as chaperones and in cell signaling. Genes coding for cyclophilins will be tagged with the commercial oligopeptide HA to confirm extracellular secretion and induction with genistein by western blot analyses. In addition, a mutant in the ppiB gene will be constructed to study the role of these cyclophilins in the symbiosis with soybean. The knowledge of the bacterial capacity to use secretion systems to be adapted to environmental conditions, such as the plant lifestyle, is growing fast. However, the role of secretion systems in beneficial plant-bacteria interactions is poorly understood. ACKNOWLEDGEMENTS This work has been supported by project BIO2011-30229-C02-01. Irene Jiménez-Guerrero work was financed with a predoctoral contract from the program IV Plan Propio of the University of Seville. REFERENCES Krishnan, H.B., et al. (2011). Appl. Environ. Microbiol. 77: 6240-6248. Margaret, I., et al. (2011). J. Biotechnol. 155: 11-19. Weidner, S., et al. (2012). J. Bacteriol. 194: 1617-1618 Yang, S., et al. (2010). Proc. Natl. Acad. Sci. USA 107: 18735-18740. 122 Session II SII-CP-07 Ocorrência e caracterização de bactérias isoladas de nódulos de amendoinzeiro (Arachis hypogaea L.) em solos paranaenses, Brasil. Andrade, D.S.1*, Cardoso, J.D.1, Pertinhez, G.N.1, Saturno, D.F., Lovato, G.M. 1, Nomura, R.B.G.1, Hungria, M.2 1 * Instituto Agronômico do Paraná, Londrina, PR, Brasil. 2 Embrapa Soja, Londrina, PR, Brasil. [email protected] RESUMO O objetivo deste trabalho foi avaliar a ocorrência e caracterizar morfofisiologicamente e geneticamente bactérias isoladas de nódulos de amendoinzeiro (Arachis hypogaea L.), em solos paranaenses, Brasil. Em amostras de 36 municípios representativos do estado Paraná foi observada nodulação do amendoim em 84,4%, independente se em áreas cultivadas, ou em florestas. Nos testes de caracterização morfofisiológica de 44 estirpes autenticadas, 75% das estirpes apresentaram taxa de crescimento rápido em meio de cultura com manitol; 25 acidificaram o meio, cinco alcalinizaram e 14 não resultaram em modificação do pH. A capacidade de produzir sideróforos foi observada em 43% das estirpes e 16% foram capazes de solubilizar fosfato de cálcio em meio de cultura. O sequenciamento do gene 16S rRNA mostrou a formação de três principais grupos, correspondentes a três filos: Alfaproteobacteria, Betaproteobacteria e Firmicutes. O gênero com maior ocorrência foi o Bacillus. INTRODUÇÃO O amendoinzeiro (Arachis hypogaea L.) é uma leguminosa oleaginosa originárioa da América do Sul, ocorrendo em regiões tropicais e subtropicais. Na maioria dos sistemas de cultivo o amendoinzeiro consegue realizar a fixação biológica de nitrogênio em quantidades adequadas para suprir suas necessidades (Thies et al., 1991). No Paraná, não se recomenda a aplicação de fertilizantes nitrogenados para a cultura. A avaliação da ocorrência de rizóbios em áreas cultivadas e sob floresta, bem como a caracterização destas estirpes pode fornecer informações sobre a filogenia de microrganismos simbióticos nativos simbiontes do amendoinzeiro. Sendo assim, o objetivo deste trabalho foi avaliar a ocorrência de rizóbios nodulantes do amendoinzeiro e analisar as características morfofisiológicas e genéticas destas bactérias simbiônticas. MATERIAL E MÉTODOS A ocorrência de rizóbio simbionte do amendoinzeiro (Arachis hypogaea L.) foi avaliada pela presença de nódulos em plantas semeadas em amostras de solos coletadas de 36 municípios do Paraná, em áreas sob cultivos de milho (Zea mays L.), pastagens e solos de mata, do Estado do Paraná, na camada 0–10 cm. Para o estudo da caracterização mofofisiológica e genética, também foram obtidas estirpes bacterianas de nódulos de plantas de amendoinzeiro de um experimento de campo, onde foram coletados nódulos de maneira aleatória e realizado o isolamento. Os isolados obtidos foram autenticados duas vezes para certificar a capacidade simbiótica no amendoinzeiro. As estirpes foram autenticadas em vasos Leonard com areia e vermiculita contendo solução nutritiva sem N mineral e a cultivar de amendoim IAC-Tatu ST. As estirpes foram avaliadas quanto à produção de sideróforos e à capacidade de solubilização de fosfato de cálcio. O DNA genômico total foi submetido à amplificação para análise do BOX-A1R e procedeu-se ao sequenciamento do gene 16S rRNA. A estirpe de Bradyrhizobium sp, SEMIA 6144 (Coleção de Cultura do laboratório da FEPAGRO, RS), autorizada para a produção de inoculante para o amendoinzeiro no Brasil, foi incluída como controle. 123 Session II SII-CP-07 RESULTADOS E DISCUSSÃO De um total de mais de 100 isolados, apenas 44 foram comprovados quanto à formação de nódulos efetivos, com coloração interna avermelhada e foram codificadas (IPR-Ah) e depositadas na Coleção de microrganismos do Instituto Agronômico do Paraná (IAPAR). A capacidade de produzir sideróforos foi observada em 43% das 44 estirpes. A capacidade de solubilizar fosfato de cálcio em meio de cultura foi observada em 16% das estirpes. A estirpe recomendada SEMIA 6144 não apresentou capacidade para produzir sideróforos nem para solubilizar fosfato de cálcio. O sequenciamento do gene 16S rRNA das 41 estirpes mostrou a presença de oito gêneros, sendo Bacillus com 63% de ocorrência (Figura 1). 2 2 2 2 2 2 Bacillus 2 2 Acinetobacter Paenibacillus Pectobacterium 7 2 63 10 Cohnella 2 Mesorhizobium 2 Stenotrophomonas Rhizobium Lysinibacillus Figura 1. Porcentagem de ocorrência dos gêneros de bactérias do total de 41 estirpes (IPR Ah) identificadas pelo sequenciamento do gene 16S rRNA. Bactérias de crescimento rápido nodulando leguminosas tropicais têm sido relatadas, o que pode ser atribuído à dupla ocupação nos nódulos, sendo sugerido o uso de antibióticos como uma estratégia para isolar apenas as de crescimento lento (Trinick, 1982). Neste estudo, todas as estirpes foram autenticadas duas vezes, então, nossa hipótese é que ocorreu simbiose entre duas bactérias no isolamento, o que significa a formação de uma colônia a partir de duas células com crescimento rápido e lento. Estes resultados mostram a necessidade de buscar estratégias para aumentar a eficiência da inoculação, evitando a competição desta alta população nativa. REFERÊNCIAS Thies, J.E., et al. (1991). Appl. Environ. Microbiol. 57:1540-1545. Trinick, M.J. (1982). In: Vincent, J.M. (Ed.), Nitrogen Fixation in Legume. Academic Press Austral ia, Sydney, pp. 229-238. 124 Session II SII-CP-08 The Type 3 secretion system effector NopP from Ensifer (=Sinorhizobium) fredii HH103 is phosphorylated by a soybean kinase. Calero, B.1*, Jiménez-Guerrero, I.1, Pérez-Montaño, F.1, Monreal, J.A.2, Ollero, F.J.1, López-Baena, F.J.1 1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012-Sevilla, Spain. 2 Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012-Sevilla, Spain. * [email protected] ABSTRACT NopP from E. fredii NGR234 is a Type 3 secreted effetor that is phosphorylated by plant kinases (Skorpil et al., 2005). E. fredii HH103 also secretes NopP and its presence is detrimental for nodulation with soybeans (López-Baena et al., 2009). However, the function of this protein in the symbiotic interaction with soybean is still unknown. The nopP gene was cloned in plasmid pGEX for in vitro expression and purification of NopP. The purified protein was used for in vitro phosphorylation and in-gel kinase assays. Results showed that the E. fredii HH103 NopP is phosphorylated by a soybean kinase that seems to be calcium dependent. INTRODUCTION Some rhizobial strains possess a specialized apparatus for protein secretion called Type 3 secretion system (T3SS), which is also present in pathogenic bacteria. This apparatus is used to deliver effectors directly into the cytoplasm of the host cells. Nodulation outer protein P (NopP) is a Rhizobium-specific type 3 effector. Phosphorylation of NopP in E. fredii NGR234 by kinases of legume roots is blocked by general inhibitors of tyrosine and serine/threonine kinases. However, this phosphorylation is not MAP kinase dependent as described for NopL (Skorpil et al., 2005). Inactivation of nopP led to an increase in the symbiotic capacity of E. fredii HH103 to nodulate Williams soybean (López-Baena et al., 2009). Results shown in this work indicate that the HH103 NopP protein was phosphorylated by a kinase obtained from soybean root extracts. This kinase is currently being characterized to try to elucidate its role in the symbiosis. MATERIAL AND METHODS Strains E. fredii HH103 RifR, E. fredii HH103 RifR nopP, and E. coli BL21 with the nopP gene cloned in plasmid pGEX were used in this study. Production of the recombinant NopP protein and in vitro phosphorylation assays were performed as described by Monreal et al. (2013). In-gel kinase assay was performed as described by Monks et al. (2001) with some modifications. RESULTS AND DISCUSSION In vitro phosphorylation assay showed that NopP was phosphorylated by a soybean root kinase (Figure 1). This kinase was calcium dependent. In-gel kinase assays were performed to identify the kinase responsible for this phosphorylation and two different bands corresponding to two possible kinases were detected. 125 Session II SII-CP-08 Figure 1. In vitro phosphorylation assay. Soybean root extracts obtained from uninoculated plants or inoculated with E. fredii HH103 RifR were mixed with the recombinant protein NopP. Lane 1, Control with recombinant NopP and GST; lane 2, molecular weight marker; lane 3, recombinant NopP with soybean root extract from plants inoculated with E. fredii HH103 RifR; lane 4, control with soybea n root extract from plants inoculated with E. fredii HH103 RifR; lane 5, recombinant NopP with uninoculat ed soybean root extract; lane 6, Control with uninoculated soybean root extract. The analysis of the genome of E. fredii HH103 confirmed the presence of another possible effector, similar to NopP, which was named NopP2. Regulation of the expression of this gene depended on NodD, TtsI and flavonoids, as confirmed by qPCR. To determine the role of all the Rhizobium-specific phosphorylated effectors in the symbiosis with soybean and elucidate whether their functions could be redundant, the double mutants nopL LacZ-GmRnopP, LacZ-GmnopP ΔnopP2, and nopL ΔnopP2, and a triple mutant nopL LacZ-GmRnopP ΔnopP2 were constructed. These mutant derivatives will be tested in nodulation assays in soybean to study their effect in symbiosis. ACKNOWLEDGEMENTS This work was financed by projects AGL2009-13487-C04 from the Spanish MEC and P11-CVI-7050 from the Junta de Andalucía. REFERENCES López-Baena, F.J., et al. (2009). Mol. Plant Microbe Interact. 22: 1445-1454. Monks, D.E., et al. (2001). Plant Cell. 13: 1205-1219. Monreal, J.A., et al. (2013). Planta. In press. Skorpil, P., et al. (2005). Mol. Microbiol. 57: 1304-1317. 126 Session II SII-CP-09 A yeast-based array to study the function of Ensifer (=Sinorhizobium) fredii HH103 Type 3 secretion system effectors in symbiosis. Ollero, F.J.1*, Jiménez-Guerrero, I.1, Pérez-Montaño, F.1, Mesa, B.2, Medina, C.2, López-Baena, F.J.1 1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. de Reina Mercedes, 6. 41012 - Sevilla, España. 2 Área de Microbiología. Centro Andaluz de Biología del Desarrollo. Universidad Pablo de Olavide/CSIC. Ctra. Utrera, Km 1. 41013-Sevilla, España. * [email protected] ABSTRACT Little is known about the functions of rhizobial Type 3 secretion system effectors in the symbiosis with their host legumes. Expression of bacterial effectors in yeast has been extensively used to study effector functions within the host cell. A yeast-based array was used to identify potential function of rhizobial effectors in the context of plantrhizobia int eractions. INTRODUCTION Many pathogenic Gram-negative bacteria encode a type 3 secretion system (T3SS), a molecular syringe evolved to deliver proteins into the host cell during infection. These virulence proteins, called type 3 effectors (T3Es), modulate several host cellular processes to promote disease. A current challenge is the determination of their virulence functions and host targets. The yeast Saccharomyces cerevisiae is currently being used as a tool to investigate bacterial T3Es due to the observation that these proteins often target cellular processes that are conserved among all eukaryotes. Kramer et al. (2007) described an approach to study bacterial effectors in yeast, which uses yeast synthetic lethal (SL) interaction data. SL is defined as the situation in which two genes that are non-essential when individually mutated cause lethality when they are combined as a double mutant. Their analysis is based on the assumption that phenotypes resulting from the activity of the T3E would resemble phenotypes of a mutation in the target gene of the effector. Accordingly, genes are defined as congruent to an effector if their sets of SL interactions overlapped with the deletion strains hypersensitive to that effector and therefore represent putative cellular targets. The major disadvantage of this approach is that it requires the screening of all 4,750 deletion strains, which limits its wide application to laboratories. Bosis et al. (2011) have developed a simple method based on the finding that it is possible to cover the majority of the interacting genes (i.e. genes having at least one known SL interaction) with 90 deletion strains. An array of yeast deletion strains fitted into a single 96-well plate covers 69% of the interacting genes with less than 2% of the deletion strains in the yeast collection. The small number of deletion strains in the array simplifies the analysis, reduces costs and facilitates the screening of a large number of bacterial T3Es in a short period of time. MATERIAL AND METHODS The E. fredii HH103 effector genes nopP, nopP2, nopL, nopM and nopC were cloned into the pGML10 vector of expression in yeast and individually transferred to the 90 deletion strains of the array. Yeast strains carrying the empty pGML10 vector were used as controls. The method described by Bosis et al. (2011) was used to identify SL interactions with some modifications. Briefly, transformed yeast strains were grown in liquid cultures in 96-well plates in inducing medium with galactose and in growing 127 Session II SII-CP-09 medium with glucose. The plate with glucose was used as a control to normalize the number of cells, and hence growth, in the inducing medium. Significant differences in the OD600nm 48h after inoculation indicated possible target genes. RESULTS AND DISCUSSION There were no differences in growth of the yeast strains expressing nopP2 and nopC. It could be possible that NopC was not a real effector but a component of the secretion machinery, as suggested by some authors (personal communication). Therefore, no metabolic route in the eukaryotic host would be affected. In the case of nopP2, despite this gene seems to be an effector because its transcription is regulated as other effectors such as NopP, this protein could not have a clear effect in the metabolism of the host or the target gene would not be included in the array. With respect to NopM, NopP, and NopL, some differences in yeast growth could be observed and possible target genes could be assigned. The effector NopM, previously described as an ubiquitin ligase (Xin et al., 2012), was used as a control to know if this method could be used with rhizobial effectors. Results obtained (Table 1) showed that the E. fredii HH103 NopM effector could be a protein involved in ubiquitination of proteins. This was in agreement with previous published results in other rhizobial strains and in animal and plant pathogens. Table 1. FuncAssociate 2.0 results assigning GO attributes for the E. fredii HH103 NopM effector. Rank N 1 3 2 3 X 83 104 LOD 1.779 1.676 p-value 0.00007419 0.0001456 GO attribute Protein ubiquitination Protein modification conjugation by small protein N = number of congruent genes that have the GO attribute. X = total number of interacting genes covered by our array that have the GO attribute. LOD = Logarithm (base 10) of the odds ratio. ACKNOWLEDGEMENTS This work was financed by projects AGL2009-13487-C04 from the Spanish MEC and P11-CVI-7050 from the Junta de Andalucía. We would like to thank Dr. Guido Sessa for providing the yeast array. Irene Jiménez-Guerrero work was supported by a predoctoral fellowship of the University of Seville. REFERENCES Bosis, E., et al. (2011). PloS One 11: e27698 Kramer, R.W., et al. (2007). PLoS Pathog. 3: e21. Xin, D.W., et al. (2012). PLoS Pathog. 5: e1002707. 128 Session II SII-CP-10 Implicación del gen bgvA en la formación de biofilm por Rhizobium tropici CIAT899. Del Cerro, P.1*, Ollero, F.J., Megías, M.2, Bellogín, R.A.1, Guasch-Vidal, B.1, PérezMontaño, F.1, Espuny, M.R.1 1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes 6, 41012-Sevilla. España. 2 Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla. Profesor García González, s/n. Sevilla. * [email protected] RESUMEN Rhizobium tropici CIAT899, bacteria altamente tolerante a diversos tipos de estrés, sintetiza factores de nodulación cuando es crecida a concentraciones de 300 mM de NaCl en ausencia del flavonoide inductor apigenina. El gen bgvA de esta bacteria, que parece estar implicado en esta actividad, se encuentra en un operón de síntesis y secreción de polisacáridos y hasta la fecha sólo se ha detectado en el genoma de CIAT899 y como una proteína hipotética en el de Agrobacterium radiobacter K84. Nuestros resultados muestran que este gen parece estar implicado en el desarrollo de biofilm de CIAT899 puesto que dos mutantes por inserción en el gen bgvA de esta bacteria, uno con Tn5-Mob y otro con el casete , desarrollan menor biofilm bajo condiciones normales y especialmente cuando crece bajo estrés salino. INTRODUCCIÓN Rhizobium tropici CIAT899 es un simbionte de judía catalogado como altamente tolerante a diversos tipos de estrés abiótico (Hungría et al.., 2003). De hecho, el análisis de su genoma ha revelado una gran variedad de rasgos que hacen que esta bacteria sea una gran colonizadora de la rizosfera (Ormeño-Orrillo et al.., 2012). Además, se ha visto que concentraciones de 300 mM de NaCl inducen la expresión de los genes nod en ausencia de flavonoides (B. Guasch-Vidal and J. Estévez et al., 2013). Se ha seleccionado y caracterizado, por inserción de un trasposón Tn5-mob, un mutante de CIAT899 denominado C9, que está afectado en la producción de factores Nod en condiciones de estrés salino (NaCl 300 mM) y sin apigenina. El gen mutado, que se registró como bgvA (GenBank HM768892) no ha sido detectado hasta la fecha en ninguna otra bacteria, con excepción de Agrobacterium radiobacter K84, donde se desconoce su función (Tesis doctoral de Guasch-Vidal, B., 2011). Este gen forma parte de un operón que se caracteriza por contener mayoritariamente genes implicados en la biosíntesis y transporte de polisacáridos superficiales bacterianos. En los rizobios, la matriz del biofilm bacteriano está compuesta principalmente por exopolisacáridos, factores de nodulación y agua (Rinaudi and Giordano, 2010). Con estos antecedentes, nos propusimos determinar la implicación de este gen en el desarrollo de biofilm, e intentar relacionarlo con posibles defectos en la producción de EPS y movilidad bacteriana. MATERIAL Y MÉTODOS Las estirpes empleadas se detallan en la Tabla 1. El medio de cultivo en todos los ensayos fue B-, al que se añadió cuando fue necesario apigenina (1 µg/ml), o NaCl (300mM) y los antibióticos adecuados. Los ensayos de biofilm se realizaron en placas de microtítulo de poliestireno, según el método de Mueller and Gonzalez (2011) y empleando los mismos medios. 129 Session II SII-CP-10 Tabla 1. Estirpes utilizadas. Estirpes CIAT899 SVQ689 SVQ689 (pMUS1066) C9 C9 (pMUS1066) Características relevantes Rhizobium tropici. Estirpe silvestre. Produce factores Nod con NaCl Mutante de CIAT899 en el gen bgvA por inserción de . Produce factores Nod con NaCl SVQ689 complementado con el gen bgvA silvestre en el plásmido pBBR1MCS-5. Mutante de CIAT899 por inserción de Tn5-Mob del gen bgvA. No produce factores Nod con NaCl Mutante C9 complementado con el gen bgvA silvestre en el plásmido pBBR1MCS-5. RESULTADOS Y DISCUSIÓN Los resultados obtenidos demuestran que existen diferencias en la formación de biofilm entre la estirpe silvestre y los mutantes SVQ689 y C9, de tal forma que los mutantes desarrollan menor biofilm en todas las condiciones (B-, B- suplementado con NaCl 300 mM y B- suplementado con apigenina). En el mutante C9 en condiciones salinas se produce un descenso aún mayor en la formación de biofilm, quizá debido a que los factores Nod forman parte de la matriz del biofilm. En el futuro se realizarán estudios de cuantificación de la producción de polisacáridos y movilidad bacteriana que pueden relacionarse con estos resultados. AGRADECIMIENTOS Este trabajo ha sido financiado por los proyectos AGL2009-13487-C04-03/AGR y AGL2012-38831del Ministerio de Economía y Competitividad de España. BIBLIOGRAFÍA Guasch-Vidal, B. (2011). Tesis doctoral. Departamento de Microbiología. Universidad de Sevilla. Guasch-Vidal, B., et al. (2013). Mol Plant Microbe Interact. 26: 451-460. Hungría, M., et al. (2003). Biol. Fertil. Soils. 39: 88-93. Lloret, L., and Martínez-Romero, E. (2005). Rev. Lat. Microbiol. 47: 43-60. Mueller K., and González, E. (2011). J. Bacteriol. 193: 485-496. Ormeño-Orrillo, E., et al. (2012). BMC Genomics 13: 735. Rinaudi L.V., and Giordano W. 2010. FEMS. Microbiol. Lett. 304: 1-11. 130 Session II SII-CP-11 Identification and characterization and of a soybean kinase that phosphorylates the Type 3 secretion system effector NopL from Ensifer (=Sinorhizobium) fredii HH103. Jiménez-Guerrero, I.1*, Pérez-Montaño, F.1, Ollero, F.J.1, Monreal, J.A.2, Cubo, M.T.1, López-Baena, F.J.1 1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012 - Sevilla, Spain. 2 Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012 - Sevilla, Spain. * [email protected] ABSTRACT Little is known about the exact role of the nodulation outer proteins (Nops) secreted by the Type 3 secretion system (T3SS) of E. fredii HH103 in the symbiosis with soybeans. One of these proteins, NopL, has been described as a phosphorylated effector that could be involved in the modulation of plant defence responses by interfering with signal transduction cascades associated to MAP kinases. This work is the first approach to identify possible soybean targets for the E. fredii HH103 NopL protein. A nopL mutant was constructed to study the symbiotic phenotype in soybean and Vigna unguiculata. The absence of NopL significantly affected symbiosis with both legumes. The nopL gene was also cloned in plasmid pGEX for in vitro expression and purification of NopL. The purified protein was used for in vitro phosphorylation and in-gel kinase assays. Results showed that the E. fredii HH103 NopL is phosphorylated by a soybean kinase that is calcium dependent. INTRODUCTION Some Gram-negative bacteria possess a specialized apparatus for protein secretion named T3SS. Symbiotic and pathogenic bacteria use the T3SS to translocate effectors, which are involved in the modulation of host defense responses, directly into the host cytoplasm. Nodulation outer protein L is a Rhizobium-specific Type 3 effector. NopL from E. fredii NGR234 is phosphorylated by plant kinases and seems to modulate host defense responses (Bartsev et al., 2004). Previous results suggest that NopL of NGR234 mimics a MAPK substrate and suppresses premature nodule senescense by impairing MAPK signaling in host cells (Zhang et al., 2011). However, the role of E. fredii HH103 NopL in the symbiotic interaction with soybean is currently unknown. MATERIAL AND METHODS Strains E. fredii HH103 RifR, E. fredii HH103 RifR nopL mutant, and E. coli BL21 with the nopL gene cloned in plasmid pGEX were used in this study. Nodulation assays on Glycine max (L.) and Vigna unguiculata (L.) were performed as described by de Lyra et al. (2006). Production of recombinant NopL protein and in vitro phosphorilation assays were performed as described by Monreal et al. (2013). In-gel kinase assay was performed as described by Monks et al. (2001) with some modifications. RESULTS AND DISCUSSION The symbiotic phenotype of the mutant strain HH103 nopL in Williams soybean showed significant differences when compared to the parental strain. Thus, nodules fresh mass and plant-top dry mass from plants inoculated with the mutant strain were higher than in plants inoculated with the parental strain. By contrast, the symbiotic 131 Session II SII-CP-11 phenotype of the mutant strain in Vigna unguiculata showed opposite results. In this plant the number of nodules and plant-top dry mass of plants inoculated with HH103 RifR nopL were lower than in plants inoculated with HH103 RifR. Therefore, NopL seems to play an important role in the symbiosis with soybean and V. unguiculata. In vitro phosphorylation assays showed that NopL was phosphorilated by a kinase present in soybean root extracts. Different kinase inhibitors are currently being used to try to characterize this plant kinase. In this sense, preliminary studies showed that NopL was phosphorylated by a calcium dependent not inducible kinase (Figure 1). On the other hand, in-gel kinase assays allowed the determination of the approximate size of the kinase involved in the phosphorylation of NopL. Figure 1. In vitro phosphorylation assay. Soybean root extracts obtained from uninoculated plants or inoculated with E. fredii HH103 RifR were mixed with the recombinant protein NopL. Lane 1, Control with recombinant NopL and GST; lane 2, molecular weight marker; lane 3, recombinant NopL with soybean root extract from plants inoculated with E. fredii HH103 RifR; lane 4, control with soybea n root extract from plants inoculated with E. fredii HH103 RifR; lane 5, recombinant NopL with uninoculat ed soybean root extract; lane 6, Control with uninoculated soybean root extract. ACKNOWLEDGEMENTS This work was financed by projects AGL2009-13487-C04 from the Spanish MEC and P11-CVI-7050 from the Junta de Andalucía. Irene Jiménez-Guerrero work was supported by a predoctoral fellowship of the University of Seville. REFERENCES Bartsev, A.V., et al. (2004). Plant Physiol. 134: 871-879. de Lyra, M.C.C.P., et al. (2006). Int. Microbiol. 9: 125-133. Monks D.E., et al. (2001). Plant Cell. 13: 1205-1219. Monreal, J.A., et al. (2013). Planta. In press. Zhang, L., et al. (2011). J. Biol. Chem. 37: 32178-32187. 132 Session II SII-CP-12 Control de movilidad y producción de EPS I en Sinorhizobium meliloti: nuevas funciones asignadas al sistema NtrY/X. Calatrava, N.*, Nogales, J., Ameztoy, K., van Steenbergen, B., Soto, M.J. Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín CSIC, 18008 Granada. * [email protected] RESUMEN Bacterial surface motility can impact colonization and infection of eukaryotic hosts and it is subject to strict control. To identify genetic determinants involved in S. meliloti surface motility, GR4-derivative transposants with an altered ability to translocate over surfaces relative to the wild type were selected. Three of these mutants contained independent Tn5 insertions that interrupted the ntrY gene coding for the putative sensor histidine kinase of the two-component system NtrY/X which has been involved in nitrogen metabolism regulation in several bacteria. Characterization of the ntrY::Tn5 mutant GNS577 revealed a pleiotropic phenotype when compared to the parental strain: i) reduced swimming and surface motilities, ii) altered cell morphology with low number of flagella, iii) mucoid phenotype after growth in minimal medium, iv) increased biofilm formation ability on abiotic surfaces, and v) impaired growth under high salt conditions. Our results indicate that the defect in motility shown by GNS577 is mainly due to reduced expression of the flagellar master regulator genes visNR whereas the mucoid phenotype is caused by EPS I over-production. GNS577 is able to develop nitrogen-fixing nodules on alfalfa plants but it is highly impaired in competitive nodulation. INTRODUCCIÓN Diversos estudios indican que la capacidad de una bacteria de desplazarse sobre una superficie puede afectar el establecimiento de interacciones con un hospedador eucariota. No obstante, el conocimiento existente sobre motilidad en superficie de los rizobios, y de cómo ésta puede afectar la colonización y establecimiento de endosimbiosis mutualista con las leguminosas es muy escaso. Nuestro grupo fue el primero en describir motilidad tipo swarming en un mutante fadD de la cepa GR4 de S. meliloti (Soto et al. 2002), mutante que resultó estar afectado en su capacidad infectiva y competitiva por la nodulación de alfalfa. Posteriormente, demostramos que la cepa Rm1021 es capaz de desplazarse sobre la superficie de medios semisólidos utilizando mecanismos dependientes e independientes de la acción flagelar, en los que la producción del sideróforo rizobactina 1021 cumple un papel esencial (Nogales et al. 2012). Con el objetivo de identificar nuevos componentes implicados en motilidad en superficie de S. meliloti, hemos llevado a cabo el aislamiento de transposantes derivados de la cepa GR4 alterados en este tipo de motilidad. En este trabajo se presenta la caracterización de uno de ellos: GNS577. MATERIAL Y MÉTODOS La cepa GR4 de S. meliloti se sometió a mutagénesis generalizada con Tn5 usando el plásmido pSUP2021. La translocación en superficie se ensayó en medio mínimo (MM) semisólido (0,6% agar Noble) y la motilidad swimming en medio Bromfield (0,3% agar bacteriológico) tal y como se describe en Nogales et al. (2012). La secuencia de ADN afectada por la inserción del Tn5 se identificó mediante PCR arbitraria y posterior secuenciación. La súper-producción de EPS I se analizó iluminando con luz UV colonias crecidas en MM conteniendo Calcoflúor (0,2 mg/mL). El fenotipo simbiótico 133 Session II SII-CP-12 (infectividad y competitividad) se analizó en plantas de alfalfa crecidas en cultivo hidropónico siguiendo la metodología descrita en Soto et al. (2002). RESULTADOS Y DISCUSIÓN El análisis de 5800 transposantes derivados de la cepa GR4 de S. meliloti nos permitió identificar 14 clones con deficiencias en translocación sobre superficie de MM semisólido. La caracterización genética de estos mutantes reveló que 3 de ellos contenían inserciones independientes de Tn5 en el gen ntrY que potencialmente codifica el sensor histidín quinasa del sistema regulador de dos componentes NtrY/X implicado en el control de genes relacionados con metabolismo nitrogenado en bacterias simbióticas fijadoras de N2. La caracterización fenotípica del mutante GNS577 reveló estar afectado no sólo en motilidad en superficie (Figure 1A) sino también en motilidad tipo swimming (Figure 1B), defectos que podrían ser debidos a la menor expresión de los genes visNR detectada en el mutante ntrY con respecto a GR4. Observaciones realizadas en TEM revelaron que GNS577 presentaba una morfología alterada (menor tamaño y más redondeada) con respecto a la cepa parental, así como un reducido número de flagelos (Figure 1C). Además, la mayor mucosidad exhibida por GNS577 en placas de MM (Figure 1D), así como la fluorescencia emitida tras iluminar con UV placas de MM conteniendo calcoflúor (Figure 1E), sugerían que este mutante producía mayores cantidades de EPS I que GR4, hipótesis que ha sido confirmada al revertir estos fenotipos en un doble mutante ntrYexoY. GNS577 presenta mayor sensibilidad a estrés salino (MM adicionado de 300 mM NaCl) (Figure 1F), y mayor capacidad de formar biofilm en superficie de vidrio que GR4 (Figure 1G). Aunque capaz de formar nódulos fijadores de N en alfalfa, el mutante ntrY muestra claros defectos en capacidad competitiva por la nodulación (Figure 1H) que pueden ser recuperados tras la sobreexpresión de Vis NR. Nuestros resultados sugieren que el sistema NtrY/X de S. meliloti participa en el control coordinado de motilidad y producción de EPS I. Figura 1. Fenotipos mostrados por GNS577 (ntrY::Tn5) en comparación con la cepa parental GR4. (Ver detalles en texto) AGRADECIMIENTOS Este trabajo ha sido financiado por MICINN (BIO2010-18005), Junta de Andalucía (Proyecto de Excelencia CVI-03541) y fondos FEDER. BIBLIOGRAFÍA Soto, M.J., et al. (2002). Mol. Microbiol. 43: 371-382. Nogales, J., et al. (2012). J. Bacteriol. 194: 2027-2035. 134 Session II SII-CP-13 Auxotrophy accounts for nodulation impairment in a Sinorhizobium meliloti mutant defective for meso-diaminopimelate biosynthesis. García-Rodríguez, F.M., Ortigosa, A., Millán V., Toro, N., Martínez-Abarca, F. * Departamento de Microbiología y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain. * [email protected] ABSTRACT Sinorhizobium meliloti establishes an effective nitrogen-fixing symbiosis with its leguminous host plant, alfalfa (Medicago sativa). A S. meliloti dapB (encoding 2,3dihydrodipicolin ate reductase) mutant has defective nodulation that is restored by the addition of diaminopimelate (DAP) to the plant growth medium. INTRODUCTION The soil bacterium Sinorhizobium meliloti establishes a nitrogen-fixing symbiosis with its legume plant partner (Medicago sativa), through a series of signal exchanges (van Rhijn and Vanderleyden 1995). Auxotrophic mutations affecting a number of aminoacid biosynthetic p athways do not affect the ability of S. meliloti to initiate nodule formation (Randhawa and Hassani 2002). However, several mutants with defects in the biosynthesis of branched-chain amino acids have been shown to display impaired initiation of nodule formation (Aguilar and Grasso 1991; Hassani et al., 2002; SanjuánPinilla 2002 ; de las Nieves Peltzer, et al., 2008). The dapB gene, encoding L-2,3-dihydrodipicolinate reductase, is required for the biosynthesis of meso-diaminopimelate (DAP; Born and Blanchard 1999). DAP is an essential component of peptidoglycan and also serves as a direct precursor of lysine. Mutations of the dapB gene impair the growth of Pseudomonas stutzeri in the absence of DAP and lysine (Rediers et al., 2003). We studied the free-living and symbiotic phenotypes of a dapB mutant of S. meliloti. MATERIAL AND METHODS dapB mutant construction. 1021-Δdap was constructed by insertion of the Sm/SpR cassette (Prentki and Krisch 1984) into the chromosome (between positions 186859 and 187585). Growth and Symbiotic phenotype. TY and MM growth media assays and plant assays were performed as described in (Soto et al., 2004). RESULTS AND DISCUSSION The S. meliloti deleted dapB (1021-Δdap)mutant displayed impaired growth in both TY and MM media, but complementation of this defect was observed when 2.5 mM DAP was added to the medium. Complementation of the mutant with the wild-type dapB ORF expressed in trans fully restored wild-type growth in media lacking DAP. We then investigated the effect of this mutation on symbiosis (Figure 1). Plants inoculated with the dapB mutant had yellow or pale green leaves, indicating a lack of nitrogen fixation. By contrast, the plants inoculated with the dapB+ parental strain or the mutant strain complemented with pKmDAP had healthy green leaves and the presence of elongated pink nodules (Figure 1A and B). Plants inoculated with the dapB mutant had white nodules, which developed on the roots later than wild-type nodules and were generally not elongated (less than 10% were elongated). Histochemical analyses of longitudinal sections of elicited nodules revealed that the nodule produced by the parental strain had a clear zonal distribution, whereas the mutant induced 135 Session II SII-CP-13 structures essentially resembling non invaded pseudonodules and displaying no nitrogen fixation. In these conditions, the nodules induced by the dapB mutant appeared at the same time as those elicited by the wild-type strain, and dry weight did not differ significantly between the two sets of inoculated plants. These results suggest that the concentration of DAP in alfalfa root exudates is limiting; however, the supplementation of this compound restored nitrogen fixation and normal infectious processes. These properties define the dapB gene as a good selective marker for screening promoters involved in early stages in the alfalfa-S. meliloti symbiosis Figure 1. Symbiosis defects of the dapB mutant. (A) Alfalfa plants 25 days after inoculation with the wt 1021 strain (panel 1), the dapB mutant (panel 2), the complemented dapB mutant (carrying pKmDAP; panel 3) and the dapB mutant in the presence of 2.5 mM DAP added to the plant growth medium (panel 4). (B) Enlarged image of the nodules elicited on alfalfa. Panel identification numbers are as described above. ACKNOWLEDGMENTS This work was supported by research grants AGR 252 (Proyecto de Excelencia de la Junta de Andalucí a), and CSD 2009-0006 of the Consolider-Ingenio Program, including ERDF (European Regional Development Funds). REFERENCES Aguilar, O.M., and Grasso, D.H. (1991). J. Bacteriol. 173: 7756-7764. Born, T.L,, and Blanchard, J.S. (1999). Curr. Opin. Chem. Biol. 3: 607-613. de las Nieves Peltzer, M., et al. (2008). Mol. Plant Microbe Interact. 21: 1232-1241 Hassani, R., et al. (2002). Indian J. Exp. Biol. 40: 1110-1120. Prentki, P., and Krisch, H,M. (1984). Gene. 29: 303-313. Randhawa, G.S. and Hassani, R. (2002). Indian J. Exp .Biol. 40: 755-764. Rediers, H., et al. (2003). Appl. Environ. Microbiol. 69: 6864-6874. Sanjuán-Pinilla, J., et al. (2002). Arch. Microbiol. 178: 36-44. Soto, M,J., et al. (2004) FEMS Microbiol. Ecol. 48: 71-77. van Rhijn, P., and Vanderleyden, J. (1995). Microbiol. Rev. 59: 124-142. 136 Session II SII-CP-14 Genomic analysis of Azospirillum brasilense Az39, the most extensively used strain for inoculant production in Argentina. Cassán, F.1*, Rivera Bottia, D.1, Molina, R.1, Revale, S.2, Vazquez, M.2, Spaepen, S.3, Vanderleyden, J.3, Perticari, A.4 1 Universidad Nacional de Río Cuarto. Córdoba. Argentina. 2 Instituto de Agrobiotecnología de Rosari o [INDEAR]. Rosario. Argentina. 3 Centre of Microbial and Plant Genetics. KU Leuven. Belgium. 4 Instituto de Microbiología y Zoología Agrícola INTA Castelar [IMYZA-INTA]. Argentina. * [email protected] ABSTRACT We sequenced and analyzed the genome of A. brasilense Az39, the most extensively used strain for inoculants production in Argentina. The genome sequence was obtained using a 454 GS FLX Titanium pyrosequencer. Genome annotation was done using the Standard Operating Procedures for Prokaryotic annotation from ISGA and from RAST annotation server. The putative plant growth promotion and related mechanisms were analyzed in A. brasilense Az39 and compared with other Azospirillum sp. strains previously sequenced. INTRODUCTION In the 1980s, an intensive program to select and identify Azospirillum sp. strains able to improve productivity of wheat and corn was started by INTA-IMYZA in Argentina. A. brasilense Az39 was selected as one of the most effective PGPR and recommended for wheat and maize inoculants formulation. After 40 years of agronomic use, numerous laboratory, greenhouse and field experiments since then have demonstrated the effectiveness of Az39 to increase growth and productivity in increasing number of crops species. In this work, we present a comprehensive analysis of the: (1) genomic features and (2) plant growth promotion and related mechanisms of Az39 in comparison with sequenced strains belonging to this genus. MATERIAL AND METHODS The genome sequence was obtained using a combined Whole Genome Shotgun and 8kb Pair-Ends strategy with a 454 GS FLX Titanium pyrosequencer at INDEAR. Replicons were identified via a PFGE analysis. Assembly was done using 454 Newbler v2.6 with a 16X genome coverage. Genome annotation was done using the Standard Operating Procedures for Prokaryotic annotation from ISGA (Hemmerich et al., 2010) and from RAST annotation server (Aziz et al. 2008). The putative plant growth promotion mechanisms of Azospirillum brasilense Az39 (16S ribosomal RNA gene accesion number JQ844453.1) were analyzed and compared with those previously obtained for Azospirillum brasilense Sp245, Azospirillum lipoferum 4B (Wisniewski-Dyé et al., 2011) and Azospirillum sp. B510 (Kaneko et al., 2010). Comparative analysis was performed by the use of KEGG (Kanehisa et al., 2012) and RAST (Aziz et al., 2012) web services. 137 Session II SII-CP-14 RESULTS AND DISCUSSION Genomic features. Table 1. General features of the A. brasilense Az39 genome, database analyzed and compared with th ose obtained for Azospirillum brasilense Sp245, Azospirillum lipoferum 4B and Azospirillum sp. strain B510. Features A.brasilense Az39 A. brasilense Sp245 A. lipoferum 4B Azospirillum sp. B510 Size GC Content [%] Number of features Number of RNAs tRNAs rRNAs Number of Coding Sequences in Subsystems non-hypothetical hypothetical not in Subsystems non-hypothetical hypothetical Number of Subsystems COG Assignement Not in COG Replicons [plasmids or chromids] 7.421.756 68,6 6760 79 68 11 6681 2717 2570 147 3964 1605 2359 467 4703 1939 5* 7.530.241 68,5 7027 105 80 25 6922 2589 2460 129 4333 1737 2596 472 4716 2206 6 6.846.400 67,7 6263 108 79 29 6155 2473 2356 117 3682 1482 2200 486 4302 1853 6 7.599.738 67,6 6886 105 79 26 6781 2683 2543 140 4098 1811 2287 484 4761 2020 6 *PFGE analysis: P1: 1.6; P2: 0.95; P3: 0.7; P4: 0.68 and P5: 0.15 Mpb Plant growth promotion and survival mechanisms. The plant growth promotion mechanisms previously described for PGPR were evaluated in genome sequence of Az39 and compared with other Azospirillum sp. genomes. Here, we included the indole-3-acetic acid biosynthesis pathways, while other mechanisms as nitrogen fixation or nitrate reduction; phytohormones and growth regulators (i.e. cytokinins, gibberellins, ethylene, abscisic acid, polyamines and nitric oxide) biosynthesis; siderophores and biofilms production; secretion system; widespread colonization island and other related mechanisms will be presented in full version of this work during the Congress. Table 2. Summary of the IAA pathways according to putative gene sequences identified in the genome of A. brasilense Sp245, Az39 and CBG497; A. lipoferum 4B, A. amazonense Y2 and Azospirillum sp. B510. IAA biosynthesis Enzyme name A. brasilense Sp245 A. brasilsense Az39 A. brasilense CBG497 A. lipoferum 4B Azospirillum sp. B510 A. amazonense Y2 IPyA indole piruvate decarboxylase aromatic amino aldehyde transferase deshydrogenase ipdC/ppdC + + + *hisC1 from A. brasilense Sp7; † IAN hisC1 * + + + + + + + nitrilase NIT1 † + + + NIT2 † + + + IAM tryptophan monooxygenase iaaM + indole acetamide hydrolase iaaH + NIT1 and NIT2 from Arabidopsis thaliana ACKNOWLEDGEMENTS This work was supported by projects granted by FONCyT, CONICET, UNRC, INTA and FWO. REFERENCES Aziz, R.K., et al. (2008). BMC Genomics 9: 75 Hemmerich, E., et al. (2010). Bioinformatics 26: 1122-1124. Kanehisa, M., et al. (2012). Nucleic Acids Res. 40: 109-114. Kaneko, T., et al. (2010). DNA Res., 17: 37-50. Wisniewski-Dye, F., et al. (2011). PLoS Genet. 7 (12). 138 Session II SII-CP-15 Two-dimensional proteomic reference map diazoefficiens strain CPAC 7 (=SEMIA 5080). of Bradyrhizobium Gomes, D.F.1, Batista, J.S.S.3*, Hungria, M.2 1 Departamento de Genética, Universidade Federal do Paraná, Curitiba, Brasil; Fellowship from CNPq. 2 Embrapa Soja, Londrina, Brasil. 3 Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, Brasil. * [email protected] ABSTRACT A two-dimensional gel electrophoresis profile was generated for Bradyrhizobium diazoefficiens CPAC 7 (=SEMIA 5080), a highly competitive strain against naturalized soil rhizobia and efficient in fixing nitrogen in symbiosis with soybean. We selected 150 spots and 124 proteins were effectively identified. The majority of the identified proteins were related to metabolic functions. INTRODUCTION Strain CPAC 7 was recently reclassified into a novel species named Bradyrhizobium diazoefficiens (Delamuta et al., 2013). Due to its outstanding efficiency in fixing nitrogen, the strain is employed in soybean (Glycine max L.) commercial inoculants in Brazil, since 1992 (Hungria et al., 2006). The genome sequencing of CPAC 7 is now in progress; so, the establishment of a proteomic reference map can add important proteinexpression i nformation into the genomic annotation process. In this study we present the two-dimensional proteomic reference map of CPAC 7 that will allow a comparative analysis with the published proteomic reference map of B. diazoefficiens, strain USDA 110 (Delmotte et al., 2010). MATERIAL AND METHODS B. diazoefficiens CPAC 7 was grown in AG medium until exponential phase. Wholecell proteins were e xtracted and separated by two-dimensional gel electrophoresis, using IPG-strips with pH range 4-7 (Gomes et al., 2012). The experiment was performed in triplicate and 150 well-defined spots, present in all three gels were randomly selected for MALDI-TOF/TOF identification. Spectra generated were searched against the public database NCBInr, Proteobacteria, using the Mascot software v. 2.3 (Matrix Science). RESULTS AND DISCUSSION Well defined and reproducible 2D gel profiles were generated (Figure 1). Among 150 protein spots randomly selected to be analyzed by mass spectrometry, 124 proteins were successfully identified. In addition to Mascot identification, we also made a prediction of the subcellular location using the software PLSpread and PsortB. Functional classification in clusters of orthologous groups (COG) was also performed, and proteins were distributed in 17 COG categories, belonging to four functional groups. Proteins related to metabolic functions were the majority, representing 39% of identified proteins (Figure 2). The first hit of most proteins was with USDA 110, defined as the type strain of B. diazoefficiens. In relation to the cellular location, 105 of the proteins are located in the cytoplasm. 139 Session II SII-CP-15 Figure 1. 2D gel profile of B. diazoefficiens CPAC 7 Figure 2. Distribution of the identified proteins into COG categories. Among the proteins identified in our study, it is worth mentioning that ATP-dependent Clp protease, GTP-binding tyrosin phosphorylated protein and electron transfer flavoprotein, besides others, were up-regulated when B. japonicum strain CPAC 15 was grown in the presence of genistein (Batista and Hungria, 2012). This reference map will be useful to add an expression point-of-view in the oncoming genome sequencing of strain CPAC 7. ACKNOWLEGMENTS Project CNPq-Repensa 562008/2010-1 REFERENCES Batista, J.S.S., and Hungria, M. J. (2012). J. Proteomics, 75: 1211-1219. Delamuta, J.R.M., et al. (2013). Int J Syst Evol Microbiol, ijs.0.049130-0v1-ijs.0.049130-0. Delmotte, N., et al. (2010). Proteomics, 10: 1391-1400. Gomes, D.F., et al. (2012). Proteomics, 12: 859-863. Hungria, M., et al. (2006). In: Nitrogen Nutrition and Sustainable Plant Productivity. 43-93. 140 Session II SII-CP-16 Emprego da metodologia de MLSA em avaliações de filogenia e taxonomia de rizóbios: estudo com Rhizobium spp. microssimbiontes de feijoeiro (Phaseolus vulgaris L.). Ribeiro, R.A.1, 2*, Delamuta, J.R.M.1, 2, Hungria, M.1, 2 1 Laboratório de Biotecnologia do Solo; Embrapa Soja. 2 Programa de Pós-Graduação em Microbiologia (Universidade Estadual de Londrina). * [email protected] RESUMO Estudos taxonômicos desempenham um papel relevante na valoração da diversidade microbiana do solo. A metodologia de MLSA (Multilocus Sequence Analysis) tem possibilitado incrementar o conhecimento da biodiversidade bacteriana do solo, bem como a descrição de novas espécies. Neste estudo, quatro genes housekeeping foram utilizados para estabelecer as relações filogenéticas entre 19 estirpes de Rhizobium spp. microssimbiontes do feijoeiro. Além de obter maior definição das relações filogenéticas, o MLSA indicou possíveis novas espécies de rizóbios ainda não descritas. INTRODUÇÃO A diversidade procariótica presente no solo é elevada, particularmente nos trópicos, mas um grande desafio consiste em classificar os microrganismos, identificando o seu potencial biotecnológico em favor de maiores rendimentos das culturas e sustentabilidade ambiental. Nesse contexto, estudos taxonômicos são fundamentais e a metodologia de MLSA aponta como uma ferramenta de grande utilidade. O objetivo deste trabalho foi o de elucidar as relações filogenéticas e a taxonomia de Rhizobium spp. microssimbiontes do feijoeiro (Phaseolus vulgaris L.), através da análise do gene 16S rRNA e de quatro genes housekeeping. MATERIAL E MÉTODOS Foram analisadas 19 estirpes de rizóbios microssimbiontes do feijoeiro, isoladas de regiões andinas, mesoamericanas e do Brasil. A extração do DNA, os primers, as condições de amplificação dos genes 16S rRNA, recA, glnII, gyrB e rpoA, bem como a purificação dos produtos de PCR, o sequenciamento e a construção das árvores filogenéticas foram realizadas conforme descrito por Ribeiro et al. (2009). RESULTADOS E DISCUSSÃO Com base na árvore do 16S rRNA, das 19 estirpes analisadas, 12 foram posicionadas no grupo de R. etli, três se posicionaram no de R. tropici e as demais foram alocadas no grupo de R. radiobacter. O baixo poder de resolução do gene 16S rRNA não permitiu boa definição da posição taxonômica das estirpes deste estudo, principalmente daquelas do grupo R. etli (Figura 1A). As sequências concatenadas e alinhadas resultaram em um fragmento comum de 1.846 nucleotídeos e foram utilizadas para a construção da árvore filogenética. Através da combinação das informações filogenéticas dos quatro genes housekeeping concatenados, obteve-se uma melhor resolução na definição das espécies, em comparação com a análise baseada em um único gene (Figura 1B). Novas linhagens, representando possíveis espécies novas foram evidenciadas na análise de MLSA. A estirpe CNPSo 669, do México, foi agrupada com a estirpe IE4771, previamente designada como R. etli; contudo, houve indicação de representar uma espécie independente de R. etli e R. phaseoli, recebendo a denominação de PEL1. A estirpe equatoriana CNPSo 679 foi posicionada independentemente como nova linhagem e denominada PEL2, enquanto as estirpes CNPSo 661, 666 e 668 formaram o grupo PEL3. As estirpes CNPSo 676, 683, 670, 671 e 672 do Equador e 659 do México 141 Session II SII-CP-16 agruparam com um bootstrap de 100% e formaram outra nova linhagem, PEL4. As estirpes WSM2304 e CCGE510, embora tenham sido identificadas como R. leguminosarum na análise do 16S rRNA, formaram dois grupos por MLSA, PEL 5 e PEL6, respectivamente (Figura 1B.). A posição taxonômica de outras estirpes foi melhor definida, por exemplo, as estirpes brasileiras CNPSo 657 e 660 agruparam estreitamente com R. leucaenae (CFN 299T) e apresentaram a inserção de 72 nucleotídeos no gene 16S rRNA, o qual é característico da espécie (Ribeiro et al., 2012). Rhizobium sp. IE 4771 (KC261566, ABRC00000000, ABRC00000000, KC261567) 100 Rhizobium sp. CNPSo 669 (KC293530, KC293516, KC293519, KC293533) A B Rhizobium sp. CNPSo 679 (JN129355, JN129310, JN129340, JN129370) 99 PEL1 PEL2 R. phaseoli CNPAF512 (AEYZ00000000, AEYZ00000000, AEYZ00000000, AEYZ00000000) R. phaseoli Ch24-10 (AHJU00000000, AHJU00000000, AHJU00000000, AHJU00000000) 100 100 R. phaseoli CIAT 652 (CP001074, CP001074, CP001074, CP001074) 100 Rhizobium sp. CNPSo 661 (JN129346, JN129301, JN129331, JN129361) 80 Rhizobium sp. CNPSo 668 (KC293529, KC293515, KC293521, KC293532) PEL3 Rhizobium sp. CNPSo 666 (JN129350, JN129305, JN129335, JN129365) 100 R. etli CFN42T (CP000133, CP000133, CP000133, CP000133) 100 R. etli CNPSo 664 (JN129348, JN129303, JN129333, JN129363) Rhizobium sp. CNPSo 672 (JN129352, JN129307, JN129337, JN129367) 100 99 Rhizobium sp. CNPSo 670 (KC293531, KC293517, KC293520, KC293534) 100 Rhizobium sp. CNPSo 671 (JN129351, JN129306, JN129336, JN129366) Rhizobium sp. CNPSo 683 (JN129356, JN129311, JN129341, JN129371) 99 PEL4 Rhizobium sp. CNPSo 676 (KC333885, KC333883, KC333884, KC333886) Rhizobium sp. CNPSo 659 (JN129344, JN129299, JN129329, JN129359) Rhizobium sp. WSM 2304 (CP001191, CP001191, CP001191, CP001191) 96 Rhizobium sp. CCGE 510 (AEYF00000000, AEYF00000000, AEYF00000000, AEYF00000000) 85 PEL5 PEL6 R. leguminosarum USDA 2671 (EU488811, EU488784, KC293526, EU488837) 100 R. leguminosarum 3841 (AM236080, AM236080, AM236080, AM236080) R. leguminosarum WSM 1325 (CP001622, CP001622, CP001622, CP001622) 96 R. pisi DSM30132T (DQ431676, JN580715, KC293522, KC293535) R. gallicum R602spT (AY907357, AF529015, AM418828, EU488840) 73 R. leucaenae CFN 299T (AJ294372, AF169583, KC293524, EU488845) 100 Rhizobium sp. CNPSo 657 (JN129343, JN129298, JN129328, JN129358) Rhizobium sp. CNPSo 660 (JN129345, JN129300, JN129330, JN129360) R. multihospitium CCBAU 83401T (EF490029, EF490040, KC293528, JF318207) 99 R. tropici CIAT 899T (AJ294373, AF169584, HQ438238, EU488833) 85 R. miluonense CCBAU 41251T (HM047131, HM047120, KC293527, JF318206) 78 Rhizobium sp. CNPSo 655 (JN129342, JN129297, JN129327, JN129357) R. lusitanum P1-7T (DQ431674, EF639841, KC293525, JF318205) R. rhizogenes K84 (CP000628, CP000628, CP000628, CP000628) R. grahamii CCGE 502T (JF424622, JF424618, AEYE00000000, AEYE00000000) 100 R. mesoamericanum CCGE 501T (JF424620, JF424617, PRJNA63213 , PRJNA63213) R. endophyticum CCGE 2052T (HM142767, JF424619, PRJNA63221, PRJNA63221) R. giardinii H52T (AM182123, EU488778, HQ438240, EU488829) R. radiobacter ATCC 19358T (FM164311, JN580718, FR695243, KC293536) 0.02 Figura 1. Ávores filogenéticas entre as estirpes de Rhizobium spp. e estirpes tipo, com base no ge ne 16S rRNA (A) e nos genes housekeeping concatenados (recA, glnII, gyrB e rpoA) (B). CONCLUSÃO Quando comparada com a análise do gene 16S rRNA, a metodologia de MLSA permitiu melhor definição taxonômica de estirpes de Rhizobium microssimbiontes de feijoeiro provenientes de regiões andinas, mesoamericanas e do Brasil. Houve indicação de novas espécies, mostrando o poder de resolução do MLSA e confirmando o potencial elevado dessa técnica para o conhecimento da biodiversidade e para a definição taxonômica de estirpes com potencial biotecnológico. AGRADECIMENTOS Projeto CNPq-Repensa 562008/2010-1. REFERÊNCIAS Ribeiro, R.A., et al. (2009). Res. Microbiol. 160: 297-306. Ribeiro, R.A., et al. (2012). Int. J. Syst. Evol. Microbiol. 62: 1179-1184. 142 Session II SII-CP-17 Relative expression of hypothetical Bradyrhizobium diazoefficiens CPAC 7. protein-related genes for Gomes, D.F.1, 2, Rolla-Santos, A.A.P. 1*, Batista, J.S.S.3, Hungria, M.1 1 Embrapa Soja, Londrina, Brazil, 2 Departamento de Genética, Universidade Federal do Paraná, Curiti ba, Brasil, 3 Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Pont a Grossa, Ponta Grossa, Brazil. * [email protected] ABSTRACT From the proteomic map of Bradyrhizobium diazoefficiens strain CPAC 7 we selected nine hypothetical protein-related genes and evaluated their relative expression by Realtime quanti tative PCR (RT-qPCR). The experiment was performed with two treatments (induced or not with 5 µM genistein). Six out of nine protein-related genes were upregulated in th e presence genistein. INTRODUCTION Bradyrhizobium diazoefficiens strain CPAC 7 (Delamuta et al., 2013) is a microsymbiont that fixes nitrogen in association with soybean (Glycine max L.) and is used in commercial inoculants in Brazil; therefore it has high ecological and economical importance. It is estimated that more than 30% of the genome of B. diazoefficiens type strain USDA 110 encodes hypothetical proteins. The hypothetical protein nomenclature is used when the existence of a gene is supported only by the prediction of gene-finding softwares, and when they do not show significant homology with any characterized gene (Batista et al., 2010). However, these proteins can bring interesting information to gene bioprospection. In these sense, our goal was to study the relative expression of some hypothetical protein-related genes from strain CPAC 7 in response to genistein. MATERIAL AND METHODS Strain CPAC 7 was grown in AG medium until exponential phase in two treatments (Induced or not with genistein 5 µM). Total RNA was isolated using Trizol and employed for cDNA synthesis using Superscript III TM reverse transcriptase (InvitrogenTM). Platinum® SYBR Green®qPCRSuperMix-UDG was used according to the manufacturer’s instructions. Amplification reactions were conducted in triplicate using a 7500 Real Time System thermocycler (Applied Biosystems). We used 16S rRNA (GeneID: 1055154) as reference gene for normalization. Relative gene expression was determined by the 2-ΔΔCT method (Livak and Schmittgen, 2001). Statistical analysis of the data was performed using the REST 2009 v.2 software, which enables the calculation of P values for each sample group and 95% confidence intervals. RESULTS AND DISCUSSION Based on an unpublished B. diazoefficiens CPAC 7 proteomic reference map of our group, we selected 18 hypothetical proteins identified and performed a preliminary functional inference using bioinformatics tools and based on the presence of function domains/motifs (Table 1). Nine genes classified as hypothetical proteins that did not fit into any functional category of COGnitor (http://www.ncbi.nlm.nih.gov/COG/old/xognitor.html), assigned as “NO related KOG”, were selected and analyzed by RT-qPCR. The analysis was performed to monitor the relative expression of these genes in response to genistein. Of the nine genes, six were up-regulated (Figure 1). Curiously, all genes coding for 143 Session II SII-CP-17 periplasmic proteins (bll0565, blr7534, bll5307 and blr0227) were differentially expressed in response to the flavonoid. Table 1. Identified hypothetical proteins of B. diazoefficiens CPAC 7. Hypothetical Cellular Functional inference protein location BJ6T_08050 Cytoplasmic Thioredoxin-like protein Bll7551 Periplasmic Blr5678 Cytoplasmic L-aminopeptidase, probably related to arginine biosynthesis Bll5131 Extracellular ATP/GTP binding site, small transmembrane domain Blr2961 Cytoplasmic Fumarylacetoacetase Blr3798 Cytoplasmic Demethylmenaquinone methyltransferase Blr5067 Cytoplasmic Blr0227 Periplasmic Blr2191 Periplasmic Histidine phosphotransferase Bll4565 Cytoplasmic Ribonuclease, high similarity with Blr3798 Bll5307 Periplasmic Blr2761 Cytoplasmic Universal stress protein UspA Blr7436 Cytoplasmic Predicted transcriptional regulator containing the HTH Bll4752 Cytoplasmic domain Bll0565 Periplasmic Bll5663 Cytoplasmic ATPase Blr3064 Cytoplasmic Succinyl-diaminopimelate desuccinylase Blr7534 Periplasmic 2 * Relative expression * 1,6 * 1,2 * * * 0,8 0,4 0 Figure 1. Gene expression analysis by RT-qPCR of nine hypothetical proteins. The differential expression of these genes in response to genistein suggests that their respective proteins are probably involved in symbiosis establishment process. ACKNOWLEGMENTS Project CNPq-Repensa 562008/2010-1 REFERENCES Batista, J.S., et al., (2010). Proteomics. 10: 3176-3189. Delamuta, J.R.M., et al. (2013). Int J Syst Evol Microbiol, ijs.0.049130-0v1-ijs.0.049130-0. Gomes, D.F., et al. (2012). Proteomics. 12:859-863. Livak K.J., and Schmittgen, T.D. (2001). Methods 25: 402-408. 144 Session II SII-CP-18 PoolSeq analysis of the selection of the Rhizobium genotypes by the legume host plant. Jorrín, B.1*, Imperial, J.1, 2 1 Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Madrid. Investigaciones Científicas (CSIC). * [email protected] 2 Consejo Superior de ABSTRACT Rhizobium leguminosarum establishes highly specific nitrogen-fixing symbioses. We have applied a Pool-Seq approach to study plant host selection of genotypes. Our results confirm, at the genomic level, previous observations regarding plant selection of specific genotypes INTRODUCTION Rhizobium leguminosarum bv. viciae establishes highly specific nitrogen-fixing symbioses with different legume genera (Pisum, Lens, Lathyrus and Vicia). The molecular bases of specificity, establishment and functioning of the symbioses have been described from the bacterial (Murray et al., 2011) and plant (Yokota and Hayashi, 2011) points of view. Thanks to the advent next generation sequencing techniques and bioinformatics tools, the genome of a large number of rhizobial mycrosimbiont strains have been sequenced. Nevertheless, there are aspects of the Rhizobium-legume interaction that are yet to be explained, and they could be relevant for efficient inoculant design. Classic studies using trap plants provided evidence that, given a choice, specific hosts select specific genotypes of rhizobia which are, apparently, particularly adapted to that host (Mutch and Young, 2004; Louvrier et al., 1996). In the Pool-Seq approach (Kofler et al., 2011), population genomics insights are gained through high-throughput DNA sequencing of pools of bacterial isolates. We have applied a Pool-Seq approach to study plant host selection of genotypes from the available genomic diversity in a well-characterized soil. MATERIAL AND METHODS A well-characterized agricultural soil (INRA Bretennieres) was used as source of rhizobia. Plants of Pisum sativum, Lens culinaris, Vicia sativa and V. faba were employed as traps. We pooled 100 nodules from each host, and the pooled DNAs were sequenced (BGI-Hong Kong; Illumina Hi-seq 2000, 180 bp PE libraries, 100 bp reads, 12 Mreads). Reads were quality filtered with FastQC and Trimmomatic. Filtered reads were mapped with Bowtie2 using Rhizobium leguminosarum bv. viciae 3841 as reference genome. Single Nucleotide Polymorphisms (SNPs) were called with VarScan (minimum coverage 20 reads, minimum frequency 0.1). Results were visualized with SeqMonk and IGV. RESULTS AND DISCUSSION An important fraction of the filtered reads were not recruited by the reference genome (Table 1), suggesting that plant soil isolates contain a large number of genes that are not present in the reference genome. Single Nucleotide Polymorphism (SNP) analysis was carried out with Pool-Seqs, and a plant-specific SNP distribution was observed within the nodDABC cluster. 145 Session II SII-CP-18 Population dissimilarities were obtained from the complete SNP genome analysis. Pairwise Euclidean distances were calculated from SNP frequencies with SPSS software (Table 2). Table 1. Reads mapped and unmapped against R. leguminosarum bv. viciae 3841. Total reads % Mapped reads % Unmapped reads PEA LENTIL FAVA VETCH 11,697,508 84.17 16.24 11,893,439 77.79 23.27 11,905,328 77.24 23.29 11,862,580 81.88 18.22 Table 2. Population dissimilarities. Pea Lentil Fava Vetch Rlv 3841 Pea Lentil Fava Vetch Rlv 3841 0 76 90 54 157 0 56 58 187 0 65 195 0 170 0 Our results confirm, at the genomic level, previous observations regarding plant selection of specific genotypes. We expect that further, ongoing comparative studies on differential Pool-Seq sequences will identify specific gene components of the plantselected genoty pes. ACKNOWLEDGMENTS Supported by the Microgen Project (Consolider-Ingenio 2010, CSB2009-00006, MCINN, Spain) to JI. REFERENCES Kofler, R., et al. (2011). Bioinformatics, 27: 343-3436. Louvrier P., et al. (1996). Appl. Environ. Microbiol. 62: 4202. Murray, J.D. (2011). Mol. Plant Microbe Interact. 24: 631. Mutch, L.A., and Young, J.P. (2004). Mol. Ecol. 13: 2435. Yokota, K., and Hayashi, M. (2011). Cell Mol. Life Sci. 68: 1341. 146 Session II SII-CP-19 Incorporación estable y expresión del gen de la diguanilato ciclasa PleD* en el genoma de bacterias que interaccionan con plantas. Romero-Jiménez, L. *, Rodríguez, D., Prada-Ramírez, H., Gallegos, M.T., Sanjuán, J., Pérez-Mendoza, D. Dpto. Microbiología del Suelo y Sistemas Simbióticos. Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Profesor Albareda 1, 18008 Granada, España. * [email protected] RESUMEN En este trabajo hemos desarrollado una serie de herramientas genéticas para la manipulación de los niveles intracelulares de c-di-GMP en bacterias. Los nuevos vectores, derivados de miniTn7, pe rmiten la integración estable del gen de la diguanilato ciclasa pleD* de Caulobacter crescentus en una localización específica del genoma de bacterias Gram-negativas. Los nuevos mini-Tn7 han sido utilizados con éxito en bacterias de los géneros Rhizobium, Sinorhizobium y Pseudomonas. INTRODUCCIÓN El c-di-GMP es un segundo mensajero implicado en la regulación de funciones bacterianas como la motilidad, virulencia, adhesión y formación de biopelículas. Los niveles intracelulares de este segundo mensajero son controlados por dos tipos de enzimas antagónicas: diguanilato ciclasas que sintetizan c-di-GMP y fosfodiesterasas que lo degradan. La sobreexpresión del gen de la diguanilato ciclasa pleD* (Aldridge et al., 2003) en el vector de expresión pJBpleD* (Pérez-Mendoza, sin publicar), provoca aumentos significativos de los niveles intracelulares de c-di-GMP y, en consecuencia, cambios fenotípicos asociados a la producción de exopolisacáridos, la formación de biopeliculas y la motilidad de bacterias que interaccionan con plantas. Sin embargo, esta construcción presenta una baja estabilidad en ausencia de presión selectiva de antibiótico, particularmente in planta. Para superar esta inestabilidad hemos construido derivados del transposón mini-Tn7 portadores de pleD*, capaces de insertarse establemente, en una sola copia y en una localización específica del genoma, sin afectar la viabilidad de las bacterias. MATERIAL Y MÉTODOS La introducción de los vectores mini-Tn7pleD* en las cepas receptoras se realizó siguiendo el protocolo utilizado por (Bao et al., 1991). Para la extracción y cuantificación de c-di-GMP se siguió una modificación del protocolo descrito por (Amikam et al., 1995). La producción de exopolisacáridos por las bacterias se puso de manifiesto utilizando los colorantes rojo congo y calcoflúor (Teather y Wood, 1982). El cultivo axénico de plantas de alfalfa se realizó según la técnica descrita por (Olivares et al. , 1980) y para el cultivo de veza y judía se siguió la técnica de (Leonard, 1943). RESULTADOS Y DISCUSION Construcción e introducción de los vectores mini-Tn7 en el genoma de bacterias. En el vector pUC18T-miniTn7T se clonó el gen pleD* en solitario o acompañado de genes de resistencia a antibióticos (kanamicina o tetraciclina). Además, se construyeron versiones de los mini-Tn7 carentes de pleD*, mediante una deleción interna de dicho gen. Se verificó la capacidad de inserción en el genoma y la estabilidad de los minitransposones e n varias especies de rizobios y en Pseudomonas syringae pv. tomato. Las frecuencias de inserción de los nuevos mini-Tn7 variaron dependiendo de la bacteria, mientras que su estabilidad en ausencia de presión selectiva llegó a ser del 100%. Determinación de los niveles de c-di-GMP e impacto del c-di-GMP en vida libre. Mediante HPLC se cuantificaron los niveles de c-di-GMP en todas las cepas portadoras pleD* en multicopia (pJBpleD*), en monocopia (mini-Tn7pleD*Km), así como de sus respectivos controles (pJB3Tc19 y Tn7::Km). Los resultados mostraron aumentos muy 147 Session II SII-CP-19 significativos de los niveles de c-di-GMP en las cepas portadoras de pleD* con independencia de su localización (vector en multicopia o insertado en el genoma). Estos elevados niveles de c-di-GMP provocaron en todos los casos un aumento en la producción de exopolisacáridos extracelulares, que pudieron ser revelados al crecer las bacterias sobre medio adicionado con rojo congo (CR) o calcoflúor (CF). Los transposantes con pleD* en monocopia tuvieron un comportamiento similar que las cepas con pleD* en multicopia, produciendo colonias rugosas rojas o fluorescentes (bajo luz ultravioleta) en presencia de CR y CF, respectivamente, en contraposición a lo observado en las cepas silvestres. Además, todas las cepas portadoras de pleD* (multicopia y monocopia), mostraron un aumento significativo de la capacidad de formar biopelículas con respecto a las cepas control. También se analizaron la motilidad "swimming" y la motilidad en superficie de las cepas portadoras del gen pleD* (pJBpleD* y transposantes Tn7::pleD*). Los resultados mostraron un descenso de ambos tipos de motilidad en las cepas portadoras de pleD* tanto en mono como en multicopia, en comparación con las cepas control. Impacto del c-di-GMP en la interacción rizobio-leguminosa. La expresión de pleD* tanto en mono como en multicopia en R. etli y R. leguminosarum provocó un aumento de hasta 160 veces en la adhesión a las raíces de sus plantas hospedadoras, Phaseolus vulgaris y Vicia sativa, respectivamente. Además se realizaron ensayos de infectividad en los sistemas S. meliloti-Medicago sativa, R. leguminosarum-V. sativa y R. etli-P. vulgaris. Solo en el caso de la interacción R. etliP. vulgari s, la simbiosis se vio claramente afectada en aquellas plantas inoculadas con cepas portadoras de pleD* tanto en mono como multicopia, observándose un tamaño reducido de la parte aérea y un menor número de nódulos que las plantas inoculadas con las cepas control. CONCLUSIONES Se han construido una serie de vectores mini-Tn7 que permiten la integración localizada y estable del gen de la diguanilato ciclasa pleD* en el genoma de bacterias Gram-negativas. La expresión de pleD* provoca una serie de cambios fenotípicos compatibles con los significativos aumentos de c-di-GMP intracelular observados. En algunos casos, estos aumentos del c-di-GMP intracelular provocaron alteraciones importantes de las interacciones de las bacterias con sus respectivas plantas hospedadoras, lo que sugiere la importancia de este segundo mensajero durante el establecimiento de asociaciones planta-bacteria. AGRADECIMIENTOS Este trabajo ha sido financiado por los proyectos BIO2011-23032 (MICIIN) y CVI-5800 (J. Andalucía) . L.R-J es perceptora de una beca predoctoral JAE-CSIC. H.P-R es perceptor de una beca predoctoral F PI. D.P-M fue perceptor de contratos postdoctorales JAE-CSIC y de la J. Andalucía. BIBLIOGRAFÍA Aldridge, P., et al. (2003). Mol. Microbiol. 47: 1695-1708. Amikam, D., et al. (1995). Biochem. J 311: 921-927. Bao, Y., et al. (1991). Gene 109: 167-168. Choi, K.H., and Schweizer, H.P. (2006). Nat. Protoc. 1: 153-161. Leonard, L.T. (1943). J. Bacteriol. 45: 523-527. Olivares, J., et al. (1980). Appl. Environ. Microbiol. 39: 967-970. Teather, R.M., and Wood, P.J. (1982). Appl. Environ. Microbiol. 43: 777-780. 148 Session II SII-CP-20 Identification of the gene responsible for a non-nodulating phenotype in chickpea. Ali, L.1, Madrid, E.2, Sánchez, R.1, Temprano, F.3, Gil, J.1, Rubio, J.4, Millan, T.1* 1 Dpto Genética, Univ Córdoba, Campus de Excelencia Internacional CeiA3, Córdoba, Spain. 2 Institute for Sustainable Agriculture, CSIC, Córdoba, Spain. 3 IFAPA Centro “Las Torres-Tomejil”, Alcalá del Río, Sevilla, Spain. 4 Área de Mejora y Biotecnología, IFAPA Centro “Alameda del Obispo”, Córdoba, Spain. * [email protected] ABSTRACT A non-nodulating chickpea mutant (PM233) was crossed by a wild type (CA2139). Segregant populations were advanced until F7 generation. Taking profit of residual heterocigositiy a pair of NILs (Near Isogenic Lines) was obtained. The polymorphic region between the NILs was localized in chickpea linkage group 5 (LG5), corresponding to Medicago truncatula chromosome 3 (Chr3). . A gene located Chr3, NSP2, was selected as a candidate gene for nodulation in chickpea. The gene (CaNSP2) consists of a 1,503 bp exon with no introns. Partial amplification in the mutant will be used to start genomic walking or inverse PCR in order to characterize this mutation. INTRODUCTION Chickpea (Cicer arietinum L.) is the second most cultivated grain legume in the world. In this crop, there have been no reports of molecular studies of the nodulation pathway. Development and study of nodulation (Nod) mutants is a very useful tool to dissect this transduction pathway. Seven nodulation mutants have been described in chickpea: PM233, PM665, PM679, PM405, PM796 and ICC435M (Davis 1988; Singh et al. 1992). The mutant PM233 carries the recessive gene rn1 (Paruvangada and Davis 1999). The aim of this study was the molecular characterization of the gene related with the non-nodulating mutation in PM233. MATERIAL AND METHODS A cross between PM233, a non-nodulating mutant Desi type, and CA2139, a nodulating Spanish Kabuli type landrace, was used for the development of a pair of NILs following Rajesh et al. (2002) scheme. The pair of NILs developed was designated as NIL7-2A (nod)/NIL7-2B (non-nod). An F3 population derived from a cross between this pair of NILs was inoculated with a suspension of Mesorhizobium sp. (isolates ISC7 and ISC11) to establish the nod/non-nod inheritance ratio and perform linkage analysis. Molecular characterization: DNA was isolated using the DNAzol method (Invitrogen). STMS markers distributed through different linkage groups (LGs) of the chickpea genetic map were chosen to detect the polymorphic region between the NILs. Using genetic information available for Medicago truncatula the nodulation signaling pathway 2 gene (NSP2)was selected to design primers to be amplified in chickpea. Six different primer combinations were tested in genomic DNA of each parental line (CA2139 and PM233). The clearest amplicon was excised from acrylamide gel, cloned and sequenced. 149 Session II SII-CP-20 RESULTS AND DISCUSSION Pairs of NILs for a given trait differ for a target region and are almost identical in the rest of the genome facilitating the identification of the gene responsible of the trait of interest. The molecular characterization of NIL7-2A (nod) and NIL7-2B (non-nod) showed polymorphisms for markers located in LG5 that corresponds to M. truncatula Chr3. The transcription factor NSP2 (MTR_3g072710) (Oldroyd and Long 2003) was selected as a candidate gene responsible for the non-nodulating phenotype. Six different primer combinations designed in M. truncatula gene were tested in PM233, CA2139 and NILs in order to amplify the gene sequence in chickpea. Only one combination gave a clear amplicon, present in CA2139 and NIL7-2A (nod) and absent in PM233 and NIL7-2B (non-nod). The full-length sequence obtained in CA2139 after assembling the reads was 1,067 bp, covering the gene except 353 bp at the 5’ end. The recently released chickpea genome sequence (Varshney et al. 2013) permitted us to obtain the complete NSP2 gene sequence. The sequence has been deposited in the GenBank (www.ncbi.nlm.nih.gov) database and assigned the accession number KC534503 for chickpea genotype CA2139 (CaNSP2). The deduced amino acid sequence of CaNSP2 contains 501 residues, with a molecular weight of 55.270 kDa. BLASTX analysis showed the highest similarity with Pisum sativum (89%) and M. truncatula (87%) (Figure 1). Characterization of the gene in PM233 mutants is necessary to be able to know the nature of this mutation and to confirm whether CaNSP2 function is similar to that reported other species. M.truncatula Pisum sativum Cicer arietinum Figure 1. Aligment of NSP2 protein among Medicago truncatula, Pisum sativum and Cicer arietinum, showing high similarity in the conserved regions of this family protein. ACKNOWLEDGEMENT This work has been supported by the project INIA contract RTA2010-00059, co-financed by EU funds (FEDER). REFERENCES Davis, T.M. (1988). J. Hered. 79: 476-478. Paruvangada, V., and Davis, T.M. (1999). J. Hered. 90: 297-299. Rajesh, P., et al. (2002). Theor. Appl. Genet. 105: 604-607. Oldroyd, G.E.D., and Long, S.R. (2003) J. Plant Physiol. 131: 1027-1032. Singh, O., et al. (1992). Crop Sci. 32: 41-43. Varshney, R.K., et al. (2013). Nat. Biotech. 31: 240-246. 150 Session II SII-CP-21 Functional characterization of Ensifer meliloti denitrification genes. Torres, M.J.1*, Rubia, M.I.1, Coba de la Peña, T.2, Pueyo, J.J.2, Bedmar, E.J.1, Delgado, M.J.1 1 Estación Experimental del Zaidin, Consejo Superior de Investigaciones Científicas (CSIC), P. O. Bo x 419, 18080-Granada, Spain. 2 Instituto de Ciencias Agrarias, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain. * [email protected] ABSTRACT Despite possessing the complete set of denitrification genes, Ensifer meliloti 1021 is considered a partial denitrifier due to its inability to grow under anoxic conditions with nitrate or nitrite as terminal electron acceptors. In this work, we have demonstrated that, under microoxic conditions, E. meliloti is able to use nitrate or nitrite as respiratory substrates to support bacterial growth and that the nap, nir, nor and nos genes are involved in microoxically nitrate respiration and denitrification. INTRODUCTION Ensifer (formerly Sinorhizobium) meliloti is the endosymbiont of alfalfa plants that also possess the genes for the complete denitrification pathway (Barnett et al., 2001). Several studies have been carried out on the regulation and symbiotic characterization of E. meliloti nap, nir, nor and nos genes (Becker et al., 2004, Bobik et al., 2006, Meilhoc et al., 2010, Horchani et al., 2011). However, up to date the role of E. meliloti denitrification genes in free-living conditions remains unknown. MATERIAL AND METHODS Bacterial strains. E. meliloti wild type (WT) strain 1021, and napA, napC, nirK, norC and nosZ mutants (RhizoGATE; Becker et al. 2009) were used in this study. Cells were grown in TY medium and further incubated in minimal medium under microoxic (2% O2) or anoxic (filled tubes) conditions. MV+-dependent nitrate reductase (MV+-NR) and nitrite reductase (MV+-Nir) activity, and Haem-staining analysis were performed as described by Bueno et al. (2010). Nitric oxide (NO) and nitrous oxide (N2O) determination. NO was measured amperometrically with an NO electrode APOLO 4000, and N 2O with a gas chromatograph type HP 4890D equipped with an electron capture detector (ECD). After RNA isolation Real-time PCR reactions were run in a 7300 Real Time PCR System. RESULTS AND DISCUSSION Nitrate-dependent growth and Nap, Nir, Nor and Nos activity under microoxic conditions. WT cells incubated under microoxic conditions without nitrate showed an OD 600 up to 0.6 (Figure 1A). Addition of nitrate to the growth medium provoked an increase in the OD 600 up to 1 (Figure 1A). Under these conditions, nitrite was produced and accumulated in the medium being consumed further by the cells (Figure 1A). Cells of the napA and nirK mutants showed a growth defect after incubation under microoxic conditions with nitrate compared to WT cells (Figure 1A). Nitrite was not observed in the napA medium, however the nirK mutant accumulated nitrite in the medium, but it was not further reduced (Figure 1B). Similarly, the norC mutant had a significant growth defect under microoxic conditions with nitrate, however the nosZ mutant showed WT growth rates (data not shown). Levels of MV +-NR, MV+-NiR, and Nor activities of the cells incubated under microoxic conditions with nitrate were strongly 151 Session II SII-CP-21 reduced in the napA, nirK and norC mutants, respectively (data not shown). Under these conditions, a nosZ mutant accumulated higher N2O levels than the WT (data not shown). B [NO2-] (mM) A A 9 8 7 6 5 4 3 2 1 0 0 12 24 36 48 60 72 84 96 108 120 Time (hours) Figure 1. (A) Nitrate-dependent growth and (B) extracellular nitrite concentration of wild type E. meliloti 1021 (s, ), and napA ( n, ), and nirK ( l, ) mutant strains. Cells were incubated under microoxic conditions in minimum medium supplemented (closed symbols) or not (open symbols) with KNO3. Expression of E. meliloti denitrification genes under anoxic conditions. Figure 2. Expression of E. meliloti dentrification genes under microoxic and anoxic conditions. By performing qRT-PCR analyses we have found that napA, nirK, norC and nosZ genes were induced not only under microoxic but also under anoxic conditions with nitrate relative to microoxic conditions without nitrate (Figure 2). In fact, these results demonstrated that anoxia and the presence of nitrate are required for maximal expression of E. meliloti napA, nirK, norC and nosZ denitrification genes (Figure 2). Furthermore, anoxically incubated cells also expressed MV+-NR, MV+-Nir, Nor and Nos activities (data not shown). However, haem staining analyses of WT cells incubated under anoxic conditions with nitrate showed a strong defect of FixP, FixO as well as NorC expression compared to microoxic conditions (data not shown). Taken together, these results suggest that the inability of E. meliloti to growth anoxically with nitrate is not due to a defect in the expression of the denitrification genes or in the activity of the denitrification enzymes. It might be possible that the low protein level could be one of the reasons of the limited growth of E. meliloti under anoxic conditions with nitrate. ACKNOWLEDGMENTS This work was supported by Fondo Europeo de Desarrollo Regional (FEDER)-cofinanced grant AGL2010-18607 and grant AGL2009-10371 from Ministerio de Economía y Competitividad. REFERENCES Barnett, M.J., et al. (2001). Proc. Natl. Acad. Sci. U.S.A. 98:9883-9888. Becker, A., et al. (2004). Mol Plant Microbe Interact 17:292-303. Becker, A., et al., (2009). J. Biotechnol. 140: 45-50. Bobik, C., et al. (2006). J. Bacteriol. 188:4890-4902. Bueno, E., et al. (2010). Environ. Microbiol. 12: 393-400. Horchani, F., et al. (2011). Plant Physiol. 155:1023-1036. Meilhoc, E., et al. (2010). Mol. Plant. Microbe Interact. 23:748-759. Torres, M.J., et al. (2013). J. Appl. Microbiol. doi: 10.1111/jam.12168. 152 Session II SII-CP-22 Genome sequence of Herbaspirillum rubrisubalbicans M1, an endophytic diazotroph and mild phytopathogen of sugarcane. Souza, E. M.*, Chubatsu, L., Cardoso, R.A., Raittz, R.T., Weiss, V.A., Monteiro, R.A., Faoro, H., Wassem, R., Baura, V.A., Balsanelli, E., Huergo, L., Muller-Santos, M., Tadra-Sfeir, M., Cruz, L.M., Pedrosa, F.O. Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, C. Postal 19046, 81531-980, Curitiba, PR, Brasil. * [email protected] INTRODUCTION H. rubrisubalbicans strain M1 is capable of developing mild disease symptoms in susceptible some varieties of sugarcane such as B4362. It is also capable of endophytic colonization of non-susceptible sugarcane, maize and sorghum without producing disease. Despite the clear symptoms observed upon inoculation of sugarcane B-4362, H. rubrisubalbicans is used as a component of a bacterial consortium recommended by the Brazilian Agricultural Research Company (EMBRAPA) as a commercial inoculant for sugarcane. This bacterial mixture can supply part of the nitrogen required by sugarcane and increase productivity, representing a positive impact on the sugarcane business by saving chemical fertilizers MATERIAL AND METHODS The H. rubrisubalbicans genome was sequenced using mate-paired reads from 454 FLX Titanium Roche Pyrosequencer and from Sanger automatic sequencing. De novo genome sequence assembly was performed using GS de novo Assembler v2.0 (Newbler; Roche). A total of 472 contigs were obtained, with 79 contigs ordered in 9 scaffolds. The scaffolds were further ordered using H. seropedicae SmR1 complete genome sequence as a reference. Potential protein-coding regions were identified by RAST. Probable functions of translation products of potential CDS were inferred using the Blast software by searching public databases RESULTS AND DISCUSSION The reads were assembled to produce a genome sequence of 5,611,430 bp encoding 4673 CDS. Among the traits potentially involved in both beneficial and pathogenic interaction of this microorganism and graminaceous plants we found genes for auxin biosynthesis, ACC deaminase, hydrolytic enzymes, adhesins and protein secretion systems. Comparison of proteins encoded by H. rubrisubalbicans M1 genome against the CAZy database revealed 1putative glycoside hydrolase of the GH8 family involved in plant cell wall degradation. Adhesins are involved in bacterial adhesion to host cells and biofilm formation. Genes encoding proteins of the T1, T3 and T4 pili were found in H. rubrisubalbicans. Furthermore, H. rubrisubalbicans has 21 genes encoding adhesins of the nonfimbrial group. Genes encoding proteins from the general secretory pathway (Sec pathway), Tat pathway and bacterial secretion systems type I, type II, type III, type V and type VI (two T6SS clusters) were found in the H. rubrisubalbicans genome. In addition, at least 10 genes encoding putative T3SS effector proteins were identified. Among these, the Hrop4 effector protein shares homology with the type III effector protein AvrPto1 from Pseudomonas syringae pv. Lachrymans. In addition, H. rubrisubalbicans M1 has at least six genes encoding proteins secreted by T6SS. 153 Session II SII-CP-22 Homologous proteins in other organisms have been shown to be involved in pathogenicity. Comparison of M1 genome sequence with that of 8 Herbapirillum spp. revealed unique regions spanning several genes, indicating putative horizontally transferred genes. The nif and T3SS gene clusters were in these variable regions suggesting that these genes are in a genomic island. The genome comparison allowed identifying the core- and pangenome of the genus, which consist of 1,526 and 13,874 genes, respectively. Functional distribution of the core-genome showed a high number of genes involved in the following COG categories: amino acid transport and metabolism; energy production and conversion; translation, ribosomal structure and biogenesis; and general function prediction only. On the other hand, the pan-genome contains a relatively low frequency of genes distributed in the COG functional categories. Conversely, the proportion of non-COG genes (genes with no homologous in COG database) is substantially higher in the pan-genome (72.6 %) than in the core-genome (15.9 %). The result suggests that genes of the pan-genome are responsible for the main the differences among the Herbaspirillum species and/or sub-groups of the genus. ACKNOWLEDGEMENTS We thank the Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio/CNPq. 154 Session II SII-CP-23 Sinorhizobium meliloti differentially expressed non-coding RNAs modulating nitrogen fixation and cell cycle progression. Robledo M.*, Frage B., Schlüter J.P., Becker A. Center for Synthetic Microbiology (SYNMIKRO), University of Marburg, Germany * [email protected] ABSTRACT In order to investigate the role of ncRNAs in post-transcriptional regulation of symbiosis-related genes, in this work we first focus on ncRNAs candidates orientated in antisense or overlapping the 5’- or 3’-untranslated regions (UTRs) of protein encoding genes located in pSymA that are required for nodule formation and nitrogen fixation. Interestingly, some transcripts accumulate differentially in response to different environmental factors, and seem to repress nif genes under conditions in which functional nitrogenase is dispensable. In addition, we study a broad conserved chromosomal trans-encoded RNA whose overexpression leads to elongated polyploid bacteroid-like cells due to interaction with mRNA derived from cell-cycle related genes at transcriptional or translational level . Therefore, this work contributes to unravel sRNA-mediated modulation of nitrogen fixation and cell cycle progression in rhizobia. INTRODUCTION Transcripts that do not encode proteins (ncRNAs) have emerged as a widespread type of regulators in all domains of life. They usually modulate translation and stability of mRNAs through base pairing. Antisense ncRNAs normally are encoded in the region that they regulate. In contrast, trans-ncRNA genes map separately from their target genes and show more limited complementarity. It is tempting to speculate that processes relevant to symbiosis are modulated by ncRNAs, especially those related to bacterial adaptation during the transition from free-living to host conditions, e.g. induction of root nodules, invasion and completion of the morphological differentiation process to become bacteroids capable of fixing nitrogen. However, nothing is hitherto known about the biological relevance of ncRNAs in symbiosis. Recently, a RNA-sequence approach delivered ~ 17,000 experimentally mapped transcriptional start sites (TSS) in S. meliloti that were assigned to protein-encoding genes or non-coding transcripts (Schlüter et al., 2013) showing an over-representation of trans- and cis-encoded ncRNAs on pSymA, specially in the region including the symbiotic genes. Besides this, sequence conservation analyses suggest strong similarities of several chromosomal trans-encoded RNAs to genomic regions in related -proteobacteria (Reinkensmeier et al., (2011), but the functions of the vast majority remain to be assigned. MATERIAL AND METHODS The following assays were basically performed as described: Northern analysis (Wilms et al., 2012), nodulation assays (Robledo et al., 2008), double plasmid assay (Sharma and Vogel, 2009) and microarray hybridizations (Bahlawane et al., 2008). RESULTS AND DISCUSSION The screening of antisense sRNA candidates that overlap partially or completely with annotated ORFs related with symbiosis lead to a great abundance of nod, fix and nif genes that harbour a sRNA encoded on the reverse strand. Between them, we chose sRNAs overlapping genes encoding essential proteins either for nodulation (NodD2) or nitrogen fixation (NifK and NifE). We confirmed seven of these candidates by Northern 155 Session II SII-CP-23 blot analysis and monitored its expression in different growth phases and media, stress shifts, luteolin and plant root exposure, nodule and low O 2, micro-oxic and conditions, inducing the expression of fix and nif genes, respectively. Hybridization signals corresponding to small RNA transcripts (≤120 nt), were reliably detected. The two longer sRNA candidates showed more than one signal, suggesting that they are processed. Three of the identified nif-related sRNAs are differentially expressed under different conditions including O2 and N2 availability, and in nodules. These asRNAs are repressed under conditions in which functional nitrogenase is required, suggesting a role in N2-fixation regulatory pathways. Furthermore, plasmid-based expression of one of this asRNA resulted in a slight decrease in nitrogen fixation. These experiments provided the first validation of rhizobial cis-acting riboregulators in response to diverse abiotic and symbiotic conditions. On the other hand, the single copy trans-ncRNA EcpR1 (elongated cell phenotype RNA1) is upregulated at late stationary phase and under several stress conditions. Flow cytometry analysis showed that after overexpression of EcpR1 the majority of cells present two or more chromosomes, suggesting that DNA replication uncoupled from cell division resulting in filamentous cells. This elongated phenotype is conserved among other rhizobia, and seems to be independent of the RNA chaperone Hfq. Using an eGFP-based reporter system we observed EcpR1-dependent altered mRNA translation rates for cell-cycle related genes. However, overexpression of an EcpR1 mutant version carrying nucleotide exchanges in the conserved loop motif predicted to bind mRNAs led to wild type-like cells and alleviated the altered translation rates. Concomitantly, 3nt-changes in the predicted target mRNA region also restored the reporter fluorescence, showing that this motif is responsible for the sRNA-mRNA interaction. Transcriptome studies revealed known and new cell cycle homologs in S. meliloti among the differentially expressed genes. Interestingly, 3´translational chromosomal integrated fusions of cell cycle related genes (Greif et al., 2010) revealed similar protein accumulation patterns in elongated EcpR1-overexpressing cells and in bacteroids. Our results suggest that EcpR1 is part of a regulatory network connecting stress adaptation and cell cycle. To our knowledge, this is the first sRNA that mediates regulation of cell cycle progression related genes in bacteria. ACKNOWLEDGES This work was supported by Humboldt and German Research Foundation (SPP 1258). REFERENCES Schlüter, J.P., et al. (2013). BMC Gen 14: 156. Reinkensmeier, J., et al. (2011). Genes 4: 925-952. Wilms, I., et al. (2012). Mol. Microbiol. 19: 5209-5217. Robledo, M., et al. (2008) PNAS 105: 7064-7069. Sharma, C.M., and Vogel, J. (2009). Curr. Opin. Microbiol. 12: 536-546. Bahlawane, C., et al. (2008). Mol. Plant Microbe Interact. 21: 1498-1509. Greif, D., et al. (2010). J. Biotechnol. 149: 280-288. 156 Session III Symbiotic plant/microbe Interactions Session III SIII-P-1 Priming plant defences by beneficial soil microorganisms Pozo, M.J.*, López-Ráez, J.A., Barea, J.M., Azcón-Aguilar, C. Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín. Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain. * [email protected] ABSTRACT Beneficial soil microorganisms are frequent in nature and confer major benefits to plant fitness and ecosystems dynamics. They are able to promote plant growth and to increase the plants’ ability to cope with biotic and abiotic stresses. In particular, some microorganisms are able to reduce damages caused by potential deleterious organisms including microbial pathogens and phytophagous insects and even parasitic plants. Several mechanisms may operate during this bioprotection, including direct interactions in the rhizosphere and plant mediated effects, in which alterations in plant nutrition and activation of plant defence mechanisms play major roles. Generally, during the interaction of the plant with the beneficial microbe a mild, but effective activation of the plant immune responses occur. This activation can lead to a primed state of the plant that allows a more efficient activation of defence mechanisms in response to attack by potential enemies. As a consequence, it is common that beneficial microorganisms increase the plant resistance to pathogens and pests, a phenomenon known as induced systemic resistance (ISR). Multiple examples illustrate that ISR relies in priming of jasmonate regulated plant defense mechanisms, as in the case of ISR triggered by arbuscular mycorrhizal fungi (AMF) and Trichoderma, and by beneficial bacteria from different genera as Pseudomonas, Bacillus and Micromonospora. INTRODUCTION Rhizosphere microbes have different trophic/living habits and establish a variety of saprophytic or symbiotic relationships with the plant, either detrimental or beneficial (Barea et al., 2013). The plant has to constantly fine-tune its immune responses to promote interactions with beneficial microbes while restricting the access to deleterious ones. Indeed, beneficial organisms interacting with plant roots, like pathogens, are confronted with the plant innate immune system and colonization success essentially depends on the evolution of strategies for mutual recognition and/or immune evasion. The modulation of plant defence responses by microbial symbionts aids establishing the delicate balance between the two partners (Pozo and Azcón-Aguilar 2007; Soto et al., 2009; Zamioudis and Pieterse 2012). As a consequence of a successful interaction, alterations in the plant primary and secondary metabolism are common and they may affect plant responses to the environment, usually increasing the plant ability to cope with stresses. The activation of the surveillance system and priming of defences is manifested not only in roots but also in aboveground plant tissues, thereby providing the plant with an ISR effective against a broad spectrum of plant pathogens and herbivorous insects below and aboveground (Pozo and Azcón-Aguilar 2007; van Wees et al. 2008, Pineda et al., 2010). Considerable research efforts have been devoted in the last decades to unravel the signalling mechanisms regulating ISR, and key regulatory elements have been identified. The role of defense signalling in the interaction between environmental conditions and the success of ISR is also a challenge for research (Pineda et al., 2013). Session III SIII-P-1 PROTECTION AGAINST PATHOGENS AND PESTS Most of the early studies on biocontrol of plant diseases by beneficial soil organisms focused on their direct ability to interact with soil borne pathogens, mainly through direct mechanisms as production of antibiotics, mycoparasitism, and/or competition for nutrients in the rhizosphere. In most cases, several of these mechanisms operate simultaneously, and in addition, changes triggered by the beneficials in the host plant also contribute to the alleviation of the damage: changes in root morphology and exudations may alter the dynamics of pathogen infection, and the activation of the plant defense mechanisms also contributes to resistance. But the enhanced resistance is not only restricted to root pathogens. Beneficial soil organisms can also impact plant interactions aboveground. The pathogens’ lifestyles have been shown to be crucial for the outcome of the interaction, especially in the biocontrol of shoot diseases by mycorrhizal symbioses (Pozo and Azcón-Aguilar 2007, Jung et al. 2012). As direct interaction between the beneficial and the pathogen is excluded in these systems, two main mechanisms drive the effect on aboveground interactions: i) the improvement of the nutritional status of the host plants and/or alterations of the source-sink relation that may help a plant overcome an herbivore attack or quickly recover after the attacker has been fought off and ii) the potentiation of plant defence mechanisms. These mechanisms may also impact herbivorous insects (Pineda et al., 2010). The final impact on insect performance will depend on the interplay between a positive effect derived from the enhanced plant growth and a negative effect derived from the induced resistance in the plant, and is largely variable depending on the type of the attacking insect. Generalist insects, able to feed on diverse plants and sensible to the plant defence mechanisms, are usually negatively affected by the presence of resistance inducers. However, specialist insects, which feed from one or only a small number of host species and show a high degree of adaptation to their hosts’ defence responses, may perform better on the symbiotic plants, probably because of the improved nutritional quality of the host (Pineda et al., 2010; Jung et al., 2012). MODULATION OF THE PLANT’S IMMUNE SYSTEM Both symbiotic and pathogenic fungi are initially recognized as alien organisms through conserved microbe associated molecular patterns (MAMPs), and the plant reacts with the activation of an immune response. This defensive response is not completely abolished but maintained to a minimum level to keep colonization under an uncritical threshold. Hence, active interference with the plant immune system is fundamental for the establishment of intimate mutualistic relationships (Soto et al., 2009, Zamidious and Pieterse, 2012). A prime example of plant immune modulation by microbes can be found in the intimate symbiotic relationship between plants and AMF, where successful recognition between both partners leads to a massive colonization of the plant’s root cortex by the fungus. AMF behave as biotrophic microorganisms and thus a quick but transient increase of endogenous salicylic acid (SA) occurs in the roots with a concurrent accumulation of defensive compounds. These initial reactions are temporally and spatially limited compared to the reaction during plant-pathogen-interactions, suggesting a role in the establishment or control of the symbiosis (García-Garrido and Ocampo 2002). In later stages AM fungi evade and manipulate the host innate immune system to promote successful colonization. Not only SA, but also the levels of other phytohormones related to defence, such as abscisic acid (ABA), ethylene (ET) and particularly jasmonic acid (JA) are altered during the plant interaction with the AM fungus (López-Ráez et al., 2010). The modulation of plant defense signalling system by AMF seems to play major roles in mycorrhiza-induced resistance. The dependence of a successful mycorrhiza on JA and SA signalling would explain the range of protection Session III SIII-P-1 conferred by this symbiosis (Pozo and Azcón-Aguilar 2007; Jung et al. 2012): mycorrhizal plants are more resistant to necrotrophs and chewing insects, which are targeted by JA-dependent defence responses, while the resistance to biotrophs is not improved, targeted by SA-regulated defences. This pattern correlates with an activation of JA-dependent defences and repression of SA-dependent ones in a well-established mycorrhiza. Similarly, JA signalling is likely to be manipulated by Piriformospora indica to suppress both early (reactive oxygen species production) and late (SAmediated responses and indole glucosinolate production) defence responses. This suggests that activation of the JA signalling pathway may be key in the control of plant interactions with beneficial endophytic fungi (Van Wees et al. 2008). The induction of resistance by beneficial microbes does not necessarily require direct activation of defence mechanisms but can result from a sensitization of the tissue to express basal defence mechanisms more efficiently after subsequent pathogen attack. This priming of the plant’s innate immune system has important fitness benefits compared to direct activation of defences (Van Hulten et al. 2006; Van Wees et al. 2008; Conrath 2009) and is common in beneficial interactions. Induction of the primed state is usually associated with a moderate accumulation of defence-related regulatory molecules, such as transcription factors or MAP kinases (Van der Ent et al. 2009; Pastor et al. 2012). For example, rhizobacteria-induced systemic resistance in Arabidopsis is related to priming of JA-dependent responses through the accumulation of MYC2, a transcription factor with a key role in the regulation of JA responses (Pozo et al. 2008). Examples of primed defence responses in mycorrhizal plants were first observed in root tissues. In tomato AMF colonization systemically protected roots against Phytophthora parasitica infection. Only mycorrhizal plants formed papilla-like structures around the sites of pathogen infection, preventing the pathogen from spreading further, and they accumulated significantly more PR proteins than non-mycorrhizal plants upon Phytophthora attack. Priming has been also involved in mycorrhizal induction of resistance against other pathogens and nematodes (reviewed in Jung et al., 2012). But the primed response is not restricted to the root system. AM symbiosis and Trichoderma colonization induce systemic resistance in tomato plants against the necrotrophic foliar pathogen Botrytis cinerea. While the amount of pathogen in leaves is significantly lower, the expression of some jasmonate-regulated, defence-related genes is higher in these plants (Jung et al., 2013, Martinez Medina et al., 2013). The use of tomato mutants impaired in JA signalling confirmed that JA is required for the induced resistance against Botrytis, confirming that ISR by beneficial fungi is similar to the well-studied rhizobacteria-induced systemic resistance (ISR) in Arabidopsis and requires a functional JA signalling pathway for the efficient induction of resistance (Pieterse et al. 1998). Recently, priming of JA dependent responses has been shown to mediate also Micromonospora induced resistance against Botrytis cinerea in tomato (Martinez-Hidalgo et al., unpublished). Thus, activation of the JA signalling pathway appears as a common mechanism in the induction of resistance by beneficial soil microorganisms. CONCLUSIONS While beneficial soil microbes may vary in their lifestyle, host range and the benefits they confer to the plant, experimental evidences point to important common points in the mechanisms mediating improved stress resistance. Interaction with the plant immune system and modulation of the jasmonate signalling pathway have been shown to be key elements in the induction of resistance. The identification of defense regulatory elements coordinating ISR is a major challenge for research and a crucial Session III SIII-P-1 step for the development of sustainable strategies for the integrated management of pests and diseases. Figure 1. Impact of beneficial soil microorganisms on plant biotic interactions. Interaction with beneficial soil microorganisms often leads to a primed state of the host plant that allows a more efficient a ctivation of defense mechanisms upon attack. As a consequence, the plant can be more resistant to herbivorou s insects and microbial pathogens below and aboveground. This priming of plant defenses relies on th e jasmonic acid (JA) signaling pathway. ACKNOWLEGMENTS This work was supported by projects AGL-2006-08029 and AGL2009-07691 of the Spanish Ministry of Science and Technology. REFERENCES Barea, J.M., et al. (2013) In: Molecular Microbial Ecology of the Rhizosphere (de Bruijn, F. Ed.) WileyBlackwell USA. pp. 29-44. Conrath, U., et al. (2006). Mol. Plant-Microbe Interact. 19: 1062-1071 Garcia-Garrido, J.M., et al. (2002). J. Exp. Bot. 53: 1377-1386. Jung, S.C., et al. (2012). J. Chem. Ecol. 38: 651-664. López-Ráez, J.A., et al. (2010). J Exp Bot 61: 2589-2601. Martinez Medina A., et al. (2013). Front. Plant-Microbe Interact. doi: 10.3389/fpls.2013.00206 Pineda, A., et al. (2010). Trends Plant Sci. 15: 507-514. Pineda, A., et al. (2013). Funct. Ecol. 27: 574-586. Pozo, M.J. et al. (2005). J. Plant Growth Regul. 23: 211-222. Pozo, M.J., and Azcón-Aguilar C. (2007). Curr. Op. Plant Biol. 10: 393-398. Pozo, M.J., et al. (2008). New Phyt. 180: 511-523. Soto, M.J., et al. (2009). Cell. Microbiol. 11: 381-388 Van der Ent, S., et al. (2009). Phytochem. 70: 1581-1588. van Hulten, M,. et al. (2006). PNAS 103: 5602-5607. Van Wees, S., et al. (2008). Curr. Op. Plant Biol. 11: 443-448. Zamioudis, C., and Pieterse C.M.J. (2012). Mol. Plant-Microbe Interact. 25: 139-150. Session III SIII-P-2 Una visión general del papel de los polisacáridos superficiales de Sinorhizobium fredii HH103 en su simbiosis con la soja y otras leguminosas. Ruiz-Sainz, J. E.*, Crespo-Rivas, J. C., Acosta-Jurado, S., Margaret, I., Buendía, A., Vinardell, J. M. Departamento de Microbiología, Universidad de Sevilla, España. * [email protected] INTRODUCCIÓN Las bacterias que hoy llamamos Sinorhizobium fredii fueron descritas por primera vez en 1982 (Keyser et al., 1982). Su descubrimiento causó una gran sorpresa en “el mundo de los rizobios” porque se demostraba que había bacterias de crecimiento rápido (1,5 a 3,0 horas de tiempo de generación) que nodulaban con la soja (Glycine max). Hasta ese momento, todas las bacterias que se aislaban de los nódulos de soja eran de crecimiento lento (6 o más horas de tiempo de generación) y se agrupaban en la especie Bradyrhizobium japonicum. Este primer trabajo, que se publicó en Science, tenía sin embargo un segundo mensaje que era poco alentador de cara a que, en un futuro, las estirpes de S. fredii fueran usadas como inoculantes comerciales para la soja: estas estirpes formaban nódulos fijadores de nitrógeno con las variedades asiáticas de soja, pero sólo inducían la formación de pseudonódulos en las variedades americanas de soja, que son las comúnmente utilizadas en muchas partes del mundo. Muchos laboratorios de investigación que iniciaron estudios sobre S. fredii dejaron de hacerlo. Esta situación cambió en 1985 cuando se describen nuevos aislamientos de S. fredii que sí son capaces de fijar nitrógeno tanto con las sojas asiáticas como con las americanas (Dowdle y Bohlool, 1985). La estirpe S. fredii HH103 pertenece a este nuevo lote de estires aisladas. S. fredii HH103 es plenamente efectivo con las sojas asiáticas, pero fija menos nitrógeno que B. japonicum USDA110 con las sojas americanas. Los estudios sobre S. fredii han seguido su curso en los últimos 30 años y, a día de hoy, podemos decir que es la especie más utilizada en los estudios de rizobios con amplio rango de nodulación. Tres son las estirpes que más se han estudiado: 1) S. fredii NGR234, aislada de Lablab y con el mayor rango de nodulación descrito (Pueppke y Broughton, 1999). Por razones desconocidas no nodula con la soja. 2) S. fredii USDA257, perteneciente al grupo de estirpes aisladas por Keyser et al. en 1982 y, por tanto, inefectiva con las variedades americanas de soja. Su rango de nodulación es muy amplio y aparece como un subconjunto del mostrado por S. fredii NGR234 (Pueppke y Broughton, 1999). 3) S. fredii HH103, capaz de formar nódulos fijadores de nitrógeno con todas las sojas probadas, aunque los niveles de fijación son mayores en las asiáticas que en las americanas hasta ahora probadas. Aparte de las sojas, el rango de nodulación de HH103 no parece diferir del de S. fredii USDA257 (Margaret et al., 2011). Se ha secuenciado el genomio completo de la estirpe S. fredii HH103 [ver póster presentado por J. M. Vinardell et al.; Weidner et al. (2012); Margaret et al. (2011)]. S. fredii HH103 es, por tanto, la bacteria mejor conocida entre aquellas que muestran un crecimiento rápido, un amplio rango de nodulación y son capaces de hacer simbiosis fijadoras de nitrógeno con todos los cultivares de soja ensayados. Session III SIII-P-2 LOS POLISACÁRIDOS SUPERFICIALES DE S. fredii HH103. Tres grupos de señales químicas bacterianas son los más estudiados en cuanto a su implicación en las simbiosis Sino/Meso/BradyRhizobium-leguminosa: I, El sistema compuesto por flavonoides (de la planta) y factores de nodulación (de la bacteria). Se puede encontrar información sobre estas señales químicas de S. fredii HH103 en los artículos Vinardell et al. (2004a) y Gil-Serrano et al. (1997). II, Las proteínas Nop (outer nodulation proteins) secretadas por el Sistema de Secreción de Tipo Tres (T3SS). Se puede encontrar información sobre el T3SS de S. fredii HH103 y sobre las Nops producidas en los artículos de Lyra et al. (2006) y López-Baena et al. (2009). III, Cuatro polisacáridos superficiales diferentes: glucanos cíclicos (GC), exopolisacáridos (EPS), polisacáridos capsulares tipo antígeno K (KPS) y el lipopolisacárido (LPS). Es a este grupo de señales de S. fredii HH103 al que dedicaremos el resto de este capítulo. Los glucanos cíclicos (GC) de S. fredii HH103. S. fredii HH103 produce beta-glucanos cíclicos (GC) no ramificados que constan de 18 a 24 residuos de glucosa, con o sin sustituciones de fosfoglicerol. Hay dos genes cromosómicos principalmente implicados en la producción de GC: ndvA y ndvB. La proteína NdvA está implicada en el transporte del GC al espacio periplásmico. La proteína NdvB, también llamada Cgs (Cyclic glucan synthase), es muy grande y estaría implicada en la todas las fases de la biosíntesis del GC. La mutación en ndvB mediante la inserción del cassette lacZΔp-GenR produce mutantes de S. fredii HH103 que son pleiotrópicos (Crespo-Rivas et al., 2009). Este mutante (SVQ562) no se mueve en los medios YMA (yeast-mannitol agar) o GYM (hipoosmótico), ni en este último suplementado con 100 mM de NaCl. Los tiempos de generación (tg) de HH103 y de SVQ562 en YMB (yeast-mannitol broth) y en GYM suplementado con 100 mM NaCl fueron iguales (aprox. 228 min). SVQ562 en medio GYM creció más lentamente (tg de 324 min, aprox.). Por tanto, como sucede con los mutantes ndvB de S. meliloti, los GC aparecen implicados en la tolerancia de la bacteria al estrés hipoosmótico. Sin embargo, el mutante SVQ562 es capaz de sobrevivir 3 días en agua destilada, cosa que no sucede con los mutantes ndvB de S. meliloti. Este hecho indica que, además de los GC, S. fredii debe tener otros mecanismos adicionales de protección frente a la hipoosmosis. SVQ562 no solamente produce más EPS que la estirpe silvestre HH103 sino que, además, este EPS contiene un mayor grado de sustituciones acetato y piruvato. El EPS producido por SVQ562 se distribuye en dos picos cromatográficos (size-exclusion chromatography) con respecto a su peso molecular, uno de unos 50-60 kDa (como el EPS de HH103) y otro de altísimo peso molecular que supera los 2.000 kDa. El gen exoA [glucosil transferasa que en S. meliloti coloca la primera glucosa en la cadena naciente (lípido-galactosa) del EPS] muestra mayores niveles de transcripción en el mutante SVQ562 respecto a HH103. El mutante SVQ562 sólo forma pseudonódulos en soja (nódulos de tipo determinado) y en Glycyrrhiza uralensis (nódulos de tipo indeterminado). Las raíces de Vigna unguiculata inoculadas con SVQ562 no muestran ninguna respuesta a nivel macroscópico. Por tanto, las mutaciones que eliminan la producción de glucanos cíclicos afectan tanto a las simbiosis formadoras de nódulos determinados como a las de Session III SIII-P-2 nódulos indeterminados. Es posible que, entre otras actividades, los GC actúen de señales para la planta. Así parece suceder con el fitopatógeno Xanthomonas campestris, cuyos GC suprimen diferentes reacciones de defensa en Nicotiana benthamiana. Los exopolisacáridos (EPS) de S. fredii HH103. La subunidad de repetición del EPS producido por S. fredii HH103 está ramificada y tiene D-glucopiranosa, D-galactopiranosa y ácido glucurónico (el poster presentado por Rodriguez-Carvajal et al. muestra la estructura química de este polisacárido). Esta estructura es igual a la descrita para Sinohizobium fredii NGR234. La producción de EPS por S. fredii HH103 se ve reducida si al medio de cultivo se le adicionan flavonoides (como la genisteína) que activen la transcripción de los genes de nodulación. Esta inhibición de la producción de EPS por flavonoides es dependiente de NodD porque los mutantes en nodD1 no muestran este fenómeno. Por otro lado, el incremento del número de copias de nolR (por clonación en un plásmido incP) provoca un aumento de la producción del EPS de S. fredii HH103. Así pues, en presencia de flavonoides, el gen nolR de HH103 actúa como un represor de la transcripción de los genes de nodulación y de la producción de proteínas Nop por el Sistema de Secreción de Tipo Tres (T3SS) y, a su vez, como un activador de la producción de EPS. Estos resultados casan bien con el hecho de que un mutante en el gen exoA de S. fredii HH103 no produzca EPS y, aparentemente, no muestre deficiencia simbiótica con la soja (Parada et al., 2006). Es más, este mutante (SVQ530) es más competitivo que su estirpe parental HH103 (productora de EPS) para nodular en la variedad Williams de soja y menos competitivo con Vigna unguiculata (cowpea). Además, el incremento del número de copias de nolR en HH103 (que entre otras cosas produce una sobreexpresión del EPS) reduc e la formación de nódulos con la soja y la aumenta con cowpea (Vinardell et al., 2004b). Estos hechos ilustran una vez más que, aunque los Brady/Sinorizobios que nodulan con la soja también nodulan siempre con cowpea, los mecanismos de nodulación, o los sistemas de percepción de señales en estas dos leguminosas, deben tener diferencias notables. Los polisacáridos capsulares tipo antígeno-K (KPS) de S. fredii HH103. Estos polisacáridos ricos en Kdo, o derivado de Kdo, sólo se han descrito y estudiado en S. meliloti y S. fredii. En S. meliloti AK631 se han definido tres regiones de genes involucrados en la producción del KPS (síntesis de la subunidad de repetición, polimerización y/o transporte). Estas regiones (llamadas rkp-1, rkp-2 y rkp-3) también se encuentran en S. fredii HH013. Las regiones rkp-1 y rkp-2 se localizan en el cromosoma y la rkp-3 en el mayor plásmido (pSfHH103e, de 2.096.125 pares de bases). La estructura química del KPS de S. fredii HH103 consiste en un homopolisacárido de un derivado del ácido pseudoamínico (Gil-Serrano et al., 1998). Esta estructura es única entre los KPS de las estirpes de S. meliloti y S. fredii analizados, que suele consistir en un disacárido en el que uno de los componentes puede ser el Kdo, el Kdn (derivados de los ácidos octulosónico y nonulosónico, respectivamente) o un derivado de este último. En S. meliloti es suficiente que la bacteria produzca EPS o un KPS simbióticamente activo para que la bacteria pueda nodular. Si no produce ninguno de los dos sólo se forman pseudonódulos en las raíces de Medicago sativa. Esta situación es diferente en S. fredii HH103 porque la no producción de KPS conlleva un deterioro de la capacidad simbiótica con la soja independientemente de que se produzca, o no, el EPS. Las raíces Session III SIII-P-2 de soja Williams inoculadas con mutantes en genes de la región rkp-1 forman muchos pseudonódulos y algunos nódulos. Estas plantas muestran claros síntomas de hambre de nitrógeno (Parada et al., 2006; Hidalgo et al., 2010; Margaret et al., 2012a). Los genes rkp-1 están involucrados en la biosíntesis de un lípido que debe intervenir en la biosíntesis y transporte del KPS. Los mutantes en la región rkp-3 (genes de la biosíntesis de la subunidad de repetición del KPS) solo inducen pseudonódulos en la soja (Margaret el al. 2012b). Estos mutantes, a diferencia de la mayoría de los genes de la región rkp-1, también tienen alterado el LPS. Los mutantes en la región rkp-2 (lpsL y rkpK) no están involucrados en la síntesis de KPS. Los mutantes de S. fredii HH103 en estos genes están afectados negativamente en la producción de EPS y biopelículas, el perfil electroforético de su LPS está alterado y tienen un retraso en la nodulación con la soja (ver póster presentado por Acosta-Jurado et al.). Aunque el KPS es importante para la capacidad simbiótica de S. fredii HH103 con la soja, los mutantes rkp no parecen estar afectados en su simbiosis con V. unguiculata. El lipopolisacárido (LPS) de S. fredii HH103. Se desconoce la estructura del LPS de S. fredii HH103. Se han mutado los genes lpsB y lpsE (glicosil transferasas que actúan en la biosíntesis del core del LPS. Los mutantes en lpsB y lpsE tienen alterado el perfil electroforético del LPS y forman muchos pseudonódulos y algunos nódulos de morfología externa normal en las raíces de soja. Las células infectadas en estos nódulos muestran claros signos de senescencia prematura, posiblemente asociada a reacciones de defensa de la planta (paredes de las células vegetales muy engrosadas y acumulación de compuestos fenólicos). Todos estos resultados indican que el LPS de S. fredii HH103 no solamente es importante en las primeras fases de la nodulación con la soja sino también en la estabilidad de los nódulos maduros (Margaret et al., 2013, aceptado con “minor revision” en PLOS ONE). AGRADECIMIENTOS Los trabajos aquí presentados han sido parcialmente subvencionados por los proyectos de Excelencia de la Junta de Andalucía P07-CVI-02506 y P11-CVI-7500 y por los proyectos BIO2008-05736-C02 y BIO2011-30229-C02-01 del Ministerio de Ciencia e Innovación. BIBLIOGRAFÍA Crespo-Rivas, J.C., et al. (2009). Mol. Plant-Microbe Interact. 22: 575-588. de Lyra, M.C.C.P., et al. (2006). Int. Microbiol. 9: 125-133. Dowdle, S.F., and Bohlool, B.B. (1985). Appl. Environ. Microbiol. 50: 1171-1176. Gil-Serrano, A.M., et al. (1997). Carbohydr. Res. 303: 435-443. Gil- Serrano, A.M., et al. (1998). Biochem J. 334: 585-594. Hidalgo, A., et al. (2010), Microbiology 156: 3398-3411. Keyser, H.H., et al. (1982). Science 215: 1631-1632. López Baena, F.J., et al. (2009). Mol. Plant-Microbe Interact. 22: 1445-1454. Margaret, I., et al. (2011). J. Biotechnol. 155: 11-19. Margaret, I., et al. (2012a). Arch. Microbiol. 194: 87-102. Margaret, I., et al. (2012b). Mol. Plant-Microbe Interact. 25: 825-838. Parada, M., et al. (2006). Mol. Plant-Microbe Interact. 19: 43-52. Pueppke, S.G., and Broughton, W.J. (1999). Mol. Plant-Microbe Interact. 4: 293-318. Vinardell, J.M., et al. (2004a). Arch. Microbiol. 181: 144-154. Vinardell, J.M., et al. (2004b). Mol. Plant-Microbe Interact. 17: 676-685. Weidner, S., et al. (2012). J. Bacteriol. 194: 1617-1618. Session III SIII-CO-1 Boron deficiency affects rhizobia cell surface polysaccharides. Quijada, N.M., Cerda, M.E., Abreu, I. *, Pérez de Nanclares, M., Bonilla, I., Bolaños, L. Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid. Madrid. * [email protected] ABSTRACT Boron (B) deficiency negatively affects the legume–rhizobia symbiotic interaction and the development of N2-fixing nodules. Many described alterations are related to plantderived carbo hydrates involved in plant–microbe interactions; however, the effects of B on the bacterial polysaccharides that are crucial for correct symbiosis are unknown. Growth of rhizobial strains was slightly affected by B deprivation; however exopolysaccharide (EPS) production and lipopolysaccharide electrophoretic profiles were altered in several strains grown in B-free media. These results provide evidence of a role of B in bacteria that can diminish the infection capacity of rhizobia. We are already analysing whether boron deficiency is affecting the expression of genes involved on bacterial cell surface polysaccharide synthesis or it is stabilizing bacteria cell surface by interacting with synthesized polysaccharides. INTRODUCTION Boron (B), a micronutrient required for plants and other organisms in micromolar concentrations plays an essential role in rhizobia-legume symbioses. As in other plant tissues, B is needed for the maintenance of nodule cell wall structure, and the involvement of B in each step of the symbiosis, from early preinfection and infection events until symbiosome development and nodule organogenesis, has now been established. The strong effect of B on legume–rhizobia symbiosis has always been related with the structure and stability of plant-derived components crucial for nodule development. As a major role, B has been involved in the modulation of cell surface interactions among legume glycoconjugates and bacterial cell surface polysaccharides important for the development of a symbiotic rather than a pathogenic interaction (Bolaños et al., 2004). Actually, induction of pathogenesis-related proteins of the PR-10 family during development of B-deficient nodules has been reported. Moreover, we recently reported that B starved Rhizobium leguminosarum apparently lacks a capsule of surface polysaccharides, leading to abortion of infection (Reguera et al., 2010), similar to the phenotypes described for mutants affected in cell surface polysaccharide production. To test this hypothesis, the effects of B deficiency on the expression of key genes related with cell surface polysaccharides, on exopolysaccharide (EPS) production and on lipopolysaccharide (LPS) structure, as well as the sensitivity of B-deprived rhizobia to several stresses is being analysed. MATERIAL AND METHODS Bacterial growth. R. leguminosarum 3841, Sinorhizobium meliloti 1021 and S. meliloti 8530 grew in defined liquid or solid (1% B-free agarose) minimal media (MM). Boron deficient media were prepared eliminating boron traces with the resin Amberlite IRA743 (Asad et al., 1997). EPS quantification. EPS were extracted from supernatant using acetone, lyophilized and weighed. Alternatively, EPS I synthesis was assayed by fluorescence in solid nutrient plates supplied with Calcofluor. Session III SIII-CO-1 LPS isolation and electrophoresis. LPS were isolated in SDS buffer, and after treated with proteinase K. Then LPS were separated by Tricine-SDS-PAGE and silver stained (Shindu et al., 1990). RESULTS AND DISCUSSION Boron deficiency affects EPS production and LPS electrophoretic profile. In a previous report (Reguera et al., 2010), we observed that rhizobia inside the infection thread apparently lacked a polysaccharide capsule, and that R. leguminosarum 3841 grew in boron-free media developed non mucoid colonies. In the present work we analyse the effect of boron deficiency on growth and in capsule formation of strains S. meliloti 1021 and 8530, because the role of B is mainly related with its interaction with carbohydrates, and the main components of the bacterial capsule and bacterial outer membrane, EPS and LPS, are enriched in sugars. Supporting that B is not essential for rhizobia we did not detected differences of growth in B-sufficient or B-deficient media. However, B-deficiency led to a 65–80% reduction in the amount of total EPS extracted from liquid cultures. Interestingly, when bacteria were platted in media supplied with Calcofluor to test EPSI production, the fluorescence was increased in the boron free plates. These results seem to be controversial, but because Calcofluor staining is not specific to EPSI, we suggest that the lack of B resulted in the increase of 1-4 bonds able to interact with Calcofluor. An analysis of sugar composition will be performed to clarify such observation. In order to deeply study the causes of the decrease of extractable EPS, we are now analysing whether boron is affecting expression of genes regulating the synthesis of both EPSI y EPSII. Concerning LPS, and similarly to what was reported as responses to different environmental changes, bacteria grown on boron deficient media show an alteration in the electrophoretic silver-stained LPS profile. Those alterations resembled some non-infective LPS mutants. As described by the EPS, we are trying to analyze if boron deficiency is altering gene expression or it is stabilizing these compounds. Moreover, we are trying to discover, if some components of the LPS are boron ligands, using an affinity chromatography with Amberlite IRA743. The results provide evidence that B is important for production of rhizobia cell surface polysaccharides essential to establish a symbiotic rather than a pathogenic-like interaction. Finally, we are studying whether alterations of bacterial cell surface in low B conditions increase sensitivity to stresses like salinity, high osmolarity o detergent pollution. In case of positive results, we could postulate that B is not essential for lab growing rhizobia, but indeed it is for environmental adaptation of the tested strains. ACKNOWLEGMENTS This work was supported by projects MINECO BIO2012-32796 and MICROAMBIENTECM Program from Comunidad de Madrid. N.M. Quijada is recipient of an Ayuda de Inicio de Estudios de Postgrado from UAM, and I. Abreu is recipient of a FPU fellowship from Ministerio de Educación (AP2010-4786) . REFERENCES Asad, A., et al. (1997). Ann. Bot. 80: 65-73. Bolaños, L., et al. (2004). Mol. Plant-Microbe Interact. 17: 216-223. Reguera, M., et al. (2010). Plant Cell Environ. 33: 2112-2120. Shindu, S.S., et al. (1990). J. Bacteriol. 172: 1804-1813. Session III SIII-CO-2 Trehalose is a key carbon regulatory player in the Legume-Rhizobium symbiosis. Sanchez, F.1*, Barraza, A.1, Estrada-Navarrete, G.1, Quinto, C.1, Merino, E.2 1 Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Autónoma de México. Av. Universidad # 2001, Col. Chamilpa. C.P.: 62210, Cuernavaca, México. 2 Departamento de Microbiologia Molecular, Instituto de Biotecnología, Universidad Autónoma de México. Av. Universidad # 2001, Col. Chamilpa. C.P.: 62210, Cuernavaca, México. * [email protected] Nacional Morelos, Nacional Morelos, ABSTRACT Trehalose metabolism during common bean (Phaseolus vulgaris L.) nodule ontology has been characterized by down-regulation of trehalase (PvTRE1) using RNAi. In addition, structural and phylogenetic analysis of trehalases suggested a high degree of evolutionary conservation of trehalose metabolism regulation. INTRODUCTION Trehalose is a ubiquitous non-reducing disaccharide that has been found accumulated in several plant-microorganism interactions (Paul et al., 2008). The nodule specific expression of PvTRE1 strongly suggests a functional role for trehalose in the symbiotic nodule (Barraza et al., 2013). The phylogenetic analysis unveiled three major branches that suggest a prokaryotic origin, a high degree for structural conservation and a marked trend for the conservation of the catalytic amino acids among trehalases from several phylogenetic origins (Barraza and Sanchez, 2013). In combination with experimental and bioinformatics approaches we were able to start unraveling the remarkable role for trehalase and trehalose in plant-microorganisms interactions and cell physiology. MATERIAL AND METHODS Rhizobium re-isolation viability assay. Bacteroid re-isolation from transgenic nodules and viability was assessed according to Müller et al. (2001). Acetylene reduction assay. Nitrogenase activity in trangenic nodules was determined by measuring acetylene reduction according to Vessey (1994). qRT-PCR measurements. Relative expression for the genes analyzed was determined using six biological replicates and data were normalized to the elongation factor 1 (EF1). Phylogenetic and structural analysis. Phylogenetic analysis were carried out with the neighbor-joining methodology coupled with 1000 bootstrap replicates. Structural analysis were performed with PyMol (DeLano, 2002). RESULTS AND DISCUSSION Rhizobium survival inside symbiotic nodules, nitrogen fixation, and trehalose content In common bean symbiotic nodules, PvTRE1 down-regulation by RNAi induced 78% increase of trehalose content. Interestingly, CFUs of re-isolated bacteria from these nodules increased by almost one order of magnitude, as well as nitrogenase activity (71%), without whatsoever negative side-effect on the aerial non-transgenic organs. In addition, the relative expression by qRT-PCR (Livak and Schmittgen, 2001), for genes directly involved in carbon metabolism and growth (SUS1, HXK1, TOR, SnRK1), autophagy (ATG3, Beclin), and nitrogen assimilation (GOGAT, GS) were assessed. In PvTRE1 silenced nodules transcript accumulation increase for SUS1 (203%), HXK1 (134%), TOR (43%), SnRK1 (81%), ATG3 (26%), GOGAT (80%) also suggests an Session III SIII-CO-2 increase in nitrogen assimilation (Barraza et al., 2013). Contrarily, for Beclin nosignificant fol d-changes were found (Barraza et al., 2013). Therefore, a positive effect on Rhizobium cell number and nitrogen fixation and assimilation; accumulation of transcripts of genes directly involved in carbon metabolism and growth and autophagy (ATG3), without the induction of Beclin 1 suggest autophagy activation towards cell recycling instead of programmed cell death. Trehalose phylogeny and structural conservation. The sequences analysis performed for trehalases from bacteria, animals, fungi and plants unveiled three major phylogenetic branches that suggest a prokaryotic origin for trehalases, and grouping plant and animal trehalases in the same branch, bacterial trehalases, and fungi trehalases (fungi acid trehalases in a discrete sub-clade). Structural analysis for all the trehalases deduced structures compared with the trehalase crystallographic structure (PDB code: 2WYN) gave RMSD values less than 0.1 Å, these results strongly suggest a structural conservation to maintain the function to regulate the trehalose content in the cell. In order to support this data, we carried a maximum likelihood analysis of natural selection codon-by-codon to determine a marked trend for synonimous substitutions mostly in the catalytic residues (Barraza and Sanchez, 2013). Altogether, the modification of trehalose metabolism in the symbiotic nodules shed light onto the mechanism that regulates the bacteroid survival, nitrogen fixation and the genes that directly orchestrate nodule physiology, and the marked trend for the structural conservation across taxonomic kingdoms to maintain the activity for trehalose content regulation. ACKNOWLEDGEMENTS This work was supported by Consejo Nacional de Ciencia y Tecnología (CB-2007-83324 and CB-20121777 44 to F. S.) with a postdoctoral fellowship (18134) to A. B. REFERENCES Barraza, A., et al. (2013). New Phytol. 197: 194-206. Barraza, A., and Sanchez, F. (2013). Plant Signal. Behav. 8 e24778. DeLano, W.L. (2002). DeLano Scientific, San Carlos, USA. Livak, K.J, and Schmittgen, T.D. (2001). Methods 25: 402-408. Müller, J., et al. (2001). J. Exp. Botany 52: 943-947. Paul, M.J, et al. (2008). Ann. Rev. Plant Biol. 59: 417-441. Vessey, J.K. (1994). Plant and soil. 158:151-162. Session III SIII-CO-3 RbohB-dependent reactive oxygen species regulates rhizobial infection, bacteroid development and nitrogen fixation in common bean. Arthikala, M.K., Montiel, J., Nava, N., Santana, O., Sánchez-López, R., Cárdenas, L., Quinto, C.* Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62271, México. * [email protected] ABSTRACT The role of RbohB during the symbiotic interaction between P. vulgaris (common bean) and Rhizobium tropici was assessed by RNAi and over-expression approaches using a hairy root system. The results obtained show that RbohB regulates the establishment and progression of the infection threads and also the number of nodules formed. Analysis of the nodule ultrastructure revealed the participation of RbohB in rhizobial colonization and in determining the number and size of bacteroids in infected cells of the bean nodules. INTRODUCTION Plant homologs of membrane-associated NADPH oxidases (Rbohs; respiratory burst oxidase homologs) have been associated with plant defense against pathogens (Torres et al. 2002). Recently, it has been reported that during the legume-rhizobia symbiosis, NADPH oxidases regulate the early steps of the bacterial infection in P. vulgaris (Montiel et al., 2012) and impair nodule functioning in M. truncatula (Marino et al. 2011). The PvRbohB transcript is the most highly expressed of the NAPH oxidases found in P. vulgaris roots and nodules. Its promoter becomes active in response to Rhizobium stimuli and was found associated with growing infection threads (Montiel et al,. 2012). In this study we present data on the effect of silencing and over-expression of PvRbohB in transgenic roots. MATERIAL AND METHODS Cloning and characterization of PvRbohB PvRbohB was cloned into pTdT-Dc-RNAi and pH7WG2D.1 binary vector respectively and expressed in P. vulgaris hairy roots. Transcripts were measured by RT-qPCR. ROS detection assay Accumulation of superoxide (O2¯) radicals was measured according to Ramel et al. 2009. Acetylene reduction assay Nitrogenase activity in transgenic nodules (at 28 dpi) was determined by measuring acetylene reduction as described by Ramírez et al. (1999). Electron microscopy of transgenic nodules Tissues were processed according to Sánchez-López et al. (2011) and examined with a Zeiss EM900 transmission electron microscope (Zeiss) at 50 kV. RESULTS AND DISCUSSION PvRbohB modulates ROS accumulation and rhizobia infection process in transgenic roots of the common bean. Herein, we cloned 3´ UTR of PvRbohB into pTdT-DC-RNAi and the open reading frame of PvRbohB into pH7WG2D.1 binary vectors for silencing and over-expression, respectively. The transgenic roots expressing the above constructs were analysed by RT-qPCR. Our results confirmed that PvRbohB-RNAi or PvRbohB-OE roots Session III SIII-CO-3 significantly down-regulated and over-expressed the transcript over the empty vector (control). The quantitative assay of NBT-formazan precipitation in transgenic roots showed a positive correlation with the PvRbohB transcript levels indicating an altered O2¯ accumulation (ROS) in silenced and over-expressed roots compared to controls. After Rhizobium tropici inoculation, the PvRbohB promoter was active at growing infection threads (ITs). At 7 dpi, the ITs prematurely aborted in the roots hairs of PvRbohB-RNAi and no such defect was seen in PvRbohB-OE roots; instead, the number of ITs significantly increased over controls. At four weeks, the total number of nodules reduced by ~80% in silenced roots and increased in over-expressed roots by ~13% compared to controls. Furthermore, nodules in silenced roots were unable to fix nitrogen (5.6±5.6 µmolC2H2/g NDW/h-1) whereas, in PvRbohB-OE nodules the efficiency increased 54% (88.6±7.1 µmolC2H2/g NDW/h-1) compared to control nodules (41.6±4.1 µmolC2H2/g NDW/h-1). Together, these results suggest that RbohB-dependant ROS production is not only required for infection thread progression but also for bacteroid development and nitrogen fixation in common bean. Analysis of the ultrastructure of transgenic nodules suggests that PvRbohB regulates the number and size of the bacteroids. To gain further insight into the architecture of PvRbohB-RNAi and PvRbohB-OE nodules, a transmission electron microscopy analysis was performed. The PvRbohB sparse silenced nodules show poorly colonized rhizobia in infected cells; in contrast, an enhanced number of bacteria per cell were found in PvRbohB over-expressed nodules. Tightly packed symbiosomes were very common in PvRbohB-OE nodules and no such phenotype was seen either in PvRbohB-RNAi or control nodules. Interestingly, the bacteroid size increased one order of magnitude, and also underwent additional volume change. Furthermore, accumulation of polyhydroxybutyrate granules increased noticeably in PvRbohB-OE nodules as compared in PvRbohB-RNAi or control nodules. Thus, we propose that PvRbohB functions in regulating size and number of bacteriods. The presence of a high number of polyhydroxybutyrate granules indicates the involvement of PvRbohB in directing carbon source to rhizobia. ACKNOWLEDGEMENTS This work was supported by Consejo Nacional de Ciencia y Tecnologìa (CB-2010-153718 to C.Q. and 132155 to L.C) with a post-doctoral fellowship (17656) to A.M.K. REFERENCES Marino, D., et al. (2011). New Phytol. 189: 580-592. Montiel, J., et al. (2012). Plant Cell Physiol. 53: 1751-1767. Ramel, F., et al. (2009). BMC Plant Biol. 9:28. Ramírez, M., et al. (1999). Mol. Plant-Microbe Interact. 12: 1008-1015. Sánchez-López R., et al. (2011). Plant Cell Environ. 34: 2109-2121. Torres, M.A., et al. (2002). Proc. Natl. Acad. Sci. USA. 99: 517-522. Session III SIII-CO-4 Quorum Sensing systems and flavonoids via NodD1 coordinate and regulate the biofilm transition in Sinorhizobium fredii SMH12, which is necessary for a successful root colonization of Glycine max cv Osumi. Pérez-Montaño, F.1*, Jiménez-Guerrero, I.1, Del Cerro, P.1, López-Baena, F.J.1, Ollero, F.J.1, Bellogín, R.A.1, Lloret, J.2, Espuny, M.R.1 1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012 - Sevilla, Spain. 2 Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid. 28049. Madrid. Spain. * [email protected] ABSTRACT The role and regulation of biofilm formation in S. fredii SMH12 during the symbiosis with Glycine max cv. Osumi has been studied. Results suggest that flavonoids, besides inducing the synthesis of Nod factors and other symbiotic molecules, potentiate the QS systems, and are necessary for the formation of biofilms in S. fredii SMH12. INTRODUCTION In all species studied until now, bacterial surface components, especially exopolysaccharides, flagella, and lipopolysaccharides are crucial for the formation of biofilms, in combination with the presence of bacterial QS signals. Biofilm formation allows soil bacteria to colonize their surrounding habitat and to survive to common environmental stresses such as desiccation and nutrient limitation. The biofilm mode of life is often crucial for survival in bacteria of the genera Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Rhizobium. However, no direct evidence has been already found that biofilm formation significantly promotes effective symbiosis with the legume host (Rinaudi and Giordano, 2010). MATERIAL AND METHODS Strains: wild type S. fredii SMH12, a nodD1 mutant, and a lactonase derivative strain were constructed. Nodulation assays on Glycine max (L.) Merrill cv. Osumi were performed as described by de Lyra et al. (2006). Colonization assays: rhizosphere attachment, epifluorescence microscopy and electronic barrier microscopy were carried out as described by Bacterial biofilm formation was studied by two complementary assays: measure of bacterial attachment to polystyrene surfaces (Pérez-Montaño et al., 2013) and observation of biofilm structure by confocal microscopy in crystal surfaces. Thin layer chromatography and quantitative RT-PCR from biofilm cultures were performed using a modified protocol described by Pérez-Montaño et al. (2011). RESULTS AND DISCUSSION In this work, the role and regulation of biofilm formation in S. fredii SMH12 during the symbiosis with Glycine max cv. Osumi has been studied. For this purpose and taking into consideration previous reports, the wild-type strain SMH12, a nodD1 mutant, and a lactonase derivative strain were constructed and tested in nodulation assays with the legume. Both strains negatively affect nodulation with respect to the wild-type strain. Three different colonization assays showed lower root colonization in both derivative strains, especially in the case of the strain that carries the lactonase gene. Furthermore, by means of adhesion assays and confocal microscopy, it was determined that in the presence of inducer flavonoids, SMH12 changed the type of biofilm formed from Session III SIII-CO-4 monolayer to microcolony (Figure 1). This process requires the presence of flavonoids and NodD1, and is coordinated by QS systems. Interestingly, these systems are activated only by inducer flavonoids. without flavonoid with nod gene inducer flavonoid WT nodD1lactonase+ Figure 1. Biofilm formation in SMH12 and two derivative strains in the presence of nod gene induce r flavonoids. Bacterial cultures were inoculated in 8-well chambers Lab-Tex Chamber Slide System (Thermo Fischer Scientific Inc., USA) and grown 4 days without shaking. Confocal microscope images were captured using a Leica TCS SP2 microscope (Leica Microsystems, Germany). In silico 3D reconstruction analysis was performed using the software Bitplane (Imaris software, Switzerland). All these results suggest that flavonoids, besides inducing the synthesis of Nod factors and other symbiotic molecules, potentiate the QS systems, and are necessary for the formation of biofilms in S. fredii SMH12. To our knowledge, this is the first report that unequivocally connect biofilm formation, root colonization and a successfully symbiosis between a rhizobial strain and its host legume. ACKNOWLEDGEMENTS This work was supported by grants AGL2009-13487-C04 from the Spanish Ministerio de Educación y Ciencia and AGR-5821 from the Junta de Andalucía, Consejería de Innovación, Ciencia y Empresa. REFERENCES de Lyra, M.C.C.P., et al. (2006). Int. Microbiol. 9: 125-133. Pérez-Montaño, F., et al. (2011). Res. Microbiol. 162: 715-723. Pérez-Montaño, F., et al. (2013). Res. Microbiol. In press. Rinaudi, L.V., and Giordano, W. (2010). FEMS Microbiol. Lett. 304: 1-11. Session III SIII-CP-01 Proteolytic control of the Bradyrhizobium japonicum transcriptional regulator FixK2. Fernández, N.1*, Bonnet, M.2, Stegmann, M.2, Maglica, Z.3, Weber-Ban, E.3, Hennecke, H.2, Bedmar, E.J.1, Mesa, S.1 1 Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain. 2 Institute of Microbiology, ETH Zurich, Zürich, Switzerland. 3 Institute of Molecular Biology and Biophysics, ETH Zurich, Zürich, Switzerland. * [email protected] ABSTRACT Here, we describe different approaches to study the posttranslational control of the Bradyrhizobium japonicum regulatory protein FixK2 by specific proteolysis, between valine 220 and leucine 221, and by general degradation mediated by ClpAP 1. Our results showed that, for both proteolytic processes, a C-terminal stretch of twelve amino acids within FixK2 protein sequence plays a crucial role. INTRODUCTION FixK2 is an important regulator involved in the control of different lifestyles of the facultative soybean endosymbiont B. japonicum. This transcription factor regulates several hundreds of genes required for microoxic, anoxic and symbiotic growth (Mesa et a1., 2008). FixK2 is an unusual member of the CRP/FNR superfamily, because it is active in vitro without an additional effector molecule (Mesa et a1., 2005) and is regulated posttranslationally by the oxidation of its singular cysteine residue (Mesa et a1., 2009). In addition to its oxidation-mediated control, FixK2 seems to be regulated by proteolysis. Despite the induction of fixK2 gene expression at low-oxygen concentrations, the steady-state levels of the FixK 2 protein remains constant regardless of the growth conditions (Mesa et a1., 2009). Further, a truncated variant is always copurified to gether with the full-length His6-FixK2 protein from FixK2-overproducing Escherichia coli cells. Moreover, this shorter FixK2 species is also present in cells of B. japonicum. In this context, it is worth mentioning that the C-terminal sequence of FNR, a FixK2-homologue in E. coli, plays a crucial role in the degradation of the inactive form of this protein which is mediated by the proteolytic system ClpXP, both in vivo and in vitro (Mettert and Kiley, 2005). MATERIAL AND METHODS DNA work and enzymatic activities were performed according to standard protocols. Protein expression and purification, and degradation assays were carried out as previously described (Bonnet et a1., 2013). In vitro transcription (IVT) assays were done according to Mesa et al. (2005). Markerless mutant strains were constructed as described elsewhere (Mesa et a1., 2009). RESULTS AND DISCUSSION FixK2 is C-terminally cleaved between residues V220 and L221 Mass spectrometric analysis revealed that the secondary form co-purified with the fulllength FixK2 is a C-terminally cleaved derivative (between V220 and L221), which lacks the last twelve amino acids. Next, we constructed a series of protein variants around the cleavage site. To limit the overall change in structure upon mutation, V220 and L221 were individually substituted to asparragine or threonine. In particular, the L221T FixK2 derivative was more prone to cleavage and showed reduced capacity to bind DNA and to activate transcription from a FixK2-dependent promoter in an IVT Session III SIII-CP-01 activation assay. Thus, the C-terminal part of FixK2 and particularly L221, have a role in FixK2 protein cleavage and activity. In order to rule out the putative function of the carboxy-terminal cleavage of FixK2 in its in vivo activity, different proteins variants are currently being tested with regard to the ability to complement a fixK2 strain. Degradation of FixK2 by ClpXP1 and ClpAP1 chaperone-proteases. In analogy to E. coli FNR, FixK2 might be proteolytically regulated by the B. japonicum Clp system. Therefore, the Clp proteins from B. japonicum, specifically ClpAP1 and ClpXP1, were purified and tested for their ability to degrade FixK2. Contrary to FNR, FixK2 is not a substrate for ClpXP1 but it was found to be a substrate for ClpAP1 in vitro (Figure 1A). This degradation was specific and was inhibited by the ClpS 1 adaptor protein (Figure 1A), indicating that FixK2 is a novel direct substrate for ClpAP1. By fusing C-terminal sequence stretches of FixK2 to green fluorescent protein (GFP), we showed that the last 12 amino acids of FixK2 play the main role in the recognition by ClpA (Figure 1B). A B Figure 1. (A) FixK2 is a direct substrate for ClpAP1. FixK2 degradation assay by ClpAP1 in the abs ence or presence of ClpS1. (B) The C-terminus of FixK2 is the main recognition site for ClpAP1. Degrada tion of different GFP derivatives was followed by fluorescence. Taken together, our results suggest the possibility that the ClpAP system is involved in the turnover of FixK2 in vivo. To confirm this hypothesis, clpA, clpP1, and clpP2 strains are currently being constructed to analyze the steady-state levels of FixK2 in different growth conditions and to elucidate whether FixK 2 degradation occurs continuously in the cells or at a particular scenario. Further, limited information is available about the role of Clp-like proteins in the symbiotic interaction of rhizobial species with leguminous plants. Hence, the putative function of Clp proteases in the symbiotic interaction B. japonicum-soybean is presently under investigation. ACKNOWLEDGEMENTS This work was supported by grants from ETH Zurich, the Swiss National Science Foundation, and the ERDF-cofinanced grant AGL2011-23383 from Ministerio de Economía y Competitividad of Spain. REFERENCES Bonnet, S., et al. (2013). FEBS Lett. 587: 88-93. Mesa, S., et al. (2005). J. Bacteriol. 187: 3329-3338. Mesa, S., et al. (2008). J. Bacteriol. 190: 6568-6579. Mesa, S., et al. (2009). Proc. Natl. Acad. Sci. USA. 106: 21860-21865. Mettert, E., and Kiley, P.J. (2005). J. Mol. Biol. 354: 220-232. Session III SIII-CP-02 Effect of temperature on ectomycorrhizal fungi associated with Pinus sylvestris L. in organic vs mineral soils. Gómez-Gallego, T.1, García-Rabasa, S.2, Flores-Rentería, D.2, Rincón, A. * 1 Group of Beneficial Plant-Microbial-Interactions, Instituto de Ciencias Agrarias, Consejo Superior de Investigaciones Científicas ICA-CSIC. 2 Museo Nacional de Ciencias Naturales MNCN-CSIC. * [email protected] ABSTRACT Ectomycorrhizal fungi of Pinus sylvestris L. were characterized in two contrasted soils. The effect of temperature on mycorrhization and fungal diversity was also tested. Eight fungi were identified by morphotyping and a total of thirteen by sequencing. Both soils had a similar number of fungi, although completely differed in the identity of the dominant ones. The number of fungi and ectomycorrhizas per seedling, together with fungal diversity significantly decreased at high temperature. Nearly half of fungi were not found forming mycorrhizas at the highest temperature and contrarily, only one fungus significantly increased in frequency with this factor. Our results emphasize the strength of environmental conditions affecting host mycorrhizal rates and structuring EM communities, which might have profound implications in forest dynamics. INTRODUCTION Pinus sylvestris is a representative ectomycorrhizal (EM) tree species highly spread in Europe. Ectomycorrhizas are the symbiotic association between roots and fungi in which basically, the fungus improves plant water and nutrient uptake in exchange of carbohydrates (Smith and Read, 1997). The EM symbiosis plays an essential role in plant nutrition and forest dynamics, which can be crucial especially under unfavourable environmental conditions such as those related to global change (i.e. drought, increased temperature) (Smith and Read, 1997). The objectives of this work were to characterize the EM fungi associated with P. sylvestris growing in two contrasted soils, and to check how different temperatures could affect the mycorrhizal status of seedlings and the diversity of their fungal associates. MATERIAL AND METHODS Soils were collected in a P. sylvestris forest (Valsaín, Segovia), in an altitudinal gradient: a) mineral soil at 1.250 m, and b) organic soil at 1.850 m. Seedlings were grown in containers filled with each soil, at three different temperatures (day/night): a) low 10/15 ºC, b) medium 15/20 ºC and c) high 20/25 ºC. A total of six treatments were established with four replicates in each. Four months after, EM percentages were assessed, and ectomycorrhizas classified by morphotypes (Agerer, 1995) and collected for DNA extraction (Rincón et al. 2007). The internal transcribed spacer region (ITS) of the rDNA was PCR-amplified with the primers ITS1F/ITS4 (Gardes and Bruns, 1993), the PCR-products digested by endonucleases (Hinf I, Msp I), and restriction patterns (RFLPs) analysed. Samples sharing identical RFLP profile were classed as unique Operational Taxonomic Units (OTU). Each OTU was sequenced and tentatively identified by comparison with sequences in the GenBank database. The relative frequencies of each fungal OTU were calculated and analysed by t-Student test (P ≤ 0.05). The EM percentages, the number of OTUs per seedling and the fungal diversity (Shannon index) were analyzed by one-way analysis of variance (ANOVA) and differences among treatments separated by the Tukey test (P ≤ 0.05). All analyses were done with the SPSS 19.0 software. Session III SIII-CP-02 RESULTS AND DISCUSSION Characterization of ectomycorrhizal fungi of organic and mineral soils Eight EM morphotypes were classified attending to morphological features, whereas sequencing allowed identifying a total of 13 different fungal OTUs. This highlighted the convenience of using a combined morphological and molecular approach for identifying EM fungi, which might have highly similar and/or cryptic characteristics leading to confusing identification (i.e. Pezizales) (Tedersoo et al., 2006). A total of 12 and 10 OTUs were respectively identified in the organic and mineral soil, with 69 % of OTUs found in both soils. Thelephora terrestris Ehrh., S. luteus and Unkown-5 were only found in the organic soil, and Unkown-9 only in the mineral one. In both soils, the fungal community structure was similar with few dominant fungi and many less frequent ones, although the specific composition clearly differed between soils. In the organic one, the most frequent fungi were: Unkown-3 (Archaeorhizomycetes), Suillus bovinus (Pers.) Roussel and T. terrestris, whereas in the mineral soil were Unkown-2 (Atheliaceae), Unknown-4, Cenococcum geophilum Fr. and Wilcoxinia sp. Fungi Unk-2 and Unk-4 significantly decreased in frequency in the organic soil compared with the mineral one (P = 0,00; P = 0,01), whereas the opposite happened to T. terrestris and S. bovinus (P = 0,04; P = 0,04). The results showed that both soils had a high potential of active EM fungal inoculum, but a totally different cohort of fungi distinctively adapted to mineral or organic conditions. Effect of temperature on seedling mycorrhization and fungal diversity The EM percentages (P=0,00), the number of fungi per sample (P=0,03), and fungal diversity (P=0,04) significantly decreased at high temperature. Reduced mycorrhization rates could indicate a lower symbiotic-dependence for nutrient uptake by the host, probably due to a enhanced photosynthetic activity at high temperature (Van derHeijden and Sanders, 2002). Nearly half of the fungi (46%) were not found forming mycorrhizas at the highest temperature, and three among them (Unk-5, Tomentella sp. and Unk-9) were only found at low temperature. The Unk-3 (Archaeorhizomycetes) was the only fungus that significantly increased in frequency in response to high temperature (P =0,04), even disappearing at the lowest one. Replacements of species and changes in the relative dominance of particular fungi denoted a variable inter-specific response pointing out to temperature as an important driver structuring EM fungal communities, which might have important consequences for the functioning of forest ecosystems (Van derHeijden and Sanders, 2002). ACKNOWLEGMENTS This work was supported by the projects S2009/AMB-1511 of the Comunidad de Madrid and CGL201129585 of the Spanish Ministry of Innovation and Science. REFERENCES Agerer, R. (1995). In: Varma A, Hock B (eds). Springer-Verlag. Berlin, Heidelberg. pp: 687-734. Gardes, M., and Bruns, T. (1993). Molecular Ecology 2: 113-118. Rincón, A., et al. (2007). Mycorrhiza 18: 23-32. Smith, S., and Read, D. (1997). Mycorrhizal Simbiosis. Academic Press. New York. pp: 605. Tedersoo, L., et al. (2006). New Phytologist 170:581-596. Van der Heijden, M., and Sanders, I., eds. (2002). Mycorrhizal Ecology. Springer-Verlag. Berlin, p p: 469. Session III SIII-CP-03 Ensifer meliloti is the preferred symbiont of Medicago arborea in eastern Morocco soils. Missbah El Idrissi, M.1, Guerrouj, K.2, Pérez-Valera, E.3, Abdelmoumen, H.2*, Bedmar, E.J.3 1 Laboratoire d’Amélioration des sols et Environnement, Ecole Normale Supérieure-Av. Mohamed Belhassan El Ouazzani-Takaddoum. BP: 5118. Rabat, Moroccco. 2 Laboratoire de Biologie des Plantes et des Microorganismes, Faculté des Sciences, Université Mohamed Premier, Oujda 6000. Morocco. 3 Department of Soil Microbiology and Symbiotic Systems. Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC). E-419, 18080-Granada, Spain. * [email protected] ABSTRACT Medicago arborea (Tree Medic) is the oldest species in the genus Medicago and the only one with a shrub habit. We carried out a polyphasic approach to identify its symbionts in different soils of the eastern Morocco. Although the high phenotypic and genetic diversity observed all the isolates belong to the species Ensifer meliloti. INTRODUCTION The main features in M. arborea are drought resistance, cold and salt tolerance, absence of summer and winter dormancy, evergreen plant, resistance to pathogens and it can reduce soil erosion (Elgin and Ostazeski, 1982; Andreu et al., 1994). De Mita et al. (2007) reported that Medicago species are nodulated by two symbiotic bacterial species of the genus Ensifer (Sinorhizobium); Ensifer medicae and/or E. meliloti but there are no data about the main symbionts of Medicago arborea species. In this work, we carried out an analysis of the phenotypic and genetic diversity of 61 bacteria isolated from this plant root nodules in different soils in Eastern Morocco identify the main symbionts of this medic tree in these areas. MATERIAL AND METHODS Sixty one bacteria were isolated from the root nodules of Medicago arborea plants grown for 3 month in four soils of eastern Morocco using the method of Vincent (1970). The isolates were first fingerprinted by ERIC PCR (Versalovic et al., 1991) to check for their genetic diversity and to avoid strains duplication. The phenotypic characterization consisted in the determination of 116 properties including physiological and cultural characteristics. PCR amplifications of 16S rRNA gene fragments were done using the two opposing primers fD1 and rD1 previously described (Weisburg et al., 2001). The pair primers nodCFn/nodCI was used for amplification of the nodC genes (Laguerre et al., 2001). Purified amplification products were subjected to cycle sequencing using the same primers as for PCR amplification, with ABI Prism Dye Chemistry, and analyzed with a 3130xl automatic sequencer at the sequencing facilities of Estación Experimental del Zaidín, CSIC, in Granada, Spain. RESULTS AND DISCUSSION The phenotypic, symbiotic and cultural characteristics analyzed allowed the description of a wide physiological diversity among the isolates. The results obtained suggest that the phenotype of these rhizobia might have evolved to adapt the local conditions. The genetic characterization consisted in an analysis of the rep-PCR fingerprints and the PCR-based RFLP of the 16S rDNA patterns. The isolates diversity was investigated by rep-PCR giving in similarity 62%, 3 clusters, 4 groups and 6 sub-clusters. The results show Session III SIII-CP-03 relationship between rep-PCR fingerprinting and sugar assimilation which are complementary in diversity investigation. The nearly complete 16S rRNA gene sequence from representative strains of each soil showed they are closely related to members of the genus Ensifer of the family Rhizobiaceae within the Alphaproteobacteria and shows the highest similitude values (99,93/100%) with Ensifer meliloti LMG 6133T (X67222). Sequencing of the symbiotic nodC gene from seven representative strains revealed they had 94.89% identity with the nodC sequence of the type strain Ensifer meliloti LMG 6133T (EF428922). Therefore, the 61 M. arborea isolates from the 4 different soils have the same phylogenetic affiliation, which proves the restricted host specificity among M. arborea species. B MI4 (JX524728) A ME5 (JX524735) MB9 (JX524731) E. meliloti LMG 6133T (X67222) MG2 (JX524730) ME21 (JX524736) ME1 (JX524733) 70 ME22 (JX524737) ME2 (JX524734) MI5 (JX524729) MB12 (JX524732) 80 E. numidicus ORS 1407T (AY500254) E. arboris LMG 14919T (AM181744) E. medicae WSM419T (CP000738) 100 E. garamanticus ORS 1400T (AY500255) 93 S. americanum CFNEI 156T (AF506513) E. fredii ATCC 35423T (D14516) E. kummerowiae CCBAU 71714T (AY034028) E. kostiensis LMG 19227T (AM181748) E. saheli LMG 7837T (X68390) 95 100 E. sojae CCBAU 05684T (GU593061) S. chiapanecum ITTG S70T (EU286550) 91 T E. mexicanus ITTG R7 (DQ411930) E. terangae LMG 7834T (X68388) ME21 (JX524727) E. meliloti LMG 6133T (EF428922) MG2 (JX524723) 100 MB9 (JX524724) MI5 (JX524722) MB12 (JX524725) MI4 (JX524721) ME5 (JX524726) E. kummerowiae CCBAU 71714T (GU994071) E. medicae USDA 1037T (EF209422) E. arboris LMG 14919T (EU123539) E. saheli LMG 7837T (GU994073) E. fredii ATCC 35423T (GU994072) E. sojae CCBAU 05684T (GU994069) R. leguminosarum bv. trifolii USDA 2071 (AF217271) 0.05 E. adhaerens LMG 20216T (AM181733) R. giardinii H152T (U86344) 0.005 Figure A. Neighbor-joining phylogenetic tree based on partial 16S rRNA sequences of strains from nodules of Medicago arborea and phylogenetically related species within the genus Ensifer. The tre e is rooted on Rhizobium giardinii H152T (U86344). T = type strain. Figure B. Neighbor-joining phylogenetic tree based on nodC sequences of strains from nodules of M. arborea and phylogenetically related species within the genus Ensifer. The tree is rooted on Rhizo bium leguminosarum bv. trifolii USDA 2071 (AF217271). T = type strain. REFERENCES Altschul, S.F., et al. (1990). J. Mol. Biol. 215: 403-410. Andreu, V., et al. (1994). Soil Use Manag. 10: 95-99 Brenner, D.J., et al. (1982). J. Clin. Microbiol. 15: 1133-1140. De Mita, S., et al. (2007). BMC Evol. Biol. 7: 210-218. Elgin, J.H., and Ostazeski, S.A. (1982). Crop Sci. 22: 39-42. Laguerre, G., et al. (2001). Microbiology 147: 981- 993. Versalovic, J., et al. (1991). Nucleic Acids Res. 19: 6823-6831. Vincent J.M. (1970). A manual for the practical study of root nodule bacteria. Blackwell Scientifi c Publications, Oxford. Weisburg, W.G., et al. (1991). J. Bacteriol. 173: 697-703. Session III SIII-CP-04 Interaction between Arbuscular Mycorrhizal Fungi and rhizobia on the growth of subclover under Mn toxicity: The role of Extraradical Mycelium. Alho, L.1*, Carvalho, M.1, Goss, M.J.2, Brito, I.1 1 ICAAM, University of Évora, Apartado 94, 7002-554 Évora, Portugal. 2 Kemptville Campus, University of Guelph, Kemptville, Ontario K0G 1J0, Canada. * [email protected] ABSTRACT When Arbuscular Mycorrhizal (AM) colonization started from an intact extraradical mycelium (ERM) its bioprotective effect on subclover was enhanced in comparison with other sources of inoculum. The presence in the soil of an intact ERM, developed previously on mycotrophic plants tolerant to Mn toxicity, resulted in the earlier colonization of subclover, reduced Mn concentration in the roots, improved development and activity of root nodules, and enhanced N acquisition. INTRODUCTION Arbuscular Mycorrhizal Fungi (AMF) can protect plants against several different abiotic stresses, including Al and Mn (Yano and Takaki, 2005; Nogueira et. al., 2007). However, a well-established AM infection is crucial for an adequate degree of protection (Garg and Chandel, 2010). When compared with other sources of AM propagules, colonization initiated from an intact ERM starts earlier and develops faster (Fairchild and Miller, 1988; Martins and Read, 1997). We hypothesized that the presence in the soil of an intact ERM developed on Mn tolerant plants, at the time of subclover seeding, could enhance the bioprotection of subclover, and its associated rhizobia, against Mn toxicity. MATERIAL AND METHODS A two-stage experiment was conducted in pots containing a sandy loam Cambisoil, where toxic levels of Mn were detected previously. In Stage 1, Mn tolerant weed species, Silene gallica L., Rumex bucephalophorus L., Lolium rigidum L. and Ornithopus compressus L. (developer plants), with different levels of mycotrophy, grew for 6 weeks. At the end of Stage 1, half of the pots were disturbed (ERM of mycotrophic developers disrupted), while glyphosate was applied to the weed shoots in the other half, thereby keeping the ERM of mycotrophic developers intact. For Stage 2 of the experiment, 6 seedlings of clover were introduced into the pots inoculated with an appropriate and effective strain of rhizobia. Three of these plants were sampled after 3 weeks to determine the arbuscular colonization of the clover, while the three remaining plants were allowed to grow for a total of 6 weeks. RESULTS AND DISCUSSION By the end Stage 1, the arbuscular colonization rate (AC) of ERM developer plants indicates different levels of mycotrophy and, therefore, different amounts of ERM were present in the soil. Developer plants did not change the availability of Mn in the soil. The AM colonization of subclover after 3 weeks was significantly greater when an intact ERM was present at the time of planting, that is, after Lolium and Ornithopus in the undisturbed treatment (Table 1). The dry matter and N content of subclover after 6 weeks was also significantly increased after the mycotrophic, than non-mycotrophic plants. AC of the clover at 3 weeks (AC3) was positively and significantly related to shoot weight at 6 weeks but inversely proportional to the Mn concentration in the roots Session III SIII-CP-04 (Figure 1A). The AC3 was significantly and positively related with N content (NS) of subclover shoots and nodule dry weight at 6 weeks (NDW) (Figure 1B), whereas Mn concentration in the roots was inversely related to NS and NDW (Figure 1C). An intact ERM, at the seeding of the subclover enhanced earlier AM colonization and with Nfixing rhizobia, w as the basis of a more effective tripartite symbiosis. This resulted from better N acquisition associated with larger and more active root nodules, the consequence of the smaller concentration of Mn in the roots. We conclude that AM colonization starting from an intact ERM greatly enhances the bioprotection granted by AMF against Mn toxicity. Table 1. Arbuscular colonization rate (AC) and Mn in soil solution of developers at the end of sta ge 1; and AC at 3 weeks, Shoot dry weigh (SDW), Nodule dry weight (NDW), Shoot N and Mn concentration, Root Mn concentration and Shoot N content of clover at 6 weeks of growth. ERM Developer Plants AC Mn in soil solution (mg/L) Silene 0.01 C 0.76 Rumex 0.01 C 0.22 Lolium 0.54 B 0.24 Ornithopus 0.73 A 0.83 Subclover Disturbance AC 3 wks Undisturbed 0.355 Disturbed 0.145 Undisturbed 0.329 Disturbed 0.219 Undisturbed 0.615 Disturbed 0.404 Undisturbed 0.682 Disturbed 0.240 SDW 6 wks (g/pot) BC 1.372 E 0.720 BD 1.246 DE 0.836 A 3.670 B 1.341 A 4.373 CE 0.707 CD DE CE BE B CE A DE NDW 6 wks (µg/nod) 24 B B 42 B B 28 B B 61 B B 418 A A 105 B B 160 B B 25 B B Concentration Content Shoot Shoot Root Shoot N Mn Mn N (g/Kg) (mg/Kg) (mg/Kg) (mg/pot) 4.9 5.0 4.9 4.8 3.2 4.6 3.6 4.6 A A A A B A B A 141 118 132 135 125 130 129 140 312 231 171 247 134 191 108 261 66 B 36 B 61 B 41 B 120 AB 59 B 155 A 33 B Figure 1. (A) Relationship between arbuscular colonization rate (AC) at 3 weeks and shoot dry weig ht (open circles) and Mn concentration in the roots (closed triangles) of subclover at 6 weeks; (B) Relationship between AC at 3 weeks and N content in the shoots of subclover at 6 weeks (crosses ) and the dry weight of nodules at 6 weeks (open circles); (C) Relationship between Mn concentration in the roots and dry weight of nodules (crosses) and N content in the shoots (open circles) at 6 weeks of clover growth. ACKNOWLEDGMENTS This work was financed by National Funds through FCT – Foundation for Science and Technology, in t he frame of the project PTDC/AGR-PRO/111896/2009. The authors wish to thank to Filipa Santos and Manuel Figo for their technical assistance and to Fertiprado for providing the subclover seeds. REFERENCES Fairchild, G.L., and Miller, M.H. (1988). New Phytologist 110: 75-84. Garg, N., and Chandel, S. (2010). Agron. Sustain. Dev. 30: 581-599. Martins, M.A., and Read, D. J. (1997). Pesqui. Agropecu. Bras. 32: 1183-1189. Nogueira M.A., et. al. (2007). Plant Soil 298: 273–284. Yano K., and Takaki, M. (2005). Soil Biol. Biochem. 37: 1569-1572. Session III SIII-CP-05 Autochthonous drought-tolerant arbuscular mycorrhizal fungi and bacteria can increase nutrients acquisition and alleviate drought stress in lavandula plants. Armada, E.1, Roldán, A.2, Azcón, R.1* 1 Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Prof. Albareda 1, 18008 Granada, Spain. 2 CSIC-Centro de Edafología y Biología Aplicada del Segura, Department of Soil and Water Conservation, P.O. Box 164, Campus de Espinardo, 30100 Murcia, Spain. * [email protected] ABSTRACT In a natural arid Mediterranean soil we determined that Lavandula dentata plants increased growth, nutrition and drought tolerance when inoculated with autochthonous bacteria or arbuscular mycorrhizal (AM) fungi and particularly with both. The highest P and K acquisition by the dual inoculated plants protected against the drought stress imposed as the decreasement of antioxidant GR, APX and CAT enzymatic activities show. The role of microbial inoculants is important in the restoration of arid ecosystems. INTRODUCTION The establishment of a plant cover based on autochthonous plant species as Lavandula is an effective strategy in the restoration of the arid degraded Mediterranean zones. Some microorganisms can promote the plant establishment in these arid soils. The arbuscular mycorrhizal (AM) symbiosis improves plant growth under extreme environments but different AM fungi differ in drought tolerance and in their ability to protect plants against water deficiency (Alguacil et al., 2003). Autochthonous microorganisms are more resistant to this environmental stress. Thus, the use of autochthonous AM fungi alone or in combination with native PGPR bacteria could decrease plant drought stress (Marulanda et al., 2006). In this study we determine the effectiveness of drought-tolerant autochthonous microorganisms in improving plant nutrition and stress tolerance tested as plant antioxidant activities. MATERIAL AND METHODS For the isolation of autochthonous bacteria or AM fungi and respective inocula production the conventional procedures were carried out using the natural Mediterranean arid soil from Murcia province (Spain). In a microcosm experiment, Lavandula was grown in this arid soil for 1 year under drought conditions (50% whc) inoculated or not with the autochthonous bacteria identified as Enterobacter sp., B. thuringiensis or Bacillus sp. A consortium of AM autochthonous fungi were also assayed associated or not to each one of the autochthonous bacteria. After one year of growth, plant biomass, nutrition and antioxidant (GR, APX, CAT and SOD) activities were evaluated following the methods proposed by Aebi (1984); Carlberg and Mannervik (1985); Amako et al. (1994); Burd et al. (2000), respectively. RESULTS AND DISCUSSION Plant parameters analyzed resulted affected by the microbial treatments applied being important the particular AM-fungi and bacteria interactions found. The plant growth increased by 123% when AM inoculated but this value was increased by the dual AM plus Bacillus sp. inoculation. These both treatments also highly increased P, K and Ca nutrition. Comparatively, K was the nutrients particularly increased by these Session III SIII-CP-05 microorganisms. The effectiveness of both Bacillus strains on K acquisition was as relevant as this of AM fungi and such K enhancement was maximized by the inoculation of both microorganisms. This is an important mechanism to increase plant drought tolerance. Concomitantly, the antioxidants APX, GR and CAT activities resulted highly reduced in these plants. These results indicate the relation between K nutrition, antioxidant responses and Lavandula adaptation to drought. The reduced APX, GR and CAT activities as affected by the microbial associations is index of drought resistance. The lower values in such enzymatic activities in response to drought represent a better drought adaptation of dually inoculated plants evidencing the lowest detrimental effect caused by drought in the inoculated plants (Benabdellah et al., 2011). Thus, inocula applied are important to promote plant growth and nutrition and also to alleviate drought stress. Effect of autochthonous bacterial strains (Enterobacter sp.; B. thuringiensis and Bacillus sp.) an d autochthonous arbuscular mycorrhizal (AM) single or dually inoculated, on shoot dry weight P, K an d Ca content (mg/plant) and APX, CAT, GR and SOD antioxidant activity in shoot, of Lavandula dentata growing in natural arid Mediterranean soil under drought. Lavandula dentata Shoot dry weight (mg) Control Enterobacter sp. B.thuringiensis Bacillus sp. (-) 655a 678a 1086c 864b APX nmol/mg prot·min (-) AM 13112d 10222c 11582cd 9938c Control Enterobacter sp. B.thuringiensis 7068a 13203d Bacillus sp. 8263ab 6913a AM 1459d 1838e 1646de 1947e P mg/plant (-) 0,62b 0,50a 0,64b 0,64b K mg/plant AM 0,78c 1,07d 1,04d 1,11d (-) 13,51a 15,03a 21,96b 19,47b AM (-) AM 19,73b 13,29b 32,71c 28,74c 9,83a 35,96c 30,56cd 16,83b 33,46c 35,22d 15,45b 44,05cd CAT nmol/mg prot·min (-) AM 1275c 1171b 1214c 1090b GR nmol/mg prot·min (-) AM 1882e 89a 920c 141a 1678d 1320c 1074d 894c 1635d 814a Ca mg/plant 370b 106a USOD nmol/ mg prot·min (-) AM 2,7a 4,2c 2,7a 4,1c 2,9ab 2,4a ACKNOWLEDGMENTS This study has been carried out in the framework of the Project AGL 2009-12530-CO2-02. REFERENCES Aebi, H. (1984). Methods Enzymol. 105: 121-126. Alguacil, M.M., et al. (2003). Physiol. Plant. 118: 562-570. Amako, K., et al. (1994). Plant Cell Physiol. 35: 497-504. Benabdellah, K., et al. (2011). Eur. J. Soil Biol. 47: 303-309. Burd, G.I., el al. (2000). Can. J. Microbiol. 46: 237-245. Carlberg, I., and Mannervik, B. (1985). Methods Enzymol. 113: 484-489. Marulanda, A., et al. (2006). Microb. Ecol. 52: 670-678. 6,6d 3,1b Session III SIII-CP-06 Análisis filogenético de la diversidad de hongos formadores de micorrizas arbusculares presentes en los distintos tipos de propágulos asociados a especies de plantas características del Parque Natural Sierra de Baza (Granada, España). Varela-Cervero, S., López-García, A., Berrio, E., Barea, J. M., Azcón-Aguilar, C.* Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín (CSIC), Granada, España. * [email protected] RESUMEN En este estudio se analiza la diversidad que albergan los distintos tipos de propágulos de hongos formadores de micorrizas arbusculares (raíces micorrizadas, esporas e hifas) asociados a especies de plantas representativas de ecosistemas semiáridos mediterráneos. La diversidad se evaluó mediante la técnica de T-RFLP. Los resultados evidencian diferencias en la diversidad de filotipos de hongos micorrícicos en los distintos tipos de propágulos. También sugieren que las estrategias de colonización y supervivencia de estos hongos difieren considerablemente y que estas diferencias tienen una base taxonómica a nivel de familia. INTRODUCCIÓN Los hongos formadores de micorrizas arbusculares (MA) son simbiontes obligados que forman asociaciones mutualistas con la mayoría de las plantas terrestres. Las hifas del hongo que se desarrollan en el suelo (micelio extrarradical) absorben y transfieren nutrientes y agua a las plantas con las que se asocian (Barea et al., 2013). La colonización micorrícica se puede iniciar a partir de tres tipos de progágulos distintos: esporas, raíces previamente micorrizadas y micelio extrarradical o hifas. Estudios recientes han mostrado una diferencia notable en la composición de hongos MA entre las esporas presentes en el suelo y las raíces micorrizadas (Hempel et al., 2007; Sanchez-Castro et al., 2012). El objetivo del presente estudio fue el análisis de la diversidad que albergan los distintos compartimentos de hongos MA (hifas, esporas y raíces micorrizadas) asociados a especies de plantas representativas de ecosistemas mediterráneos semiáridos. MATERIAL Y MÉTODOS Se muestrearon las raíces y el suelo adyacente de cinco especies representativas (Rosmarinus officinalis, Thymus zygis, Thymus mastichina, Genista cinerea, Lavandula latifolia) de un ecosistema mediterráneo del Parque Natural Sierra de Baza (Granada). Se separaron los tres tipos de propágulos mediante tamizado húmedo y decantación y se analizó la diversidad de hongos MA presente en cada uno de ellos mediante T-RFLP (Terminal Restriction Fragment Length Polymorphism). Esta técnica permite detectar presencia o ausencia de filotipos de hongos MA en las muestras analizadas (LópezGarcía et al., 201 3). Para el análisis mediante T-RFLP se utilizaron cebadores específicos para amplificar una región del gen del ARN ribosómico 18S. RESULTADOS Y DISCUSIÓN Se detectaron un total de 16 filotipos distintos de hongos MA, pertenecientes a 6 familias diferentes (Glomeraceae, Diversisporaceae, Pacisporaceae, Paraglomeraceae, Claroideoglomeraceae y Scutellosporaceae). No se detectaron diferencias significativas en la diversidad de filotipos encontrados en las raíces de las distintas especies botánicas Session III SIII-CP-06 estudiadas, ni en la de los propágulos presentes en sus correspondientes rizosferas. Sin embargo, sí que se observaron diferencias importantes en la diversidad de hongos MA presente en los distintos tipos de propágulos micorrícicos. En todos los propágulos micorrícicos predominaban los filotipos pertenecientes a la familia Glomeraceae. Sin embargo, mientras que estos representaban más del 80 % del total de filotipos detectados en raíces, solo representaban el 36 % de los encontrados en esporas y algo más del 50 % de los detectados en hifas (Tabla 1). Por el contrario, los filotipos pertenecientes a otras familias de hongos MA se encontraron preferentemente en hifas o esporas. Así, los de Pacisporaceae y Paraglomeraceae eran más abundantes en esporas, mientras que los de Diversisporaceae, Claroideoglomeraceae y Scutellosporaceae se detectaban con mayor frecuencia en esporas e hifas. Estas tendencias generales se observaron en todos los filotipos pertenecientes a la misma familia, lo que permite concluir que los hongos MA presentan estrategias vitales y de colonización diferentes y que estas diferencias tienen una base filogenética. Sugieren, además, que la taxonomía de los hongos MA tiene una base funcional. Los resultados obtenidos confirman la existencia de distintas estrategias vitales en hongos MA (Hart et al., 2001) y que estas son independientes de la planta hospedadora implicada en la simbiosis. Ello tiene implicaciones importantes en el diseño de inoculantes con una composición y estructura optimizada para maximizar su eficacia. Tabla 1. Frecuencia de detección de diferentes familias de hongos MA en los distintos tipos de propágulos de hongos micorrícicos. Glomeraceae Diversisporaceae Pacisporaceae Paraglomeraceae Claroideoglomeraceae Scutellosporaceae Raíces 81,3 a 9,2 b 0,5 b 2,7 b 1,9 b 4,4 b Esporas 36,0 a 20,9 ab 10,2 b 16,0 b 7,1 b 9,8 b Hifas 50,9 a 25,1 b 3,4 c 6,4 c 6,1 c 8,1 c Datos en la misma columna que no comparten una letra en común difieren significativamente de acuerdo a la prueba del 2. AGRADECIMIENTOS Este trabajo ha sido subvencionado por el proyecto CGL2009-08825 (Ministerio de Ciencia e Innovación). BIBLIOGRAFÍA Barea, J.M. et al. (2013). In: Beneficial Plant-Microbial Interactions: Ecology and Applications ( Rodelas, B., and Gonzalez-Lopez, J., eds.). CRC Press. ISBN: 9781466587175. Hart, M.M., et al. (2001). Mycologia 93: 1186-1194. Hempel, S., et al. (2007). Environ. Microbiol. 9: 1930-1938. López-García, A., et al. (2013). Plant Soil DOI 10.1007/s11104-013-1625-0. Sanchez-Castro, I., et al. (2012). J. Arid Environ. 80: 1-9. Session III SIII-CP-07 Influencia de la composición de la cubierta vegetal sobre los hongos micorrícico-arbusculares y la estabilización de un suelo degradado en un ecosistema mediterráneo. Cornejo, P.1, 2, Ferrol, N.1, Barea, J.M.1*, Azcón-Aguilar, C.1 1 Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, 18008 Granada, España. 2 Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile. * [email protected] RESUMEN En un sistema de mesocosmos se analizó la influencia de la composición de la cubierta vegetal sobre las densidades de propágulos de hongos formadores de micorrizas arbusculares (HMA) y la estabilización del suelo durante 3 años. Se observaron combinaciones de plantas que produjeron una mayor cobertura vegetal, una mayor densidad de propágulos fúngicos y mayores índices de estabilización de suelos, que sugieren la utilización de combinaciones sinérgicas de plantas en procesos de estabilización de suelos degradados. INTRODUCCIÓN Numerosos estudios muestran que una colonización MA efectiva favorece procesos de estabilización de suelos degradados, debido a diversas causas, como el crecimiento de micelio fúngico y producción de glomalina, que favorecen la agregación de las partículas del suelo y, por ende, evitando procesos erosivos (Curaqueo et al., 2011). En ambientes mediterráneos predominan plantas arbustivas, muchas de las cuales dependen en gran medida de la formación de MA para establecerse y crecer (Barea et al., 2011). Sin embargo, poco se conoce acerca del efecto en la estabilización de suelos cuando estas plantas crecen en combinación, como una forma de restituir una adecuada diversidad de la cubierta vegetal en estos ecosistemas degradados. Este estudio reporta resultados de tres años de estudio en diversas asociaciones vegetales usando plantas del matorral mesomediterráneo, inoculadas con una mezcla de HMA, sobre diversos parámetros del crecimiento vegetal, fúngico, y la estabilización de un suelo degradado. MATERIAL Y MÉTODOS Se establecieron mesocosmos utilizando un suelo degradado de la Sierra de Baza (Granada, España) inoculados con una mezcla de seis especies de HMA del género Glomus, en los que se hicieron crecer cuatro plantas de distintas especies del matorral mediterráneo (lavanda-Lavandula latifolia, retama-Retama sphaerocarpa, romeroRosmarinus officinale y mejorana-Thymus mastichina) de forma monoespecífica o en combinación de dos especies, por un período de tres años. Cada tres meses se evaluó la superficie de cobertura en cada mesocosmos, las densidades de propágulos (esporas por 100 g de suelo, micelio y raíz colonizada) y diversos parámetros físico-químicos del suelo (pH, P disponible, glomalina fácilmente extraíble, formación de agregados estables al agua -AEA-), y se analizó la relación existente entre las diversas variables analizadas en función de las asociaciones vegetales establecidas. RESULTADOS Y DISCUSIÓN Los resultados mostraron una marcada influencia del aumento de densidad de los propágulos de HMA sobre el crecimiento vegetal (datos no mostrados) y sobre la mejora de las características del suelo (Figura 1). En particular, la formación de AEA correlacionó fuertemente con los aumentos de la densidad de micelio a través del Session III SIII-CP-07 tiempo, aumentando en algunos casos desde cerca de un 40% inicial a valores comprendidos entre 60 y 80% al final del estudio (Figura 1A y B). Por su parte, el crecimiento vegetal correlacionó fuertemente con el aumento de estructuras fúngicas (Figura 1 C), pero también fue posible observar efectos sinérgicos en algunas asociaciones de especies vegetales (lavanda-mejorana, lavanda-retama, mejoranaretama), así como ot ras combinaciones que resultaron menos compatibles (en especial con romero), probablemente por efectos alelopáticos. Th Th-Ret 3,2 Lav Th-Ros Ros Lav-Ros Th-Lav Ret-Ros 3 C A 2 2,4 1,6 0,8 0,0 100 B 80 AEA (%) Ret Lav-Ret PC AM fungal propagules (64.5%) Hifas HMA (m g-1) 4,0 60 40 Ret-Ros Lav-Ros 1 Lav-Ret Th-Ros 0 Th-Ret Th-Lav -1 Ros Ret -2 Lav -3 Th -3 20 -2 -1 0 1 2 PC soil properties (59.7%) 0 Año 1 Año 2 Año 3 Figura 1. Influencia de la composición de la comunidad vegetal sobre la evolución del crecimiento fúngico y estabilización de las propiedades de un suelo degradado. A) Evolución de la densidad de hifas de HMA; B) Evolución de la formación de agregados estables al agua (AEA); C) Relación entre las componentes principales obtenidas para las variables de crecimiento de HMA (esporas, hifas, colonización) y mejora de las características del suelo (pH, P disponible, Glomalina, AEA). Abrevi aturas: Thymus mastichina (Th); Lavandula latifolia (Lav); Retama sphaerocarpa (Ret); Rosmarinus officinal e (Ros). Las combinaciones que presentaron los mejores incrementos globales, tanto en el aumento de los propágulos de HMA, crecimiento vegetal, como en la mejora de las características del suelo fueron lavanda y mejorana, tanto solas como en combinación, lo que sugiere que una alternativa para mejorar los procesos de estabilización de suelos degradados, además de la utilización de plantas inoculadas con HMA, es la elección de especies vegetales compatibles, que puedan actuar sinérgicamente al ser utilziadas en combinación. AGRADECIMIENTOS Estudio financiado por CICYT-EU (Proyectos REN2000-1506/GLO y REN2003-00968/GLO. Pablo Cornejo agradece las becas MAE-AECI (España) y grupo Santander-Universia (Banco Santander, Chile). BIBLIOGRAFÍA Barea, J.M., et al. (2011). J. Arid Environ. 75: 1292-1301. Curaqueo, G., et al. (2011). Soil Till. Res. 113:11-18. Session III SIII-CP-08 Salt tolerance evaluation in Casuarina glauca: impact on the photosynthetic apparatus. Batista-Santos, P.1*, Graça I.1, Semedo J.2, Lidon, F.3, Alves, P.1, Scotti, P.2, Pais, I.2, Ribeiro, A.I.1, Ramalho, J.C.1 1 Centro de Ambiente, Agricultura e Desenvolvimento (BioTrop), Instituto de Investigação Científica Tropical (IICT), Qta Marquês, Av. República, 2784-505 Oeiras, Portugal. 2 Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV), Qta Marquês, Av. República, 2784-505 Oeiras, Portugal. 3 Dept. Ciências e Tecnologia da Biomassa, Faculdade de Ciências e Tecnologia / Univ. No va de Lisboa, Campus da Caparica, 2829-516 Caparica, Portugal. * [email protected] ABSTRACT Salt stress is one of the major abiotic stresses that imposes negative effects on plants and consequently on plant productivity. Casuarina glauca belongs to the group of actinorhizal plants that establish N2-fixing root-nodule symbiosis with actinomycetes of the genus Frankia. It is commonly found in the saline soils of the coastal zones, presenting an outstanding ability to cope with environmental constraints. In this context, we are presently evaluating to which extent is C. glauca able to cope with salt stress and what is the relationship of such ability with the symbiotic capacity. In this work stage, we are analysing the impact of salt on the photosynthetic pathway by means of morphological and physiological methodologies. In general, C. glauca tolerates high levels of salt (up to 600 mM). The stressed plants present reductions in relative water content (RWC) suggesting a salinity induced water stress, associated with a decrease in the plant vigor (decreased growth). Similarly, leaf gas exchanges and fluorescence of chlorophyll a parameters declined with salt stress imposition. The results pointed to the impairment of photosynthesis with the salt imposition, probably related to stomatal and non-stomatal limitations. INTRODUCTION Actinorhizal plants, a group of woody plants belonging to eight different families, are capable of high rates of N2 fixation due to their capacity to establish root-nodule symbiosis with N2-fixing actinomycetes of the genus Frankia. These plants are able to grow in impoverished and disturbed soils and are important elements in plant communities worldwide. They have a great capability for adaptation and can be used for fuel, wood production, agro-forestry, and land reclamation (Diem and Dommergues, 1990). According to (Wand et al., 2003), drought and salinity are becoming particularly widespread in many regions, and may cause serious salinization of more than 50% of all arable lands by the year 2050. Thus, salt tolerance is becoming increasingly more relevant in agro-forestry systems. Besides being considered the model of actinorhizal plants, C. glauca is commonly found in saline soils of the coastal zones and is widely used to recover marginal soils and to prevent desertification. Under this context, we are presently evaluating the impact of salinity in C. glauca and its association, if any, with the symbiotic capacity. MATERIAL AND METHODS Plants were grown in hydroponic culture and nodulated by Frankia Thr according to standard procedures available in our lab. Six month-old plants were transferred to walkin growth c hambers (10000 EHHF, ARALAB, Portugal) where they were kept at 26/25 ºC (day/night), 12 h photoperiod, 65-70% RH, ca 380 µL L-1 of external CO2 and ca. Session III SIII-CP-08 500 µmol m-2 s-1 of irradiance. Nodulated and non-nodulated plants were subjected to salt stress (0 mM, 200 mM, 400 mM and 600 mM NaCl). Relative water content (RWC) and membrane permeability were determined in branchlets samples of 9 pieces (1.5-2 cm long each), respectively, according to (Albouchi et al., 2003) and (Campos et al., 2003). Gas exchange measurements and Chlorophyll a fluorescence parameters were determined as referred in (Batista-Campos et al., 2011). Each evaluation was performed in 4-5 replicates. RESULTS AND DISCUSSION One of the common symptoms of salinity stress in many plants is the decrease of the relative water content (RWC). In C. glauca, the exposure to increased NaCl levels decreased the RWC of both nodulated and non-nodulated plants. This reduction reflected the water stress induced by salinity and was probably associated with a decrease in plant vigor (Greenway and Munns, 1980). Concomitantly, leaf net photosynthetic rate (Pn) and stomatal conductance to water vapour (gs) also declined with the gradual salt imposition in both nodulated and non-nodulated plants. The results suggest that the plants responded to increased NaCl concentrations by decreasing gs, in order to avoid further dehydration, thus limiting Pn (and therefore growth) as the CO2 supply (Ci) to carboxylation sites was reduced. However, the impairment of photosynthesis would also be related to non-stomatal limitations, linked to limitations on PSII photochemical efficiency, thylakoid electron transport and membrane selectivity. ACKNOWLEGEMENTS This work was supported by Fundação para a Ciência e a Tecnologia, through the project PTDC/AGRFOR /4218/2012 and by the grants SFRH/BPD/78619/2011 (P. Batista-Santos) and SFRH/BD/ 41589/2007 (I. Graça), both co-financed by the Portuguese PIDDAC program and European Social Fund, under the 3rd framework program. REFERENCES Albouchi, A., et al. (2003) Science et changements planétaires/Sécheresse 14: 137-42. Batista-Santos, P,. et al. (2011). J. Plant Physiol. 168: 792-806. Campos, P.S., et al. (2003) J. Plant Physiol. 160: 283-92. Diem, H.G., and Dommergues, Y.R. (1990). In C. R. Schwintzer and J. D. Tjepkema (ed.), The biology of Frankia and actinorhizal plants. Academic Press, San Diego. pp. 317-342. Greenway, H. and Munns, R. (1980). Annu. Rev. Plant Physiol. 31: 149-190. Wang, W., et al. (2003). Planta 218: 1-14. Session III SIII-CP-09 Rhizosphere symbiots valorisation: Common bean-rhizobia symbiosis adaptation to P deficiency in Ain Temouchent agro-ecosystem in Algeria. Benadis, Ch.1*, Bekki, A.1, Lazali, M.2, Drevon, J.J.2 1 Laboratoire de biotechnologie des rhizobia et amélioration des plantes LBRAP. BP n°16 Faculté des sciences d’Oran. ALGÉRIE. 2 INRA, UMR Eco & Sols, Place Viala F-34060, Montpellier, France. * [email protected] ABSTRACT Leguminous have a high environmental, dietary and socio-economic importance especially for the African countries. Despite of their interest, during these last years, their culture is decreasing caused by biotic and abiotic stresses: Temperature variations, impairment of Mediterranean soils in minerales especially phosphorus. The ability of a symbiotic association with rhizobia allows the biological nitrogen fixation resulting can be exploited to improve plant growth and fertility soil. The inoculation by rhizobia plays an important role in improving and increasing the potential of fixing the atmospheric nitrogen through increasing number and weight of nodules. In this context that is our study whose objective is to select an efficient rhizobia and bean genotypes to improve growth and production of this very important beans and adapt it to address constraints in particular soil phosphorus deficiency. INTRODUCTION The biological nitrogen fixation in symbiosis established with bean-rhizobia and nodule formation provides annually to the plant and soil an amount of nitrogen equivalent to chemically synthesized in the fertilizer industry (Oliveira et al., 1998). Inside Common beans are often considered as a poor nitrogen fixer (ISOI and Yoshida, 1991). A study conducted by CIAT has also shown that 60% of cultivated soils beans are deficient in P (Vadez, 1996).Witch are our challenges in this work: Improve symbiotic potential of Phaseolus vulgaris in adverse conditions and Increase its adaptation to infertile soils with Reasoned Inoculation that is study of natives rhizobia from selected agroecosystem to preservate of biodiversity and agroecosystem functions. MATERIAL AND METHODS Six recombinant inbred lines, namely RILs: (CIAT) and one common bean variety widely cultivated in Algeria. Lines 115, 104 and 75 have been characterized as Pefficient whereas 147, 83 and 29 have been categorized as P-inefficient based on plant growth and seed yield in relation to the availability of P. This RIls was sowing both in vitro trapping and in natura multilocus test for 20 plots from Ain Temouchent agroecosystem chose in northwest of Algeria. After 45 days after transplanting the nodules are collected, according to macroscopically aspect 40 strains was selected to PCR-RFLP analysis amplified 16S rDNA genes. Total DNA was extracted as Laguerre (1992) described. Aliquots of PCR products were digested with restriction endonucleases. The following enzymes were used: Msp I and Nde II. All strains were tested in glass house at hydroponic cultural condition. The preparation begins before sowing, by sterilization of the seeds (Tajini et al., 2009; Bargaz et al., 2010). After, seeds were cultivated in bottles 1 -1 warped with aluminum foil to maintain the rooting darkness condition, and contain the same nutriment solution changed weekly, (Vadez, 1996; Adelson et al., 2008). Session III SIII-CP-09 RESULTS AND DISCUSSION Small collection of isolates revealed an interesting diversity. For 40 strains studied five were identified as Rhizobium etli, 3 R. leguminosarum, 11 R. gallicum, 1 R. loti, 1 R. ciceri and 6 Agrobacterium and 10 strains remaining will be sequenced to be identified. For this strain studied, 29 can establish a nodule and 17 and 2 strains were more efficiency that was identified as R. etli. Figure 1. Data are means of five replicates of nodules number after 45 days transplanting (A) in n atura and (B) in vitro for 20 plots from Ain Temouchent agroecosystem. Bo îte à M o u s ta c h e s p a r Gro u p e s M oy enne M o y e n n e ± Erre u r-Ty p e M o y e n n e ± Ec a rt-Ty p e Va ri a b l e :No d u l e s n u m b er 450 400 350 No d u l e s nu m b e r 300 250 200 150 100 50 0 -5 0 2 4 6 8 11 13 16 18 20 22 24 26 29 1a 10G 15a 28a 30a 33a 33c s tra i n Figure 2. Data are means of five replicates of nodules number after 45 days transplanting in vitro in hydroaeroponic cultural system. AKNOWLEDGEMENT This work was supported by Oran University for the stay of BENADIS Chahinez in Borj cedria (Tunisi a) and Montpellier (France), the Grand Federative Project Fabatropimed of Agropolis Montpellier Franc e and PNR of Algeria. The authors thank Adnane Bargaz for his help and Sabrine Saidi for her technic al assistance. REFERENCES Adelson, P.A., et al. (2008). Plant Soil. 312: 129-138. Bargaz, A., et al. (2010). Plant Soil. 61: 602-611. Isoi, T,. et al. (1991). Sci. Plant Nutr. 37: 559-563. Laguerre, G., et al. (1992). FEMS. Microbiol. Ecol. 101: 17-26. Olivera, M., et al. (2004). Physiol plant. 121: 79-84. Tajini, F., et al. (2009). BMC Plant Biol. 9: 73-81. Vadez, V., et al. (1996). Plant Physiol. Biochem. 34: 871-878. Session III SIII-CP-10 The genotypic variability of beans affects the growth parameters, the nodulation and the microorganisms which initiate nodulation. Benadis, Ch.1, Bekki, A.1*, Abed, N.H.2, Ouzane, H.2, Irekti, H.2 1 Laboratoire de biotechnologie des rhizobia et amélioration des plantes LBRAP. BP n°16 Faculté des sciences d’Oran. Algérie. 2 Division Biologie du sol. Institut National de la Recherche Agronomiqu e d'Algérie 2, Rue Frères Ouadek, El Harrach, Alger. Algérie. * [email protected] ABSTRACT The exploitation of rhizobia-legume symbiosis, the choice of suitable varieties of legumes and reasoned inoculation will contribute to a better management of ecosystems and ecological preservation of the environment and biodiversity. The practice of these symbioses in rotation with cereal crops will reduce the need of chemical fertilizers and lead to a good functional agroecosyteme complex for a sustainable agriculture. In our study the economic outputs of this strategy in a targeted agroecosystems mass production of pulses will be tested. In natura, six contrasting lines of bean (provided by the International Centre for Tropical Agriculture. Colombia) and a local varietal, the most planted by farmers, will be tested on 20 plots located in four different regions of Ain Temouchent agroecosystem. INTRODUCTION Food legumes are of great importance due to their high nutritional value, making them an essential component in the food and feed in the world. On the other hand, they are a way to improve soil fertility and associated crops yields and thus are of interest in establishing a sustainable agriculture. Despite their agronomic benefit, in Algeria they are poorly cultivated and represent only 1% of the total UAA and 2-3% of the cultivated grains, and the efforts given to culture surface intensification have often failed to achieve the desired results. In the western region of Algeria, Ain Témouchent is considered as the first legumes production granary in Algeria. One of the less cultivated legumes, bean remains in fourth position after chickpea, faba bean and lens; farmers are reluctant to this culture because of its low yields. It is in this context that our Bean project is focused, in order to use different bean genotypes inoculated with rhizobia and then to choose the most effective Rhizobium-legume symbiotic couples adapting to a specific settlement for sustainable and organic agriculture. MATERIAL AND METHODS Stations of Observation. 20 agroecosystem stations selected in Ain Témouchent, northwest of Algeria, are targeted in four areas: Sidi Ben Adda, Hammam Bouhjar, Ain Tolba and Terga. In each station, a plot is sown in multisite with testing contrasting 6147 lines: 115, 104, 83, 34, 29 and a local line (Loc). Growth nodulation and yield Measuring. 12 plants are harvested by digging up roots in a volume of 20 cm square at the flowering stage. Sampling is carried out in five different randomly chosen points in the plot. On each plant, the aerial part is separated from the roots at the cotyledonary node. The number of nodules, nodule dry and aerial part weight was measured after 48 hours of drying in an oven at 70 ° C for each plant. Analysis of soil and irrigation water. At each station, soils are sampled at planting. Session III SIII-CP-10 RESULTS AND DISCUSSION The results (Figures 1 and 2) show that genotypic variability affects the growth parameters, the nodulation and the microorganisms which initiate nodulation with seeded lines at the targetted parcels (Vadez et al., 1996; Bargaz et al., 2010). Nodulation is limited in In natura conditions (Figure 2) and plots where high dry matter yield was observed also had a significant nodulation (Tajini et al., 2009). The hierarchical classification similarity index grouped the physicochemical properties of soils plots into two main groups (Figure 3). A large variability between plots shows that even if the plots are in the same microclimate, other factors had an influence on the physico-chemical characteristics such as T and previous crops. AKNOWLEDGEMENTS This work was supported by Oran University for the stay of BENADIS Chahinez in Borj cedria (Tunisi a) and Montpellier (France), the PNR of Algeria and financial support of LBRAP. REFERENCES Bargaz, A., et al. (2010). Plant Soil 61: 602-611. Tajini, F., et al. (2009). BMC Plant Biol. 9: 73-81. Vadez, V., et al. (1996). Plant Physiol. Biochem. 34: 871-878. Session III SIII-CP-11 Phosphoenol pyruvate phosphatase transcript in nodule cortex of Phaseolus vulgaris. Bargaz, A.1*, Lazali, M.2, Ghoulam, C.3, Drevon, J.J.2 1 Swedish University of Agricultural Sciences, Department of Biosystems and Technology, PO Box 103, 2 3 SE-230 53 Alnarp, Sweden. INRA, UMR Eco&Sols, 1 Place Viala, F34060, Montpellier, France. Equipe de Biotechnologie Végétale et Agrophysiologie des Symbioses, Faculté des Sciences et Techniques Guéliz, BP 549, 40000, Marrakech, Maroc. * [email protected] ABSTRACT Increases of acid phosphatases (APases) enzymes are among mechanisms which lead to increase efficiencies both of N2 fixation and nodule respiration under P deficiency. Our findings have revealed that activities and differential expression of phosphoenol pyruvate phosphatase (PEPase) transcripts were positively correlated with increases both of the rhizobial symbiosis efficiency (EURS) and nodule O 2 permeability. The induced enzyme activity and the marked transcript localization of this APase in nodule cortex would control nodule respiration and contribute to adaption of nodulated legumes to low P availability. INTRODUCTION In nodules, expression of large number of Glycine max APase “GmPAP” genes was mainly detected under P-deficiency (Li et al., 2011). This organ was reported to be strongly enriched in highly tissue-specific genes (Libault et al., 2010; Severin et al., 2010). Overall, most APases are nonspecific hydrolyzing Pi from a broad spectrum of Pi mono-esters and may have different functions as described for a soybean APase gene GmPAP3 whose expression alleviates oxidative stress caused by salinity and osmotic constraint (Li et al., 2008). The involvement of APases in tolerance to various abiotic and biotic constraints, led us to assume that PEPase may have a multiplicity of functions as well as those related to phosphate metabolism and should provide new insights into adaptation to P-deficiency. Thus, the present work hypothesizes that differential expression in the nodule cortex, of PEPase may contribute to low P adaptation and may be involved in the regulation of nodule respiration. MATERIAL AND METHODS Nodules of about 3 mm diameter of each RIL corresponding to 50 ± 05 mg of nodule fresh weight (FW) were carefully detached at 42 days after transplanting (DAT) and immediately frozen in liquid nitrogen and stored at -80 °C until use for PEPase activity assay. Sample preparation and fixation for in situ reverse transcription polymerase chain reaction (RT-PCR) were set according to the protocol described by Molina et al. (2011). The method involves in situ amplification of specific nucleic acid sequences on nodule sections, followed by fluorescence detection of the localized PCR product via epifluorescence micr oscopy. RESULTS AND DISCUSSION To our knowledge, this study is the first to reveal that the PEPase transcription was induced by P-deficiency with differential expression among nodule tissues. The differential localization of transcripts encoded for PEPase in the outer cortex and infected zone under P-deficiency (Figure 1) opens new insights into understanding the physiology of N2-fixing legumes as well as requirements for N2 fixation and regulation of nodule permeability to O2 diffusion. Under P-deficiency, the correlation between Session III SIII-CP-11 PEPase enzyme activity or transcript localization in infected cells and EURS or N 2 fixation (Figure 2) suggest that this APase is involved in nodule metabolism that is linked to N2 fixation and the overall N2-dependent growth of the legume in hydroaeroponics. The marked increase in PEPase transcripts in the nodule cortex of Pdeficient nodu les not only suggests an increase in intracellular Pi scavenging but also opens up a challenge to understand whether such sub-localization is involved in the scavenging of Pi from extracellular organophosphates. Figure 1. In situ localization of PEPase transcripts (green spots) in nodules of common bean RIL115 and RIL147 grown under a sufficient (250 P) versus a deficient (75 P) P supply. InC, infected cell; IC, inner cortex; IZ, infected zone; OC, outer cortex; UC, uninfected cell; VT, vascular trace parenchyma. Figure 2. Efficiency of use of rhizobial symbiosis of common bean RIL115 and RIL147 inoculated with R. tropici CIAT899 and grown under a sufficient (open circles) versus a deficient (filled circles) P supply. Data represent individual values of 14 replicates harvested at 42 DAT. Ndw, Nodule dry weight. ACKNOWLEGMENTS. This work was supported by the FABATROPIMED project financed by Agropolis Fondation under the reference ID 1001-009. REFERENCES Li, W.Y.F., et al. (2008). New Phytol. 178: 80-91. Li, C., et al. (2011). Ann Bot. 109: 275-285. Libault, M., et al. (2010). Plant J. 63: 86-99. Severin, A.G., et al. (2010). BMC Plant Biol. 10: 160. Session III SIII-CP-12 P-deficiency increases phytase activity and O2 uptake per N2 reduced in Phaseolus vulgaris L. Lazali, M.1, 2, 3*, Abadi, J.1, Ounane, S.M.2, Bargaz, A.4, Pernot, C.1, Drevon, J.J.1 1 Institut National de la Recherche Agronomique (INRA), UMR 1222 Ecologie Fonctionnelle & Biogéochimie des Sols et Agroécosystèmes, INRA-IRD-CIRAD-SupAgro. Place Pierre Viala, 34060 Montpellier, France. 2 Ecole Nationale Supérieure Agronomique (ENSA), Département de phytotechnie. Avenue Hassan Badi, El Harrach 16200 Alger, Algérie. 3 Université de Khemis Miliana, Faculté des Sciences de la Nature et de la Vie & des Sciences de la Terre. Route Theniet El Had, Soufay, 44225 Ain Defla, Algérie. 4 Swedish University of Agricultural Sciences (SLU), Department of Biosystems and Technology, Box 103, SE-23053 Alnarp, Sweden. * [email protected] ABSTRACT To understand the mechanisms used by legumes to improve their PUE for SNF under Pdeficiency, six R ILs of P. vulgaris were inoculated with Rhizobium tropici CIAT899, and grown at two levels of P supply. Our results have revealed that the increases of APases and phytases activities under P-deficiency varied among RILs of P. vulgaris and were positively correlated with increases both of the nodule conductance to O2 diffusion and the EURS. This may represent an adaptive mechanism for N 2-fixing legumes to respond to low P availability, by increasing the utilization and the uptake of P for SNF . INTRODUCTION Phosphorous (P) is one of the most important macronutrients involved in many physiological and biochemical processes in plants (Tran et al., 2010). However, P concentration in soil solution is often low because of a strong P fixation by organic compounds, free Fe or Al oxides. Therefore, low P availability is a major constraint to plant growth and development. While the induction of APase is a universal response to P-deficiency in plants (Duff et al., 1994), the physiological role of these enzymes in nodule O2 permeability of N2 fixing legume is crucial but still not fully understood. In this context, recent studies have reported that high expression of large number of PAP genes in the nodules of Glycine max is considered as an adaptive mechanism to tolerate P-deficient conditions (Li et al., 2012). The present work hypothesizes that the differences among common bean RILs for phytase activity may be involved in the regulation of nodule respiration and can contribute to low P soils adaptation. MATERIAL AND METHODS This study was carried out using six recombinant inbred lines, namely RILs 147, 115, 104, 83, 34 and 29 obtained from a cross of two parental lines, namely BAT477 and DOR364 originated from CIAT. Root nodules were induced by Rhizobium tropici CIAT899, and grown in hydroaeroponic culture under P-sufficiency (250 µmol P plant−1 week−1) versus P-deficiency (75 µmol P plant −1 week−1) supply. At 42 days after transplantion, nodules of about 3 mm diameter of each RIL corresponding to 60 mg of fresh weight were carefully detached to roots after measuring O2 uptake by nodulated roots (Conr) and nodule conductance to O2 diffusion. Plants were harvested at the stage of pod setting and separated in shoots, roots and nodules. After drying for 3 days at 70° C, plant and nodule dry weight was measured and used to determine the efficiency in use of the rhizobial symbiosis (Drevon et al., 2011). Session III SIII-CP-12 RESULTS AND DISCUSSION The increases of phytase activity (Figure 1A) in nodules under P-deficiency were accompanied with an increase in their conductance to O2 diffusion (Figure 1B) and have shown several genotypic variations among the tested common bean RILs. The positive correlation between phytase enzyme activity and nodule O 2 permeability (data not shown) substantiates a physiological role of phytase in regulating nodule O 2 diffusion. Although, the induced of APase activity in plants during P-deficiency has been widely recognized. Phytases might play a major role for internal plant metabolism rather than for obtaining P from the soil phytate. Furthermore, phytase expression in nodule cortex (data not shown) and the regulation of the nodule respiration since this zone, more particularly the inner cortex, which is postulated to be a physical barrier for the regulation of nodule permeability to gas diffusion (Witty and Minchin 1998) and osmotic conditions (Schulze and Drevon 2005). Figure 1. Phytase activity (A) and conductance to O2 diffusion (B) in nodules of six common bean R ILs inoculated with R. tropici CIAT899 and grown under sufficient (white) versus deficient (grey) P su pply. Data are mean and standard deviation of seven replicates harvested at 42 DAT. In summary, the present study suggests that the higher increased of phytases activity in the nodules of all RILs under P-deficiency, indicating that N2-fixing legumes can enhance P utilization within the nodules to tolerate low P conditions. ACKNOWLEGMENTS This work was supported by the Great Federative Project FABATROPIMED of Agropolis Fondation under the reference ID 1001-009. REFERENCES Drevon, J.J., et al. (2011). Proc. Environ. Sci.9: 40–46. Duff, S.M.G., et al. (1994). Physiol. Plant. 90: 791–800. Li, C., et al. (2012). Ann. Bot. 109: 275–285. Schulze, J., and Drevon, J.J. (2005). J. Exp. Bot. 56:1779–1784. Tran, H.T., et al. (2010). Plant Sci. 179: 14–27. Witty, J.F., and Minchin, F.R. (1998). J. Exp. Bot. 49:1015–1020. Session III SIII-CP-13 Effect of inoculation with selected rhizobia on symbiotic nitrogen fixation and Faba bean grains yield in north Tunisia agro-ecosystems. Sifi, B.1, 2, 3*, Maazaoui, H.1, Drevon, J.J.2 1 Laboratore des Sciences et Techniques Agronomique, Institut National Reverche Agronomique de Tunisie, rue Hédi Karray, 2080 Ariana, Tunisie. 2 Faculté des sciences de Bizerte, Tunisie. 3 INRA , UMR Eco&Sols - Ecologie Fonctionnelle & Biogéochimie des Sols & des Agroécosystèmes, 2 Place Pierre Viala, F34060, Montpellier, France. * [email protected] ABSTRACT In order to improve of agro-ecosystem productivity based on cereal and legumes rotation, fourteen Rhizobium strains were tested in hydroponic culture with faba bean ²Bachar variety² in glasshouse. The selected rhizobia were used to inoculate faba bean in thirteen farmers’ fields during two years. Inoculation with the different isolates enhanced nodulation and improved production parameters. Furthermore, Mat strain was the most preferment compared to the tested rhizobia. Results were confirmed by those obtained experiments carried out in farmer’s fields. Inoculation of faba bean with ²Mat² Rhizobium strain increased significantly nodulation and grains yield. The variability of faba bean nodulation and yield was related to soils fertilities. INTRODUCTION North Tunisia agro-ecosystems were based on cereal monoculture. Introduction of legumes and inoculation with specific Rhizobium strain can improve soil fertility and productivity. In recent decades there has been a great deal of interest in enhancing symbiotic nitrogen fixation during the production of legumes for sustainable agriculture. Reduction of nitrogen fixation was due to soil fertility will often be related to either an excess of soil nitrates or a deficiency of some essential plant growth nutrient. Moreover, the majority of soils were found to have very small concentrations of soluble phosphorus and this was related to poor nodulation and (Amijee and Giller, 1998). Investigations conducted in Mediterranean areas revealed the absence of Rhizobium leguminosarum in many soils (Dhamane et al., 1994). In such cases, the improvement of nodulation and symbiotic nitrogen fixation requires not only the supply of fertilizers but also the application of efficient rhizobial inoculants for legumes crops. MATERIAL AND METHODS The experiment was designed for the evaluation of nodulation potential of a collection of local Rhizobium strains modulating faba bean under hydroponic culture in glasshouse conditions. The experiment was designed to assess the effect inoculation of faba bean with preferment selected rhizobia in farmer’s fields in Mateur, north of Tunisia. RESULTS AND DISCUSSION Inoculation has ameliorated nodulation, but the number and biomass of nodule was variable a cording field and significantly correlated with soil fertility (Figure 1). Faba bean nodulation number and quality was significantly variable in farmer’s fields. Session III SIII-CP-13 Nod.pl-1 Dry W g.pl-1 Figure 1: Effect of inoculation with rhizobia on faba bean nodules number. Figure 2: Effect of inoculation with rhizobia on faba bean root dry weight (g.pl-1) Faba bean plant root dray weight was significantly variable a cording the farmer’s field trials (Figure 2). The root dry weight was correlated with nodules number and variable throw the farmer field’s zone. Dray weight shoot of inoculated faba bean plant was significantly variable a cording the farmer’s field trials. The shoot dry weight was correlated with nodules number and variable throw the farmer field’s zone (Figure 3). Dry W.g.pl-1 Figure 3: Effect of inoculation with rhizobia on faba bean shoot dry weight (g.pl-1). g.m-2 Figure 4: Effect of inoculation with rhizobia on faba bean grains yield (g.m-2) Faba bean average yield was significantly variable (200 to 420 g.m-2) from field to other. Grains yield was not significantly correlated with plant nodulation root and shoot biomass production. REFERENCES Amijee, F., and Giller, K.E. (1998). African Crop Sci. J. 6: 159-169. Dahmane, A., et al. (1994) In: Facteurs limitant la fixation symbiotique de l’azote dans le bassin méditerranéen. Montpellier, 6-8 avril 1994. Ed. INRA, Paris 1995 (Les colloques, No 77). p175-182. Session III SIII-CP-14 Integrating soil microbial community context in plant response to mycorrhizal symbionts. Carvalho, L. *, Correia, P., Meleiro, A.I., Carolino, M., Dionisio, F., Cruz, C. Centre for Environmental Biology, Faculty of Sciences, University of Lisbon. Campo Grande, Bloco C 2, 1749-016 Lisbon, Portugal. * [email protected] ABSTRACT We found that more beneficial mycorrhizal species to a host plant, in terms of phosphorus and nitrogen nutrition, did not coincide when in isolation and when interacting with a bacterial community, showing that plant growth response to mycorrhizal fungi is rhizospheric-associated bacterial community dependent. INTRODUCTION Arbuscular mycorrhizal fungi (AMF) are likely the most abundant plant mutualists, improving phosphorus and nitrogen plant nutrition in exchange of carbon. Each plant simultaneously associates with several species of AMF that vary in symbiont quality, that is, in the delivery of benefit they provide to the host plant (Klironomos et al., 2003), which could threaten the stability of mycorrhizal mutualisms. Recent research indicate that plants can favor more-beneficial against less-beneficial AMF symbionts (Bever et al., 2009; Kiers et al., 2011). Studies testing mycorrhizal symbiont quality have been conducted using plants and AMF in isolation, but plant response to mycorrhizal fungi may be biotic community context dependent (Hoeksema et al., 2010). In nature, plants interact with other symbiotic and associative soil microbial symbionts involved in plant nutrition, and these plant-AMF-microbe interactions may influence plant response to each AMF species. Here, we investigated whether the nutritional effects of different AMF species to the host plant (symbiont quality) are dependent on soil biotic community context, particularly on the presence of plant-growth promoting bacteria. MATERIAL AND METHODS Maize plants were inoculated with one of four AMF species, alone or together with a community of plant-growth promoting rhizobacteria involved in phosphorus and nitrogen plant nutrition; non-mycorrhizal control treatments were included. Plant growth, phosphorus and nitrogen content, nitrogen isotope rations (15N), and mycorrhizal colonization were assessed after two months of growth under greenhouse conditions. RESULTS AND DISCUSSION Mycorrhizal benefits in providing phosphorus and nitrogen to maize plants vary with AMF taxa, but variation in AMF identity effect was influenced by the presence of a rhizospheric bacterial community (Figure 1). For instance, AMF species conferring more nutritional benefits when inoculated alone, provided similar or lower benefits than other species when AMF were inoculated together with a bacterial community. Plant benefits from AMF relatively to non-mycorrhizal controls were generally highest in nitrogen rather than in phosphorus contents, and synergistic effects between AMF and rhizospheric bacteria depended on mycorrhizal species. Overall, shoot 15N signature of maize plants was influenced by AMF taxa. Session III SIII-CP-14 Figure 1. Phosphorus and nitrogen content of maize plants inoculated with one from four AMF specie s, alone or together with a bacterial community of six PGPR. This study indicates that the presence of a soil microbial community differentially influences symbiotic function of mycorrhizal fungal species to host plants, which can have important evolutionary and ecological implications for the stability of the mutualism between plants, and for the design of effective microbial consortia to be applied in agriculture. ACKNOWLEDGEMENTS This work was supported by the project PTDC/AGR-PRO/115888/2009 and SFRH/BPD/33633/2009 (to LC) of the Fundação para a Ciência e a Tecnologia of Portugal. REFERENCES Bever, J.D., et al. (2009). Ecol. Lett. 12: 13-21. Hoeksema, J.D., et al. (2010). Ecol. Lett. 13: 394-407. Kiers, E.T., et al. (2011). Science 333: 880-882. Klironomos, J.N. (2003). Ecology 84: 2292-2301. grant Session III SIII-CP-15 Identificación y caracterización de sistemas de transporte de hierro en el hongo micorrícico arbuscular Rhizophagus irregularis. Tamayo, E., Ferrol, N.* Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, 18008, Granada. * [email protected] RESUMEN Se han identificado en el genoma del hongo micorrícico arbuscular Rhizophagus irregularis genes que codifican proteínas implicadas en la síntesis de sideróforos y en la vía reductiva de captación de hierro (Fe). La caracterización del gen RiFTR1 indica que codifica una permeasa de Fe de alta afinidad cuya expresión se inhibe en presencia de niveles tóxicos de Fe y que se expresa mayoritariamente en el micelio intrarradical. INTRODUCCIÓN Los hongos disponen de dos vías principales de captación de hierro, una dependiente de sideróforos y otra reductiva, consistente en la reducción extracelular de Fe con la subsecuente absorción del ion Fe2+ mediante un complejo de transporte de alta afinidad formado por una oxidasa multicobre (FET3) y una permeasa de Fe (FTR1) (Johnson, 2008). Los hongos micorrícicos arbusculares (hongos MA), pertenecientes al phylum Glomeromycota, establecen simbiosis mutualistas con las raíces de la mayoría de las plantas. El hongo ayuda a la planta a adquirir nutrientes minerales del suelo y a cambio la planta le cede al hongo compuestos carbonados. En condiciones de deficiencia en Fe, las micorrizas arbusculares (MA) contribuyen a la adquisición de este metal por la planta y en condiciones de toxicidad las plantas micorrizadas son más tolerantes al exceso de Fe (Smith and Read, 2008). Sin embargo, los mecanismos de homeostasis de Fe en MA se desconocen actualmente. El objetivo del presente trabajo es identificar y caracterizar los sistemas de absorción de Fe en el hongo MA Rhizophaugus irregularis. MATERIAL Y MÉTODOS Material Biológico Se utilizaron cultivos monoxénicos de R. irregularis desarrollados en placas de Petri compartimentadas en presencia de diferentes dosis de Fe. Identificación de genes Se realizó mediante búsquedas en las bases de datos del transcriptoma y del genoma de R. irregularis (Tisserant et al., 2012). Las secuencias génicas completas se obtuvieron mediante RT-PCR y/o RACE. Caracterización de RiFTR1 El análisis funcional se realizó en la cepa Δftr1 de Saccharomyces cerevisiae. Los análisis de expresión génica se realizaron mediante técnicas de qRT-PCR. RESULTADOS Y DISCUSIÓN Identificación de genes candidatos implicados en transporte de hierro En el genoma de R. irregularis se han identificado genes que codifican proteínas implicadas en la biosíntesis de sideróforos y en la vía reductiva de absorción de hierro. En concreto, se han identificado dos genes, RiNRPS1 y RiNRSP2, que presentan homología (>40%) a los genes SidC de varios hongos que codifican sintetasas de péptidos no ribosomales, RiFRE1 que presenta la mayor homología con la reductasa férrica cloroplastídica FRO7 de Arabidospis, RiFTR1 y RiFTR2 que codifican posibles permeasas de Fe y los genes RiFET1-3 que presentan la mayor homología (>50 %) con Session III SIII-CP-15 oxidasas multicobre de varios hongos. Estos resultados sugieren que los hongos MA podrían usar tanto la vía reductiva como la de producción y secreción de sideróforos para la adquisición del hierro que necesitan para su desarrollo. Con el fin de caracterizar ambas vías actualmente estamos caracterizando varios de los genes identificados y más concretamente, los implicados en la vía reductiva. Caracterización de la permeasa de Fe RiFTR1 La expresión del gen RiFTR1 en la cepa Δftr1 de S. cerevisiae, incapaz de crecer en condiciones de deficiencia de Fe, revirtió el fenotipo mutante (Figura 1). Estos resultados permiten concluir que RiFTR1 codifica una permeasa de Fe de alta afinidad. Figura 1. Análisis funcional de RiFTR1 en S. cerevisiae. El análisis de la regulación de la expresión de RiFTR1 en el micelio extrarradical de R. irregularis por Fe mostró que la expresión de este gen se inhibía en presencia de altas concentraciones de este metal. La observación de que la estructura fúngica en la que este gen se expresa mayoritariamente es el micelio intrarradical sugiere que FTR1 está implicada en la adquisición de hierro en la interfase simbiótica y, por tanto, una participación del hierro en el desarrollo de la simbiosis MA. AGRADECIMIENTOS Este trabajo ha sido financiado por el Programa Nacional de Proyectos de Investigación del Ministe rio de Economía y Competitividad, Proyecto AGL2012-35611. BIBLIOGRAFÍA Johnson, L. (2008). Mycol. Res. 112: 170-183. Tisserant, E., et al. (2012). New. Phytol. 193: 755-769. Smith, S.E., and Read, D.J. (2008). Mycorrhizal Symbiosis. Academic Press. London, UK. Session III SIII-CP-16 Structure of the exopolysaccharide isolated from Sinorhizobium fredii HH103. Rodríguez-Carvajal, M.A.1*, Acosta-Jurado, S.2, Rodriguez-Navarro, D.N.3, CrespoRivas, J.C.2, Marg aret, I.2, Sanjuán, J.4, Soto, M.J.4, Vinardell, J.M.2, Ruiz-Sainz, J.E.2, Gil-Serrano, A.1 1 Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla. Sevilla, Spain. 2 Departamento de Microbiología Vegetal y Biología Molecular, Facultad de Biología, Universidad de Sevilla. Sevilla, Spain. 3 Centro Las Torres-Tomejil (IFAPA). Alcalá del Río (Sevilla), Spain. 4 E stación Experimental del Zaidín, CSIC, Departamento de Microbiología del Suelo y Sistemas Simbióticos. Granada, Spain. * [email protected] ABSTRACT The structure of the exopolysaccharide isolated from Sinorhizobium fredii HH103 has been determined on the basis of mass spectrometry and NMR studies. Its repeating unit consists of a branched nonasaccharide with the following structure: This structure is identical to that found in the exopolysaccharide isolated from Rhizobium sp. strain NGR234. INTRODUCTION Sinorhizobium fredii HH103 is a fast-growing rhizobial strain that is able to nodulate legumes that develop determinate nodules, e.g., soybean, and legumes that form nodules of the indeterminate type. Different bacterial surface polysaccharides, such as K-antigen capsular polysaccharides (KPS) and cyclic glucans (CGs) are relevant for these symbiotic interactions. In this work, we present the determination of the structure of the S. fredii HH103 exopolysaccharide (EPS). MATERIAL AND METHODS Isolation and purification of EPS: Culture medium was treated with proteinase K, concentrated up to 20% and three volumes of cold ethanol were added. The resulting precipitate was separated by centrifugation, re-dissolved in water and purified by dialysis against distilled water at 4 °C. Monosaccharide composition (Chaplin, 1982) and their absolute configurations (Gerwig et al., 1978) were identified on GLC-MS analysis. Samples of polysaccharide having carboxyl groups previously reduced (Rodriguez-Carvajal et al., 2008) were methylated by using the method of Ciucanu and Costello (2003). Hydrolysis, reduction with NaBD4, and acetylation were performed according to Kim et al. (2006), yielding the corresponding partially methylated and acetylated alditols (PMAAs), which were analysed by GLC-MS. Partial hydrolysis: A solution of the exopolysaccharide in 0.5M TFA was heated for 1 h at 100 °C and then dialysed against water. The diffusate was concentrated and the residue was chromatographed on Bio-Gel P-2 using water as eluent. Fractions Session III SIII-CP-16 containing low molecular-weight oligosaccharides were chromatographed on Silica-gel with a gradient of acetonitrile/water to elute the oligosaccharides (Fauré et al., 2006). Lithium degradation: The polysaccharide was stirred in ethylenediamine with lithium metal as described in Fernández de Córdoba et al. (2008), and then purified by size exclusion chromatography on Biogel P-2. NMR: Samples were deuterium-exchanged several times by freeze-drying from D2O and then examined in solution (1-5 mg/750 µL) in 99.98% D2O. Spectra were recorded at 303 K and 333 K on a Bruker AV500 spectrometer operating at 500.20 MHz ( 1H) and 125.8 MHz (13C). Standard Bruker sequences (DQFCOSY, TOCSY, ROESY, HSQC, HMBC, and HMQC-TOCSY) were used for 2D experiments (Parella et al., 2004). RESULTS AND DISCUSSION Although standard monosaccharide analysis shows that the polysaccharide is composed by D-Glc and D-Gal (5:2), the study of the oligosaccharides obtained by partial hydrolysis indicates that it also is composed by D-GlcA. Methylation analysis allow identifying the units: Õ3)-D-Galp, Õ3)-D-6-d2-Glcp (from a glucuronic acid unit), Õ4)-D-Glcp (coeluting with Õ4)-D-6-d2-Glcp), Õ6)-D-Glcp, Õ4,6)-D-Glcp, and Õ4,6)-D-Galp in a ratio close to 1:1:4:1:1:1. Furthermore, the occurrence of Õ3) and Õ4)-linked uronic acids makes applicable the degradation of EPS with lithium in ethylenediamine [8], yielding a polysaccharide (EPS-Li) that was studied by NMR. Moreover, the study by NMR of the polysaccharide recovered after partial hydrolysis allowed the identification of, besides the units previously identified in EPS-Li, a trisaccharidic branch composed of two units of -D-GlcA and a unit of -D-Gal bearing a pyruvate group as ketal. Finally, NMR of the undegraded exopolysaccharide also shows signals from acetyl groups located on ~50% of non-reducing terminal galactose residues (at O-2 and O-3). This structure has the same carbohydrate backbone of the exopolysaccharide isolated from Rhizobium sp. strain NGR234 (Djordjevic et al., 1986; Staehelin et al., 2006). ACKNOWLEDGEMENTS We thank the Andalusian Consejería de Innovación, Ciencia y Empresa (Proyecto de Excelencia CVI2506) and the Spanish Ministerio de Educación y Ciencia (MEC) (AGL2009-13487-C04-02) for financial support. We also thank the Centro de Investigación, Tecnología e Innovación (CITIUS) of the University of Seville for NMR and MS facilities. REFERENCES Chaplin, M.F. (1982). Anal. Biochem. 123: 336-341. Ciucanu, I., and Costello C.E. (2003). J. Am. Chem. Soc. 125: 16213-13219. Djordjevic, S.P., et al. (1986). Carbohydr. Res. 148: 87-99. Fauré R., et al. (2006). J. Org. Chem. 71: 5151-5161. Fernandez de Cordoba, F.J, et al. (2008). Biomacromolecules 9: 678-685. Gerwig, G.J., et al. (1978). Carbohydr. Res. 62: 349-357. Kim, J.S., et al. (2006). Carbohydr. Res. 341: 1061-1064. Lau, J.M., et al. (1987). Carbohydr. Res. 168: 219-243. Parella, T. (2004). Pulse Program Catalogue. NMR Guide v4.0. Bruker Biospin. Rodriguez-Carvajal, M.A., et al. (2008). Carbohydr Res 343: 3066. Staehelin, C., et al. (2006). J. Bacteriol. 188: 6168-6178. Session III SIII-CP-17 First evidence for interlinked control of surface motility and biofilm formation in Sinorhizobium meliloti. Amaya-Gómez, C.V.1, Hirsch, A.H.2, Soto, M.J.1* 1 Dpto. Microbiología del Suelo y Sistemas Simbióticos. Estación Experimental del Zaidín (CSIC), 180 08 Granada, Spain. 2 Department of Molecular, Cell, and Developmental Biology and Molecular Biology Institute, University of California-Los Angeles, CA 90095-1606, USA. * [email protected] ABSTRACT Swarming motility and biofilm formation are surface-associated behaviors, which in some bacteria are subjected to inverse co-regulation. In Rhizobium, these two opposite lifestyles are largely unexplored. In the current study, we investigated whether factors known to influence swarming motility in Sinorhizobium meliloti have an impact on its capability to form biofilms. We show that siderophore rhizobactin 1021 (Rhb1021), together with flagella, are required for both surface motility and biofilm formation. In addition, our results support the existence of control mechanisms that inversely coregulate these two different lifestyles in S. meliloti to allow for optimal plant root colonization and in which iron, and the fadD and rirA genes are involved. INTRODUCTION The majority of bacteria spend most of their lifecycle associated to surfaces behaving either as motile cells or sessile communities named biofilms. Swarming is a mode of surface translocation dependent on rotating flagella and is characterized by the rapid and coordinated movement of multicellular groups of bacteria. Several studies have revealed the existence of a link between biofilm formation and swarming motility in pathogenic bacteria (Verstraeten et al., 2008). However, little information is available about these opposite surface-associated processes in rhizobia. Our previous work revealed that S. meliloti Rm1021 and GR4 strains show different swarming motility phenotypes. Whereas Rm1021 spreads over semisolid surfaces using flagella-dependent andindependent mechanisms, GR4 is non-motile under the same conditions (Nogales et al., 2010, 2012). In both strains, a mutation in the fadD gene promotes surface translocation by yet unidentified mechanisms (Nogales et al., 2010). A transcriptomic analysis of S. meliloti under swarming-inducing conditions led to the discovery that biosynthesis of the siderophore rhizobactin 1021 (Rhb1021) is essential for surface translocation of Rm1021 (Nogales et al., 2010, 2012). Moreover, the global iron response regulator RirA and levels of iron in the media, were shown to participate in the control of swarming in Rm1021. The aim of this work was to investigate the existence of a possible connection between swarming motility and biofilm formation in S. meliloti by comparing the biofilm formation ability of GR4 and Rm1021 and analyzing the impact of iron and genes such as fadD, rhb, and rirA on the development of biofilms. MATERIAL AND METHODS S. meliloti wild-type strains GR4 and Rm1021, fadD mutants of GR4 and Rm1021 (GfadD and RmfadD, respectively) and rhbA and rirA mutant derivatives from Rm1021 were analyzed for biofilm formation on two different abiotic surfaces (PVC and glass) and on alfalfa roots. Biofilm formation on abiotic surfaces was assessed by growing cells in Minimal Media (MM). The biofilm formed on PVC was stained with crystal violet and quantified by measuring the optical density. Biofilm formation on glass and alfalfa roots was studied by confocal laser scanning microscopy (CLSM) using green fluorescence protein (GFP)-labeled strains. Session III SIII-CP-17 RESULTS AND DISCUSSION The defects in biofilm development on glass shown by the rhbA mutant regardless of iron concentration (Figure 1A), demonstrate that siderophore Rhb1021, which is essential for Rm1021 surface motility, plays an important role in biofilm formation and it is not exclusively related to iron acquisition. The surfactant properties inherent to Rhb1021 might be responsible for its function in the two surface-associated phenotypes. In addition, several results obtained during this study provide the first evidence in Rhizobium of an inverse co-regulation of surface motility and biofilm formation: i) Strain GR4, which exhibits stricter control over surface motility under laboratory conditions, is more efficient in biofilm establishment on abiotic and root surfaces than strain Rm1021, which exhibits greater surface motility (Figure 1A, and C); ii) fadD and rirA mutations, which are known to promote S. meliloti surface translocation, interfere with the development of mature biofilms on glass surfaces, regardless of iron levels (Figure 1A); and iii) high-iron conditions that inhibit swarming motility in S. meliloti promote biofilm formation by inducing the development of thicker and sponge-like structured biofilms (Figure 1A, and B). S. meliloti fadD, rhb and rirA mutants were affected in plant root colonization (Figure 1C). These results indicate that components essential for biofilm formation and control mechanisms that inversely co-regulate swarming motility and biofilm development in S. meliloti participate to allow for optimal plant root colonization. Figure 1. A) CLSM images showing the thickness of 3- and 10-day-old biofilms developed by GR4 or Rm1021 and their derivative mutant strains on chambered cover glass slides in response to low (22 µM FeCl3) and high (220 µM FeCl3) iron availability. The biofilm thickness is represented by the xz p lanes. Structured biofilms developed in MM containing 220 µM of FeCl 3 are indicated with an asterisk (*) . B) CLSM images showing the architecture of 3-day-old biofilms developed by GFP-labelled GR4 cells on glass after growth in MM containing different concentrations of FeCl3. Bars, 15 µm. C) CLSM images of biofilms established on alfalfa root surfaces 3 dpi. Bars 70 µm. ACKNOWLEDGMENTS This work was supported by projects BIO2007-62988 and BIO2010-18005 of the Spanish MICINN. REFERENCES Verstraeten, N., et al. (2008). Trends Microbiol. 16: 496-506. Nogales, J., et al. (2010). BMC Genomics 11: 157 Nogales, J., et al. (2012). J. Bacteriol. 194: 2027-2035. Session III SIII-CP-18 Symbiotic phenotype of different rhizobial species on Lotus japonicus Gifu and Lotus burttii. Rodríguez-Navarro, D.N.1*, Temprano, F.1, Velázquez, E.2, Ruiz-Sainz, J.E.3 1 IFAPA, Centro Las Torres-Tomejil. 41200-Alcalá del Río, Sevilla (España). 2 Departamento Microbiología y Genética, Universidad de Salamanca, 37007 Salamanca (España). 3 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, 41012 Sevilla (España). * [email protected] ABSTRACT We have study the symbiotic behavior of strains belonging to Rhizobium, Sinorhizobium (Ensifer), Mesorhizobium, and Bradyrhizobium on Lotus japonicus Gifu and L. burttii species. Extra-slow-growing bradyrhizobia were also included. In comparison with L. japonicus plants, L. burttii nodulated with a wider range of rhizobial species. These differences in nodulation range might be related to the fact that bacterial entry through root-epidermis cracks might be more common in L. burttii than in L. japonicus (see poster Rodríguez-Navarro et al.: “Lotus japonicus Gifu and L. burttii responses to inoculation with a collection of S. fredii HH103 mutants affected in symbiotic signals”). INTRODUCTION Lotus japonicus is the model legume currently used for studying the formation of determinate (spherical) nodules. S. fredii HH103 is a fast-grower microsymbiont of soybean that also forms nitrogen fixing nodules with Lotus burttii (Sandal et al., 2012). This strain, however, only forms pseudonodules and ineffective white nodules with L. japonicus Gifu, indicating that L. burttii and L. japonicus show clear differences in symbiotic partner preference. In addition, Mesorhizobium loti appears to be an infrequent microsymbiont of Lotus spp. and rhizobial strains belonging to Mesorhizobium spp. and to Rhizobium spp. have been isolated from Lotus spp. nodules (Estrella et al., 2009). All these facts prompted us to investigate a set of Rhizobium, Sinorhizobium (Ensifer), Mesorhizobium, and Bradyrhizobium strains for their symbiotic capacity with L. japonicus and L. burttii. MATERIAL AND METHODS Surface sterilization of L. japonicus Gifu and L. burttii seeds and nodulation tests in square plastic boxes were carried our as described by Sandal et al. (2012). RESULTS AND DISCUSSION Results of this work are presented in Table 1. Although there are previous works describing rhizobial strains isolated from nodules of Lotus spp. plants growing in different soils of various countries (Lorite et al., 2010) to our knowledge this is the first attempt to evaluate the symbiotic capacity of a range of rhizobia on the model legume L. japonicus. Our results show that a wide variety of strains belonging to Rhizobium, Sinorhizobium (Ensifer), Mesorhizobium, and Bradyrhizobium isolated from legume species non-related with the Lotea tribe were also able to induce L. japonicus and /or L. burttii responses to inoculation. These root responses varied from only swellings or pseudonodules to nitrogen fixing (Fix+/- or Fix+) nodules. Session III SIII-CP-18 Table 1. Lotus japonicus and L. burttii responses to inoculation with different Rhizobium, Sinorhi zobium (Ensifer), Mesorhizobium and Bradyrhizobium strains. Genus Specie Strain L. japonicus L. burttii leguminosarum W290/TA1 Nod+, Fix+/Nod+, Fix+/bv trifolii leguminosarum ISL9 NodNod+ bv. viciae giardinii bv H152T Nod+, FixNod+, Fix+/giardinii etli CE3 Nod+, Fix+/Nod+, Fix+/Rhizobium gallicum R602T nd Nod+, Fix+/indigoferae CCBAU71714T NodNod+/-, Fixhainanense CCBAU57015T NodNod+/-, Fixloessense CCBAU71903T NodNod+/-, Fixundicola NodNod+/- Fix+/sullae CC1337 Nod+/-, Fixnd Rhizobium species that are Nod- with L. japonicus and L. burttii: R. galega HH103 Nod+, FixNod+/Fix+ Sinorhizobium fredii SMH12 Nod+, Fix+ Nod+, Fix+ NGR234 Nod+/-, FixNod+/-, FixSinorhizobium species that are Nod- with L. japonicus and L. burttii: S. mel iloti, S. medicae, S. kostiense, S. morelense australicum LMG24608T Nod+/-, FixNod+, FixMesorhizobium loti NZP2235 Nod+/Fix+ Nod+/Fix+ plurifarium LMG11892T NodNod+, Fix+ (1) temperatum CCBAU11018T Nod+, FixNod+, Fix+ (1) Mesorhizobium species that are Nod- with L. japonicus and L. burttii: M. amorphae, M. camelthorni, M. chacoense, M. robiniae. Bradyrhizobium canariense BTA-1T nd Nod+, Fix+ jicamae nd Nod+ liaoningense nd Nod+ pachyrhizi PAC48T nd Nod+, Fix+/Bradyrhizobium species that are Nod- with L. japonicus and/or L. burttii: B. japonicus, B. elkanii, B. spp. (peanut), B. betae. Nod-: absence of macroscopic root responses. Nod+/-: swellings, pseudonodules or a few white nodul es are formed. Nod+: nodules of normal external morphology are formed. Fix-: absence of nitrogen fixa tion. Fix+/-: weak or very weak nitrogen fixation. Fix+: presence of effective pink nodules. (1): acetyl ene reduction was measurable. nd: not determined ACKNOWLEDGMENT This work was supported by grants of Andalusia Government, P07-CVI-02506 and P11-CVI-7500. We thank Dr. Niels Sandal for providing Lotus seeds. REFERENCES Estrella, M.E., et al. (2009). Appl. Environ. Microbiol. 75: 1088-1098. Lorite, M.J., et al. (2010). Proceedings XIII-SEFIN, pp.23-24, Zaragoza (Spain). Madsen, L.H., et al. (2010). Nature. Doi: 10.1038/ncomms1009. Sandal, N., et al. (2012). DNA Res. 19: 317-323. Session III SIII-CP-19 Los efectores secretados a través del T3SS suprimen la respuesta de defensa en soja inducida por Ensifer (=Sinorhizobium) fredii HH103. Cubo, M.T.*, Jiménez-Guerrero, I., Pérez-Montaño, F., Ollero, F.J., López-Baena, F.J. Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. de Reina Merced es, 6.41012-Sevilla, España. * [email protected] RESÚMEN La función de los efectores secretados a través del sistema de secreción de tipo 3 (T3SS) simbiótico es aún desconocida. Por su similitud con efectores de patógenos, los efectores rizobianos deberían también ser capaces de suprimir respuestas de defensa de la leguminosa para favorecer la infección. Este trabajo pretende determinar qué respuestas defensivas se disparan en la soja en presencia de un rizobio compatible, Ensifer fredii HH103, y si la presencia del T3SS hace que esta estirpe sea capaz de nodular esta planta más eficientemente. INTRODUCCIÓN Muchas bacterias patógenas Gram-negativas utilizan el T3SS para inyectar efectores al interior de la célula hospedadora e inducir la enfermedad. Las plantas se defienden induciendo una respuesta de defensa mediada por el reconocimiento de ciertas moléculas del patógeno. Los efectores alterarían rutas de señalización en el hospedador para suprimir estas defensas. Sin embargo, algunos efectores son reconocidos por receptores específicos, disparándose una respuesta de defensa más fuerte que bloquea la infección. Los mecanismos de defensa de las plantas incluyen la acumulación de calosa y la producción de ROS, NO y proteínas PR y suelen estar asociados a la producción de hormonas vegetales como el ácido salicílico (SA), ácido jasmónico (JA) y etileno (ET). E. fredii HH103 (desde ahora HH103) es una estirpe de amplio rango de nodulación que posee el T3SS y que es capaz de secretar una serie de proteínas, denominadas Nop, al medio extracelular en respuesta a flavonoides. El papel de estos efectores en la simbiosis es aún desconocido aunque algunos se han caracterizado bioquímicamente y se ha demostrado que juegan un papel importante en la nodulación de ciertas leguminosas. Al inicio de la infección los rizobios son reconocidos como patógenos, pero la respuesta de defensa es débil y transitoria. Sin embargo, en la interacción simbiótica HH103-soja, se observa un aumento de la expresión del gen GmPR1 en la raíz en presencia de esta bacteria. Esta expresión aumenta tras inocular con un mutante que no secreta Nops. Es de destacar que se ha observado una restricción en la nodulación de la soja dependiente del T3SS. Un ejemplo sería la especificidad de cultivar que impide la nodulación de variedades americanas de soja por sólo algunas estirpes de E. fredii. MATERIAL Y MÉTODOS Los ensayos de nodulación en soja cv. Williams82 se realizaron según el método descrito por de Lyra et al. (2006). La expresión de genes de soja por qPCR se cuantificó según el método descrito por López-Baena et al. (2009). La determinación de las hormonas vegetales, mediante HPLC, y la detección de acumulaciones de calosa se realizaron siguiendo cualquier de los métodos descritos en la literatura. RESULTADOS Y DISCUSIÓN López-Baena et al. (2009) determinaron que el gen de soja PR1 se inducía en raíces inoculadas con HH103 en los estadíos iniciales de la nodulación. Esta inducción era Session III SIII-CP-19 mayor en ausencia de proteínas Nop. Para determinar si esta respuesta de defensa se asociaba a una reducción en el número de nódulos se realizó una cinética de nodulación que determinó que las plantas inoculadas con un mutante en el T3SS sufrían un retraso claro en la formación de nódulos. Este menor número de nódulos se correlacionaba con una menor expresión de los genes NIN, indicando que se estaban formando un menor número de tubos de infección. En este trabajo se estudió la expresión de genes relacionados con la patogénesis como el gen PR1 y dos peroxidasas a 2, 4 y 8 d.p.i. La expresión de PR1 volvió a ser mayor en raíces de plantas inoculadas con el mutante en T3SS pero en este caso, y a diferencia de los resultados previamente publicados, se aumentó la densidad celular del inóculo. Este aumento en el número de células provocó un adelanto en la respuesta de defensa. Las dos peroxidasas estudiadas tuvieron un pico de expresión a 2 d.p.i. y posteriormente fue decreciendo. Al igual que ocurría con la expresión de NIN, la transcripción de estos genes fue menor en plantas inoculadas con el mutante en el T3SS, indicando que probablemente se estuviera induciendo la formación de un menor número de nódulos. La expresión de PR1 es dependiente de SA por lo que se estudió si se estaba produciendo un aumento de la concentración de esta hormona en las raíces. Además de SA, se cuantificaron otras hormonas vegetales como el ile-JA y el ácido abscísico (ABA) a 2, 4 y 8 d.p.i. Los resultados indicaron un aumento en la concentración de SA en las plantas inoculadas con el mutante en el T3SS, indicando que efectivamente un aumento en la expresión de PR1 se asociaba a un aumento en la concentración de SA. Por otro lado, los niveles de JA se mantuvieron similares en todos los tratamientos a 2 y 4 d.p.i. Sin embargo, a los 8 días se produjo un aumento drástico en la concentración de la hormona en las plantas inoculadas con la estirpe parental HH103. En cuanto al ABA, a pesar de no observarse diferencias entre tratamientos a los diferentes tiempos, sí hubo un aumento claro de su concentración a los 2 días para posteriormente ir disminuyendo con el tiempo. Una vez determinado que se estaba produciendo una respuesta de defensa que estaba siendo suprimida por los efectores de HH103, se intentó determinar qué molécula bacteriana estaba induciendo esta respuesta de la planta. Los polisacáridos de superficie suelen estar involucrados en la inducción de respuestas de defensa primarias en patógenos por lo que estudiamos su papel en la inducción de la formación de depósitos de calosa. Los resultados indicaron que si bien no había diferencias en la deposición de calosa entre la estirpe silvestre y el mutante en el T3SS, en las raíces de plantas inoculadas con los mutantes en superficie apenas se observaban depósitos. Esto podría indicar que la superficie de la bacteria es responsable del reconocimiento inicial del rizobio como agente patógeno. AGRADECIMIENTOS Este trabajo ha sido financiado con los proyectos AGL2009-13487-C04 del MEC y P11-CVI-7050 de la Junta de Andalucía. El trabajo de Irene Jiménez-Guerrero ha sido financiado con un contrato predoc toral del IV Plan Propio de la US. REFERENCIAS de Lyra, M.C.C.P., et al. (2006). Int. Microbiol. 9: 125-133. López-Baena, F.J., et al. (2009). Mol. Plant Microbe Interact. 22: 1445-1454. Session III SIII-CP-20 Pode Bradyrhizobium japonicum, em combinação com diferentes densidades de plantio de soja alterar componentes do rendimento? de Luca, M.J.1*, Nogueira, M.A.2, Hungria, M.2 1 Universidade Estadual de Londrina; Brasil, Londrina, PR. 2 Embrapa Soja, Brasil, Londrina, PR. * [email protected] RESUMO A campo, baixas densidades de plantas de soja, de 80.000 pl./ha, estimulam a formação de grãos por planta, mas não a produtividade; contudo, diferentes estirpes de Bradyrhizobium não afetam esse parâmetro. Em casa de vegetação, densidades de 80.000 pl./ha incrementam o número de vagens/pl. em relação à densidade de 320.000 pl./ha, com diferenças entre estirpes. INTRODUÇÃO Baixas densidades de plantas resultam em incrementos na disponibilidade de luz na parte inferior das plantas, onde a radiação é baixa e a abscisão de flores e vagens pequenas é elevada, resultando em um aumento na retenção de vagens (Johnston et al., 1969). Há relatos de que a relação infravermelho/vermelho afeta a ultra-estrutura dos cloroplastos, a partição de carboidratos para as células, a eficiência fotossintética e a concentração de vários metabólitos (Kasperbauer, 1987), bem como a nodulação e a fixação biológica do nitrogênio (FBN) (Lie, 1964; 1969). O objetivo deste trabalho foi o de verificar o efeito de diferentes densidades de plantio de soja, em combinação com diferentes estirpes de B. japonicum, em componentes de rendimento. MATERIAL E MÉTODOS Foram realizados ensaios em casa de vegetação e a campo, em Londrina, PR, Brasil, na estação experimental da Embrapa Soja. Utilizou-se a cultivar de soja BRS 284, de hábito de crescimento indeterminado. Em ambos os experimentos foram avaliadas duas densidades de semeadura, de 80.000 pl./ha e de 320.000 pl./ha. Sementes de soja foram inoculadas com as estirpes de Bradyrhizobium japonicum SEMIA 5079 (=CPAC 15) e CNPSo 2050, na dose de 1,2 milhões de células/semente. Foram incluídos controles não inoculados sem N mineral e, a campo, também com N mineral. No experimento em casa de vegetação foram utilizados vasos de 50 x 23 x 16 cm com uma mistura de terra e areia (3:1) esterilizada. No tratamento simulando 80.000 pl./ha cada vaso continha duas plantas separadas por 25 cm, enquanto no de 320.000 pl./ha, oito plantas separadas por 6 cm. O delineamento experimental foi em blocos ao acaso, com 30 repetições por tratamento. O ensaio foi coletado em R5. Avaliou-se o número total de vagens por planta. Os ensaios de campo foram conduzidos por três anos (2010, 2011 e 2012), em parcelas de 4 m x 10 m, com desenho experimental em blocos ao acaso, com seis repetições. Em V6 foram avaliados o número de grãos/pl. e o rendimento; avaliaram-se, também, parâmetros de fixação biológica do nitrogênio, como número (NN) e massa (M) de nódulos secos por planta e teor de N dos tecidos por digestão sulfúrica. As análises estatísticas foram realizadas com o software Infostat (Di Rienzo et al., 2009). RESULTADOS E DISCUSÃO No experimento em casa de vegetação, houve maior formação de vagens por planta na menor densidade, bem como diferenças entre estirpes, sendo maior com a estirpe CNPSo 2050 em ambas densidades (Tabela 1). Na maior densidade de plantas, os tratamentos 4 e 6 foram iguais. Uma hipótese é de que baixas densidades e a estirpe Session III SIII-CP-20 CNPSo 2050 afetaram o balanço de CKs. Em soja, CKs podem incrementar a relação fonte/dreno, promovendo a divisão celular nos ovários jovens e redirecionando o movimento dos assimilados para os ovários em desenvolvimento, diminuindo a taxa de aborto de vagens. Tabela 1. Número de vagens por planta. obtido em ensaio em casa-de-vegetação. Embrapa Soja (Londrina-PR) Tratamento 1 T1 T2 T3 T4 T5 T6 113,3 b2 49,5 c 102,6 b 44,5 c 138,7 a 57,8 c n° de vagens/pl. 1 Tratamentos: T1) testemunha (sem inoculação e sem N mineral), 2 pl./vaso; T2) testemunha 8 pl./vas o; T3) SEMIA 5079, 2 pl./vaso; T4) SEMIA 5079, 8 pl./vaso; T5) CNPSo 2050, 2 pl./vaso; T6) CNPSo 2050, 8 pl./vaso. 2 Letras distintas indicam diferenças significativas (Teste de Fisher, p<0,10). No experimento de campo, baixas densidades de plantas (80.000 pl./ha) estimularam a formação de grãos por planta, mas não foram observadas diferenças no rendimento (Tabela 2). Também não foram observadas diferenças entre estirpes. Em baixas densidades houve um incremento nos parâmetros de NN/pl. e MNS/pl., mas não se detectaram diferenças entre estirpes. Tabela 2. N° grãos/pl.; teor de N na parte aérea [TEORN (%)], número de nódulos por planta (NN/pl. ), e massa de nódulos secos por planta (MNS mg/pl.) obtido em um ensaio conduzido no campo experimental da Embrapa Soja (Londrina – PR). Rendimento Tratamento (kg/ha) N° grãos/pl TEORN (%) NN/pl MNS mg/pl T1 2473 a1 343,6 a 3,2 a 81,8 a 264,8 a T2 2798 a 127,5 b 3,1 a 54,8 b 222,0 bc T3 2592 a 354,1 a 3,3 a 48,9 b 123,1 d T4 2805 a 127,5 b 3,3 a 33,7 c 102,4 d T5 2371 a 334,3 a 3,1 a 71,7 a 251,0 ab T6 2655 a 135,8 b 3,2 a 46,4 b 196,7 c T7 2427 a 329,4 a 3,0 a 70,1 a 242,4 ab T8 2644 a 129,4 b 3,1 a 49,6 b 208,1 c 1 Tratamentos, T1) Testemunha sem inoculação, 80.000 pl/ha; T2) Testemunha sem inoculação, 320.000 pl/ha; T3) T + fertilizante nitrogenado (200 kg de N/ha, parcelados em duas aplicações), 80.000 pl /ha; T4) T + fertilizante nitrogenado (200 kg de N/ha, parcelados em duas aplicações), 320.000 pl/ha; T 5) CNPSo 2050, 80.000 pl/ha; T6) CNPSo 2050, 320.000 pl/ha. T7) SEMIA 5079, 80.000 pl/ha; T8) SEMIA 5079, 320.000 pl/ha. 2 Letras distintas indicam diferenças significativas (Teste de Fisher, p<0,10). AGRADECIMENTOS Trabalho executado com recursos de Embrapa Soja, INTA (Instituo Nacional de Tecnologia Agropecuaria, Argentina) e CNPq-Repensa (562008/2010-1). REFERENCIAS Beaufils, E.R. (1973). Soil Science, Bulletin N° 1. Di Rienzo, J.A., et al. (2009). InfoStat versión. FCA, Universidad Nacional de Córdoba. Johnston, T.J,. et al. (1969). Crop Sci. 9:577-581. Kasperbauer, M.J. (1987). Plant Physiol. 85: 350-354. Lie, T.A. (1964). Wageningen University, Wageningen, 89 p. Lie, T.A. (1969). Plant Soil 30:391-404. Session III SIII-CP-21 Effects of glyphosate-resistant gene and herbicides on biological nitrogen fixation symbiotic efficiency and grain productivity of soybean in Brazil. Hungria, M.1, Mendes, I.C.2, Nakatami, A.S.1, 3, Reis-Junior, F.B.2, Fernandes, M.F.4* 1 Embrapa Soja, Londrina, PR, Brazil. 2 Embrapa Cerrados, Planaltina, DF, Brazil. 3 Pos-doc fellowsh ip from CNPq. 3 Embrapa Tabuleiros Costeiros, Aracaju, SE, Brazil. * [email protected] ABSTRACT We reported the effects of transgenic glyphosate resistant soybean cultivars (GRC), glyphosate and weed management strategies (glyphosate + GRC vs. conventional herbicides + non-GRC) on biological nitrogen fixation (BNF), symbiotic efficiency (SyEf) and grain yield (SGY) in soybean. Six variables related to BNF were pooled and analyzed as SyEf. Data were obtained in field trials in six sites in Brazil and three growing seasons. Small effects of GRC and glyphosate where observed on SyEf in some sites. SyEf did not differ between the two weed management strategies. SGY was higher in the glyphosate + GRC management in three out of the six sites. INTRODUCTION Glyphosate-resistant soybean cultivars (GRC) were released in Brazil in 2003 and, in 2010, have already accounted for 86% of the cultivated area in the country. In 2010, it was estimated that the inoculation of soybean with N2-fixing bacteria led to savings of US$ 7 billion/year due to the non-use of N fertilizers. The widespread use of glyphosate and GRC has raised concerns whether the biological N fixation (BNF) would be affected. Results on the effects of glyphosate or GRC on BNF are inconsistent (Bohm et al., 2009; Kremer and Means, 2009; Zablotowicz and Reddy, 2004). When deleterious effects of glyphosate are observed on the BNF variables, they have not been accompanied by decreases in soybean grain yields (Bohm et al., 2009). In this work we reported the results of experiments carried out between 2003 and 2006 in six major producing areas of Brazil. Our objective was to evaluate the effects of GRC, glyphosate and weed management strategies on the BNF symbiotic efficiency and grain productivity in soybean. MATERIAL AND METHODS The experiments were set up under no-till in the 2003/2004; 2004/2005, and 2005/2006 growing seasons at six sites in Brazil: Passo Fundo (RS); Ponta Grossa (PR); Londrina (PR); Uberaba (MG); Planaltina (DF); and Luiz E. Magalhães (BA). Trials were conducted in a RBD, with 5 treatments x 3 cultivars, with 6 replicates. Treatments were 1) GRC + glyphosate; 2) GRC + conventional herbicides; 3) non-GRC + conventional herbicides; 4) GRC + hand weed control; 5) non-GRC + hand weed control. Three pairs of cultivars, each including the non-GRC and its nearly-isogenic GRC, were cropped in each site. Soybean was inoculated with Bradyrhizobium elkanii SEMIA 587 and B. diazoefficiens SEMIA 5080. At the V4 stage, plants were evaluated for nodule dry weight (NDW), and at R2, for NDW, shoot total N and N concentration, total Nureide and percentage of N-ureide. SyEf was described by these six variables pooled and analyzed by multivariate techniques. SGY was determined at harvest. Means contrasts were used to evaluate the effects of transgenic trait, type of herbicide in GRC, and weed management strategies on SGY and SyEf. Multiresponse permutation procedures (MRPP, Mielke and Berry, 2000) were used to test for differences in SyEf between treatments. RESULTS E DISCUSSION Biological Nitrogen Fixation Symbiotic Efficiency (SyEf). SyEf was affected by the type of cultivar (Contrast 1) in four out of six areas, and by the type of herbicide (Contrast 2) only in Passo Fundo (Table 1). In all these cases, effects were very small, as observed by the chance-corrected within-group agreements (A Session III SIII-CP-21 values) (see footnotes in Table 1). SyEf did not differ between the two weed management strategies (Contrast 3, Table 1). Table 1. Statistical significance (p) and effect size (A value)1 of means contrasts2 comparing tra nsgenic GR trait, type of herbicide and weed management strategy on BNF symbiotic efficiency in six areas in Brazil. p values (A values) Site Contrast 1 Contrast 2 Contrast 3 Passo Fundo 0.040 (<0.01) 0.005 (0.023) 0.069 (<0.01) Ponta Grossa 0.100 (<0.01) 0.251 (<0.01) 0.418 (<0.01) Londrina 0.003 (0.011) 0.073 (<0.01) 0.611(<0.01) Uberaba 0.716 (<0.01) 0.470 (<0.01) 0.473 (<0.01) Planaltina 0.009 (0.013) 0.484 (<0.01) 0.408 (<0.01) L. E. Magalhães 0.047 (<0.01) 0.179 (<0.01) 0.554 (<0.01) 1 A value represents the chance-corrected within-group agreement. When A equals zero, in a scale fro m 0 to 1, the within-group heterogeneity of the samples equals that observed by chance. 2 Contrast 1 compares GR and non-GR cultivars; Contrast 2 compares glyphosate and conventional herbicide; Contrast 3 compares GR-cultivars + glyphosate and non-GR cultivars + conventional herbicide. Grain Yield. GRC reduced SGY only at Passo Fundo (-21 %). In GRC, the use of glyphosate resulted in SGY increases (+ 8 to 33%) in four out of the six areas evaluated. SGY was higher in the glyphosate + GRC-based weed management in three of the six regions (+10 to 12%) (Table 2). Table 2. Soybean grain yield as affected by gliphosate resistant cultivar (GRC) (Contrast 1, C1), herbicide type (Contrast 2, C2) and weed management strategy (Contrast 3, C3) in six major producing areas i n Brazil Passo Ponta Luis E. Fundo Grossa Londrina Uberaba Planaltina Magalhães C1 GRC 859 2545 2230 2957 3378 2550 Non-GRC 1094 2512 2249 3023 3442 2572 p <0.01 ns ns ns ns ns C2 GRC with glyphosate 1089 2747 2389 2939 3572 2766 GRC with conventional herbicide 815 2474 2200 2931 3405 2448 p <0.01 <0.05 <0.05 ns ns <0.05 C3 GRC + glyphosate 1089 2747 2389 2939 3572 2766 Non-GRC + conventional herbicide 1067 2466 2178 2984 3387 2462 p ns <0.05 <0.01 ns ns <0.05 ACKNOWLEGMENTS Project CNPq-Repensa 562008/2010-1 REFERENCES Kremer, R., and Means, N. (2009). Eur. J. Agron. 31: 153-161. Zablotowicz, R., and Reddy, K. (2004). J. Environ. Qual. 33: 825-831. Bohm, G.M.V., et al. (2009). Soil Biol. Biochem. 41: 420-422. Mielke, P.W., and Berry, K.J. (2000). Permutation methods: a distance function approach. Springer, NY, USA, 352 pp. Session III SIII-CP-22 Diversidade genética de bactérias que colonizam nódulos radiculares de Phaseolus vulgaris L. cultivado em campo e em casa de vegetação. Oliveira-Francesquini, J.P.1*, Hungria, M.2, Glienke, C.1, Kava-Cordeiro, V.1, GalliTerasawa, L.1 1 Departamento de Genética, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, Paraná, Brasil. 2 Embrapa Soja, Londrina, Paraná, Brasil. * [email protected] RESUMO Foi realizado o sequenciamento parcial dos genes 16S rRNA e glnII de seis isolados de nódulos radiculares de feijoeiro comum (Phaseolus vulgaris L.), sendo três de plantas cultivadas a campo (LGMB10, LGMB57 e LGMB58) e três de plantas cultivadas em casa de vegetação (LGMB73, LGMB88 e LGMB99). Foi observada uma preferência de colonização de acordo com o experimento avaliado. INTRODUÇÃO A análise da diversidade genética de bactérias capazes de nodular o feijoeiro comum auxilia no processo de desenvolvimento de tecnologias de baixo custo para a produção desta cultura, como o desenvolvimento de inoculantes contendo espécies fixadoras de nitrogênio. A associação entre a planta e o microssimbionte obedece uma estreita relação, regulada geneticamente. No entanto, bactérias nativas isoladas a partir de nódulos de Phaseolus vulgaris mostram uma diversidade genética considerável, sugerindo que várias espécies de Rhizobium podem se associar com o feijoeiro (Eardly et al., 1995; Piñero et al, 1988). Ainda, variações na diversidade em condições de campo e em solos diluídos foram observadas por Alberton et al. (2006) e Oliveira et al. (2011), baseadas no estudo de marcadores moleculares. No presente trabalho, essas diferenças foram demonstradas através da análise de sequências de DNA. MATERIAL E MÉTODOS Sequenciamento do gene 16S rRNA A amplificação inicial e o sequenciamento do gene foram realizados de acordo com Menna et al. (2006). Sequenciamento do gene glnII A amplificação inicial e o sequenciamento do gene foram realizados de acordo com Stepkowski et al. (2005). Análise filogenética Para cada gene, as sequencias obtidas foram alinhadas com sequencias de linhagens já identificadas, dentre elas as linhagens tipo. O alinhamento foi realizado no programa MAFFT (http://mafft.cbrc.jp/alignment/server/) e a construção da árvore filogenética no programa MrBayes (Ronquist and Huelsenbeck, 2003), utilizando o GTR (General time-reversible) como modelo evolutivo. RESULTADOS E DISCUSSÃO Por meio da análise da árvore filogenética gerada com os dados do 16S rRNA (dados não mostrados) não foi possível inferir uma classificação precisa para os isolados a nível de espécie, uma vez que alguns deles formaram grupos com mais de uma linhagem tipo. Por outro lado, a árvore gerada com os dados do glnII (dados não mostrados) apresentou uma melhor resolução para as espécies avaliadas, dando um bom suporte na análise das sequencias concatenadas (Figura 1). Session III SIII-CP-22 Apesar das linhagens de Rhizobium leguminosarum, R. etli e R. phaseoli não estarem bem resolvidas nesta análise, pode-se observar que os isolados de plantas cultivadas em casa de vegetação formaram um grupo com 100% de suporte com a espécie R. miluonense e os isolados provenientes do experimento a campo agruparam (100% de suporte na Inferência Bayesiana) com R. leguminosarum, o que oferece um forte indício para sua classificação. Desta forma, a especificidade de colonização de acordo com as condições ambientais fornece subsídios para o desenvolvimento de inoculantes comerciais com linhagens que sejam capazes de se adaptar ao ambiente em que serão utilizadas. A caracterização genética através do gene 16S rRNA tem sido utilizada como ferramenta de classificação para bactérias, no entanto sua análise isolada não é suficiente para diferenciar espécies estreitamamente relacionadas. Sendo assim, outros genes têm sido propostos para auxiliar na realização de análises filogenéticas, dentre eles o glnII (Gevers et al., 2005). No presente estudo, foi possível realizar uma melhor discriminação entre as espécies, baseada na análise conjunta das duas sequências gênicas. No entanto, a avaliação de um maior número de genes, além de um estudo polifásico, é recomendada para uma classificação mais precisa. Figura 1. Árvore filogenética baseada nas sequencias de glnII e 16S rDNA de rizóbios isolados de nódulos radiculares de feijoeiro comum e linhagens de referência. A árvore foi gerada no programa MrBayes, utilizando GTR como modelo evolutivo. AGRADECIMENTOS Este trabalho teve apoio do CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil), CNPq-Repensa (56008/2010-1). REFERÊNCIAS Alberton, O., et al. (2006). Soil Biol. Biochem. 38: 1298-1307. Eardly, B.D., et al. (1995). Appl. Environ. Microbiol. 61: 507-512. Menna, P., et al. (2006). Syst. Appl. Microbiol. 29: 315-332. Gevers, D., et al. (2005). Nat. Rev. Microbiol. 3: 733-739. Oliveira, J.P., et al. (2011). World J. Microbiol. Biotechnol. 27: 643-650 Piñero, D., et al. (1988). Appl. Environ. Microbial. 54: 2825-2832. Ronquist, F., and Huelsenbeck, J.P. (2003). Bioinformatics. 19: 1572-1574. Stepkowski, T. et al. (2005). Appl. Environ. Microbial. 71: 7041-7052. Session III SIII-CP-23 Symbionts of Mimosa spp. in ultramafic soils in central Brazil. Reis Junior, F.B.1*, James, E.K.2, Hertel Júnior, C.R.1, Lopes, A.A.C.1, Alves, M.R.P.1, Souza, L.M.1, Baura, V.A.3, Mendes, I.C.1, Andrade, L.R.M.1 1 Embrapa Cerrados. Planaltina, Distrito Federal, Brazil. 2 The James Hutton Institute. Dundee, Scot land. 3 Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná (UFPR). Curitiba, Paraná, Brazil. * [email protected] ABSTRACT In this study we investigated the diazotrophic symbionts associated with Mimosa in the metal-rich soils from Barro Alto – GO, Brazil. Our results indicated Burkholderia spp. as their symbionts in this peculiar environment, and that these symbioses could probably play an important role in the restoration of ultramafic areas degraded by mining activity. INTRODUCTION Recent studies have shown that species of Mimosa, native to the Brazilian Cerrados, form symbiosis with diazotrophic bacteria known as -rhizobia, particularly Burkholderia spp., and seems to play a prominent role in N cycling in this ecosystem (Reis Junior et al., 2010). These same studies suggest that environmental characteristics rather than the host species are responsible for determining the distribution of Burkholderia species. Previous reports have shown that acidic soils, which is not the case of the ultramafic areas, could be more conducive for the survival of Burkholderia symbionts. The neutral to basic pH is more favorable for Cupriavidus, another symbiont of Mimosa spp., but not yet found in Brazil nodulating these plants in natural systems. Cupriavidus is also known as a metal-loving genus (Klonowska et al., 2012), so it might be expected to encounter endemic Mimosa spp. that have co-evolved with similar bacteria in the ultramafic soils, as these contain an excess of metals such as cobalt, chromium, copper, and especially nickel. The aim of this study was to investigate the diazotrophic symbionts associated with M. somnians and M. claussenii in the peculiar metal-rich soils of Barro Alto – GO, Brazil. This is the first step in a bigger project that aims to give greater knowledge in the symbiosis between diazotrophs and the legumes that grow in these soils with high metal concentrations. In addition, the results obtained can provide information to assist in restoration programs of areas affected by Ni mining. MATERIAL AND METHODS Two sites were chosen for this study corresponding to different Ni bioavailabilities, and where M. somnians and M. claussenii were observed to grow. Nodules were sampled from Mimosa collected in the above sites, and these were taken for “rhizobial” isolation and microscopy according to standard protocols (Reis Junior et al. 2010). Nodules that were sub-sampled for microscopy were analyzed by immunogold labeling with antibodies specific to the genera Burkholderia and Cupriavidus (Reis Junior et al. 2010). The 16S rRNA from two isolates of M. claussenii that morphologically represented a large part of the isolates were amplified and sequenced for bacterial identification. Symbiotic efficiency was assessed using plants of M. pudica grown in pots containing a mixture of sand and vermiculite (50/50) and supplemented with nitrogen-free nutrient solution. The dry weight of shoots and roots were measured at 45 days post inoculation in a greenhouse. Session III SIII-CP-23 RESULTS AND DISCUSSION (A) (B) (C) Figure 1. Light micrographs of M. claussenii (A) and M. somnians (B) nodules from ultramafic soil that have been immunogold-labeled with an antibody against B. phymatum, and of M. claussenii with an antibody against C. taiwanensis (C). The majority of the nodules examined from both sites were found to be effective in appearance when examined under the light microscope. It was shown that almost all Mimosa nodules reacted positively with the B. phymatum antibody (Figs. 1A and 1B). No nodules reacted with the antibody against C. taiwanensis (Fig 1C). The 16S rRNA sequencing of the two isolates of M. claussenii confirmed that the isolates belong to the Burkholderia genus. In general, inoculation promoted significant increases in shoot (up to 2100%) and root (up to 400%) dry matter production of M. pudica, which indicates that the symbiosis was efficient and shows its importance for plant growth (Figs. 2A and 2B). (A) (B) Figure 2. M. pudica inoculated with isolates from M. claussenii (47) and M. somnians (43) from sit es with lower (A) and higher (B) Ni availability, respectively, compared with uninoculated control (1 ). Recent studies have shown that as with nodulation, the degree of endemism apparently has no effect on the general choice of Burkholderia as a symbiont by the Mimosa spp. (Reis Junior et al., 2010). Our preliminary results indicate that even with soil characteristics apparently not suitable for these bacteria, such as the ultramafic soils from this study, Burkholderia remains as the main and preferred symbiont of Mimosa in Brazil. Additionally, we can suggest that this symbiosis could be an important factor for the restoration of ultramafic areas degraded by mining activity. ACKNOWLEGMENTS This work was supported by Anglo American Brazil, National Counsel of Technological and Scientific Development (CNPq) and INCT-FBN. REFERENCES Klonowska, A. et al. (2012). FEMS Microbiol. Ecol. 81: 618-635. Reis Junior, F.B., et al. (2010). New Phytol. 186: 934-946. Session III SIII-CP-24 Cloning, Expression and Characterization of a Chitinase class III from Casuarina glauca nodules. Graça, I.1, 2*, Liang, J.1, 3, Guilherme, M.2, Ferreira-Pinto, M.3, Ribeiro, A.1, 3, Pereira, A.S.2 1 Centro BIOTROP/IICT, Oeiras, Portugal. 2 Requinte/CQFB, Departamento de Química, FCT/UNL, Monte da Caparica, Portugal; 3ITQB/UNL, Oeiras, Portugal. * [email protected] ABSTRACT Actinorhizal plants have become increasingly important as climate changes threaten to remake the global landscape over the next decades. These plants are able to grow in poor and disturbed soils and are important elements in plant communities worldwide. Besides that, most actinorhizal plants are capable of high rates of nitrogen fixation due to their capacity to establish root nodule symbiosis with N 2-fixing Frankia strains. Nodulation is an ontogenic process that requires a sequence of highly coordinated events. One of these mechanisms is the induction of defence-related events, whose precise role is not clear. Here we report on the biochemical and biological characterization of a class III chitinase from Casuarina glauca (CgCHI3) activated specifically in nodules as compared with roots and leaves. According to our results, this protein seems to be involved in nodule development, most likely in the formation of the infection thread or in any other cell modification to accommodate the symbiotic bacteria. INTRODUCTION Actinorhizal plants are a group of perennial dicots distributed over 8 different families and 25 genera. These plants are essentially woody shrubs or trees (Benson and Silvester 1993). Ecologically, the majority of actinorhizal plants are pioneers on nitrogen-poor open sites, being of great economical and ecological relevance in the recovery and stabilisation of degraded ecosystems (Diem and Dommergues 1990). Like in the case of legumes, actinorhizal symbiosis results in the formation of a new organ, the root nodule, where bacteria are hosted and N 2 fixation takes place (Pawlowski and Bisseling 1996). Nodulation is a highly complex and coordinated process that involves the induction and suppression of several genes and their respective products. Amongst others, defence genes are activated during nodulation, which may be involved in nodule protection or development (Fortunato et al. 2007; Santos et al. 2010). Chitinases are an important group of enzymes up-regulated during N2-fixing rootnodule symbioses wh ere they might play multiple roles (reviewed by Santos et al. 2008). Here we report on the biochemical and biological characterization of CgCHI3, a class III chitinase activated specifically in Casuarina glauca nodules induced by Frankia, and discuss its possible role during the symbiotic process. MATERIAL AND METHODS Recombinant protein expression. The Open Reading Frame of cgchi3 (accession number: EF134410) was cloned into the pET-28a expression vector (Novagen) and expressed in Escherichia coli strain BL21 (DE3) for protein expression. BL21 (DE3) cells containing the pRec-cDNA-ORF were grown in 1L LB medium at 37ºC, during 2h. The expression of the recombinant protein (CgCHI3) was then induced by IPTG during 3h. CgCHI3 was purified from inclusion bodies at 15ºC as described by Kirubakaran & Sakthivel (2007) with slight Session III SIII-CP-24 modifications: -mercaptoethanol was replaced by the redox buffer GSSG/GSH. Biochemical assays included chitinase, chitosanase, glucanase, lisozymatic and cellulase activities. Biological assays against bacteria (Frankia alni, Rhizobia, E.coli, Paracoccus denitrificans and Bacillus subtilis) and phytopathogenic fungal species, Botritys cinerea (Jatropha curcas), Colletotrichum gloeosporioides (Manihot esculenta), Furasium oxysporum (Solanum lycopersicum), Trichoderma viridae (Solanum lycopersicum), were performed with the disc diffusion method (Bauer et al 1996). RESULTS AND DISCUSSION The recombinant CgCHI3 (32 kDa) was mainly produced as insoluble inclusion bodies. Therefore, it was further solubilized, purified and refolded in an urea gradient. The purified protein was unable to use chitin and chitosan as substrates, but has successfully metabolized -1,4-endoglucanase, -1,3-glucan and lysozyme. Biological assays with symbiotic Frankia and rhizobia did not affect the symbiotic process, suggesting that CgCHI3 is not directly involved in the infection process. Additionally, CgCHI3 was unable to impair or promote growth of pathogenic bacteria as well as pathogenic fungi. According to Fortunato et al (2007), cgchi3 is an early nodulin gene, being expressed in the meristem and in the uninfected cortical cells of young nodules, and is probably associated with the process of nodule development. Our results support this hypothesis, suggesting that most likely CgCHI3 is involved in the formation of the infection thread or in any other cell modification to accommodate the symbiotic bacteria. ACKNOWLEDGEMENTS This work was supported by grants PPCDT/AGR/55651/2004 and PTDC/AGRFOR/4218/2012 from Fundação par a a Ciência e Tecnologia (FCT), co-financed by the European Fund FEDER, and by FCT grant SFRH/BD/41589/2007, co-financed by the Portuguese PIDDAC program and European Social Fund, under the 3 rd framework program. REFERENCES Bauer, A.W. et al. M (1996). Am. J. Clin. Pathol 45: 493-496. Benson, D.R., and Silvester, W.B. (1993). Microbiological Reviews, Washington. 57: 297-319. Diem, H.G., and Dommergues, Y. (1990). In:, The Biology of Frankia and Actinorhizal Plants, Academ ic Press Inc., New York. Schwintzer C.R., Tjepkema J.D., eds, pp. 317-342. Fortunato, A, et al. (2007). Physiol. Plantarum 130: 418-426 Pawlowski, K., and Bisseling, T. (1996) The Plant Cell 8: 1899-1913. Santos, P., et al. (2010).. Symbiosis 50: 27-35. doi:10.1007/s13199-009-0031-0 Santos, P., et al. (2008). Plant Biotech. 25, 299-307. doi:10.5511/plantbio technology.25.299. Singh, A., et al. (2007). Protein Expr. Purif. 56:100-109. Session III SIII-CP-25 Negative short-term salt effects on the soybean-B. japonicum interaction and partial reversion by calcium addition. Muñoz, N.1, 2, Rodriguez, M.1, Robert, G.1, 2, Lascano, R.1, 2* 1 Instituto de Fisiología y Recursos Genéticos Vegetales, Centro de Investigaciones Agropecuarias-IN TA, Camino a 60 Cuadras Km 5 y ½, Córdoba Argentina. 2 Cátedra de Fisiología Vegetal, Facultad de Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 299, Córdoba Argentina. * [email protected] ABSTRACT The short-term (2 h) effects of salt stress (50 and 150 mM NaCl), on early events of soybean-B. japonicum interaction were analyzed determining the following parameters in root hair with o without calcium addition: deformation, apoplastic superoxide radical production (O2.-), root hair death and sodium/potassium ion content. We also analyzed whether this short-term salt stress influenced later formation of crown and noncrown nodules determining number and weight of nodules. The negative effect of salt stress on root hair deformation, O2.- production, ionic homeostasis, root hair death and crown nodulation were attenuated by the addition of 5 mM CaCl2. INTRODUCTION Soybean-rhizobia symbiotic interaction is severely affected by salt stress. Our group has characterized early negative effects of salt stress on root hair deformation and on the production/degradation of reactive oxygen species (ROS), which are fundamental responses during early events of legume-rhizobia interaction. Decreased root hair deformations and root hair death were observed 2 h post inoculation in saline treatments. Some of the responses observed in combinations of salt stress and the rhizobia are similar to pathogenic interactions. The addition of calcium, a common agricultural practice to reverse salt effects, also has a positive effect on the soybeanrhizobia s ymbiotic interaction. The positive effect of calcium addition on soybean under saline conditions is first reported. MATERIAL AND METHODS Early effects of salt stress treatments (50 and 150 mM NaCl) were evaluated determining deformation, apoplastic superoxide radical production (O 2 -), root hair death and sodium/potassium ion content (Muñoz et al., 2012) with or whitout the calcium addition (5 mM CaCl2). The influences of this short-term salt stress on later stages were evaluated determining formation of crown and noncrown nodules (number and weight of nodules). RESULTS AND DISCUSSION Short-term salt stress on ionic homeostasis, root hair deformation and death: effect of calcium addition. Na+/K+ ratios in root hair was increased in a dose-dependent manner during saline treatments and independently of the symbiont presence, and did not change in osmotic treatments. This change in the Na + /K+ ratios is due to sodium influx from the extracellular medium to the intracellular environment and not to an outflow of potassium and these responses were partially reverted with the addition of calcium. Root hair deformation was not affected under 50 mM NaCl treatment, although it was significantly delayed. Under 150 mM NaCl, root hair deformation was significantly reduced. Interestingly, these responses were partially reverted with the addition of Session III SIII-CP-25 calcium. Root hairs death was evaluated by observation of nuclear morphology. Loss of nuclear integrity (chromatin condensation and DNA fragmentation) was observed in 50 mM saline treatments inoculated and 150 mM regardless of the symbiont presence. The differences observed in the nucleus morphology, particularly associated with the increase of nucleus with chromatin condensation and the decrease of nucleus with DNA fragmentation in saline treatments supplemented with calcium, with respect to the unsupplemented, indicates that the addition of calcium to saline treatments inhibited or at least delayed death process (Figure 1). This inhibition or delay in the death progress, given by calcium addition, could help to sustain the rhizobia infection and subsequent nodule formation. Short-term salt stress on nodulation: effect of calcium addition. The negative effect of short-term saline treatments on crown nodule formation was dose-dependent and was produced by the ionic component of salt stress, since the number of nodules remained unaltered in the osmotic controls of these treatments (Figure 2). This result suggests that ionic homeostasis of root hair during the early event of symbiotic interaction has a key role on later events, such as nodule formation. The addition of calcium helped to partially reverse the negative effects of salt on nodule formation. Figure 1. Percentages of nuclei with chromatin condensation or DNA fragmentation after two hours under different treatments: NaCl (50 or 150 mM), and NaCl (50 or 150 mM) supplemented with 5 mM CaCl2, all of them inoculated and noninoculated with B. japonicum. Figure 2. Nodule number in soybean plants treated at germinated seed stage for two hours with: NaCl (50 or 150 mM), Sorbitol (100 or 300 mM) and NaCl (50 or 150 mM) supplemented with 5 mM CaCl2 all of them inoculated with B. japonicum. ACKNOWLEDGEMENTS This work was supported by grants from the Agencia de Promoción Científica y Tecnológica, Argentin a, and the Instituto Nacional de Tecnología Agropecuaria (INTA). REFERENCES Muñoz, N., et al. (2012). Environ. Exp. Bot. 78: 76-83. Session III SIII-CP-26 Expresión de supresores de muerte celular en raíces en cabellera de soja: efectos bajo condiciones de estrés y en la interacción con Bradyrhizobium japonicum. Robert, G.1, 2*, Muñoz, N.1, 2, Lascano, R.1, 2 1 Instituto de Fisiología y Recursos Genéticos Vegetales (IFRGV-INTA), Camino a 60 Cuadras Km 5 ½, X5020 ICA, Córdoba, Argentina. 2 FCEFyN, Universidad Nacional de Córdoba, Cátedra de Fisiología Vegetal. Avda. Vélez Sarsfield 290. CP 5000, Córdoba, Argentina. * [email protected] RESUMEN La expresión del supresor de muerte celular programada, Ced-9, del nemátodo Caenorhabditis elegans en raíces en cabellera de plantas de soja (Glycine max), retrasó los procesos de muerte celular inducidos por i) inoculación con Bradyrhizobium japonicum bajo condiciones de estrés salino moderado (NaCl 50 mM) y ii) estrés salino severo (NaCl 150 mM), los cuales mostraron características de procesos de muerte ordenada y necrótica, respectivamente. Asimismo, paradójicamente la expresión de Ced-9 inhibió la nodulación. En este trabajo se presentan evidencias de que estos efectos podrían estar relacionados con alteraciones en el proceso de autofagia. INTRODUCCIÓN Los supresores de muerte celular de origen animal no presentan homólogos en el reino vegetal, no obstante, la expresión de estos genes ectópicos afectan los eventos de muerte/vida en las plantas. Un aspecto importante a destacar es que si bien se ha evaluado la conservación de función de éstas proteínas entre los reinos, no se han esclarecido los mecanismos a través de los cuales ejercen sus efectos en las plantas. En este trabajo, evaluamos posibles mecanismos de acción a través de los cuales CED-9 podría estar ejerciendo sus efectos sobre los eventos de muerte y la nodulación en raíces en cabellera de soja. MATERIAL Y MÉTODOS Las raíces en cabellera fueron obtenidas mediante la infección con Agrobacterium rhizogenes K599 y fueron sometidas a los distintos tratamientos durante 3 h. La determinación del contenido de malondialdehido (MDA) se realizó según Heath y Packer (1968). Los niveles de ATP se determinaron según Rodriguez et al. (2010) y el contenido de iones se cuantifico por HPLC. El análisis de expresión de genes fue realizado mediante PCR tiempo real. RESULTADOS Y DISCUSIÓN Efectos de la expresión del supresor de muerte celular Ced-9 en raíces en cabellera sometidas a condiciones de estrés. Raíces en cabellera fueron sometidas a condiciones de estrés inductoras de muerte, previamente identificadas por nuestro grupo (Muñoz et al., 2012): inoculación con B. japonicum en presencia de estrés salino moderado (50 mM NaCl) y estrés salino severo (150 mM NaCl). Asimismo, estos eventos de muerte ocurrieron en presencia y ausencia de generación de especies activas del oxígeno (EAO), respectivamente. Se evaluaron los niveles de malondialdehido (MDA), los contenidos de ATP y de los iones Na+, K+ y Ca2+, y se analizó actividad autofágica a través del estudio de las formas lipidizadas de la proteína ATG8 (ATG8-PE) mediante western blot. Los resultados obtenidos sugieren que bajo condiciones de inoculación en presencia de estrés salino, los eventos de muerte celular en raíces en cabellera silvestres ocurren de manera Session III SIII-CP-26 ordenada (incrementos de ATP y de actividad autofágica, Figura 1) y presentan daño oxidativo (altos niveles de MDA y generación de EAO). Contrariamente, bajo condiciones de estrés salino severo, los niveles de ATP y MDA no mostraron diferencias respecto al tratamiento control, pero presentaron alteraciones en la homeostasis iónica, principalmente pérdida de K+, sugiriendo eventos de muerte celular no ordenada o necrótica. Bajo estos tratamientos, la expresión de Ced-9 inhibió o al menos retardó la muerte celular en las raíces en cabellera. Bajo las condiciones de estrés por B. japonicum + 50 mM NaCl, las raíces Ced-9 no presentaron incrementos en el contenido de MDA y la actividad autofágica se vio afectada (Figura 1); mientras que en los tratamientos de 150 mM NaCl, la expresión de Ced-9 previno la pérdida de K+ y Ca2+ e incrementó los contenidos de ATP. Estos resultados demuestran el rol del supresor de muerte celular de origen animal en el control de la homeostasis iónica y del metabolismo celular en las plantas, como así también su participación en el control del proceso de autofagia. Número de nódulos Efectos de la expresión del supresor de muerte celular Ced-9 en raíces en cabellera sobre la nodulación. Por otro lado, se estudiaron los efectos de la expresión de Ced-9 sobre la nodulación de las raíces en cabellera. Curiosamente, las raíces Ced-9 tuvieron inhibida su capacidad de nodulación y mostraron una reducción significativa en los niveles de expresión de genes claves del proceso de autofagia. Con el fin de evaluar la participación de autofagia en la interacción simbiótica, se evaluó la nodulación en raíces en cabellera con silenciamiento post-transcripcional de ATG6, proteína clave para la inducción del proceso. Estas raíces presentaron una disminución dramática en el número de nódulos (Figura 2), postulando así la importancia del proceso de autofagia en la interacción soja-B. japonicum. 60 50 40 30 20 10 0 k599 vacía Figura 1. Evaluación de los niveles de proteína ATG8 en raíces en cabellera K599-vacía y pBECed-9 mediante anticuerpos anti-AtATG8a. ATG6i Figura 2. Nodulación en raíces en cabellera silvestres (K599 vacía) y con silenciamiento post-transcripcional de Atg6 (ATG6i) BIBLIOGRAFÍA Rodriguez, M., et al. (2010). J. Plant Physiol. 167: 1137-1144. Muñoz, N., et al. (2012). Environ. Exp. Bot. 78: 76-83. Heath, R., and Packer, L. (1968). Arch. Biochem. Biophys. 125: 180-198. Session III SIII-CP-27 Efecto del contenido de taninos condensados (TC) sobre la formación de nódulos en especies del género Lotus. Escaray, F.J.1*, Estrella, J.1, 2, Paolocci, F.3, Damiani, F.3, Ruiz, O.A.1 1 IIB-INTECH/CONICET-UNSAM, Chascomús, Bs. As., Argentina. 2 Investigadora adjunta, Comisión de Investigación Científica (CIC). 3 National Research Council, Plant Genetics Institute-Perugia ( CNRIGV), Perugia, Italy. * [email protected] RESUMEN Cuatro genotipos de L. corniculatus cv. Leo, con diferente contenido de TC en raíces, se inocularon con dos cepas de rizobios de estrecho rango de nodulación (Mesorhizobium loti NZP2213 y Bradyrhizobium loti NZP2309). No se observó interacción entre los genotipos y los tratamientos de inoculación para todos los parámetros medidos, demostrando que el contenido de TC no es un factor determinante para la nodulación en L. corniculatus. INTRODUCCIÓN Algunos autores han sugerido que la composición y los niveles de taninos condensados (TC) presentes en raíces de plantas del género Lotus forman parte de los mecanismos de determinación del rango de hospedante para la nodulación en la simbiosis mutualista con rizobios (Pankhurst et al., 1982; Cooper and Rao, 1992). Sin embargo, estos autores basaron sus resultados en estudios realizados con diferentes especies de Lotus, con lo cual, no se puede descartar que hayan participado otros factores especie-específicos además de los TC. Para poner a prueba la hipótesis de que los TC en el género Lotus, son factores determinantes para la formación de nódulos, nos propusimos realizar ensayos de nodulación con cepas de rizobios que tienen distinto rango de hospedante y plantas isogénicas de L. corniculatus que presentan diferentes niveles de TC en sus raíces. MATERIAL Y MÉTODOS Se utilizaron clones de cuatro genotipos de L. corniculatus cv. Leo transformados con la construcción CaMV35S-Sn, que expresa el gen Sn, el cual codifica para un factor de transcripción relacionado con la síntesis de TC (Robbins et al., 2003; Paolocci et al., 2007). Dos de esos genotipos tienen bajo contenido de TC en raíces (Sn6 y Sn9), uno tiene alto contenido de TC (Sn10) y uno transformado con el vector vacío que tiene contenido intermedio (121.1). Los clones fueron cultivados durante 30 días bajo condiciones controladas, en jarras Leonard que contenían arena:perlita (1:1) e irrigadas con solución Evans 1x sin nitrógeno. Se aplicaron tres tratamientos: a) inoculación con la cepa M. loti NZP2213, b) inoculación con la cepa B. loti NZP2309 y c) sin inocular (controles); con 6 repeticiones biológicas. La inoculación se realizó 10 días después de la siembra con 1 ml del cultivo de la cepa en YEM líquido (DO = 0,8). La tinción y cuantificación de TC se realizó de acuerdo a la metodología descripta por Escaray et al., (2012). RESULTADOS Y DISCUSIÓN Al finalizar el ensayo se pudo observar que todos los clones de los genotipos cultivados bajo condiciones control o inoculados con la cepa B. loti presentaban un aspecto clorótico en comparación a los inoculados con la cepa M. loti. Por otra parte, se observó un mayor peso seco y longitud de tallo principal en aquellos genotipos inoculados con la cepa M. loti; sin embargo, no observó interacción entre los factores genotipo x tratamiento aplicado (p>0,5). Session III SIII-CP-27 Bajo la condición control ningún clon presentó nódulos. Por su parte, se observó que todos los clones inoculados con la cepa M. loti, independientemente del nivel de TC que expresaban en raíz, formaron nódulos de tamaño normal y color rosado. Por su parte, los clones inoculados con la cepa B. loti formaron nódulos de menor tamaño, color blanquecino y en número marcadamente mayor (Figura 1A y 1B). Considerando que el aspecto rojizo de los nódulos se encuentra relacionado generalmente con su capacidad fijadora de nitrógeno (Lodwig et al., 2003) y que la inoculación con cepas deficientes para la fijación de nitrógeno (denominadas “Fix -“) genera un fenotipo generalmente hipernodulante (Lohar and VandenBosch, 2005), el fenotipo de nodulación observado, junto con los parámetros evaluados en la parte aérea, indican que la inoculación con la cepa B. loti forma nódulos Fix- en los cuatro genotipos evaluados. Independientemente del contenido basal de TC en raíces, se observó un mayor contenido de estos compuestos cuando la inoculación se realizó con la cepa de B. loti y un menor contenido en aquellas inoculadas con M. loti (Tabla en la Figura 1). Se pudo observar a través de la tinción de TC en raíces noduladas, que existe una mayor acumulación de estos compuestos en el nódulo formado por la cepa B. loti (Figura 1C y D), resultados concordantes con el trabajo de Pankhurst et al. (1979). Figura 1. Clones nodulados y tinción y contenido de TC en raíces con nódulos. Clones del genotipo Sn10 inoculado con M. loti NZP2213 (A y C) y B. loti NZP2309 (B y D). C y D: el color azul indica la presencia de TC (tinción con DMACA:HCl). A la derecha se muestra la tabla con el contenido de TC e n raíces. En función de los resultados obtenidos se puede concluir que el contenido de TC de L. corniculatus no es un factor determinante del rango de hospedante para la formación de nódulos, en la simbiosis mutualista con rizobios. BIBLIOGRAFÍA Cooper, J.E., and Rao, J.E. (1992). Plant Physiol. 100: 444-450. Escaray, F.J., et al. (2012). Phytochem. Lett. 5: 37-40. Lodwig, E.M., et al. (2003). Nature 422: 722-726. Lohar, D.P., and VandenBosch, K.A. (2005). J. Exp.Botany 56: 1643-1650. Pankhurst, C.E., et al. (1979). J. Exp.Botany 30: 1085-1093. Pankhurst, C.E., et al. (1982). J. Gen. Microbiol. 128: 1567-1576. Paolocci, F., et al. (2007). Plant Physiol. 143: 504-516. Robbins, M.P., et al. (2003). J. Exp.Botany 54: 239-248. Session III SIII-CP-28 Alteraciones en el proceso de nodulación en ausencia y presencia de fuente de nitrógeno externa de un mutante deficiente en el transporte de nitrato de la leguminosa Lotus japonicus. García-Calderón, M.1*, Pal’ove-Balang, P.2, Pérez, C.M.1, Betti, M.1, Marquez, A.J.1 1 Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Química, Universidad de Sevilla, C/ Profesor García González, 1, 41012, Sevilla. 2 Institute of Biology and Ecology, P. J. Šafárik University, Mánesova 23, SK-04001 Košice, Slovak Republic. * [email protected] RESUMEN Diferentes estudios de nodulación se realizaron utilizando plantas silvestres y mutantes resistentes a clorato (Ljclo1) a dos estadíos diferentes tras la inoculación con Mesorhizobium loti. El proceso de nodulación de las plantas mutantes fue similar al de las plantas silvestres en ausencia de fuente de nitrógeno externa, sin embargo, la biomasa específica de nódulos respecto a la de raíz disminuyó considerablemente en plantas mutantes cuando se cultivaron en presencia de nitrato o amonio en el medio de riego. Estos resultados sugieren la existencia de alteraciones en el proceso de señalización por nitrógeno relacionado con la nodulación de las plantas. INTRODUCCIÓN Estudios recientes han mostrado que una amplia variedad de mecanismos regulatorios están implicados en la modulación por nitrógeno de la organogénesis del nódulo en plantas leguminosas (Omrane and Chiurazzi, 2009). Si los efectos represivos del NO 3 se deben al i ón por sí mismo, a los productos de su asimilación o a ambos, no está claramente demostrado (Morere-LePaven et al., 2011). Un mutante resistente a clorato (Ljclo1) fue identificado de la leguminosa modelo Lotus japonicus, capaz de crecer considerablemente bien durante una semana en medios con una concentración entre 0,510 mM KClO3, mi entras que las plantas silvestres no eran capaces de sobrevivir en estas condiciones. Estudios previos habían indicado que dicho mutante posee niveles normales de nitrato reductasa pero está afectado en el transporte de nitrato y/o clorato de baja afinidad (Márquez et al., 2005). Estas plantas mutantes fueron utilizadas para examinar el proceso de nodulación en ausencia y presencia de distintas fuentes de nitrógeno. MATERIAL Y MÉTODOS El cultivo de las plantas así como la inoculación con rizobios se realizó como se describe en García-Calderón et al. (2012). Las plantas control se regaron con medio Hornum (Handberg and Stougaard, 1992). Se utilizaron medios conteniendo KNO 3 o NH4Cl como única fuente de nitrógeno a las concentraciones indicadas en cada experimento. 3 mM de KCl se añadió al medio de amonio para compensar la falta de K. RESULTADOS Y DISCUSIÓN Se realizaron diferentes estudios de nodulación de plantas silvestres y mutantes a dos estadíos de tiempo tras la inoculación (45 y 65 días). Se determinó el número total y el peso fresco de nódulos, la relación de peso fresco de nódulos por número de nódulos así como el peso fresco de nódulos por peso fresco de raíz. No se obtuvieron diferencias significativas de estos parámetros ni a 45 ni a 65 días cuando el proceso de nodulación se realizó en ausencia de fuente de nitrógeno externa. Sin embargo, sí se obtuvieron diferencias cuando los experimentos se realizaron en presencia de nitrato o amonio. Se observó una fuerte inhibición de la ratio peso fresco de nódulos por peso fresco de raíz Session III SIII-CP-28 tanto en plantas silvestres como mutantes Ljclo1, a todas las concentraciones examinadas. Los resultados obtenidos en plantas mutantes mostraron una mayor inhibición del proceso de nodulación a concentraciones de 1-2 mM de nitrato comparado con plantas silvestres. De hecho, no se obtuvieron nódulos a 45 días en plantas mutantes en presencia de 1 ó 2 mM de nitrato, mientras que sí se obtuvieron en las plantas silvestres (resultados no mostrados). Además, los datos obtenidos después de 65 días tras la inoculación mostraban una inhibición del 70% en la ratio de peso fresco de nódulos por peso fresco de raíz en plantas mutantes a 1 mM de nitrato, mientras que en el caso de plantas silvestres esta inhibición fue sólo del 30% (Figura 1). El reducido número de nódulos presentes a concentraciones de 2 mM de nitrato fue casi el doble en plantas silvestres que en mutantes. También se obtuvieron diferencias en las plantas mutantes Ljclo1 respecto a las plantas silvestres cuando se estudió el proceso de nodulación en presencia de 1-2 mM de NH4+, aunque en menor grado que en presencia de nitrato. Por tanto, las diferencias observadas en el proceso de nodulación no parecen ser una respuesta específica del mutante en presencia de nitrato sino más bien es indicativo de un defecto general de la señalización por N característica del mutante Ljclo1. Experimentos de qRT-PCR han demostrado alteraciones en los niveles de expresión de algunos genes implicados en la asimilación de nitrógeno en respuesta a la presencia/carencia de nitrógeno, sugiriendo también un posible defecto en la señalización por nitrógeno en las plantas mutantes Ljclo1. 0.8 WT Ljclo1 Pfresco Nódulos/Pfresco raíz 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 sin N 0,5mM NO 3 1mM NO 3 2mM NO 3 + 0,5mM NH 4 + 1mM NH 4 + 2mM NH 4 Figura 1. Ratio del peso fresco de nódulos por peso fresco de raíz de plantas silvestres y mutante s Ljclo1 de Lotus japonicus a 65 días tras la inoculación. Los valores representan la media de siete réplicas biológicas ± error standard. indica diferencias significativas entre plantas WT y mutantes, determinadas mediante el test de Student (P<0,05). AGRADECIMIENTOS Agradecemos la financiación de la Junta de Andalucía al grupo BIO-163 y proyecto CVI-P10-6368 así como de la Unión Europea (proyecto EXPERT). BIBLIOGRAFÍA García-Calderón, M., et al. (2012). Mol. Plant Microbe Interact. 25: 211-219. Handberg, K., and Stougaard, J. (1992). Plant J. 2: 487-496. Márquez, A.J. et al. (2005). J. Exp. Bot. 56: 1741-1749. Morere-Le Paven, M.C., et al. (2011). J. Exp. Bot. 62: 5595-5605. Omrane, S., and Chiurazzi, M. (2009). Plant Signal. Behav.4: 1066-1068 Session III SIII-CP-29 Phylogenetic diversity and structure of sebacinoid fungi associated with plant communities along an altitudinal gradient. Garnica, S.*, Riess, K., Bauer, R., Oberwinkler, F. Eberhard Karls Universität Tübingen, Institute of Evolution and Ecology, Plant Evolutionary Ecolog y, Auf der Morgenstelle 1, 72076-Tübingen, Germany. * [email protected] ABSTRACT The phylogenetic diversity and patterns in the structure of Sebacinales communities from eight vegetation types with different degrees of disturbance along an altitudinal gradient were studied. Ecosystem disturbances and environmental factors proved to be the major forces influencing the diversity and structure of Sebacinales communities. INTRODUCTION Sebacinales form a wide spectrum of symbiotic associations with plants, including orchioid, ericoid and arbutoid mycorrizas; ectomycorrizas; endophytic associations with herbs and interactions with mosses (Oberwinkler et al. 2013). Only few of these fungi have been isolated and cultivated on standard media showing a great potential for enhancing plant growth under controlled conditions. In order to better understand the diversity and how the Sebacinales communities are structured in relatively undisturbed plant communities as well as communities with various degrees of disturbance (Garnica et al. 2013), we addressed the following specific questions: What are the phylogenetic diversity and structural patterns that characterise the Sebacinales communities in the studied plant communities? Do soil factors, altitude and/or plant communities affect the phylogenetic structure of the Sebacinales communities? MATERIAL AND METHODS In our study, we analysed the phylogenetic diversity and structure of the Sebacinales communities from 456 thalli or roots from eight different vegetation types under various degrees of disturbance along an altitudinal gradient in the Bavarian Alps (Germany). To amplify the 3' region of the 18S region, intergenic transcribed spacers (ITS1 and ITS2), the 5.8S ribosomal subunit and the D1/D2 regions of the nucLSU of Sebacinales, we used various sets of specific and universal fungal primers. Phylogenetic, diversity and community structure analyses were performed using several approaches. RESULTS AND DISCUSSION A total of 264 sebacinoid sequences spanning the ITS, 5.8S and D1/D2 domains (approx. 1300 bp) of the 28S rRNA gene were amplified by mainly using a nested PCR approach. a 97% sequence similarity, 73 molecular taxonomic units (MOTUs) of Sebacinales were detected from 70 host species belonging to 44 plant families. A total of 26 MOTUs proved to be singletons. Although Sebacinales were shown to occur in low abundance in the roots of the plants, these microorganisms are phylogenetically very diverse and widely distributed in the ecosystems analysed. Ordination analyses showed that the land use, pH and humus content strongly influenced the diversity and composition of Sebacinales. In most cases, the Sebacinales communities in ecosystems with extreme soil conditions or with extremely large anthropogenic use exhibited significant phylogenetic clustering, whereas in undisturbed plant communities, no trends were observed. These findings Session III SIII-CP-29 suggest that disturbance of the ecosystems and environmental forces have influenced the diversity and structure of Sebacinales communities at local and spatial scales. Finally, we discuss the promising potential of locally adapted Sebacinales strains and their use as biofertilizers, e.g. in the stimulation of the growth of crop plants and/or in the restoration of degraded ecosystems. ACKNOWLEGMENTS This research was supported was supported by the German Research Foundation (grant OB 24/30). REFERENCES Garnica, S., et al. (2013). FEMS Microbiol. Ecol. 83: 265-78. Oberwinkler, F., et al. (2013). Mycol. Progress 12: 1-27. Session III SIII-CP-30 Grain yield of cowpea in Ghana responds to Rhizobium inoculation. Atakora, W.K.1, Guimarães, A.P.2*, Fosu, M.1 Boddey, R.M.2, Xavier, G.R.2 1 Embrapa Agrobiologia, BR 465, km 07, Seropédica, 23891-000, Rio de Janeiro, Brazil. 2 Savanna Agricultural Research Institute (SARI), P.O. Box 52, Tamale, Northern Region, Ghana. * [email protected] ABSTRACT Over a two-year period two replicated plot experiments were performed at the SARI filed station in Tamale northern Ghana and 31 unreplicated on-farm trials in the nearby districts. The field station experiments tested three different Rhizobium strains on a polymer based carrier. Without inoculation, grain yields were between 1060 kg ha -1 (Exp.1) and 1370 kg ha-1 (Exp 2) and inoculation increased these to over 2000 kg ha -1 in both experiments. These results were confirmed on the trials on smallholder´s properties where inoculation with two different Rhizobium strains increased average grain yields by 430 to 560 kg grain ha -1 at the June planting and between 495 and 570 kg ha-1 at the August planting. INTRODUCTION Cowpea (Vigna unguiculata, Walp.) is the foremost grain legume crop in Africa where the crop is estimated to occupy over 11 million ha (4 million ha in Nigeria alone) with a mean yields between 450 and 500 kg grain ha -1. In Ghana the crop occupies ~200,000 ha with mean grain yield of~450 kg/ha. Cowpea nodulates well in almost all soils of the tropics and other regions and until recently both Brazil and in Africa it was assumed that as Rhizobium capable of nodulating this crop were found in the soils of these regions it was not worth inoculating the crop with selected strains. However, results from trials in Brazil over the last decade have shown frequent, significant and considerable increases in grain yield to inoculation of selected Rhizobium strains. The objective of the present study was to test on a field station in Northern Ghana and in smallholder properties in the same region the technology of the inoculation of cowpea with strains of Rhizobium selected in Brazil with inoculants made with the CMC polymer used as carrier. MATERIAL AND METHODS In August 2011 a set of un-replicated trials were established on 15 smallholder properties with four treatments: non-inoculated with zero and 40 kg N of ammonium sulphate and two inoculated with Rhizobium strains BR 3267 and BR 3299, Rhizobium strains from Brazil with no phosphate addition. Similar on-farm trials, 16 each, were planted in June and August 2012 with the same four treatments and all plots were amended with 60 kg P ha-1 as triple super phosphate Two field experiments were established with seven treatments in a randomized block design (5 replicates) at two different sites at the SARI field station planted in June and August of 2012, respectively, again with phosphate addition (60 kg P ha-1). The treatments consisted of inoculation with three selected, BR 3262, BR 3267 and BR 3299 and a fourth treatment with a mixture of the strain BR 3267 and BR 3299, and three non-inoculated treatments fertilized with 0, 40 and 80 kg N, respectively, as ammonium sulphate. Harvests for assessing nodule weight were made at 35 days after planting (DAP) and for final dry matter accumulation and grain yield at approximately 70 DAP. Session III SIII-CP-30 RESULTS AND DISCUSSION For the on-farm trials nodule weight and grain yield responded positively to the application of all Rhizobium inoculants. In the first set of trials of 2011 when P was not added grain yields increased from an average for all trials of 675 kg to 859 and 1062 kg grain ha-1 for the strains BR 3299 and BR 3267, respectively. In the on-farm trials in 2012 where P fertilizer was applied nodule dry weight increased from 322 to 482 and 522 mg plant-1 for strains BR 3299 and BR 3267 respectively, but N fertilizer reduced nodule weight to 138 mg plant -1 (Table 1). Mean grain yield was increased from 1162 to 1722 and 1594 kg grain ha -1 In the second set of on-farm trials planted in August similar results were obtained the Rhizobium inoculants increased yields from 891 to approximately 1400 kg ha -1. The field station experiments also showed large responses of nodule mass and grain yield to Rhizobium inoculation, such that yields increased from approximately 1300 kg grain ha-1 to over 1650 kg ha-1 at the June planting and from 1000 to over 2000 kg grain ha-1. Table 1. On farm trials first planting 2012. Nodule weight at 35 days after planting and yield parameters a final harvest (70 DAP) Treatment Non Inoculated 40 Kg N ha-1 Strain 3267 Strain 3299 CV (%) 35 DAP Nodule weight mg DM plant 322 B 138 C 522 A 482 A 23 -1 DM shoot Final Harvest 70 DAP Dry weight pods Grain yield -1 -------------------------- kg ha ------------------------2510 B 1882 AB 1162 B 5844 A 1185 B 1086 B 2563 B 3724 A 1722 A 2401 B 2105 AB 1594 A 11 18 35 Values are means of eleven replicates. Means within the same column followed by the same letter ar e not different at p< 0.0 5 (Student ‘t’ test) In all cases N fertilizer had a highly significant positive effect on total shoot dry matter but this was not reflected in grain yield. The large increases in grain yield obtained in 2 field experiments and 22 on-farm trials to the application of the polymer-based inoculant of selected Rhizobium strains show that an extremely small investment (US$ 2 to 3 per ha) between 300 and almost 1200 kg of extra grain can be harvested which illustrates the huge potential of this simple technology (Fernandes Júnior et al., 2012). ACKNOWLEDGEMENTS This work was funded by the Africa/Brazil Agricultural Innovation Marketplace supported by Embrapa , The Bill and Melinda Fates foundation, DfID (UK). REFERENCES Fernandes Júnior, P.I., et al. (2012). African J. Biotech. 11: 2945-2951. Session III SIII-CP-31 Pseudomonas fluorescens enhances photosynthesis and improves bioactive profiles and antioxidant potential in blackberry (Rubus sp. Var. Lochness) in field conditions. García-Seco, D., Bonilla, A., Algar, E., García-Villaraco, A., Gutiérrez Mañero, F.J. *, Ramos Solano, B. Faculty of Pharmacy, Universidad CEU San Pablo, Ctra. Boadilla del Monte Km 5.3, 28668 Boadilla de l Monte, Madrid, Spain. * [email protected] ABSTRACT Inoculation of P.fluorescens N21.4 to blackberry plants (Rubus spp. var Lochness) throughout its phenological cycle in field conditions resulted in improved photosynthetic yield during vegetative growth. This improvement was also detected in fruits that showed improved antioxidant potential and higher flavonoid and anthocyanin contents. INTRODUCTION Blackberry (Rubus spp. var Lochness) is an aggregate fruit, composed of small drupelets, belonging to the Rosaceae family. Blackberry represents an important food for human health in prevention of chronic diseases due to the presence of numerous bioactive compounds. These bioactive compounds (anthocyanins, phenolic acids and flavonoids all with antioxidant activity) are products of secondary metabolism designed to provide maximal adaptation for the plant against environmental changes; therefore, secondary metabolism is highly inducible and is subjected to strong fluctuations due to environmental conditions (Boué et al., 2008), and therefore, the potential benefits for health through the diet are not consistent. Among the factors that are able to trigger secondary metabolism are beneficial bacteria able to induce systemic resistance (Ramos Solano et al., 2010b). This lack of reproducibility may be overcome by means of elicitation during plant growth, which will be reflected in secondary metabolites contained in the fruit (Capanoglu, 2010). In a previous study, this strain demonstrated its ability to trigger secondary metabolism with a low number of plants (García-Seco et al., 2013). The aim of the present study was to confirm this ability in a larger sample area, evaluating photosynthesis, as a marker of systemic resistance, and fruit quality to confirm the potential of this strain to palliate changes in concomitant to environmental changes. MATERIAL AND METHODS Pseudomonas fluorescens N21.4 (CECT 7620) was isolated from the rhizosphere of Nicotiana glauca (Ramos Solano et al., 2010a). Three greenhouses (360 plants) of R. fruticosus var. Lochness were defined within the production area. Plants were grown from November through May under natural light conditions. Four blocks were defined in each greenahouse, N21.4 inoculated plants, N21.4 plus the usual chemical treatments, and the non-inoculated controls (Control and Chemical-Control). Inoculations were delivered by soil drench every two weeks. Total flowering buds and branches were recorded at two time points and photosynthesis was analysed by a Fluorescence Monitoring System (FMS; Hansatech). Fruits were harvested and bioactive contents were determined at the maximum point of production. The total phenolic content was determined with Folin-Ciocalteau reagent (Sigma-Aldrich, St Louis, MO) by colorimetry with modifications (Xu and Chang, 2007). Total flavonoid content was measured by the aluminum chloride colorimetric Session III SIII-CP-31 assay (Zhishen et al., 1999). Total anthocyanin content was determined quantitatively by the pH differential method of Giusti and Wrolstad (2001). The radical scavenging activity of R. fruticosus extract against DPPH free radical (antioxidant potential) was measured using the method of Brand-Williams et al. (1995). Epicatechin was determined by Capillar Electrophoresis according to Piovenzal et al. (2013). RESULTS AND DISCUSSION Photosynthesis was improved in the first sampling time (January) on inoculated plants as compared to controls, as indicated by the significantly lower values of Fo, and greater PSII, and Fv/Fm. These physiological values were consistent with higher number of flowering branches and flowering buds per branch. However, during the second sampling time (May, maximum production), this significant difference disappeared, indicating that inoculated plants had accelerated growth probably due to increased photosynthesis, a mechanism that has been proposed for growth promotion of some PGPR strains (Zang et al., 2008). Fruits from plants inoculated with N21.4 showed increased antioxidant potential, and higher flavonoids and anthocyanins. Total flavonoids levels in controls were under 65 mg catechin equivalents per 100 g FW, while inoculated fruits were over 80 mg catechin equivalents (100 g FW), being these differences significant. However, the flavonoid (+)-epicatechin concentration was higher on inoculated plants but not significant. Total antocyanins were arround 90 mg of cyanidin-3-glucoside equivalents in controls and increased up to 120 mg of cyanidin-3-glucoside equivalents, on inoculated fruits. ACKNOWLEDGEMENTS. The authors would like to thank Ministerio de Ciencia e Innovación for granting DGS (BES-201003805 7) and projects AGL 2009-08324, CM S2009/AMB-1511, and Agricola El Bosque, S.L. REFERENCES. Boué, S.M., et al. (2008). J. Agric. Food Chem. 57: 2614-2622. Brand-Williams, W., et al. (1995). Lebensm. Wiss Technol. 28: 25-30. Capanoglu, E. (2010). Trends Food Sci. Technol. 21: 399-407. Garcia-Seco, D., et al. (2013). Agron. Sustain. Dev. 33: 385-392. Giusti, M., and Wrolstad, R. (2001). Current protocols in food analytical chemistry. pp 121-129. Piovenzal et al. (2013). Electrophoresis, in press. Ramos Solano, B., et al. (2010a). Plant soil 334: 189-197. doi: 10.1007/s11104-010-0371-9 Ramos Solano, B., et al. (2010b). J Agric Food Chem 58:1484–1492. doi: 10.1021/jf903299a Xu, B.J., and Chang, S.K.C. (2007). J. Food Sci. 72: 159-166. Zang, H., et al. (2008). Plant J 56: 264–273. doi: 10.1111/j.1365-313X.2008.03593.x Session III SIII-CP-32 Improving opium poppy yield through MAMPs from selected beneficial bacterial strains in field trials. Bonilla, A.1, Garcia-Seco, D.1, Muñoz Ledesma, F.J.2, Lucas, J.A.1*, Ramos Solano, B.1, Gutiérrez Mañero, F.J.1 1 Faculty of Pharmacy, Universidad CEU San Pablo, Ctra. Boadilla del Monte Km 5.3, 28668 Boadilla del Monte, Madrid, Spain. 2 ALCALIBER I+D+i, S.L.U. Ctra. Carmona-El Viso del Alcor, Km.1,8. 41410 Carmona, Sevilla, Spain * alucg[email protected] ABSTRACT Three beneficial PGPR strains were inoculated at two different physiological status of Papaver somniferum and in both status, in experimental plots in field conditions in spring 2010 and 2011. Foliar applications of two strains resulted in increased yield due to an increase in opium poppy capsule weight. The increases detected under Chryseobacterium balustinum Aur 9 were due to increased seed production while Stenotrophomonas maltophila N5.18 caused an increase in straw, not in seeds. The effects of N5.18 at the end of the flowering period was consistent in the two experiments. This was coupled to increases in alkaloids since they accumulate mainly in the capsule straw and also to an increase in photosynthetic yield. INTRODUCTION Field production of opium poppy is the only economically feasible way to obtain morphinane alkaloids, being morphine the best analgesic to date. Legal production of Papaver sominferum worldwide is carried out in Australia, France, Spain and Turkey, with Spain accounting for 73 MT of 440.3 (INCB, 2012), being under strict control by the Ministerio de Sanidad. Benzylisoquinoline alkaloids (BIAs) are a large and structurally diverse group of nitrogenous compounds related with plant defence. Among the Papaveraceae family only Papaver somniferum is able to produce significant amounts of alkaloids accumulated only in the capsules. However, due to the inducible nature of secondary metabolism, alkaloid levels change according to environmental conditions, and therefore, yield is highly compromised due to different factors. This lack of reproducibility may be overcome by the means of elicitation with PGPR (Ramos Solano et al., 2010). Plants possess an active secondary metabolism that can be actively expressed in response to stress. Hence, triggering plant’s metabolism by the means of a Microbial Associated Molecular Pattern (MAMP) (Erbs and Newman, 2012) or a plantgrowth-promoting r hizobacteria (PGPR) could increase levels of secondary metabolites The aim of this study was to evaluate the effects of inoculating 3 PGPR strains in Papaver somniferum plants to enhance secondary metabolism of BIAs. To evaluate the systemic stimulation of poppy metabolism yield parameters were determined, as well as photosynthesis and alkaloid contents, as metabolic markers of the induction. MATERIAL AND METHODS Field trials were conducted on 15 m 2 subplots within two Papaver somniferum production areas, as indicated by Alcaliber I+D+I. Three bacterial strains were used Aur9 (Chryseobacterium balustinum), N21.4 (Pseudomonas fluorescens) and N5.18 (Stenotrophomonas maltophilia); each of them was delivered by foliar spray right before flowering stem grows (cabbage state) and two weeks after when flowers are formed (flowering state), and in both status. Photosynthesis was measured 2 weeks after inoculation. When plants were dryed, one subsample (1 m2) was defined within each Session III SIII-CP-32 plot and plant number, capsule number were recorded. Capsules were brought to the lab, seeds were separated from straw and weight was determined. Alkaloids were extracted and determined by HPLC. ANOVAs were used to determine statistical significance of data and LSD RESULTS AND DISCUSSION Among the three strains assayed, only N5.18 delivered at the flowering state was able to trigger Papaver somniferum metabolism, causing significant increases as compared to non-inoculated control plants. The most affected parameter was total capsule weight, due to significant increases in straw and seed dry weight. This increase in biomass is supported by the improvement in photosynthetic parameters, a mechanism reported for some PGPR to improve plant yield (Zhang et al., 2008; Zulak et al., 2008) Total alkaloid contents (mg/g straw) were not significantly increased by this strain, and there was only an increasing trend in morphine. Despite the reported role of BIAs as phytoalexins (Facchini et al., 1996), our results are based only in 4 alkaloids (morphine, codein, thebain and oripavine) so the possibility of other BIAs being increased may not be ruled out. Consistently, an increase in sanguinarine after Botrytis PAMPs (PathogenAssociated M olecular Patterns) has been reported (Facchini et al., 1996). Furthermore, after elicitation with certain MAMPs or PAMPs, metabolic pathways leading to phytoalexins may be detoured to one specific branch by triggering specific branching points (Rühmann et al., 2013). This becomes especially relevant in alkaloid biosynthetic pathways which are highly compartimented in different tissues and subcellular fractions (Facchini et al., 1996). Considering effects on biomass increase by MAMPs of N5.18, together with alkaloids results in an encouraging result for production since the increase in total straw will result in higher alkaloid yields when considering surface under production. ACKNOWLEDGEMENTS The authors thank Alcaliber I+D+I, for funding and technical help. This research was supported by Ministerio de Ciencia y Tecnología AGL2009-08324 and CAM S-0505/AMB/000321. REFERENCES Erbs and Newman.(2012). Mol. Pl. Pathol. 13: 95-104. Facchini P.J., et al. (1996). Plant Physiol. 111: 687-697. INCB (2012). ISBN: 978-92-1-048152-6. Ramos-Solano B., et al. (2010). J. Agri. Food Chem., 58:1484-1492. Rühmann S., et al. (2013) Pl. Biol. Biochem. http://dx.doi.org/10.1016/j.plaphy.2013.03.011. Zulak K.G., et al. (2008). BMC Plant Biol. 8: 5-23. Zang et al. (2008). Plant J. 56: 264–273. doi: 10.1111/j.1365-313X.2008.03593.x. Session III SIII-CP-33 Rhizobial cellulase CelC2 and its Ensifer homolog play an important role in plant and nodule development. Menéndez, E.*, Robledo, M., Velázquez, E., Martínez-Molina, E., Rivas, R., Mateos, P.F. Department of Microbiology and Genetics. CIALE. University of Salamanca. Spain. * [email protected] ABSTRACT Cellulolytic enzymes are widespread in bacteria establishing root-nodule symbioses. Genes encoding these cellulases are present in all rhizobia genera. Among them, celC gene from Rhizobium leguminosarum bv trifolii ANU843 encodes a ß1-4D endoglucanase (GH8), called CelC2, that is shown to be essential for primary symbiotic infection in legume host roots and shows host specificity for Trifolium repens root hair tips (Robledo et al., 2008). Furthermore, there are celC orthologs in the genera Rhizobium and Ensifer (Robledo et al., 2011, 2012). Here we report the effects of rhizobial CelC2 and its E. medicae WSM419 homolog in plant and nodule development. INTRODUCTION Among others, Rhizobia-legume symbiotic association is one of the most important plant-microbe interactions and provides the most efficient assimilable Nitrogen source for agricultural crops. The nitrogen-fixing endosimbiotic bacteria can invade their plant hosts though colonization of intercellular epidermal spaces, crack entry at emerging lateral roots or intracellular infection of root hairs. The infection process is tightly regulated in legumes to ensure appropriate bacterial symbiont penetration to establish a nitrogen-fixing, intracellular state within the host. Preliminary studies have shown rhizobial cellulase CelC2 is essential in primary symbiotic infection and its tightly regulated production is required for root hair and nodule functional development in its host, Trifolium repens (Robledo et al., 2008, 2011). Besides that, few are known about cellulases implications in other symbiotic systems, as the model interaction MedicagoEnsifer. In t his study we show the role of CelC2 and its Ensifer homolog when are homo/heterologous expressed in this model system. MATERIAL AND METHODS Bacterial strains and plant material. M. truncatula Jemalong line A17 seeds were used as plant material. E. meliloti 1021, E. medicae WSM419, A. rhizogenes strain ARqua1 and its derivatives were used as bacterial inoculants. Engineering celC-expressing and gfp-tagged derivative strains. Construction of E. meliloti 1021 and E. medicae WSM419 derivative strains was performed introducing vector pJZC2 (Robledo et al. 2008) and pEMC2 in wild-type strains by triparental matting. To generate GFP-expressing bacteria, plasmid pHC60 (Cheng and Walker, 1998) was introduced into wild type and derivative strains as described. A.rhizogenes-mediated root transformation. Seedlings were transformed with A. rhizogenes strain ARqua1 carrying the appropriate binary vector using standard protocols (Boisson-Dernier et al., 2001). Composite plants were inoculated with 1 mL of E. meliloti 1021 suspension (OD600 0.5). Nodules were collected 4 weeks after inoculation and sectioned for inmunoblotting assays. Session III SIII-CP-33 RESULTS AND DISCUSSION CelC2 heterologous expression in Medicago and Ensifer has similar phenotypes. To analyze heterologous expression effects, we have introduced the rhizobial celC gene into E. meliloti 1021 and M. truncatula A17, which contain no homologs in their genomes. E. meliloti 1021C2+ produces a delay in plant development as shown by plant shoot length and dry weight, in Nitrogen-free conditions. Nodule sections showed abnormal morphology and premature senescence. Moreover, ANU843 celC overexpression in E. medicae WSM419 has similar results (Table 1). At the same time, CelC2 root-transformed M. truncatula plants and inoculated with E. meliloti 1021 showed similar phenotype to wild type plants inoculated with the CelC2 derivative strain (Table 1). Although nodule inmunohistological sections show CelC2 expression in the whole host root, only infection zone is affected, mainteining rest of root structures intact. celC of E. medicae WSM419 overexpression enhances plant development in Medicago. Moreover, we have introduced R. leguminosarum bv trifolii ANU843 and E. medicae WSM419 celC in wild type WSM419 strain. Greenhouse assays show a significative increase in plant development when inoculated with its own cellulase overproducing derivative, as revealed by plant shoot lenght data (Table 1) Table 1. Differences in M. truncatula plant shoot length (cm) 3 months after inoculation, when gro wn under N2-free conditions (±S.E.*) E. meliloti 1021 Uninoculated 0,02±0,001 a Wild-type 16,1±0,98 b 1021C2+ 7,8±0,51 c E. medicae WSM419 Uninoculated 3,95±0,17 a Wild-type 28,74±0,65 b EV 27,69±0,52 b celC+ (843) 12,04±1,05c celC+ (WSM) 34,57±0,57 d M. truncatula EV 4,3±0,44 a EV 1021 4,97±0,87 a celC 3,11±0,31 b celC1021 3,13±0,3 b Values followed by the same letter in each treatment are not significantly different from each oth er at P = 0.05 according to Fisher’s Protected LSD (Least Significant Differences). S.E. = Standard Error. *From each treatment 3 replicates with at least 35 plants each were analyzed. ACKNOWLEDGEMENTS M.E. acknowledges FPI-MICINN PhD fellowship. This work was supported by MICINN AGL200803360 and AG L2011-28227 grants and Junta de Castilla y León project SA306A11-2 . REFERENCES Boisson-Dernier, A., et al (2001). Mol. Plant Microbe Interact. 14: 695-700. Cheng, HP and Walker, G.C. (1998). J. Bacteriol. 180: 5183-91. Robledo, M., et al (2008). Proc. Natl. Acad. Sci. USA 105: 7064-7069. Robledo, M., et al (2011). Mol. Plant Microbe Interact. 24: 798-807. Robledo, M., et al (2012). Microbial Cell Factories 11: 125. Session III SIII-CP-34 Ocorrência de fungos micorrízicos arbusculares na cultura da palma no semiárido de Pernambuco. Mergulhão, A.C.E.S.*, Silva, M.L.R.B., Figueroa, C.S., Lyra, M.C.C.P. Instituto Agronômico de Pernambuco - IPA. Laboratório de Genoma-Avenida General San Martin, 1371, Bongi, CEP. 52761000 Recife-Pernambuco. Brasil. * [email protected] RESUMO A palma forrageira tem grande importância econômica no Nordeste do Brasil por ser o alimento principal dos animais e por possuir alta capacidade de adaptar-se a região semiárida. Os fungos micorrízicos arbusculares (FMA) são importantes por beneficiarem o crescimento de culturas de interesse econômico, aumentando absorção de nutrientes de baixa mobilidade no solo, o que resulta em vegetais mais tolerantes a entraves bióticos e abióticos. A falta de conhecimento sobre a ocorrência dos FMA na cultura da palma é um enigma que precisa ser investigado para entender melhor a íntima relação que desenvolvem estes fungos com a planta hospedeira. Este trabalho teve como objetivo ampliar os conhecimentos sobre a ocorrência e diversidade dos FMA coletados da rizosfera de palma forrageira como também de palma frutífera em duas áreas dos municípios de Caruaru (CA) e Arcoverde (AR) no Agreste e Sertão de Pernambuco respectivamente. Maior esporulação e ocorrência de FMA ocorreram na rizosfera de palma em Arcoverde quando comparado a Caruaru. Foram identificadas 22 espécies de FMA nas áreas estudadas. O gênero Glomus foi o mais comumente encontrado nas áreas estudadas podendo assim ser testado em benefício do solo e das palmas forrageiras e frutíferas no Agreste e Sertão de Pernambuco. INTRODUÇÃO Os fungos micorrízicos arbusculares (FMA) destacam-se pela grande importância nos habitats ecológicos ocupados pelas plantas. Estes fungos promovem melhoria no crescimento dos hospedeiros pelo aumento da absorção de nutrientes e contribuem consequentemente para a sua sobrevivência. Devido a essas características e à ampla distribuição, os FMA desempenham importante papel no equilíbrio das comunidades. Esses simbiontes proporcionam ao vegetal maior rapidez no crescimento, melhoria geral no estado nutricional e na produção de biomassa (Cavalcante et al., 2009). A cultura da palma forrageira no Nordeste do Brasil constitui um dos suportes básicos de subsistência da pecuária leiteira, sobretudo em zonas áridas e semiáridas (Mergulhão et al., 2012). Estudos com esta cultura e FMA são necessários, visto que, o conhecimento sobre quais fungos micorrízicos está presente na cultura da palma não é conhecido. Essas informações podem ser utilizadas para a seleção de isolados eficientes para aplicação em processos biotecnológicos de interesse agrícola ou ambiental na cultura da palma. Desta forma esse trabalho teve como objetivo ampliar os conhecimentos sobre a ocorrência e diversidade dos FMA coletados da rizosfera de palma forrageira como também de palma frutífera em duas áreas dos municípios de Caruaru (CA) e Arcoverde (AR) no Agreste e Sertão de Pernambuco respectivamente. MATERIAL E MÉTODOS Foram realizadas coletas de solos em 15 plantas de palma forrageira como também de palma frutífera (existentes no Banco Ativo de Germoplasma (BAG) do Instituto Agronômico de Pernambuco - IPA) em duas áreas dos municípios de Caruaru (CA) e Arcoverde (AR) no Agreste e Sertão de Pernambuco respectivamente (Tabela 1). Em cada área amo st ras de so lo (5 -20 cm de profundidade) foram co let adas preferencialmente na rizosfera das plantas, sendo os pontos definidos aleatoriamente. Os esporos de FMA foram extraídos do solo por peneiramento úmido (Gerdemann e Nicolson, 1963) e centrifugação em água e sacarose a 50% (Jenkins, 1964), contados Session III SIII-CP-34 em microscópio estereoscópico e também montados em lâminas com PVLG ou reagente de Melzer + PVLG (1:1) e observados ao microscópio. Para identificação taxonômica foram consultados Schenck e Pérez (1990) e a home page http://invam.caf.wvu.edu. O número total de esporos foi submetido à análise de variância (ANOVA) considerando os quinze pontos amostrais, sendo sete pontos em Arcoverde e oito pontos em Caruaru como repetições. As médias das diferentes áreas foram comparadas pelo teste de Tukey (P≤0,05) utilizando o programa Sanest (Zontal et al., 1984), para minimizar a variância o número de esporos foi transformado em Log (X+1,5). RESULTADOS E DISCUSSÃO A quantidade de esporos de FMA em Arcoverde foi maior (média de 4,9 esporos g -1 de solo) do que na área de Caruaru com média de 2,6 esporos g -1 de solo (Tabela 1). Valor inferior a estes foram encontrados em área de caatinga nativa na Bahia por Silva et al. (2001) onde o número de esporos foi de 1,6 g -1 de solo. Segundo Dodd et al. (1983) apesar da quantidade de esporos no solo não fornecer o valor real de sua infectividade, pode dar indicação do nível populacional dos FMA. Foram identificadas vinte e duas espécies de FMA, sendo quatorze espécies de FMA encontradas em AR e oito espécies em CA. O maior número de espécies (8) de FMA identificadas pertence ao gênero Glomus e representam 36% do total de espécies encontradas. A diversidade de espécies de FMA neste trabalho foi considerada média, porém são necessários mais estudos para permitir o registro inclusive daquelas espécies ainda não conhecidas para a ciência. Tabela 1. Espécies vegetais (Palmas forrageiras e frutíferas), fungos micorrízicos e número de esp oros registrados em duas áreas Arcoverde (AR) e Caruaru (CA) no Agreste e Sertão de Pernambuco. * Médias originais, transformadas em Log (X+1,5) para análise de variância, CV(%)=30 CONCLUSÕES Maior ocorrência de FMA foi verificada na rizosfera de palmas em Arcoverde. O gênero Glomus foi o mais comumente encontrado nas áreas estudadas podendo assim ser testado em benefício do solo e das palmas forrageiras e frutíferas no Agreste e Sertão de Pernambuco. REFERENCIAS Cavalcante, U.M.T., et al. (2009). Aspectos da simbiose micorrízica arbuscular. 28 p. Dodd, J., et al. (1983). Israel J. Botany 32: 10-21. Gerdemann, J.W., and Nicolson, T.H. (1963). Transactions of the British Mycological Society, 46: 2 35244. Jenkins, W.R. (1964). Plant Dis. Rep. 48: 692 p. Mergulhão, A.C.E.S., et al. (2012). Pes.Agropecu. Pernambucana 17: 78-82. Schenck N.C., and Pérez, Y. (1990). Manual for the identification of VA mycorrhizal fungi, 3 ed. Silva, G.A., et al. (2001). Rev. Bra. Bot. 24: 135-143. Zonta, E.P., et al. (1984). Sistema de análise estatística para microcomputadores. Session III SIII-CP-35 Bactérias diazotróficas associadas a cladódios de palma: solubilização de fosfato inorgânico e tolerância à salinidade. Silva, M.L.R.B.*, Figueroa, C.S., Mergulhão, A.C.E.S., Lyra, M.C.C.P. Instituto Agronômico de Pernambuco-IPA. Laboratório de Genoma. Avenida General San Martin, 1371 Bongi Recife. Pernambuco. Brasil. * [email protected] RESUMO A cultura da palma forrageira no Nordeste do Brasil tem uma grande importância econômica por ser o alimento principal dos animais na região semiárida. Dessa forma, para propiciar boas condições de desenvolvimento e produtividade, deve-se dar grande importância ao suprimento de fósforo. Esse trabalho teve como objetivo selecionar bactérias pela sua capacidade de solubilização de fosfato inorgânico e crescimento em meio de cultura livre de fonte nitrogenada sob diferente concentrações de NaCl. A capacidade de solubilização de fosfato foi avaliada pelo índice de solubilização (IS). Para avaliação da influencia de sal sobre o crescimento bacteriano em meio de cultura livre de fonte nitrogenada cultivadas em meio NFb semissólido, acrescido de 2,5 e 5,0 de NaCl. As bactérias diazotróficas associadas à palma, cultivadas no agreste e sertão de Pernambuco, apresentaram a capacidade de solubilizar fosfato inorgânico e crescer em meio de cultura livre de fonte nitrogenada sob concentrações de 2,5% e 5% de NaCl, tornando-se candidatas a promoção de desenvolvimento vegetal em solos salinos. INTRODUÇÃO A cultura da palma forrageira no Nordeste do Brasil tem uma grande importância econômica por ser o alimento principal dos animais na região semiárida. Dessa forma, para propiciar boas condições de desenvolvimento e produtividade, deve-se dar grande importância ao suprimento de fósforo. De acordo com Kuklinsky-Sobral et al. (2004) a habilidade das bactérias endofíticas em relação à solubilização do fosfato inorgânico vem despertando grandes interesses. Uma vez que as bactérias endofíticas durante o processo de colonização, pode inicialmente colonizar superficialmente o hospedeiro e, consequentemente, fornecer o nutriente fósforo para o desenvolvimento das plantas. As bactérias solubilizadoras de fósforo, quando utilizadas como biofertilizante, propiciam muitas vantagens para a planta e sua produtividade. Desta forma esse trabalho teve como objetivo selecionar bactérias pela sua capacidade de solubilização de fosfato inorgânico e o crescimento em meio de cultura livre de fonte nitrogenada sob diferente concentrações de NaCl. MATERIAL E MÉTODOS Solubilização de fosfato inorgânico. Para a solubilização, os isolados foram crescidos em 5 mL do meio DYGS liquido (Rodrigues Neto et al., 1986) durante 48 h, a 28º C, sob agitação de 150 rpm. À capacidade de solubilização de fosfatos, foi realizada segundo Verma et al. (2001), modificado (Santos et al., 2012) fosfato insolúvel (10g/l de glicose; 5 g/l de NH4Cl; 1 g/l de NaCl; 1g/l de MgSO4.7H2O; 4g/l de CaHPO4; 15 g/l de agar; pH 7,2). Em cada placa, foi colocado 5 µL do crescimento bacteriano com três repetições em pontos equidistantes. As placas foram incubadas a 28 °C por 72 h e, em seguida, foi verificada a presença de área solubilizada (capacidade de solubilizar) e feita à medição do diâmetro dessa área e da colônia. A partir destes dados, foi obtida a relação entre os diâmetros do halo e da colônia, utilizado na avaliação do potencial de solubilização. Session III SIII-CP-35 Salinidade sobre o crescimento bacteriano. A influência da salinidade sobre a capacidade de bactérias diazotróficas em se desenvolver em meio de cultura livre de fonte nitrogenada foi avaliada por meio da inoculação de colônias isoladas em meio NFb semissólido (Döbereiner et al., 1995), acrescido dos seguintes tratamentos: 0% de NaCl; e as concentrações 2,5 e 5% de NaCl designadas para caracterizar bactérias halo tolerantes (Santos et al., 2012). Os experimentos foram realizados em duplicata, incubados a 28°C por oito dias, sendo o experimento repetido mais uma vez. O crescimento bacteriano foi avaliado observandose a presença o u ausência de uma película típica (halo) do crescimento bacteriano no interior do meio de cultura. RESULTADOS E DISCUSSÃO Quando avaliado o índice de solubilização (IS), foi observado que os 11 isolados bacterianos capazes de solubilizar fosfato inorgânico, in vitro, expressaram valores de IS variáveis, indicando diferenças quanto ao potencial de solubilização em meio sólido. Cinco isolados diazotróficos endofíticos apresentaram valores de IS baixos entre 1,00 a 2,00, enquanto que 06 isolados apresentaram IS médios entre 2,00 a 3,43. Os isolados LG-P2 e NFB P64 foram consideradas não solubilizadoras por não apresenta a formação de halo no meio com fosfato após 18 dias de crescimento (Figura 1). O entendimento da capacidade e da eficiência de microrganismos, em solubilizar fosfatos pode levar à seleção de isolados com alto potencial de uso para a inoculação em plantas (Souchie et al., 2005). Dentre as bactérias diazotróficas avaliadas, 100% formaram película de crescimento bacteriano no meio de cultura no tratamento de 0% de NaCl, 90% em 2,5% e 60% em 5% de NaCl. Estes micro-organismos halo tolerantes, utilizam, em geral, estratégias de osmoadaptação flexíveis que lhes permitem responder rapidamente a flutuações de salinidade do meio exterior, como a acumulação de íons inorgânicos em concentrações elevadas, mantendo dessa forma a integridade celular (Santos et al., 2012), possibilitando a sobrevivência destes em solos salinos e sódicos (Stamford et al., 2005). O mecanismo de promoção de crescimento vegetal por microrganismos endofíticos ainda necessita de mais estudos para um melhor entendimento dos fatores envolvidos. A B Figura 1 . (A) NFB -P33 bactéria solubilizadora de fosfato inorgânico, (B) LG P2 bactéria não solubilizadoras por não apresenta a formação de halo no meio de cultura. CONCLUSÕES Bactérias diazotróficas associadas à palma, cultivadas no agreste e sertão de Pernambuco, apresentaram a capacidade de solubilizar fosfato inorgânico e crescer em meio de cultura livre de fonte nitrogenada sob concentrações de 2,5% e 5% de NaCl, tornando-se candidatas a promoção de desenvolvimento vegetal em solos salinos. REFERENCIAS Döbereiner, J., et al. (1995). Como isolar e identificar bactérias diazotróficas de plantas não-le guminosas. 60p. Kuklinsky-Sobral, J., et al. (2004). Environ. Microbiol. 6: 1244-1251. Rodríguez, H., and Fraga, R. (1999). Biotech. Adv. 17: 319-339. Santos, I. B., et al. (2012). Biosci. J. 28: 142-149. Souchie, E. L., et al. (2005). Pesqui. Agropecu. Bras. 40: 1149-1152. Stamford, N.P., et al. (2005). Microbiota dos Solos Tropicais. 399 p. Verma, S.C., et al. (2001). J. Biotech. 91: 127-141. Session III SIII-CP-36 Genotypic characterization of natural rhizobial populations isolated from pea and lentil in two eco-climatic subhumid and semi-arid zones in Algeria. Riah, N.1, 2*, Djekoun, A.1, Heulin, K.2, Laguerre, G.2 1 Laboratoire de Génétique, Biochimie et Biotechnologies Végétales.Université Mentouri Constantine, Algérie. 2 CIRAD, UMR 113 Symbioses Tropicales et Méditerranéennes, F-34398 Montpellier, France. * [email protected] ABSTRACT The genetic diversity of 237 isolates from root nodules of pea and lentil plants sampled in six study sites representing two contrasting eco-climatic conditions subhumid and semi-arid in East Algeria. Rhizobial isolates were characterized by PCR/RFLP of 16S23S ribosomal i ntergenic region (16S-23S IGS) and of nodD-F symbiotic region, and sequence analysis 16S ribosomal DNA gene. 26 IGS/nod genotypes assigned to the Rhizobium leguminosarum species were identified. The diversity of genotypes is mainly accounted for by the diversity of the IGS types. The structure of the population is affected by the host plant of origin but even more by the geographic effect, which is especially marked in the semi-arid zone. INTRODUCTION In Algeria food and forage legumes are, with cereals, major challenges of agriculture. Legume plants have the capacity to enter a nitrogen fixing symbiosis with rhizobial soil bacteria. Water déficit, rain irregularity and salt constraint represent major limitations for plant growth and agronomic production in Mediterranean zone, particularly in Algeria (L’Taief et al., 2009). Rhizobial adaptation to drough stress is variable (BenRomdhane et al., 2009). In this work we examined structure and genetic diversity of natural rhizobial populations isolated from pea and lentil in study sites representing two contrasting eco-climatic conditions subhumid and semi-arid in East Algeria. MATERIAL AND METHODS The isolates were characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR-RFLP), amplified DNA fragments with restriction enzyme HaeIII (Laguerre et al., 2003). Two DNA regions were targeted using a chromosomal marker, the intergenic spacer region between 16S and 23S rDNA (IGS) and a functional marker, the nodulation gene region nodD-F. The rDNA 16S gene was amplified using universal primers fD1 and rD1 as described by Laguerre et al. (1994). and primer 16S-1080r (Zakhia et al., 2006). RESULTS AND DISCUSSION Rhizobial population genetic diversity. A total of 237 bacteria were isolated from root nodules of proteaginous pea, forage pea and lentil, approximately 80 isolates from each host plant. PCR-RFLP analysis distributed these isolates in 14 IGS types and 10 nod types. The combination of these haplotypes allowed to classify the isolates into 26 distinct IGS/nod genotypes. The diversity of IGS/nod genotypes is mainly accounted for by the diversity of the IGS types with 5 predominant types representing a total of 87% isolates. Session III SIII-CP-36 Genetic Structure of the rhizobial populations. Rhizobial genotype distributions in eco-climatic zone, in different sites and from host plant of origin were analysed by AMOVA for comparison. Differentiation is high between the populations from both zones, due to their contrasted dominant genotype frequencies. Significant differences are also observed between sites in a given ecoclimatic zone. The structure of the population is affected by the host plant of origin but even more by the geographic effect, which is especially marked in the semi-arid zone. Some dominant genotypes were observed only within the populations of the cultivated lentil in semi-arid zone. The host plant of origin does not influence the distribution of the IGS types. 16S rRNA Sequence-based phylogeny. The partial sequences of the 16S rRNA representing IGS/nod genotype, show little variation. These sequences have high (99% to 100%) similarity with sequences of Rhizobium leguminosarum strains available in the databases. From the phylogenetic tree Algerian isolates may be regarded as Rhizobium leguminosarum (Figure. 1). 0.01 B. japonicum ATCC 10324T E. morelense Lc04T E. terangae LMG 6463 100 E.meliloti LMG 6133T 89 E. saheli LMG 7837T R.sullae IS123T 95 R.indigofera CCBAU 71042T R .mongolense USDA 1844T 72 R. gallicum R602spT 97 76 R. yanglingense SH22623T Rhizobium sp. BLR195 (Lens culinaris, Bangladesh) 71 Rhizobium sp. BLR29 (Lens culinaris, Bangladesh) 94 R.etli CFN42T 89 R.pisi DSM 30132T (Pisum sativum) R.phaseoli ATCC 14482 R.etli WzP15 (Pisum sativum, China) 76 R.etli WzP3 (Pisum sativum, China) R.fabae CCBAU 33202T (Vicia faba, China) 72 Ag. rhizogenesT 100 R. lusitanum P1-7T R. hainanense I66T 100 99 R. tropici CIAT8899T Isolate CL4 85 Isolate BP25 and 21 other isolates T 79 R. leguminosarum bv. viciae USDA 2370 (Pisum sativum, USA) 82 R. leguminosarum bv. trifolii ATCC 14480 R. leguminosarum bv. viciae XtP1 (Pisum sativum, China) Isolate SL15 Figure 1. Phylogenetic tree (neighbour-joining) based on 907 bp alignment of nucleotide sequences of the 16S rDNA. Only bootstrap probability values of ≥70% (over 100 replicates) are indicated at the branching points. The Algerian isolates are shown in bold. The letter “T” indicates the type strai n of the species. The sequence of isolate BP25 is identified with sequences of 21 other Algerian isolates. The host plant of origin and geographic origin of the strains are indicated between parentheses. B.: Bradyrhizobium; E.: Ensifer; R.: Rhizobium; Ag.: Agrobacterium. The scale bar indicates the number of substitutions per site. REFERENCES L’Taief, B., et al. (2009). Biotechnol. Agron. Soc. Environ. 13: 537-544. Laguerre, G., et al. (2003). Appl. Environ. Microbiol. 69: 2276-2283. Laguerre, G., et al. (1994). Appl. Environ. Microbiol. 60: 56-63. Zakhia, F., et al, (2006). Microb. Ecol. 5: 375-393. Ben Romdhane, S., et al. (2009). Soil. Biol. Biochem. 41: 2568-2572. Session III SIII-CP-37 Native bradyrhizobia isolated from Lupinus mariae-josephae possess an essential T3SS for symbiosis. Durán, D.1, Pastor, V.1, Zehner, S.2, Göttfert, M.2, Imperial, J. Argüeso, T.1* 1, 3 , Rey, L.1, Ruiz1 Departamento de Biotecnología (ETS de Ingenieros Agróomos) and Centro de Biotecnología y Genética de Plantas (CBGP). Universidad Politécnica de Madrid. 28040 Madrid, Spain. 2 TU Dresden, Institute of Genetics, Dresden, Germany. 3 CSIC-Madrid, Spain. * [email protected] ABSTRACT Analysis of the genome sequence of bradyrhizobia strains isolated from root nodules of Lupinus mariae-josephae revealed the presence of a type III secretion system (T3SS). Mutagenesis of ttsI gene that codes for the transcriptional activator (TtsI) resulted in the formation of white, non-fixing nodules in L. mariae-josephae. The T3SS cluster includes a gene coding for a NopE-like protein with an autocleavage motif. The NopE protein is an effector in the Bradyrhizobium-soybean symbiosis (Wenzel et al., 2010). The autocatalytic properties of the purified NopE-like protein have been studied. INTRODUCTION Lupinus mariae-josephae is an endemic lupine found in basic soils from a small area in Eastern Spain. Endosymbiotic bacteria that form nodules with L. mariae-josephae have been isolated from this area and characterized by multilocus phylogenetic analysis as belonging to Bradyrhizobium genus. These bradyrhizobia gather in six different operational taxonomic units (OTUs) unrelated to any other isolated from lupines (Duran et al. 2013). Draft genomic sequences from representative strains corresponding to the different OTUs have been obtained and analyzed in this work. MATERIAL AND METHODS Genome draft. Genome sequencing was performed with a HiSeq2000 instrument (500bp library, PE100, 100 x coverage) at BGI (Beijing Genomics Institute, Shenzhen, China), and the reads were assembled into several contigs and scaffolds using the Short Oligonucleotides Alignment Program (SOAPdenovo). Construction of a ttsI mutant. For mutant construction, a DNA fragment containing an internal deletion of ttsI gene was cloned into plasmid pK10mobSac and then conjugated to the wild-type strain. NopE expression and purification. Plasmid pT7.7 was used for the expression of NopE-Strep in E. coli. Cloning of LmjC nopE gene, modified to code for a C-terminal Strep-tag, was performed by PCR using specific primers. NopE was purified by StrepTactin affinity chromatography. RESULTS AND DISCUSSION Bradyrhizobia isolated from Lupinus mariae-josephae contains a T3SS. Analysis of the genome drafts of several isolates from L. mariae-josephae (Lmj strains) revealed the presence of a type III secretion system (T3SS) in a cluster of about 30 genes. Genes belonging to this cluster encode the transcriptional activator TtsI, structural components of the secretion apparatus, secreted proteins including a NopElike found amo ng a few uncharacterized proteins of plant -associated bacteria (Schirrmeister et al. 2011), as well as hypothetical and unknown proteins. The highest Session III SIII-CP-37 gene conservation was observed with the genes encoding T3SS structural components of B. diazoefficiens USDA110, B. japonicum USDA6 and B. elkanii USDA61. However, the genes of non-structural proteins from Lmj strains are not conserved in other bacterial species. T3SS of Bradyrhizobium sp. LmjC is essential for symbiosis with L. mariae-josephae. TtsI has been described as the positive regulator of the T3SS. In order to ascertain the relevance of T3SS in Lmj strains, a ttsI mutant was generated in strain LmjC. Symbiosis of L. mariae-josephae with the mutant appeared to be severely impaired, since plants were significantly smaller than those inoculated with the wild type strain and since nodules produced by the mutant were white and unable to reduce acetylene. This suggests that the T3SS from strain LmjC is required for effective symbiosis with L. mariae-josephae. Autocleavage of purified LmjC NopE protein in the presence of different cations. NopE-like protein from LmjC strain presents just one autocatalytic motif (DUF1521) unlike NopE1 and NopE2 proteins secreted by the T3SS of B. diazoefficiens that contain two cleavage sites that play a role in the symbiotic host-range definition (Schirrmeister et al. 2011). Consistent with this trait, LmjC NopE protein, purified from E. coli, was autocleaved in two fragments of the predicted size in the presence of Ca 2+, Cu2+, Cd2+, Zn2+ and Mn2+. In contrast, autocleavage did not take place in the presence of Ni2+, Co2+ or Mg2+. Site-directed mutagenesis of the DUF1521 motif of LmjC NopE in E. coli abolished the in vitro self-cleavage. The role of NopE in LmjC strain is unknown, and the behavior of the NopE-DUF1521 mutant in LmjC strain is required. ACKNOWLEDGMENTS This work was supported by FBBVA (Contract BIOCON08-078 to TRA), MICINN (CGL2011-26932 to JI), and UPM (AL12-PI+D05 to LR). REFERENCES Duran, D. et al. (2013). Syst. Appl. Microbiol. 36: 128-136. Schirrmeister, J., et al. (2011). J. Bacteriol. 193: 124-129. Wenzel, M., et al. (2010). MPMI 23: 124-129. Session III SIII-CP-38 Which phosphatases may contribute to P use efficiency for N 2 fixation in the rhizobial symbiosis with legumes ? Drevon, J.J.1*, Amenc, L.1 , Pernot, C.1 , Abadie, J.1 , Blair, M.2, Lazali, M.1, Bargaz, A.1, 3 , Ghoulam, C.4, Ounane, S-M.5, Zaman-Allah, M.1, 6 1 INRA, UMR Eco&Sols, 1 Place Pierre Viala, 34060 Montpellier, France. 2 Cornell University, 242 Emerson Hall, Ithaca, NY 14853, USA. 3 Swedish University of Agricultural Sciences (SLU), Box 103, SE-23053 Alnarp, Sweden. 4 Faculté des Sciences et Techniques Guéliz, BP 549, 40000 Marrakech, Maroc. 5 ENSA, Département de phytotechnie. Avenue Hassan Badi, El Harrach 16200 Alger, Algérie. 6 ICRISAT, International Crops Research Institute for the Semi-Arid Tropics, Patancheru, India. * [email protected] ABSTRACT In order to assess the contribution of phosphatases activity to P use effeciency for N 2 fixation in legume nodules, an in situ RT-PCR methodology was used to localize and quantify the transcripts of candidate phosphatases genes in nodules of common bean in hydro-aeroponic culture under deficient vs sufficient P supply. The transcript localization of phytase, phosphoenolpyruvate-, fructose 1,6 bisphosphate- and trehalose 6-phosphate- phosphatases was found to be tissue specific and to differ among Pase genes, P treatment and legume genotype. It is concluded that these phosphatases contribute differently to the use of organic P for N2 fixation and the increase in nodule permeability to O2 under P deficiency. INTRODUCTION Symbiotic nitrogen fixation (SNF) by legumes may provide an ecologically acceptable complement or substitute for mineral nitrogen fertilizers that farmers cannot afford for economic limitation or try to minimize for environment sake. However, P deficiency is a major limiting factor for legume-rhizobia symbioses, particularly in acidified or calcareous soils. Nevertheless the expression of legume SNF potential under P deficiency may be improved (Pereira and Bliss, 1987; Vadez et al., 1999). In this study we have compared the expression of various acid phosphatases (APases) in nodules of common bean recombinant inbred lines (RILs) that were previously selected for their contrast in P use efficiency (PUE) for SNF (Drevon et al., 2011). MATERIAL AND METHODS Common bean RILs were selected and grown in hydro-aeroponic culture as described by Hernandez and Drevon (1991). The in situ RT-PCR was performed as described in Bargaz et al. (2012) on sections of standard nodules with 3 mm diameter that were harvested at flowering stage, and immediately fixed. Specific primers for APase genes were designed online at the National Center of Biotechnology Information (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi) with the known mRNA sequence of the homologous genes of Medicago and Glycine spp. RESULTS AND DISCUSSION All candidate genes of APases were intensely marked by fluorescence in the layers of inner-cortex cells, i.e. the tissue localized between vascular traces and the infected zone of nodule (Figure 1). This reveals an intense metabolism of organic P in this tissue. It may supply Pi for the energy-demanding cell turgescence for permeability of nodule cortex to gases (Serraj et al., 1995; Schultze and Drevon, 2005) and/or for the catabolism of sucrose from vascular traces into organic acids for bacteroids in the infected zone. Session III SIII-CP-38 The higher increase in phytase signal (Lazali et al., 2013) and activity (Araújo et al., 2008) under P deficiency for the RIL115 compared to the less efficient RIL147 suggests that the use of phytate in nodule cortex may contribute to the adaptation of the rhizobial symbiosis to low-P soils. In the outer-cortex, the expression of TPP might contribute to the control of various abiotic constraints by trehalose release (Bargaz et al., 2013), and that of PEPase suggests Pi mobilization from senescent cells (Bargaz et al., 2012). Both TPP and PEP genes expression increased under P deficiency. In the infected zone these genes may supply Pi for bacteroidal metabolism. In the middle cortex, surrounding the internal cortex, the PEPase activation under P deficiency would not only supply additional Pi but also reduce active oxygen species through a non-enzymatic oxidative decarboxylation of pyruvate by hydrogen peroxide (Bargaz et al., 2012). Figure 1. Transcript localization of phytase (left), phosphoenolpyruvate phosphatase (center), tre halose 6-phosphate phosphatase (right) in nodule transversal section of the efficient common-bean RIL115. In conclusion, the increase of APases transcripts under P-deficiency predicts not only an optimal intracellular Pi scavenging, but also opens up a challenge to understand whether their specific ti ssular localization in nodules is also related with responses to environmental constaints, such as oxidat ive stress, and the osmo-regulation of nodule gas diffusion. ACKNOWLEGMENTS This work was supported by the Great Federative Project FABATROPIMED of Agropolis Fondation under the reference ID 1001-009 and the EU AVERROES scholarship programme and INCOMED Aquarhiz project for the internships of Adnane Bargaz and Mainassara Zaman-Allah in Montpellier, a nd AUF-PCSI 63113PS012 of Algeria-French cooperation for the internship of Mohamed Lazali. REFERENCES Araújo, A.P., et al. (2008). Plant Soil 312: 129-138. Bargaz, A, et al. (2012). J. Exp. Bot. 63: 4723-4730. Bargaz, A, et al. (2013). Planta: DOI 10.1007/s00425-013-1877-1. Drevon, J.J., et al. (2011). Proc. Environ. Sci. 9: 40-46. Hernandez, G. and Drevon, J.J. (1991). J. Plant Physiol. 138: 587-590. Lazali, M., et al. (2013). Planta: DOI: 10.1007/s00425-013-1893-1. Pereira, P.A.A. and Bliss, F.A. (1987). Plant Soil 104: 79-84. Serraj, R., et al. (1995). Plant Cell Environ. 18: 455-462. Vadez, V., et al. (1999). Euphytica 106: 231-242. Session III SIII-CP-39 nod gene inducers released by Mimosa flocculosa seeds and its effects on common bean growth and yield under Brazilian cerrado soil conditions. Mercante, F.M.1*, Otsubo, A.A.1, Cunha, C.O.2 1 Embrapa Western Agriculture, Dourados, MS, Brazil. Technological Development-CNPq, Brasília, DF, Brazil. * [email protected] 2 National Council for Scientific and ABSTRACT The nod gene-inducing effect of M. flocculosa seed rinse (SR) was assayed by betaglucuronidase act ivity of Rhizobium tropici CIAT899 harbouring plasmid pGUS32 (containing Sinorhizobium sp. BR816 nodABC promoter fused to the gusA reporter gene); SR of Phaseolus vulgaris cv. Pérola was used as a positive control. M. flocculosa SR was also tested as a rhizobial inoculant additive in a common bean field trial; the commercially recommended strains R. tropici CIAT899 and Rhizobium sp. PRF 81 were used in inoculants. For both rhizobial strains assayed, significant gains in common bean grain yield were obtained when M. flocculosa SR was used as an inoculant additive. INTRODUCTION Common bean (Phaseolus vulgaris), as well as other leguminous crops, is able to establish a symbiotic relationship with rhizobia. The early steps of symbiotic nodule formation by rhizobia on plants require the coordinate expression of several nodulation (nod) gene operons, which are induced by molecules released from seeds and plant roots, mainly flavonoids (Peters et al., 1986; Ghasem et al., 2012). Our previous studies (Mercante and Franco, 2000) revealed that a mixture of seed rinses from common bean and M. flocculosa had synergistic effect on nod gene inducing activity; this synergistic effect was observed with R. tropici strains CIAT899 and F98.5, as well as with R. etli strain CFN42. The aim of this study was to evaluate the effects of M. flocculosa SR, alone or in combination with common bean SR, on nod gene activity of R. tropici CIAT899/pGUS32; comparing in vitro nod gene expression with the effects of SR (as an inoculant additive) on common bean growth and grain yield under Brazilian Cerrado soil conditions. MATERIAL AND METHODS Seed rinse preparation. M. flocculosa SR was obtained from previously scarified (H2SO4 for 5 minutes) seeds (10 g), by adding 100 mL of sterile distilled water and incubating, in the dark, at 30°C for 24 h, under agitation (80 rpm). Common bean SR was obtained by adding 100 mL of sterile distilled water to 100 g of seeds and incubating, in the dark, at 30°C for 24 h, under agitation (80 rpm). SR was filter-sterilized (0.22 µm Millipore filters) before use. Expression of nod genes in Rhizobium tropici CIAT899. The expression of nod genes was studied in Rhizobium tropici CIAT899/pGUS32 and was evaluated by measuring the activity of the enzyme -glucuronidase, using as substrate p-nitrophenyl--D-glucuronide at a concentration of 1000 µg mL -1. Inductions were carried out by incubating 20 µL of bacteria culture with 180 µL of diluted filtersterilized S R. After a induction period of 48 h at 30°C, reactions were carried out at 37°C, after addition of 100 µL of "GUS buffer", as described by Mercante and Franco (2000). Session III SIII-CP-39 Grain yield under field conditions. Experiment was conducted in a Brazilian Cerrado Oxisol, in the City of Dourados, Mato Grosso do Sul State, Brazil. Treatments consisted of: (1) control (without rhizobia inoculation and N fertilizer), (2) N-control (80 kg ha-1 of N-urea : 20 kg ha-1 at sowing; 30 kg ha-1, 20 days after emergency-DAE; 30 kg ha-1, 40 DAE), (3) inoculation with R. tropici CIAT899; (4) inoculation with Rhizobium sp. PRF 81. Seeds of Phaseolus vulgaris cv. Pérola were inoculated with a peat-based rhizobial inoculant (109 cells of rhizobia per gram of peat). Rhizobial inoculants were added to common bean seeds in the proportion of 500 g to 50 kg of bean seeds. Subsequently, seeds were treated with different combinations of SR, in the proportion of 300 mL kg -1 of common bean seeds. RESULTS AND DISCUSSION The highest values of -glucuronidase activity were observed when common bean SR was used alone, followed by the 1:1 (v:v) combination of common bean SR and M. flocculosa SR (Figure 1A). Even though the combination of M. flocculosa SR and common bean SR did not result in a synergistic induction of nod genes in R. tropici CIAT899 in this assay; the use of M. flocculosa SR, at a concentration of 10% (10 g of M. flocculosa seeds in 100 mL of sterile distilled water), significantly increased common bean grain yield when seeds were inoculated with either CIAT899 or PRF 81 strains, up to around 3000 kg ha-1 (Figure 1B). In addition, the use of M. flocculosa SR significantly increased common bean grain yield supplied with mineral N, but did not affect control plants. Interestingly, in earlier experiment, common bean nodulation was not inhibited by suppling mineral N if M. flocculosa SR was used as an inoculant additive (Mercante and Franco, 2000). (A) (B) Figure 1. Activity of -glucuronidase enzyme, resulting from induction of nod genes in strain CIAT8 99 (A) and grain yield of common bean inoculated with CIAT899 or PRF 81 strains (B). Values are means of 6 replicates. Different letters on the bars indicate contrast by Duncan test, 5%. ACKNOWLEGEMENTS The study was partially supported by National Council for Scientific and Technological Development (CNPq) and Fabio M. Mercante is a research fellow from CNPq. REFERENCES Ghasem, F., et al. (2012). J. Agr. Sci. Tech. 14: 1255-1264. Mercante, F.M., and Franco, A. A. (2000). R. Bras. Ci. Solo 24: 301-310. Peters, N.K., et al. (1986). Science 233: 977-980. Session III SIII-CP-40 Diversidade de fungos micorrízicos arbusculares e colonização do solo em sistema de cultivo consorciado cafeeiro (Coffea arábica L.) e seringueira (Hevea brasiliensis). Colozzi-Filho, A.1*, Bugatti, E.P.1, Garbossi, A.1, Scaramal, A.2, Machineski, O.1, Carrenho, R.3 1 Laboratório de Microbiologia de Solos, Instituto Agronômico do Paraná, Londrina-Pr., Brasil. 2 Bol sista do Laboratório de Microbiologia do Solo-IAPAR. 3 Departamento de Agronomia, Universidade Estadual de londrina, Londrina, Pr. Brasil. * [email protected] RESUMO Avaliou-se a diversidade de Fungos Micorrizicos Arbusculares (FMA) e a produção de micélio externo total (MET), em solo sob cultivo de cafeeiro e seringueira, em monocultivo ou consórcio. Foram identificadas trinta espécies de FMA no sistema, distribuídas em seis gêneros. A maior diversidade foi observada na seringueira solteira. A menor diversidade observada no cafeeiro solteiro. Quanto menor a densidade de seringueiras no cafeeiro, maior a diversidade de FMA no solo. A maior colonização do solo por FMA foi observada no tratamento com maior densidade de raízes. O cultivo consorciado cafeeiro-seringueira proporcionou maior diversidade de FMA e influenciou positivamente a atividade dos FMA para produção de MTE no solo. INTRODUÇÃO O cultivo do cafeeiro (Coffea arabica L.) sombreado com seringueira (Hevea brasiliensis) pode reduzir o stress de cultivo a pleno sol, aumentando a produção do cafeeiro além de proporcionar renda extra com a extração do látex da seringueira. Os fungos micorrízicos arbusculares (FMA) são de ocorrência natural no solo e estabelecem relações de simbiose mutualística com o cafeeiro. O cultivo da seringueira nas entrelinhas do cafeeiro pode alterar a diversidade e a ocorrência dos FMA no solo, com efeito sobre a simbiose micorrízica e até sobre a produtividade do cafeeiro. MATERIAL E METODO As avaliações foram realizadas em um experimento conduzido em campo desde 2001, na Estação Experimental do Instituto Agronômico do Paraná (IAPAR) em Londrina-PR, em um latossolo vermelho distroférrico textura argilosa. Os tratamentos são 3 espaçamentos de seringueira entre as linhas de cafeeiro (13.0m;16.9m e 22.1m por 4m de distancia entre as seringueiras na linha do cafeeiro), mais os controles cafeeiro e seringueira solteiros, nos espaçamentos de 2.5mx1.3m e 8.0mx2.5m, respectivamente. O delineamento experimental é de blocos casualizados com 4 repetições. Amostrou-se o solo na profundidade de 0-20 cm. Os esporos foram extraídos do solo por peneiramento úmido e centrifugação em água e sacarose e identificados morfológicamente sob microscópio a nível de gênero e espécies. A extração do micélio externo total (MET) foi realizada segundo a metodologia descrita por Melloni et al. (1999). Determinaram-se os índices de diversidade de Shanon. RESULTADOS E DISCUSSÃO Foram identificadas trinta espécies de FMA no sistema, distribuídas em seis gêneros. A maior diversidade (20 espécies) foi observada na seringueira solteira. A menor diversidade (13 espécies) foi observada no cafeeiro solteiro. Quanto maior o espaçamento de seringueiras no cafeeiro, maior a diversidade de FMA no solo. As espécies Acaulospora aff. Sieverdingii, A. koskei, A. mellea, A. morrowiae, A. Session III SIII-CP-40 scrobiculata, Diversispora spurca, Glomus claroideum, G. etunicatum e G. macrocarpum tiveram ocorrência generalizada, independente dos tratamentos. Por outro lado, observou-se também que 11 espécies apresentaram ocorrência restrita em apenas 1 tratamento. O cafeeiro possui alta dependência micorrízica, na fase de mudas (Siqueira e Colozzi-Filho, 1986) e também em plantas adultas a campo (Cardoso et al., 2003). Lopes et al. (1983), Nogueira, 2007) identificaram vinte e duas espécies de FMA em raízes de cafeeiro, os gêneros mais encontrados foram: Acaulospora e Glomus. O MET foi superior no tratamento com o menor espaçamento de seringueira em consórcio, diferindo significativamente dos demais tratamentos. O tratamento com a seringueira solteira foi o que apresentou o menor comprimento do MET (Figura 1). O cultivo consorciado cafeeiro-seringueira proporcionou maior diversidade de FMA e influenciou positivamente a atividade dos FMA para produção de MTE no solo. Figura 1. Hifas de fungo em monocultivo de cafeeiro, seringueira ou em cultivo consorciado de ambos com diferentes densidades de seringueira. AGRADECIMENTO Ao Programa CAFÉ do IAPAR. REFERENCIAS Cardoso, E.J.B.N,. et al. (2003). Rev. Bra. Ciên. Solo 27: 415-423. Colozzi Filho, A. e Nogueira, M.A. (2007). Microbiota do solo e qualidade ambiental, p. 39-56. Melloni, R., et al. (1999). Rev. Bra. Ciên. Solo 24: 767-775. Rillig, M.C., et al. (2006). New Phytol. 171: 41-53. Siqueira, J.O. e Colozzi Filho, A. (1986). Rev. Bra. Ciên. Solo 10: 207-211. Session III SIII-CP-41 Effect of mycorrhizal inoculation on wheat and barley genotypes differing in Al-tolerance growing at phytotoxic Al level. Meier, S., Curaqueo, G., Seguel, A., Aguilera, P., Cornejo, P., Borie, F. * Scientific and Technological Bioresources Nucleus BIOREN-UFRO, Universidad de la Frontera, P.O Box 54-D, Temuco, Chile. * [email protected] ABSTRACT The aim of this study was to test the effect of arbuscular mycorrhizal (AM) inoculation on wheat and barley genotypes differing in Al tolerance when growing at phytotoxic Al level. The experiment was carried out in a growth chamber using soiless substrate, Altolerant and Al-sensitive plants genotypes, a native mycorrhizal inoculum and a mineral solution additioned or not with 200 uM of Al. After five weeks of growing, plants were harvested and biomass, root colonization, mycorrhizal spores, glomalin and Al in shoots and roots were measured. Results showed that spore number increased in all cultivars when subjected to Al following the same trend that glomalin production. Al-sensitive wheat cultivar “Tukan” as well the Al-sensitive barley cultivar “Scarlett” significantly increased the glomalin content suggesting an important role in Al detoxification in the rhizosphere. INTRODUCTION Chilean cereal production is chiefly supported in acidic soils from volcanic origin. Soil acidity is one of the main constraints for the productivity of agricultural plants at worldwide level. The excess of protons ( H+) in soils involves an increase of phytotoxic Al levels together a decrease in the availability of P, Ca, Mg and Mo. Therefore, plant roots growing in acid soils subjected to such stressful environments reduce seriously their capacity of nutrient acquisition (Tang and Rengel, 2003) thus affecting crop productivity. For overcoming the negative effects on productivity of acid soils farmers apply some agricultural management including lime application, phosphate fertilizers and/or the use of Al tolerant plant cultivars. Some of such practices are sometimes economical unfeasible (lime) or have potential environmental risks (P-fertilizers). Accordingly, it appears important to deep in how to increase Al tolerance in cultivars habitually used in local extensive agriculture. However, a key factor such as the presence of associated microorganisms in its rhizosphere has not been considered. The AM symbiosis plays an important role protecting the roots from deleterious Al effects conferring to the plant hosts a higher Al tolerance through an enhanced P, Ca or Mg acquisition (Marschner, 1995) as well as an increased exudation of organic anions with Al chelation capacity (Klug and Cumming 2007; 2009). In both mechanisms a decrease on the deleterious effects of Al in the rhizosphere is produced. In addition, Aguilera et al. (2011) have evidenced the Al capacity to form stable chelates with glomalin (expressed as GRSP), a glycoprotein released by AM fungal structures, suggesting another way of decreasing the toxicity of free Al. More recently, Seguel et al. (2013) have reviewed the role of AM on decreasing Al phytotoxicity in acidic soils depicting an integrated model for induced Al resistance to plants. In consequence, the aim of this study was to determine the effect of mycorrhizal inoculation on grow wheat and barley cultivars differing in Al tolerance when subjected to high Al phytotoxic level. Session III SIII-CP-41 MATERIAL AND METHODS The experiment was carried out in 300 mL polypropilene pots containing vermiculiteperlite (2:1) as substrate inoculated or not with AM fungi spores from a pot culture with sudangrass originated from a soil with 70% Al-saturation (300-500 spores). Wheat seeds of Al-tolerant genotype “Porfiado” and Al-sensitive “Tukan” together barley seeds of Al-tolerant genotype “Aurora” and Al-sensitive “Scarlett” were sown in each pot. Additionally, the Al-tolerant wheat genotype “Atlas 66” was included as a comparison treatment. Seedlings were irrigated with modified Hoagland mineral solution during two weeks. Lately, Al was added to mineral solution (0-200 µM as Al2(SO4)3) and plants grown for other 3 weeks. At harvesting, biomass production, spore number, root colonization (%), glomalin (Wright and Upadhyaya, 1996) and Al in shoots and roots were measured. The experiment was carried out with four replicates. RESULTS AND DISCUSSION It is recognized that Al damage is mainly produced at root level, particularly rootlets by shortening and thickening of them interfering in nutrient absorption. Hence, for testing Al tolerance or sensitivity is better to use root length instead root weight. Consequently, we did not found significant differences between cultivars. In general, as species, barley is more Al-sensitive than wheat; however, barley “Aurora” appears to have a high Al tolerance sometimes similar or higher than wheat cultivars. Arbuscular mycorrhizal spore number increased significantly in all the cultivars tested when plants were subjected to Al. The lowest spore number were obtained in “Atlas” cultivar probably due to this is a foreign cultivar not habitually cropped in volcanic acid soils and non adapted to such conditions. In addition, results showed that GRSP content increased in all cultivars when subjected to Al following the same trend that spore number, which reinforces the role played by this glycoprotein in conferring higher Al tolerance to host plants when they live in symbiosis with AM fungi. Al-sensitive wheat cultivar “Tukan” as well the Al-sensitive barley cultivar “Scarlett” increased in a higher degree the GRSP levels suggesting that its role in Al detoxification in the rhizosphere is more important in Al-sensitive than Al-tolerant plants. This statement deserves more and deeper studies in the near future. ACKNOWLEDGEMENTS. This study was supported by CONICYT Grant 1100642. REFERENCES Aguilera, P., et al. (2011). Soil Biol. Biochem. 43:2427-2431. Klugh, K., and Cumming, J. (2007). Tree Physiol. 27: 1103-1112. Klugh, K., and Cumming, J. (2009). Soil Biol. Biochem. 41: 367-373. Marschner, H. (1995). Academic Press Inc. San Diego, CA, 889p. Seguel, A., et al. (2013). Mycorrhiza 23: 167-183. Tang, C., and Rengel, Z. (2003). Handbook of Soil Acidity. Marcel Dekker, N.Y, pp-57-81. Wright, S., and Upadhyaya, A. (1996). Soil Sci., 161: 575-586. Session III SIII-CP-42 Origin of arbuscular mycorrhizal fungi determines plant development and resource distribution between Phaseolus vulgaris L. and Oriza sativa in intercropping. Ramanankierana, H.1*, Rasamiarivelo, A.V.2, Razafimbelo, T.3, Becker, T.4, Razakatiana, A.T.E.2, Manitriniaina, H.3, Rabeharisoa, L.3, Randriambanona, H.1, Drevon, J.J.4, Duponnois, R.5 1. Laboratoire de Microbiologie de l’Environnement, Centre National de Recherches sur l’Environnement , BP 1739 Fiadanana Antananarivo. Madagascar. 2 Laboratoire de Biotechnologie-Microbiologie, Faculté des Sciences BP 906, Université d’Antananarivo. Madagascar. 3 Laboratoire des Radioisotopes, Université d’Antananarivo BP 3383 Antananarivo. Madagascar. 4 IRD, UMR 210 Eco&Sols, CIRADINRA-Sup Agro, 2 place Viala, Batiment 12, 34060, Montpellier Cedex France. 5 Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM) UMR 113 IRD/CIRAD/SupAgro/UM2/USC INRA, TA A-82/J 34398 Montpellier cedex 5 France. * [email protected] ABSTRACT To illustrate the effect of arbuscular mycorrhizal fungi (AMF) origin on plant development and resource distribution between rice and bean in intercropping, three groups of AMF were used from soil rhizospheric of these two plants. The combination of AMF from rice and common bean stimulated the development of both cultivated plants and increase the concentration of P and N mineral on aerial part. Our work suggests that the origin of AMF contributes to increase yield and to reduce the use of organic, mineral or chemical amendment in rice-bean intercropping. INTRODUCTION Plant species coexistence constitutes one of biological tools which can be valorized to improve soil fertility management. However, environmental factors determining the distribution of resource between these plant species are few documented. This study shows the effect of arbuscular vesicular mycorrhizal fungi origin on plant development and resource distribution between P. vulgaris and O. sativa planted separately or together in low fertility ferralitic soil of Madagascar. MATERIAL AND METHODS Plant and fungal material. The plants and AMF used in this study occur in a field culture situated on Malagasy highland plateau where common bean P. vulgaris L. and O. sativa are usually cultivated on red ferralitic soil. The three used AMF groups were collected from root system of P. vulgaris and/or O. sativa cultivated separately or associated in a field [(F1) from P. vulgaris, (F2) from O. sativa and (F3) a combination of the two AMF groups]. Each of the three AMF groups was used to inoculate P. vulgaris and/or O. sativa cultivated together or separately in 2 litter plastic pot culture containing 1950 g of an autoclaved soil mixture of sand and ferralitic soil (50:50 v:v). Ferralitic soils were amended or not with organic amendment and/or phosphate fertilizer (20 Kg P ha-1 of manure or M20, 20 Kg P ha-1 of triple superphosphate ferlitizer or TSP 20, and both manure and triple superphosphate or TSP 20_M20). Control was established with ferralitic soil without amendment (manure or triple superphosphate). The plants were maintained in the glasshouse conditions (daylength of 12 h per day) and no other nutrients were given during the experiment. Session III SIII-CP-42 Plant development, resource distribution, and mycorrhizal/rhizobial infection assessment. The plants were harvested after 60 days growing time. For each plant, the mycorrhizal rate was assessed by the method of Phillips and Hayman (1970) and the number of nodule was counted for each common bean individual plant. Dried plant material was ground in a ball mill, mixed thoroughly, and P and N concentration of the shoots of each plant species were determined. Phosphate concentrations were determined by the molybdate blue ascorbic acid method (Watanabe and Olsen, 1965) and N concentration with HCN analyser. The total dry eight of each plant was determined (85°C, 24 hours). RESULTS AND DISCUSSION Inoculation with one of the three AMF groups and fertilization with TSP 20 and/or M 20 change the biomass produced in each pot culture with an increasing of plant development in co-culture system. Co-culturing common bean and rice in the pots amended or not with manure or triple phosphate stimulated significantly the development of biomass (Figure 1). The effect of the AMF group on the development of plant was more significant for the combination of rice and common bean AMF than for the use of the two AMF groups separately. Figure 1. Development (total biomass) of rice and common bean inoculated with the three AMF groups : Common bean in monoculture; in co-culture. Common bean in co-culture; Rice in monoculture ; Rice Phosphorus and N mineral content of co-culturing plants inoculated with the combination of rice and common bean AMF was significantly higher than those measured on plant inoculated with the two groups of AMF separately. The occurrence of the two groups of AMF stimulated the establishment of symbioses structure (mycorrhizas and nodules of nitrogen fixing bacteria) on root systems of the two cultivated plants. ACKNOWLEGMENTS This work was supported by the project Fabatropimed (Agropolis Foundation under the reference ID 1001-009) REFERENCES Phillips, J.M., and Hayman, D,S. (1970). Trans Br Mycol Soc 55: 158-160. Watanabe, F.S., and Olsen, S.R. (1965). Soil Sci. Soc.Am; Proc. 29: 67- 678. Session III SIII-CP-43 Asociación Española de Leguminosas De Ron, A.M.1, De la Cuadra, C.2, Millán, T.3* 1 Dpto de Recursos Fitogenéticos. Misión Biológica de Galicia. CSIC.Pontevedra. 2 Centro de Recursos Fitogenéticos. INIA. Apdo. 1045 Alcalá deHenares. Madrid. 3Dpto de Genética, Universidad de Córdob a. Campus de Rabanales. Córdoba * [email protected] RESUMEN LaAsociación Española de Leguminosas (AEL), es una entidad sin ánimo de lucro, constituida para la promoción de las Leguminosas en España. Trata de contribuir a la coordinación de la investigación con el sector agrícola español, promover foros de discusión con los interlocutores sociales, defender la variabilidad y fomentar nuevos usos de las leguminosas. INTRODUCCIÓN LaAEL fue creada como resultado de la iniciativa de un grupo nacional de investigadores de leguminosas. La idea partió de la Primera Conferencia Técnica sobre Leguminosas Grano celebrada en 1992 en Palencia reuniendo a 90 personas interesadas en la producción y el consumo de las leguminosas ibéricas procedentes del campo de la investigación, del mundo empresarial y de la administración pública. Ocho años después, dentro de las actividades de la 3ª Conferencia sobre Leguminosas Grano celebrada en Valladolid, surgió la decisión de constituir una Asociación, entendida como un puente desde la investigación hacia productores, empresas y administración. La AEL fue constituida en febrero del 2000 y registrada en mayo del mismo año. OBJETIVOS Los objetivos de esta asociación son: a) Promover el desarrollo de las leguminosas en España. b) Coordinar la investigación con el sector agrícola español. c) Servir de foro de discusión de investigación y sector con los interlocutores sociales. d) Defensa de la variabilidad de las leguminosas. e) Fomento de nuevos usos de las leguminosas. ACTIVIDADES Esta asociación ha organizado cinco jornadas, una cada tres años, a partir del año 2000, y las Jornadas de Difusión de Nutrición y Salud en Madrid (2002).las contribuciones presentadas en las diferentes jornadas se han publicado en Actas de la asociación y otras publicaciones (http://www.leguminosas.es/actas-de-la-ael.html). La amplia distribución geográfica de los miembros de la AEL ha permitido poner en contacto los grupos de investigación de diferentes áreas con el objetivo común de las leguminosas. Esta asociación tiene también una proyección internacional, ha participado en el apartado de difusión de resultados en los proyectos Europeos PHASELIEU y GRAINLEGUMES y ha colaborado en la organización de congresos internacionales como EUFABA y ASCOCHYTA2012. La AEL está conectada con instituciones nacionales, como los Ministerios de Educación y Agricultura, Alimentación y Medio Ambiente, asociaciones de productores de semillas y de desarrollo rural. Pertenece a la COSCE (Confederación de Sociedades de Científicos de España , http://www.cosce.org/ ) y a la Plataforma Tecnológica de Session III SIII-CP-43 Agricultura Sostenible (http://www.agriculturasostenible.org), foro de encuentro entre los agentes del sistema ciencia-tecnología-empresa en el ámbito del sector agrario. La amplia distribución geográfica de sus miembros ha permitido poner en contacto los grupos de investigación de diferentes áreas pero con el objetivo común de las leguminosas. DIFUSIÓN Los objetivos y actividades de la asociación se difunden a través de su página web (http://www.leguminosas.es/) donde se pueden descargar publicaciones relacionadas (revista Grain Legumes, descriptores, catálogos, recetarios) y las Actas de las cinco reuniones organizadas por la asociación. A través de nuestro blog (http://leguminosas.wordpress.com/) se pueden comentar las noticias de actualidad relacionadas con leguminosas. FUTURO Entre nuestros objetivos para el futuro se contempla la organización de un Foro sobre Leguminosas y Jornadas Técnicas (Sur y Norte de España). Pretendemos estrechar la relación con otras sociedades afines, como la SEFIN, y asociaciones relacionadas con la nutrición y alimentación. También pretendemos ampliar nuestra relación con países vecinos como Portugal. Session IV Associative interaction plant/beneficial microbe Session IV SIV-P-1 Biotechnological applications of the plant-microorganism interaction: health, environment and agriculture. Gutierrez Mañero, F.J.* Universidad CEU-San Pablo. Facultad de Farmacia. Madrid. España. * [email protected] Nowadays, the rhizosphere is an ecosystem, although it was not considered as such until the late 20th century. It’s a unique ecosystem, probably the largest in the Earth, of outmost complexity since three partners (plant, soil and microorganisms) are involved. Understanding of this system calls for the previous knowledge of each of the three parts, to then learn about their interaction. Today, the complex system of microbial communities associated to plant roots is considered the second genome of the plant, with a key role on its physiology and health (Berendsen, 2012). As a matter of fact, research in the plant microorganism interaction is currently one of the hot-spots in biology research. The study of the rhizosphere is resulting in an enormous array of biotechnology-based applications with successful results for the food market, agriculture, pharmacy and environment. A multidisciplinary approach is absolutely necessary to study the rhizosphere, calling for multiphase methodologies involving all areas of knowledge in biology. All approaches, from the most basic knowledge starting at biophysics and biogeochemistry, to molecular levels, namely communication strategies, metabolic adaptations and multitrophic interactions; and finally, studying the physiology of complex systems such as symbiotic relations (mycorrhiza and N fixation), growth promotion and plant fitness. Each of the before mentioned approaches to the rhizosphere, with no exception, have contributed to considerable improvements of knowledge from a basic or applied point of view. The rhizosphere is probably the area where the most specialized signal exchange between plant and microorganism takes place. The first communication process studied within this area was the symbiotic interaction, and from there on, it was extended to plant-pathogenic microorganism and plant-beneficial microorganism interactions, specifically with Plant Growth Promoting Rhizobacteria (PGPR) (Kloepper et al., 1989). This study extends to communication among microorganisms in order to organize their relations with other living organisms within the rhizosphere, including other microorganisms and extending to the plant and how its physiology is affected. Identification and knowledge about bacterial communication has contributed to understand some mechanisms used by certain PGPR to improve plant fitness. The interaction between plants and beneficial bacterial strains is one of the most complex processes known due to the exchange of information between two organisms that do not share the same cellular compartment. The relevance of this exchange is such that the more accessible partner, the microorganism, has become the target for manipulation, in order to manipulate the most complex partner, the plant, in our benefit. 261 Session IV SIV-P-1 Figure 1. Model of the interactions among beneficial, commensal and pathogenic microorganisms, and within these and the plant. Effects of microorganisms on the plant. Based on the above, and considering the actual knowledge of the interaction mechanism that deals with: Defensive systems in the plant Plant secondary metabolism involved in defense Physiology of plant growth and development Communication and elicitation systems Signal transduction mechanisms Microbial metabolism involved in cell to cell communication outside the plant (basically quorum sensing) Microbial metabolism involved in biogeochemical cycles A number of biotechnology based procedures are currently being used, all of them based on this knowledge of the plant-microorganism interaction. Some are currently being used with great profit, and are named below. Agrifood technology, including plant production both, from the classical agronomic point of view, that is evaluating yield improvment, and from a modern point of view, that is considering production with low chemical inputs, environmentally friendly and sustainable agriculture and evaluating food quality. Pharmaceuticals and nutraceuticals, improving bioactive concentration in foods and phytopharmaceuticals in medicinal plants. Phytorrizorremediation, use of biological systems to clean up toxic waste and recovery of degraded or polluted ecosystems. 262 Session IV SIV-P-1 A. B. Figure 2. A. Workplan to isolate beneficial microorganisms from rhizosphere systems. B. Factors th at affect plant’s secondary metabolism, the potential biotechnological targets to improve the plant’s adaptative capacity to stress and applications. The rhizosphere of wild plants has been shown as an excellent source of specialized PGPR due to the co-evolution processes that have taken place throughout the years (Gutierrez Mañero et al., 2003). Some rhizosphere strains are able to trigger plant’s metabolism, a process known as elicitation. Biological elicitation is a useful strategy to improve plant production (biomass) and/or to trigger secondary metabolism, which is especially relevant for the pharmaceutical and food industry (Zhao et al., 2005; Boué et al., 2009; Nosov, 2012). The use of PGPR to trigger secondary metabolism is an extremely useful tool to increase concentration of bioactive compounds in the plant, which results in a raw material of high quality, that can be used either as a food product, naturally rich in bioactive compounds, or for the pharmaceutical industry, to develop new drugs or food supplements. Systemic induction of plant secondary metabolism involved in defense has been shown in a number of plant species (van Loon, 2007). However, the nature of the molecules involved in triggering the response is not clear yet. Several bacterial determinants have been proposed and their role demonstrated in induction of systemic resistance in several plant species, such as bacterial cell wall lipopolysaccharides (LPS) or flagellin, the structural protein from bacterial flagelum (Ramos Solano et al., 2008b; van Wees et al., 2008). This fact suggests that there is certain specificity on the recognition process that is mediated by plant receptors able to trigger a signaling cascade to activate defense mechanisms. A common feature of elicitors is that they are conserved components of cell surface, or metabolites released by the microorganism (siderophores, antibiotics, biosurfactants, etc.), and a single microorganism may show different elicitors. Currently, elicitors from pathogenic microorganisms are termed PAMPs, from pathogen-associated molecular patterns, or MAMPs from microbe-associated molecular patterns (Erbs and Newman, 2011). 263 Session IV A. SIV-P-1 B. Figure 3. A. Biotechnological applications of the plant-microorganism interaction targetted to pla nt secondary metabolism. B. Metagenomic approach to the rhizosphere: isolation of genes from noncultu rable rhizosphere microorganism should result in obtention of effective molecules beneficial to plant fitness, avoiding the use of the microorganism. REFERENCES Berendsen, R.L., et al. (2012). Trends in Plant Science, 17: 478-486. Boué, S.M., et al. (2009). J Agr Food Chem, 57: 2614-2622. Erbs, G., and Newman, M.-A. (2011). Molecular Plant Pathology, 13: 95-104. Gutiérrez Mañero et al. (2003). J.Plant Physiology, 160: 105-113. Kloepper, J.W., et al. (1989). Trends Biotechnol, 7: 39-43. Nosov, A.M. (2012). Applied Biochemistry and Microbiology. 48: 609-624 Ramos Solano, B., et al. (2008b). Phytopathology, 98: 451-457. van Loon, L.C. 2007. Eur J Plant Pathol, 119: 243-54. Van Wees, S. C., et al. (2008). Current Opinion in Plant Biology, 11: 443-448. Zhao, J., et al. (2005). Biotechnol Adv, 23: 283-333. 264 Session IV SIV-P-2 Aspectos bioquímicos y moleculares de la interacción Delftia-RizobioAlfalfa: un lenguaje de señale s químicas. Castro-Sowinski, S.* Sección Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de la República, Igua 4 225, Montevideo, Uruguay * [email protected]; [email protected] Las rizobacterias pueden ejercer efectos neutros, beneficiosos o desfavorables sobre el crecimiento y desarrollo de las plantas. Estos efectos son el resultado de un intercambio de señales donde existe un reconocimiento mutuo de moléculas difusibles producidas por la planta y el microorganismo. Comprender esta compleja red de señales constituye una gran oportunidad para utilizar esta información para el diseño de estrategias que mejoren la productividad y sustentabilidad de los cultivos. Las plantas y los microorganismos producen flavonoides, fitohormonas, receptores de membrana e intracelulares, nodulinas, lectinas, enzimas, lipoquito-oligosacáridos, exopolisacáridos, aminoácidos, ácidos grasos, vitaminas y moléculas volátiles, entre otras, como parte del diálogo molecular. La interacción más estudiada es la que establecen las plantas y las rizobacterias promotoras del crecimiento vegetal (PGPR, del inglés Plant Growth Promoting Rhizobacteria) diazótrofas, siendo los ejemplos más remarcables las interacciones establecidas con miembros de los géneros Azospirillum y Rhizobium. La estrategia de señalización entre los rizobios y las leguminosas se basa principalmente en la producción de carbohidratos, tales como los Factores de Nodulación y exopolisacáridos (EPS), entre otros. Pero las raíces y los microorganismos también producen cócteles específicos de moléculas, diferentes a carbohidratos, que son fundamentales durante una interacción exitosa. Las raíces secretan fitohormonas y flavonoides (que actúan como moléculas atrayentes) que inician el diálogo químico del par simbiótico. Los rizobios reconocen los flavonoides por su unión a receptores de membrana conocidos como NodD, quienes funcionan como un sensor ambiental y activador transcripcional de los genes que se encuentran aguas debajo de la caja nod. En respuesta, los rizobios producen y secretan los Factores de Nodulación (lipoquitooligosacárido), quienes actúan como ligandos de receptores ubicados en los pelos radicales emergentes. Estos receptores se conocen como NFR-LK (por su siglas en inglés, Nod Factor Receptor-like kinase). Luego del reconocimiento NFR-LKligando, se produce la depolarización de la membrana plasmática como consecuencia de un flujo alterado de iones, seguido de oscilaciones periódicas en los niveles intracelulares de calcio (calcium spiking o firma de calcio). El calcio actúa entonces como un segundo mensajero que afecta una gran variedad de eventos celulares. Luego del reconocimiento del Factor de Nodulación, se activan varios receptores quinasa que funcionan activando una cascada de transducción de señales que controlan la progresión del hilo de infección, la organogénesis del nódulo y la fijación de nitrógeno. Se produce entonces la deformación de los pelos radicales, se inicia la formación del hilo de infección, se rearregla el citoesqueleto, se produce la expresión de las nodulinas tempranas, y finalmente se desarrolla el nódulo (Figura 1). Algunos rizobios producen proteínas involucradas en su especificidad por el hospedero y su eficiencia simbiótica. Tal es el caso de los sistemas de secreción del tipo III, T3SS, capaces de inyectar proteínas (conocidas como Nops, nodulation outer proteins) 265 Session IV SIV-P-2 directamente dentro de la célula hospedera. Por ejemplo, NopL y NopP interfieren con las vías de señalización de la planta, actuando como un efector positivo que ayuda a la formación del nódulo. Otras proteínas Nop contribuyen a la supresión de la respuesta inmune de la planta, facilitan la liberación de los rizobios desde el hilo de infección, inician la simbiosis, y participan en la persistencia del bacteroide. Figura 1. Vista simplificada de la interacción rizobio-leguminosa. a) la planta secreta flavonoide s que inducen la expresión de los genes nod de rizobios, con producción del Factor Nod (FN). b) Percepci ón del FN por parte de los receptores NFR-LK de raíz, oscilación de los niveles de Ca2+ que produce la sí ntesis localizada de citoquinas (CK). CK induce la producción de ENOD40 y la cascada de señalización que activa la respuesta simbiótica y la organogénesis del nódulo. c) deformación de los pelos radicale s y formación del hilo de infección (HI); los rizobios se movilizar por el HI. d) los rizobios penetra n a las células corticales, y se liberan en el citoplasma del hospedero, transformándose en simbiosomas, u bicados dentro del bacteroide. e) se sintetiza el péptido CLE en el nódulo, y reconocimiento del mismo por los recpetores específicos LRR-RLK; producción del inhibidor SDI que regula el número de nódulos (AON) . f) los nódulos indeterminados producen el péptido NCR, que induce la diferenciación del bacteroide . Adaptado de Morel y Castro-Sowinski (2013). El rizobio se traslada por el hilo de infección hasta que penetra a la célula vegetal por endocitosis, y se rodea de la membrana peribacteroidea, de origen vegetal. Las fitohormonas y péptidos señal de origen vegetal (ENOD40, CLE, NCR) inducen entonces la organogénesis del nódulo, la proliferación celular y dediferenciación, y diferenciación del bacteroide. El péptido CLE actúa como una señal que asciende desde las raíces hacia sus receptores LRR (leucine-rich repeat), quienes controlan múltiple aspectos del desarrollo del nódulo y la producción a nivel de las hojas de un inhibidor (SDI, shoot-derived inhibitor) que regula el número de nódulos de la raíz, el cual forma parte de la vía de señalización de autorregulación de la nodulación (AON, autoregulation of nodulation), que controla la hipernodulación (Figura 1). El Factor de Nodulación, así como la citoquina (mensajero secundario que se sintetiza en las células epidérmicas, y se traslada a la células corticales iniciando la formación del nódulo primordial) activan la expresión de los genes correspondiente a las nodulinas 266 Session IV SIV-P-2 tempranas, como ENOD12 y ENOD40. Esta última participa en la movilización de nutrientes a las células corticales y la maduración del tejido del nódulo, entre otras. Las leguminosas como Medicago, Pisum, Vicia y Trifolium desarrollan meristemas apicales activos que producen nódulos indeterminados, consecuencia de la diferenciación irreversible mediada por el péptido NCR (nodule-specific cystein-rich). Las células del hospedero producen los NRCs, y estos interfieren con el ciclo celular del rizobio, afectando la diferenciación terminal de la bacteria. La simbiosis rizobio-leguminosa puede favorecerse por la presencia de otras PGPR, diferentes a los rizobios, que producen fitohormonas que estimulan el crecimiento de la raíz, producen un cambio cualitativo y/o cuantitativo del perfil de flavonoides secretados por las plantas, y/o producen sustancias que solubilizan nutrientes de baja biodisponibilidad en el suelo (principalmente fósforo), entre otras. Probablemente, la producción de fitohormonas sea el mecanismo que mejor explica este efecto, pero se postula que el efecto se debería de la suma de diferentes mecanismos. En el caso de la coinoculación de leguminosas con rizobios y Azospirillum spp., o Pseudomonas spp., se induce la síntesis de flavonoides por las raíces de las leguminosas. Sin embargo, no parece ser estrictamente necesaria la presencia del microorganismo. La aplicación de exudados libres de bacterias a las leguminosas produce un efecto similar a la coinoculación. La lista de metabolitos capaces de mejorar la asociación plantamicroorganismo inclu ye también: 1) las vitaminas que actúan como suplementos nutricionales para los rizobios; b) enzimas hidrolíticas que ayudan al rizobio durante su penetración al pelo radical o atacan hongos fitopatógenos, c) ácidos que solubilizan fosfatos e incrementan su biodisponibilidad. En los últimos años han sido publicados varios trabajos que posicionan a las bacterias del género Delftia como PGPR (diazótrofo con actividad biocontroladora). Estas bacterias fijan nitrógeno (actividad reductora de acetileno) en vida libre cuando se las ensaya en medio semisólido libre de nitrógeno suplementado con vanadio; producen sideróforos y la auxina ácido indol-3-acético (AIA); promueven el crecimiento de alfalfa, trébol y soja en condiciones con bajo riego de nitrógeno; y actúan como bacterias que asisten durante la interacción entre los rizobios y la leguminosa, aumentando el número de nódulos por planta, la velocidad de nodulación y el peso seco de la planta, en condiciones gnotobióticas. Este efecto también se evidenció cuando los ensayos de plantas se realizaron en condiciones de invernáculo, utilizando tierra:arena:vermiculita como soporte. Curiosamente, estas bacterias resisten Cr(VI) y Pb(II), dejando pendiente el estudio de su potencial uso en la promoción del crecimiento de plantas bioacumuladoras de metales pesados. Con el objetivo de contribuir al entendimiento del diálogo químico involucrado en la comunicación entre las leguminosas y sus PGPR, se estudió la composición de las moléculas secretadas por plantas de alfalfa y las bacterias en condiciones de hidroponia. Se cultivaron plantas de alfalfa y se inocularon y coinocularon con Sinorhizobium meliloti U143 (inoculante comercial) y Delftia sp. JD2, y se colectaron las soluciones de hidroponia (desde ahora, rizodeposiciones) a varios tiempos. La composición de las rizodeposiciones se analizó por cromatografía gaseosa y líquida, acopladas a espectrometría de masas y FID. Se destaca la gran producción de ácidos grasos, principalmente ácido lignocérico (C24:0; derivado de la degradación de lignina), así como una secreción aumentada de luteolina en las rizodeposiciones de plantas coinoculadas, comparada con las rizodeposiciones obtenidas durante la inoculación 267 Session IV SIV-P-2 simple con U143. Las grandes diferencias se detectaron a los 4 días de inoculación, con disminución de la concentración de señales ya a los 7 días de inoculación. El aumento en la secreción de luteolina puede explicarse porque los flavonoides tienen un efecto importante en las primeras etapas de la interacción, induciendo la expresión de los genes de nodulación y la atracción de los microorganismos hacia las raíces; además, protegen a las células en división del daño oxidativo (poder antioxidante). También se detectó la secreción aumentada de flavonoides no involucrados directamente en la inducción de la expresión de los genes de nodulación. Este tipo de flavonoides suelen actuar como parte del mecanismo de contención de un fenotipo hipernodulante. Se detectaron terpenos (como el antioxidante limoneno), posiblemente involucrados en la interacción microorganismo-planta. Se evidenció la presencia de triptófano (precursor de la síntesis de AIA) en los exudados colectados a los 4 días de inoculación (sin detección de AIA), mientras que a los 7 días de inoculación se detectó AIA, concomitantemente con la desaparición del triptófano, posiblemente debido a su utilización durante la biosíntesis de la auxina. Finalmente, las rizodeposiciones (libres de microorganismos por filtración) producidas durante la coinoculación, mostraron efectos promotores del crecimiento de alfalfa, sugiriendo que las mismas contienen sustancias promotoras. La detección de otros metabolitos será discutida durante la comunicación oral de María Morel (Morel, M.A., Cagide, C., Dardanelli, M.S., Castro-Sowinski, S.: Caracterización de la interacción Delftia-Sinorhizobium en alfalfa.) CONTRIBUCIONES Y AGRADECIMIENTOS Los resultados acá presentados son el resultado de trabajos de grado y posgrado (Mag. María Morel, Lic. Victoria Braña, Est. Célica Cagide) financiados por Pedeciba y ANII. Se agradece a la Dra. MS Dardanelli (Universidad Nacional de Río Cuarto, Río Cuarto, Argentina), quien supervisó varios de los estudios realizados sobre las rizodeposiciones. BIBLOGRAFÍA Morel, M.A., and Castro-Sowinski, S. (2013). En: Plant microbe symbiosis-Fundamentals and Advances , NK Arora (Ed.), Springer India. DOI 10.1007/978-81-322-1287-4_6 (en prensa). Morel, M.A., et al. (2012). En: Crop Plant, Dr A. Goyal (Ed.) ISBN: 978-953-51-0527-5, InTech, DOI : 10.5772/37413. http://www.intechopen.com/books/crop-plant/legume-crops-importance-and-use-ofbacter ial-inoculation-to-increase-production. Morel, M.A., et al. (2011). Arch. Microbiol. 193: 63-68. Mortier, V., et al. (2012). En: Plant signaling peptides. H.R. Irwing, and C. Gehring (Eds.) DOI 10.1007/978-3-642-27603-3_8. Ubalde, M.C., et al. (2012). Curr. Microbiol. 64: 597-603. 268 Session IV SIV-CO-1 Evaluation of Micromonospora as a potential biocontrol agent. Martínez-Hidalgo, P. 1, 2*, Martínez-Molina, E. 1, 2, Pozo, M.J.3 1 Departamento de Microbiología y Genética, Universidad de Salamanca, España. 2 GIR "Interacciones mutualistas planta-microorganismo", Unidad Asociada al IRNASA. CSIC, Salamanca. España. 3 Departamento de Microbiología y Sistemas Simbióticos, Estación Experimental del Zaidín. CSIC, Granada, España. * [email protected] ABSTRACT In this study the potential for biological control of Micromonospora has been analysed, in the context of the new agricultural sustainability that has been outlined by the European Union and accepted by our country. Results show that Micromonospora effectively protects tomato plants from grey mould, caused by Botrytis cinerea. The quantitative analysis for gene expression in the defensive routes of the plant shows that Micromonospora increases immune response of the plant in what is now known as priming. The defensive response of the plant inoculated with Micromonospora is different and stronger than the plants not inoculated and it only appears when the pathogen is present. INTRODUCTION Agriculture derived from the green revolution that is defined by the use of pesticides fertilizers and herbicides of chemical origin, together with genetic improvement of plant germoplasm, produced an increase in agricultural productivity. Decades ago, the cost and risks derived of this kind of agriculture were revealed and as a consequence, a new agricultural revolution is starting to develop in which probiotic microorganisms are an alternative to chemicals. However, it is necessary to make an adequate analysis, evaluation and selection of the strains used in order to obtain the desired effect. In our laboratory, strains of Micromonospora have been isolated from healthy plant nodules in a variety of genera of leguminous plants. The main goal of this study was to determine their function as probiotic bacteria as there is scarce information about them even though their biotechnological potential and also their impact in this new agriculture are relevant. MATERIAL AND METHODS Plant culture, mRNA extraction, gene expression analysis by means of RT-PCR (qPCR) and the statistical analysis were performed as shown in López-Raez et al. (2010). RESULTS AND DISCUSSION Studies performed with Micromonospora to determine its effectiveness in tomato plant defence against Botrytis gave excellent results. This effectiveness was observed in whole plant and experiments in which leaves were detached before applying the pathogen (detached leaf assay). It became clear that this microorganism, when inoculated in plant roots, is capable of reducing the symptoms the fungi causes in leaves (Figure 1). Results for the quantitative analysis of gene expression in genes used as markers for defence pathways in tomato, showed that the bacteria does not have a direct effect in the induction of said defence mechanisms, nor in jasmonic or salicylic regulated response. The pathogen (Botrytis) triggers the expression of the salicylic acid pathway (Figure 2). 269 Session IV SIV-CO-1 Figure 1. Effect of Micromonospora in plant defence against Botrytis. A) Diameter of the necrotic halo in treatments using whole plants. B) Necrosis caused by Botrytis in detached leaves. Treatments: C: C ontrol, inoculated only with Botrytis. PR18: Inoculated with Micromonospora strain PR18 a month before infection with Botrytis. B5: Inoculated with Micromonospora strain B5 a month before infection wit h Botrytis. PR181D: Inoculated with PR18c a day before infection with Botrytis. B51D: Inoculated wit h B5 a day before infection with Botrytis. Figure 2. Quantitative PCR results for PR1, LOXA and PinII normalized against EF (Elongation Facto r). Treatments: C: Uninoculated plants. PR18: Inoculated with Micromonospora strain PR18. C+Bot: Inoculated only with Botrytis. PR+Bot: Inoculated with Micromonospora strain PR18 and Botrytis. Micromonospora inoculation was performed a month before detaching the leaves and infecting them wi th Botrytis. Plants treated with Micromonospora and later infected with Botrytis cause an activation of LOXA and PinII genes (from the jasmonic acid pathway) and confers resistance against this pathogen (El Oirdi et al., 2011) while downregulating the salicylic acid pathway, which is the same one that becomes upregulated when Micromonospora is not present (Figure 2). In conclusion, Micromonospora has a priming effect in tomato when inoculated in the root. Defensive response against Botrytis is higher when a previous treatment with Micromonospora was applied but it is only triggered when the pathogen is infecting the plant. According with the results, Micromonospora is a good candidate for biocontrol in order to protect crops against phytopathogenic fungi. Using this bacteria also has the advantage of being able to be inoculated while sowing, since the results did not vary when the plants were inoculated with Micromonospora one day or one month before Botrytis, which means that the effectiveness of Micromonospora inoculation lasts at least one month since its application. ACKNOWLEDGEMENTS This work has been done thanks to the funds from the Ministerio de Ciencia y Tecnología and the Ju nta de Castilla y León. P.M-H. received a predoctoral fellowship from the CSIC (Programa JAE). REFERENCES López- Raez, J.A., et al. (2010). J. Exp. Bot. 61: 2589-2601. El Oirdi, M., et al. (2011). Plant Cell 23: 2405-2421. 270 Session IV SIV-CO-2 Movimiento y quimiotáxis en Pseudomonas fluorescens F113, influencia en una colonización competente de la rizosfera de las plantas. Baena, I., Muriel, C., Martín, I., Jalvo, B., Hernanz, A., González de Heredia, E., Barahona, E., Navazo, A., Redondo-Nieto, M., Martínez-Granero, F., Lloret, J., Rivilla, R., Martín, M. * Departamento de Biología. Facultad de Ciencias. Universidad Autónoma de Madrid. Madrid. España. * [email protected] RESUMEN Se presenta el análisis de la regulación de la funcionalidad del flagelo a través del diGMPc y el posible papel de tres sistemas de quimiotaxis encontrados en la secuencia de P. fluorescens F113. INTRODUCCIÓN Pseudomonas fluorescens F113 fue aislada de la rizosfera de remolacha y su capacidad para producir compuestos con actividad antibiótica y antifúngica la convierte en una estirpe de interés en biocontrol. Los usos de F113 en sistemas integrados planta/microorganismo dependen de la eficacia de la colonización de la rizosfera. Uno de los caracteres de interés en la colonización competitiva de la rizosfera es la capacidad de movimiento (Capdevila et al., 2004; Martínez-Granero et al., 2006). El conocimiento adquirido sobre la regulación del movimiento en F113 (Redondo-Nieto et al., 2008; Navazo et al., 2009; Barahona et al., 2010; Martínez-Granero et al., 2012) nos ha permitido diseñar estirpes que se mueven mucho más y que presentan muy disminuida la capacidad para formar biopelículas (Barahona et al., 2010). Estas estirpes hipermóviles son más competitivas y presentan sustancialmente mejorada la capacidad de biocontrol en dos patosistemas distintos (Barahona et al., 2011). En P. fluorescens F113, hemos observado la existencia de dos aparatos flagelares uno típico de todas las Pseudomonas y otro similar al de A. vinelandi y existen al menos seis señales (5 ambientales y 1 citoplasmática) que independientemente regulan el movimiento a nivel de síntesis de cada uno de los aparatos flagelares y de su funcionalidad (Navazo et al., 2009; Martínez-Granero et al., 2012). Por otro lado, sabemos que el movimiento quimiotáctico es esencial para asentarse y colonizar competentemente la rizosfera de las plantas por parte de las rizobacterias (de Weert et al., 2002). Las pseudomonas en general cuentan con dos sistemas de quimiotaxis. Sin embargo, tras la secuenciación del genoma de F113 (Redondo-Nieto et al., 2013) hemos observado la existencia de tres sistemas de quimiotaxis en P. fluorescens F113. MATERIAL Y MÉTODOS Obtención de mutantes, mediante mutagénesis por inserción. Obtención de proteína de fusión en el extremo C terminal. Fusión de eCFP al extremo 3’ de flgZ en el plásmido pVLT31. Análisis de localización mediante microscopio confocal espectral LEICA SP5 con objetivo 63x y laser de Argón a 458 nm. SIDI-UAM Complementación de los mutantes con los cósmidos correspondientes aislados de la librería genómica de F113. Análisis de movimiento en placas con 0.3% de agar. Experimentos de anoxia realizado en jarras anaeróbicas Análisis de competitividad inoculando la misma proporción de estirpe silvestre y competidor en la base del tallo de la plántula recién germinada. Tras dos semanas de 271 Session IV SIV-CO-2 colonización se recogen las rizobacterias del ápice de la raíz y se analizan las UFC en medio selectivo. RESULTADOS Y DISCUSIÓN Dos diguanilatociclasas y una fosfodiesterasa regulan la funcionalidad del flagelo. La molécula señal diGMPc está implicada en la señalización que regula la funcionalidad del flagelo. Existen dos diguanilatociclasas (SadC y WspR) y una fosfodiesterasa (BifA) cuyos mutantes están afectados en movilidad y por tanto están implicadas en el mantenimiento de los niveles de diGMPc en relación con el movimiento flagelar. El genoma de F113 cuenta con una proteína flagelar FlgZ que tiene un dominio PilZ que detecta diGMPc y responde interaccionando con la proteína FliG del motor flagelar. Su expresión está regulada por el activador transcripcional de síntesis del aparato flagelar de Pseudomonas FleQ y su localización en la bacteria depende de los niveles de diGMPc celulares. Cuando los niveles de diGMPc son altos como consecuencia de la acción diguanilatociclasa de SadC y WspR, FlgZ se une al motor flagelar probablemente frenando el movimiento. Cuando los niveles de diGMPc son bajos como consecuencia de la acción de la fosfodiesterasa BifA la proteína se encuentra dispersa por la célula favoreciendo el movimiento. F113 tiene tres sistemas de quimiotaxis necesarios y no intercambiables. Los mutantes obtenidos en cada una de las histidín quinasas (CheA) de los tres sistemas presentan distintos fenotipos de movilidad cuando el análisis se realiza tanto en aerobiosis como en anaerobiosis. Los sistemas quimiotácticos 1 y 3 tienen una mayor importancia en la colonización competitiva de la alfalfa por F113. Cabe señalar que los mutantes en CheA1 y CheA3 son inmóviles cuando los experimentos son llevados a cabo en anaerobiosis. Creemos que la capacidad para realizar quimiotaxis en presencia de oxígeno o en anaerobiosis es muy importante para el mantenimiento de la competitividad por la colonización de un nicho ecológico cambiante como es el rizosférico. AGRADECIMIENTOS Este trabajo ha sido financiado por los proyectos BIO2009-08254, BIO2012-316034 y Microambiente CM. BIBLIOGRAFÍA Barahona, E., et al. (2010). Environ. Microbiol. 12: 3185-3195. Barahona, E., et al. (2011). Appl. Environ. Microbiol. 77: 5412-5419. Capdevila, S., et al. (2004). Microbiology SGM 150: 3889-3897. De Weert, S., et al. (2002). Appl. Environ. Microbiol. 56: 1173-1180. Martínez-Granero, F., et al.(2005). Microbiology SGM 151: 975-983. Martínez-Granero, F., et al. (2006). Appl. Environ. Microbiol. 72: 3429-3434. Martínez-Granero, F., et al.(2012). PLoS ONE Vol 7: e31765. Navazo et al. (2009). Microbial Biotech. 2: 489-498. Redondo-Nieto, M., et al. (2008). J. Bacteriol. 190: 4106-4109. Redondo-Nieto, M., et al. (2012). J. Bacteriol. 194: 1273-1274. Redondo-Nieto, M., et al.(2013). BMC Genomics 14: 54. 272 Session IV SIV-CO-3 Caracterización de la interacción Delftia-Sinorhizobium en alfalfa. Morel, M.A.1*, Cagide, C.1, Dardanelli, M.S.2, Castro-Sowinski, S.1, 3 1 Unidad de Microbiología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE ), Av. Italia 3318, Montevideo, Uruguay. 2 Departamento de Biología Molecular, Universidad Nacional d e Río Cuarto, Río Cuarto, Argentina. 3 Sección Bioquímica y Biología Molecular, Facultad de Ciencias , Universidad de la República, Montevideo, Uruguay. * [email protected] RESUMEN Durante la co-inoculación de alfalfa con Sinorhizobium meliloti y Delftia sp. se produce un aumento significativo del crecimiento vegetal, comparada con la inoculación simple, probablemente debido al aumento en la secreción de flavonoides (especialmente luteolina) y glucosa. Cambios en la secreción de otros metabolitos pueden también explicar la promoción del crecimiento de alfalfa. Los mismos serán discutidos en la sesión de póster. INTRODUCCIÓN Muchas veces, la co-inoculación, con dos o más microorganismos promotores del crecimiento vegetal (MPCV), produce un incremento del rendimiento vegetal de leguminosas, comparado con la inoculación simple con rizobios. Algunos ejemplos son la co-inoculación con bacterias de los géneros Bacillus y Azospirillum (Morel et al.,. 2012). Las bacterias del género Delftia son -Proteobacterias y algunas de ellas, descritas como MPCV, son capaces de fijar nitrógeno en vida libre, producir ácido indol-3-acético (AIA) y sideróforos (Han et al., 2005; Morel et al., 2011; Ubalde et al., 2012). Actualmente, existe al menos un registro comercial de inoculante conteniendo Delftia acidovorans, formulado para el mejoramiento en la producción de soja y canola (Brett Young™). Los objetivos de esta propuesta fueron: 1) demostrar la capacidad del aislamiento nativo Delftia sp. JD2 de promover el crecimiento de alfalfa, en ensayos de co-inoculación con S. meliloti U143 (inoculante nacional), en condiciones de invernadero; y 2) analizar la composición química de las rizo-deposiciones, obtenidas en condiciones de hidroponia, durante la co-inoculación de las plantas. MATERIAL Y MÉTODOS Estudios en invernáculo. Se determinó el peso seco de la parte aérea y radicular y el largo de raíz principal de plantas de alfalfa co-inoculadas con U143 y JD2, y los controles correspondientes (inoculación simple y sin inoculación), luego de 45 días de crecimiento en invernáculo en macetas conteniendo una mezcla de tierra:arena:vermiculita (2:2:1). Estudios analíticos sobre exudados radicales. Se analizó la concentración por espectrofotometría y la composición por cromatografía de: flavonoides y AIA (LCMS/MS), azúcares y ácidos grasos (GC-FID) y aminoácidos (GC-MS), en los líquidos de hidroponia obtenidos luego de 4 dias de crecimiento de plantas de alfalfa coinoculadas con U143 y JD2 (y los controles correspondientes). Los ensayos se realizaron por triplicado y los datos se analizaron estadísticamente por ANOVA utilizando PAST software versión 2.16 (Hammer et al., 2001). RESULTADOS Y DISCUSIÓN La producción de materia aérea de alfalfa se incrementó en un 10% cuando se realizó la co-inoculación con U143 y JD2, comparado con la inoculación simple con rizobios (Figura 1). Bajo déficit de nitrógeno, JD2 indujo el aumento del desarrollo del sistema radicular (Figura 1), probablemente debido a la producción de AIA. Sin embargo, no se 273 Session IV SIV-CO-3 detectó aumento del tamaño del sistema radical durante la co-inoculación, ni en plantas fertilizadas con nitrógeno. 500 c 800 b b 700 b 600 Peso seco (mg) Peso seco aéreo (mg) 400 d 300 c a acd 200 b d 500 400 a ad 300 200 100 100 JD2 -N 2 3-JD U14 JD2 U14 3 Con trol JD2 N U14 3-JD 2 JD2 U14 3 N Con trol N 0 0 Figura 1. Peso seco de parte aérea (izquierda) y raíz (derecha). Control y N: plantas sin nitrogen ar y nitrogenadas, respectivamente, sin inocular; U143 y JD2: plantas inoculadas con U143 y JD2, respec tivamente; U143-JD2: plantas co-inoculadas. Todas las inoculaciones fueron bajo déficit de nitrógeno. Se detectaron cambios en la composición de azúcares exudados entre los diferentes tratamientos. Es de destacar que durante la co-inoculación solo se detectó glucosa, el azúcar con mayor contenido energético, entre los analizados (Tabla 1). Tabla 1. Composición en azúcares de las rizo-deposiciones. Azúcar Ribosa Galactosa/Celobiosa Glucosa U143 15±2 19±3 66±5 JD2 25±4 29±3 46±3 U143+JD2 ----100 A excepción de la naringenina (precursora de otros flavonoides más complejos), durante la co-inoculación se produjo un aumento de la secreción de los flavonoides analizados, resaltando el aumento en luteolina en un factor de 150 (Tabla 2). Tabla 2. Composición (µM) en flavonoides, AIA y triptófano (Trp) de las rizo-deposiciones. Apigeni na, Api; Crisina, Cri; Genisteína, Geni; Naringenina, Narg; Luteolina, Luteo; Morina, Mor; Naringina, Nar. AIA U143+JD2 JD2 U143 0.009 Trp Api Cri Geni Narg Mor Nar Luteo 0.15 18 19 17 0.06 3.07 - 0.3 0.16 0.3 4 0.7 2.2 0.15 - 0.09 0.3 6 10 6 11 0.02 - 0.11 9 Basados en el aumento de secreción de luteolina durante la co-inoculación, se continuó con la evaluación del efecto de la luteolina y/o de las rizo-deposiciones de alfalfa sobre las proteínas secretadas por Delftia sp. JD2. Estos resultados y otros serán discutidos y mostrados en la sección de póster durante el congreso. AGRADECIMIENTOS A PEDECIBA, ANII, Amsud-Pasteur. BICLIOGRAFÍA Hammer, O., et al. ( 2001). Palaeontología Electrónica 4:9. Han, J., et al. (2005). Syst Appl Microbiol 28: 66-76. Morel, M.A., et al. (2011). Arch. Microbiol. 193: 63-68. Morel, M.A., et al.(2012). Crop Plant, Dr. A. Goyal (Ed.), In Tech, DOI: 10.5772/37413 Ubalde, M.C., et al. (2012). Curr Microbiol 64: 597-603. 274 Session IV SIV-CO-4 Estrés salino, calidad nutricional y comportamiento postcosecha, en lechuga inoculada con Azospirillum. Fasciglione, G. *, Casanovas, M., Yommi, A., Quillehauqui, V., Roura, S., Barassi, C.A. Unidad Integrada: Facultad de Cs. Agrs. (UNMdP) -EEA Balcarce (INTA). Ruta 226 Km 73,5 CC 276 (7620) Balcarce, Argentina *Becaria CONICET. * [email protected] RESUMEN La inoculación de semillas de Lactuca sativa, cv. Elisa con A. brasilense Sp245 revirtió la disminución en la emergencia debida a la salinidad e incrementó la partición a raíces. A cosecha, la supervivencia, el peso fresco y los contenidos de agua, ascorbato y clorofila, fueron significativamente mayores en las plantas inoculadas, manteniéndose este efecto durante la poscosecha. Asimismo, la inoculación redujo el Potencial de Browning en plantas estresadas, lo cual podría atribuirse a la mayor capacidad antioxidante de las plantas inoculadas debida, en parte a los mayores niveles de ascorbato. INTRODUCCIÓN. Revertir los efectos negativos de la salinidad y aumentar el valor nutricional de los productos se encuentran entre las demandas tecnológicas de la producción hortícola (Fasciglione et al., 2012). A pesar de que se ha avanzado en el estudio de inoculantes a base de Azospirillum (PGPR, del inglés Plant-Growth Promoting Rhizobacteria) para cultivos extensivos, poco se sabe de su aplicación en producciones intensivas (Barassi et al., 2007). El objetivo del presente trabajo fue evaluar si la inoculación con Azospirillum podría actuar como paliativo del estrés salino, así como mejorar la calidad nutricional y el comportamiento postcosecha de plantas de lechuga. MATERIAL Y MÉTODOS 1.Ensayo plántulas. Semillas de lechuga controles (C) e inoculadas con 109 células de A. brasilense.semilla-1 (I) se sembraron en bandejas plásticas de 66 celdas conteniendo sustrato comercial. Se regó por capilaridad con soluciones de NaCl: 0, 40, 80 y 120 mM. A los 30 días desde la siembra (dds) se evaluó el porcentaje de emergencia (PE) y los pesos secos de la parte aérea y de la raíz, calculándose la partición aérea (PA) y a raíces (PR). 2.Ensayo a cosecha y poscosecha. Plántulas C e I provenientes de los tratamientos 0 y 40 mM de NaCl se trasplantaron a macetas hasta la cosecha (95 dds), momento en el que se determinó: supervivencia (S), peso fresco aéreo (PFA), contenido de clorofila (CF), contenido relativo de agua (CRA) e índices representativos de calidad nutricional: Potencial de Browning (PB), contenido de Ácido Ascórbico (AA) y capacidad antioxidante total (AAnt). Para el ensayo de poscosecha, las plantas se envasaron en bolsas de polietileno y se almacenaron durante 20 días a 4°C y 98% HR. A los 10 y 20 días de poscosecha (dpc) se determinaron: el CRA y los índices nutricionales ya mencionados. RESULTADOS Y DISCUSIÓN 1.Ensayo plántulas. El PE disminuyó al incrementarse el nivel de salinidad, independientemente del nivel de inóculo (datos no mostrados). La inoculación revirtió este efecto negativo de la salinidad, incrementando el PE en un 12%. Las plántulas I creciendo con 80 y 120 mM de NaCl, evidenciaron una mayor PR (Tabla 1). 2. Ensayo a cosecha y poscosecha. La salinidad redujo el PFA y el CRA e incrementó la AAnt y el contenido de AA, en ambos niveles de inóculo (datos no mostrados). Independientemente del nivel de salinidad, la inoculación incrementó tanto la S (15%) 275 Session IV SIV-CO-4 como el PFA (18%), ambos factores determinantes del rendimiento del cultivo. Los principales componentes que determinan la calidad en lechuga, la turgencia, el color y la ausencia de pardeamiento, se ven afectados durante el almacenamiento. Al analizar CF, CRA y AA fueron significativos (p≤0.05) los incrementos asociados al factor inóculo (Tabla 2). El mayor CRA de las plantas I podría deberse a que la mayor PR observada en las plántulas incremento el desarrollo radical en etapas posteriores (Tabla 1). Si bien el contenido de CF, CRA y AA disminuyó significativamente en todos los tratamientos durante la poscosecha, los valores de las plantas I fueron siempre significativamente superiores a las C (Tabla 2), relacionado con un producto de mayor calidad. Tabla 1. Partición a raíces (%) a los 30 dds de plantas de lechuga C ó I creciendo sin ó bajo tres niveles de estrés salino. Letras diferentes indican diferencias entre los niveles de inóculo para cada niv el de estrés salino según test LSD (p<0,05). Partición a Raíces (%) Inóculo 0 40 Na Cl (mM) 80 120 C 23,81+1,67a 21,14+1,54a 23,13+0,75b 12,61+0,54b I 23,34+2,03a 22,01+0,79a 27,86+1,47a 19,65+1,05a La ocurrencia de estreses abióticos induce incrementos en el contenido de fenoles en tallos de la lechuga, estos se oxidan y generan pardeamiento y manchas marrones, disminuyendo la calidad comercial. Al analizar el PB fue significativa la interacción Inóculo y Salinidad (p< 0.05), ya que la inoculación redujo el PB sólo en las plantas estresadas (Tabla 3). Esta reducción posiblemente se asocie al contenido foliar de AA que fue superior en las plantas I (Tabla 2). Asimismo, la inoculación incrementó la AAnt únicamente a cosecha (datos no mostrados). Tabla 2. Evolución del CRA y de los contenidos de AA y de CF en plantas de lechuga I o C durante l a poscosecha. Los valores son promedio de los obtenidos en condiciones de salinidad 0 y 40 mM de NaC l. Letras diferentes indican diferencias significativas dentro de cada nivel de inóculo, según test L SD (p<0.05). CRA (%) AA (mg.100g PS-1) CF (mg.100g PS-1) C I C I C I Días de poscosecha 0 10 20 71,7+2,1b 73,1+1,6b 68,8 +1,0b 75,2+0,9a 76,5+1,1a 73,9+1,6a 178,7+13,2b 138,5+5,7b 71,7+2,1b 286,9+18,1a 205,2+15,9a 75,2+0,9a 769,2+30,9b 630,3+24,7b 562,8+35,4b 869,6+27,1a 733,6+32,3a 679,5+33,3a Tabla 3: Evolución del PB durante la poscosecha en plantas de lechuga I o C, creciendo a 0 ó 40 mM de NaCl. Letras diferentes indican diferencias significativas dentro de cada nivel de inóculo, según el test LSD. Potencial de Browning (Abs 320 nm/g PF) 0 dpc 10 dpc 20 dpc NaCl C 0,03+0,003b 0,18+0,004a 0,22+0,02a 0 mM I 0,04+0,002a 0,18+0,003a 0,19+0,02a C 0,06+0,003a 0,26+0,003a 0,46+0,01a 40 mM I 0,04+0,003b 0,17+0,001b 0,25+0,02b BIBLIOGRAFÍA Barassi, C.A., et al. (2007). Global Science Books. Dynamic Soil, Dynamic Plant 1: 68-82. Fasciglione G. et al. (2012). J. Sci. Food Agric. 92: 2518-2523. 276 Session IV SIV-CP-01 Bioprospecção de Bactérias Isoladas de Milho para Promoção de Crescimento de Plantas. Ikeda, A.C.1*, Szilagy-Zecchin, V.J.1, Hungria, M.2, Kava-Cordeiro, V.1, Glienke, C.1, Galli-Terasawa, L.V.1 1 Departamento de Genética, Universidade Federal do Paraná, Curitiba, Brasil. 2 Embrapa Soja, Londri na, Brasil. * [email protected] RESUMO Isolados bacterianos associados a raízes de milho identificados por sequenciamento parcial do gene 16S RNAr foram avaliados em testes de promoção de crescimento vegetal. Também foram conduzidos testes in vitro para a capacidade de produção de sideróforos, solubilização de fosfato, produção de AIA, FBN e produção de enzimas líticas. Cinco isolados apresentaram resultados promissores na caracterização enzimática e nos testes de atividade promotora de crescimento e, portanto, poderão ser avaliados in vivo quanto a parâmetros de crescimento vegetal em ensaios em casa de vegetação. INTRODUÇÃO Associações entre raízes de gramíneas e bactérias podem trazer benefícios de incremento na produtividade do milho. Bactérias presentes no solo também podem promover indiretamente o crescimento vegetal, por inibição de fito-patógenos ao produzirem enzimas líticas e, de forma direta, atuam em processos de FBN e solubilização de fosfato, na produção de substâncias análogas a fitormônios, como a auxina, e compostos queladores de ferro (sideróforos) (Araújo et al., 2010). O sequenciamento do gene 16S RNAr é uma ferramenta útil para a identificação da diversidade e estudos filogenéticos de bactérias. MATERIAL E MÉTODOS Os 24 isolados bacterianos provenientes de fragmentos radiculares de milho, pertencem à coleção do LABGEM, UFPR-Curitiba, Brasil. O DNA genômico foi extraído de acordo com Raeder e Broda (1985). A reação de sequenciamento do gene 16S RNAr foi realizada de acordo com Weisburg et al. (1991) e a qualidade das sequências foi avaliada pelo programa Phred com edição no programa BioEdit versão 7.0. As sequências editadas foram comparadas no GenBank pelo programa BLAST em busca de alinhamentos significativos. A avaliação da produção das enzimas líticas (celulase, quitinase e pectinase), a detecção da atividade esterásica, a produção de sideróforos, de ácido indol acético (AIA), a solubilização de fosfato e o potencial de fixação biológica de nitrogênio (FBN), foram realizados de acordo com Cattelan (1999). RESULTADOS E DISCUSSAO Os resultados dos testes in vitro estão apresentados na Tabela 01. Os isolados que apresentaram os maiores índices de produção de AIA e melhor desempenho nos testes qualitativos foram identificados como pertencentes ao gênero Bacillus sp. Isolados desse gênero são amplamente descrito em estudos com promoção de crescimento em plantas (Wahyudi et al., 2011). Os isolados LGMB137, LGMB196 e LGMB216 apresentaram resultados positivos em pelo menos cinco dos sete testes conduzidos e podem ser utilizados em ensaios de casa de vegetação, ainda que a produção de AIA apresente índices baixos, em comparação com o valor máximo observado (22,77 µg/ml). Araújo e Guerreiro (2010) observaram que a maioria dos isolados de Bacillus sp. que promoveram crescimento em milho não são os maiores produtores in vitro de 277 Session IV SIV-CP-01 AIA, uma vez que a adição de auxina microbiana pode alterar o nível ótimo da auxina endógena, causando inibição do crescimento da planta. Os isolados LGMB189 e LGMB222 apresentaram o segundo e o terceiro maiores níveis de produção de AIA e resultado positivo para pelo menos cinco dos sete testes realizados, sendo assim promissores na promoção de crescimento in vivo. A capacidade de solubilizar fosfato está associada ao aumento da disponibilidade de fósforo para a planta e da eficiência de fixação biológica de nitrogênio. Na cultura de milho, a utilização de bactérias com resultado positivo para FBN e solubilização de fosfato, colabora para a diminuição do uso de fertilizantes fosfatados e nitrogenados, reduzindo custos para o produtor e com menor impacto ambiental. Também, a produção de sideróforos disponibiliza ferro para a planta e inibe, por competição, a colonização de fito-patógenos. Assim, bactérias com resultado positivo para a produção de enzimas líticas podem ser promissoras para o biantagonismo, uma vez que essas enzimas podem quebrar compostos da parede celular de fito patógenos (Hernández, 2004; Singh, 2013). Concluindo, os isolados LGMB137, LGMB189, LGMB196, LGMB216 e LGMB222 foram selecionados para avaliação de desempenho em ensaios in vivo por parâmetros de crescimento vegetal em casa de vegetação. Tabela 1. Resultados dos testes in vitro de promoção de crescimento e identificação por sequenciam ento parcial do gene 16S RNAr de isolados bacterianos isolados de raízes de milho. AGRADECIMENTOS À Semilia-Genética e Melhoramento pelo material genético. O trabalho foi parcialmente financiado p elo Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq-Microrganismos Facilitadores (557746/2009-4) e CNPq-Repensa (562008/2010-1). REFERÊNCIAS Araújo, W.L., et al. (2010). CALO, 169p. Araújo, F.F. e Guerreiro, R.T. (2010). Ciên, Agrotec. 34: 837-844. Cattelan, A.J. (1999). Embrapa Soja, 36p. Hernández, A. (2004). Ver. Colom. Biotec. 6: 6-13 Raeder, U., and Broda, P. (1985). Lett. Appl. Microbiol. 1: 17-20. Singh, J.S. (2013). Resonance. 275-281. Wahyudi, A.T., et al. (2011). J. Microbiol. Antimicrob. 3: 34-40. Weisburg, W.G., et al. (1991). J. Bacteriol. 173: 697-703. 278 Session IV SIV-CP-02 Caracterización de una metilobacteria aislada de la superficie del grano de arroz. Gallego Parrilla, J.J., Alías-Villegas, C., Díaz-Olivares, I.M., Gutiérrez Alcántara, R., Madinabeitia-Peiró, N., Bellogín, R.A. *, Espuny, M.R. Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes 6, 41012-Sevilla. España. * [email protected] RESUMEN. De granos de arroz que desarrollaban un color rosa en algunas plántulas que crecían en tubos en condiciones asépticas se aisló una bacteria que crecía en AMS con metanol (0,5%) como fuente de carbono formando colonias de un color rosa intenso. Esta bacteria, a la que se denominó RS, tiene características promotoras del crecimiento vegetal como solubilización de fosfatos y producción de AIA, que se traducen en una mejora en la germinación y alargamiento de la raíz de plantas de arroz. Además es productora de acil homoserina lactona y puede crecer hasta en concentraciones 800 mM de NaCl. La amplificación, secuenciación y análisis del gen que codifica el ARN16S determinó que esta bacteria presenta un 96,29 % de similitud con Methylobacterium aquaticum. INTRODUCCIÓN. El género Methylobacterium incluye un grupo de bacterias metilotrofas facultativas pigmentadas de rosa que crecen con compuestos de un solo carbono como formiato, formaldehído o metanol como única fuente de carbono. Las especies de Methylobacterium están ampliamente distribuidas en la naturaleza (suelo, agua, polvo, sedimentos, aire, ambientes hospitalarios, etc.) y son abundantes en la filosfera de las plantas. Methylobacterium coloniza las partes aéreas y subterráneas de las plantas como epi- y endófita. Tras la colonización, expresan los genes implicados en la metilotrofía beneficiándose del metanol producido por las plantas. Se han publicado diversos artículos donde se describe el papel como promotora del crecimiento vegetal a través de mecanismos directos o indirectos, como la producción de fitohormonas o enzimas que modulan el crecimiento vegetal, la secreción de compuestos implicados en biocontrol o en la supresión de la enfermedad. Curiosamente esta bacteria ha sido aislada de aguas potables y otros ambientes relacionados con la actividad humana y esto es debido a que son altamente resistentes a cloro (Hiraishi et al.,1995). MATERIAL Y MÉTODOS. Para el crecimiento de RS se utilizaron los medios AMS con metanol o King B. Para el estudio de las propiedades promotoras del crecimiento se emplearon ensayos de solubilización de fosfatos en NBRIP (Shekhar, 1999), de producción de ácido indol acético (AIA) (Patten and Glick, 2002), producción de sideróforos (Alexander and Zuberer, 1991). El crecimiento a distintas concentraciones de NaCl (100, 300, 600, 800 y 1000 mM) se llevó a cabo en King B líquido midiendo la absorbancia a 600 nm cada 24 h durante 3 días. La producción de acil-homoserina lactonas se realizó usando dos biosensores, Chromobacterium violaceum CV026 (McClean et al., 1997), y Agrobacterium tumefaciens NT1 (Cha et al., 1998). Para estudiar el efecto de esta bacteria sobre las plantas de arroz se usaron semillas descascarilladas de arroz (Oryza sativa “Puntal”) desinfectadas con hipoclorito sódico. Las plantas se cultivaron en cámaras iluminadas. El test índice de germinación de arroz se realizó adaptando el método de Belimov et al. (2001) con algunas modificaciones. Se 279 Session IV SIV-CP-02 realizaron recuentos del índice de germinación cada 12 horas durante 5 días. El ensayo de alargamiento de raíces de arroz se llevó a cabo siguiendo el método adaptado de Poonguzhali et al (2008). Tras 15 días de cultivo en condiciones asépticas en el interior de un recipiente de cristal cerrado en cámara iluminada, se extrajeron las plantas y se midieron las raíces. RESULTADOS Y DISCUSIÓN. Durante la realización de ensayos que se realizaban para el estudio de las propiedades como PGPR de algunas bacterias, se observó que algunos granos de arroz tomaban un color rosa-rojizo al cabo de unas dos semanas, a pesar de que las semillas de arroz habían sido previamente desinfectadas con un tratamiento de 30 minutos de hipoclorito sódico. De esos granos se aisló una bacteria que por su coloración podría ser una metilobacteria facultativa pigmentada de color rosa (PPFM) por lo que se probó su crecimiento en AMS con metanol como fuente de carbono, comprobándose que crecía en este medio. Dado que se había aislado de plantas de arroz que presentaban aspecto saludable se estudió si presentaba algunas propiedades que beneficiaran a la planta y que pudieran clasificarla como PGPR. De este modo se comprobó que produce AIA, solubiliza fosfatos, presenta actividad ureasa, siendo, además, productora de AHL detectadas en los ensayos con los dos biosensores (CV026 y NT1 pZLR4). Sin embargo, no produce sideróforos y no presenta actividad celulasa. Por otra parte, no acelera la germinación pero sí promueve el desarrollo de un mayor número de raíces a corto plazo y un alargamiento radicular a más largo plazo. Se ha descrito que las especies de Methylobacterium producen considerables cantidades de AIA y de citoquininas que estimulan la germinación de semillas y el desarrollo de las plantas y algunas presentan ACC desaminasa (Madhaiyan et al, 2006). Esta bacteria presenta una identidad del 96.296 % con Methylobacterium aquaticum, lo que podría significar que se trate de una nueva especie. AGRADECIMIENTOS. Este trabajo ha sido financiado por los proyectos AGL2009-13487-C04 del Ministerio de Ciencia y Tecnología del Gobierno Español y de Excelencia P10-AGR5821 de La Junta de Andalucía. BIBLIOGRAFÍA. Alexander, D.B., and Zuberer, D.A. (1991). Biol. Fert. Soils 12: 39-45. Belimov, A.A., et al. (2001). Can. J. Microbiol. 47: 642-652. Cha, C., et al. (1998). Mol. Plant-Microbe Interact. 11: 1119-1129. Hiraishi, A., et al.(1995).Appl. Environm. Microbiol. 61: 2099-2107. McClean, K.H., et al., (1997). Microbiology 143: 3703–3711. Madhaiyan M., et al. (2006). Planta 224: 268–278. Patten, C.L., and Glick, B.R. (2002). Appl. Environm. Microbiol. 68: 3795-3801. Poonguzhali, S,. et al. (2008). Appl. Microbiol. Biotechnol. 78: 1033–1043. Shekhar, C. (1999). FEMS Microbiol. Lett. 170: 265-270. 280 Session IV SIV-CP-03 Resistencia a metales pesados de bacterias aisladas de leguminosas de las Marismas del Odiel y del entorno del Río Tinto. Lara-Dampier, V.1, Acera-Mateos, P.1, Alías-Villegas, C.1, Gómez-Cárdenas, A.J.1, Temprano, F.2, Camacho, M.2, Bellogín, R.A.1, Espuny, M.R.1* 1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes 6, 41012-Sevilla. España. 2 IFAPA Centro Las Torres-Tomejil. Sevilla. España. * [email protected] RESUMEN Se ha analizado la resistencia a distintos metales pesados (plata, cadmio, cobalto, cromo, cesio, cobre, níquel, arsénico, zinc y mercurio) a 50 rizobios aislados de nódulos y a otras bacterias asociadas a los mismos de leguminosas recolectadas de distintas localizaciones de las Marismas del Odiel y del entorno del Río Tinto. La mayoría de los aislamientos de la misma zona presentaron valores de tolerancia a metales semejantes, y en algunos casos alcanzaron los valores considerados como de resistencia. Algunas de los aislamientos seleccionados crecieron a las siguientes concentraciones milimolares de metales: As 7-10; Cd 0,5; Co 0,5-1; Cr 0,3-0,5; Cs 40; Cu 2-3; Hg 10-50 (micromolar); Ni 2; Zn 1-2. La mayoría de los aislamientos presentaron resistencia a varios metales. Cinco aislamientos del grupo de los no rizobios produjeron sideróforos, apareciendo, en algunos casos, pigmentos difusibles en los medios con determinados metales. INTRODUCCIÓN. Los métodos que emplean plantas para descontaminar suelos afectados por la presencia de metales pesados (fitorremediación) son menos costosos que los métodos fisicoquímicos y de menor o nulo impacto ecológico (Dary et al., 2010). La fitorremediación depende de la capacidad de la planta para solubilizar y/o movilizar metales pero presenta como inconveniente que consume mucho tiempo. Así, la mayoría de las plantas, excepto las hiperacumuladoras de metales, no son rentables para fitorremediación debido a su poco y lento desarrollo en los ambientes contaminados. Para hacer más rentable la fitorremediación en estas circunstancias habría que utilizar bacterias promotoras del crecimiento vegetal por cualquier mecanismo, directo o indirecto, que, además, fueran resistentes a metales, ya que estas bacterias juegan un papel importante modificando la disponibilidad y toxicidad de los mismos (Rajkumar et al., 2010; Rajkumar et al., 2012). MATERIAL Y MÉTODOS. Los aislamientos se realizaron en medio YMA con Rojo Cong o después de la desinfección de los nódulos con hipoclorito de sodio. Tras su purificación en el mismo medio se eliminaron redundancias seleccionando sólo uno de los clones que presentaron el mismo perfil ERIC (cebadores ERIC1 y ERIC2). Posteriormente se distinguieron dos grupos de bacterias, aquellas que amplificaron el gen nodC (nodCF2 y nodCI, Laguerre et al., 2001) y que nodularon en la leguminosa de la que se aisló o en otra similar, y aquellas bacterias que no lo hicieron, que fueron consideradas como no rizobios. A todas ellas se les estudió la resistencia a metales en las siguientes formas: HgCl2, CdCl2, CoCl2, NiCl2, AgNO3, ZnSO4·7H2O, CuSO4·5H2O, K2Cr2O7, Na2HAsO4.7H2O, CsCl. Se prepararon soluciones concentradas (1 M, 1mM o 0,1 mM según los casos) que se esterilizaron por filtración. A partir de ellas se prepararon placas de TY o YMA3 añadiendo los volúmenes correspondientes cuando el medio estaba a 50º C. Se colocaron gotas de 10 microlitros de cultivos en TY o YM3 de los distintos aislamientos en fase logarítmica tardía. El crecimiento se observó a los 5, 7 y 14 días. El estudio de la 281 Session IV SIV-CP-03 producción de sideróforos se realizó en placas de microtítulo usando 100 µl de sobrenadante de 7 días de las bacterias en medio líquido (TY y medio mínimo M9) y 100 µl de reactivo de CAS modificado; siguiendo el método de Alexander y Zuberer (1991). RESULTADOS Y DISCUSIÓN Algunas de las áreas de las Marismas del Odiel y del entorno del Río Tinto presenta contaminación por metales pesados derivada de la actividad minera ancestral que se realiza en esta zona de la franja pirítica del suroeste de la península Ibérica y por otras actividades antropogénicas, como los dragados de canales de zonas industriales. Por ello, estas áreas fueron seleccionadas para aislar rizobios de nódulos de leguminosas y otras bacterias asociadas a estos nódulos que crecieron en las placas de aislamiento. Los aislamientos se hicieron de: Ornithopus compressus y Lotus corniculatus, de una zona recuperada pero contaminada por metales (Zn, Pb, As y Hg) de las Marismas del Odiel; de Medicago marina y Medicago sp, de una zona arenosa de las Marismas del Odiel cuyos análisis no manifestaron metales pesados, y de Trifolium sp y Medicago polymorpha del entorno de Río Tinto, también contaminada con metales pesados (Cu, Ni y Hg). Los resultados preliminares mostraron que muchos de los aislamientos pueden crecer a concentraciones de metales pesados que se consideran de resistencia (1 mM en los casos de Cu2+, Ni2+, Zn2+ y 0,5 mM en los casos de Co 2+ y Cr3+, Brim et al., 1999; Nieto et al., 1987) y, en ocasiones, superiores. Cinco aislamientos del grupo de los no rizobios produjeron sideróforos. Además, estos mismos aislamientos produjeron pigmentos amarillos difusibles en las placas de algunos metales, lo que sugiere que pueda tratarse de sideróforos inducidos por dichos metales. AGRADECIMIENTOS Este trabajo ha sido financiado por el proyecto de Excelencia P10-AGR5821 de La Junta de Andalucía . BIBLIOGRAFÍA Alexander, D.B., and Zuberer, D.A .(1991). Biol. Fertil. Soils 12: 39-45. Brim, H., et al. (1999). Syst. Appl. Microbiol. 22, 258–268. Dary, M., et al. (2010). J. Hazard. Mater., 177, 1–3, 323-330 Laguerre, G., et al. (2001). Microbiology 147: 981-993. Rajkumar, M, et al. (2010). Trends Biotechnol. 28: 142. Rajkumar, M, et al. (2012). Biotechnol. Adv. 30: 1562-1574. 282 Session IV SIV-CP-04 Comparación de las características microbiológicas del suelo en huertos de aguacate bajo manejos orgánico y convencional. Bárcenas-Ortega, A.E.1*, Chávez-Bárcenas, A.T.1, García-Saucedo, P.A.1, OlaldePortugal, V.2, Tulai s-Alvarado, C.A.1, Zavala-Gómez, A.1 1 Facultad de Agrobiología “Presidente Juárez” de la Universidad Michoacana de San Nicolás de Hidalg o, México, 2 CINVESTAV Unidad Irapuato, México. * [email protected] RESUMEN Para determinar el impacto del manejo convencional sobre la microbiota del suelo, se compararon algunas características microbiológicas en huertos de aguacate ‘Hass’ bajo manejos orgánico y convencional en el municipio de Uruapan, Michoacán, México. En los huertos bajo manejo orgánico se encontró mayor cantidad de microorganismos del suelo (bacterias, hongos y actinomicetos) que en los huertos con manejo convencional; lo mismo ocurrió con el número de esporas de hongos micorrizógenos arbusculares (HMA), superándolos en un 130%. INTRODUCCIÓN El aguacate es el cultivo más importante de Michoacán por su impacto sobre la economía regional, pero es también el principal responsable de la pérdida de cobertura forestal debido al cambio de uso de suelo, pérdida de la biodiversidad, modificación micro-climática y uso indiscriminado e intensivo de agroquímicos. El componente microbiológico es muy importante en el funcionamiento de los ecosistemas y constituye un marcador biológico potencialmente útil para evaluar sus perturbaciones (Calvo et al., 2008). El objetivo de la investigación fue: comparar las características microbiológicas en árboles de aguacate ‘Hass’ bajo manejos orgánico y convencional en el municipio de Uruapan, Michoacán, México, para determinar el impacto del manejo convencional sobre la microbiota del suelo. MATERIAL Y MÉTODOS El estudio se realizó en cuatro huertos establecidos por pares, uno con manejo orgánico frente a otro con manejo convencional. Se efectuaron muestreos de suelo en el área de goteo del árbol. El número de unidades formadoras de colonias (UFC) de bacterias, hongos y actinomicetos en los suelos se obtuvo mediante la técnica de dilución seriada y vaciado en placa con medio selectivo (Alvarez et al., 2000), extracto de sueloglucosa-agar para bacterias, medio Martin (1950) para hongos y para actinomicetos el medio agar Czapeck-Dox (Merck). Para extraer las esporas de HMA del suelo se utilizó el protocolo de tamizado húmedo y decantación propuesto por Gerdemann y Nicolson (1963), seguido de centrifugación en sacarosa (Walker y Mizecw, 1982); las esporas obtenidas se montaron en preparaciones usando alcohol polivinílico glicerol y se identificaron y contaron usando microscopio compuesto. RESULTADOS Y DISCUSIÓN En los huertos bajo manejo orgánico se encontró mayor cantidad de microorganismos del suelo (bacterias, hongos y actinomicetos) que en los huertos con manejo convencional (Figura 1A), lo que se traduce en un mayor equilibrio; lo mismo ocurrió con el número de esporas de hongos micorrizógenos arbusculares, superándolos en un 130% (Figura 1 B). 283 Session IV SIV-CP-04 Media de N° esporasen 100g ss MEDIAS DEL No DE UFC • 104/g ss-1 A 80 70 60 50 40 A 30 B 20 10 0 ORG 900 B 800 700 600 500 A 400 300 200 B 100 0 ORGÁNICO CONV CONVENCIONAL MANEJO MANEJOS Figura 1. Comparación de medias de: A) UFC de microorganismos (bacterias, hongos y actinomicetos) y B) número de esporas de HMA, encontradas en huertos de aguacate con dos tipos de manejo en Uruapan, Mich. Letras distintas indican diferencias estadísticas significativas. Las diferencias entre los dos tipos de manejo coinciden con Sánchez et al. (2009) que encontró que la biomasa y actividad microbiana del suelo fueron significativamente mayores en cultivos con manejo orgánico en comparación con aquellos manejados de manera convencional. Esto puede deberse a los contenidos de materia orgánica que fueron más altos en los huertos con manejo orgánico y a la no utilización de agroquímicos como fertilizantes, herbicidas, plaguicidas y otros insumos de síntesis química. Acuña et al. (2006) mencionan que los efectos de prácticas agrícolas, así como los producidos por fertilizantes y sistemas de cultivo, pueden ser evaluados a partir de las determinaciones de la biomasa microbiana, su actividad metabólica y el conteo de las poblaciones microbianas más importantes de la microflora del suelo, por su parte Vandermer (1995) afirma que, la agricultura orgánica trata de cerrar el ciclo de nutrientes en sus granjas, proteger la calidad del medio ambiente y mejorar las interacciones biológicas beneficiosas y los procesos, además, puede reducir algunos efectos negativos atribuidos a la agricultura convencional y tiene beneficios potenciales en la mejora de la calidad del suelo. AGRADECIMIENTOS A CONACYT, México, por financiar el proyecto. BIBLIOGRAFÍA Acuña, O., et al. (2006). Memorias XII Reunión ACROBAT, Santa Catarina, Brasil. 222-233. Álvarez S., et al. (2000). Agrociencia 34: 523-532. Calvo, P., et al. (2008). Estudio de las poblaciones microbianas de la rizosfera del cultivo de pa pa (solanum tuberosum) en zonas altoandinas. Ecología Aplicada. Departamento Académico de Biología. Universidad Nacional Agraria La Molina, Lima, Perú. Pp 141-148. Gerdemann, J.W., and Nicolson, T,H. (1963). Trans. Br. Mycol. Soc. 46: 235-244. Sánchez, M., et al. (2006). Actividad y biomasa microbianas como indicadores de materia orgánica e n sistemas de cultivo de maracuyá (Passiflora edulis) en Toro, Valle del Cauca, Colombia. Revistas.unal.edu.co Vandermeer, J. (1995). Ann. Rev. Ecol. Syst. 26: 201-224. Walker, C., and Mizecw, M.(1982). Can. J. Bot. 60: 2518-2529. 284 Session IV SIV-CP-05 Efecto de la inoculación foliar y radicular de bacterias PGPR en el cultivo de Aguaymanto (Physalis peruviana). Flores, L., Ogata, K. *, Zúñiga, D. Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso, Dpto. Biología, Universidad Nacional Agraria La Molina. Lima -Peru. Web: www.lamolina.edu.pe/lmt. * [email protected] RESUMEN Se utilizaron cuatro cepas aisladas del cultivo de aguaymanto. Dos de las cepas incrementaron la germinación y el porcentaje de semillas pubescentes in Vitro. Por otro lado, la cepa Aa25, inoculada radicularmente, incremento significativamente la altura y el número de hojas de la planta, en conjunto con la inoculación foliar de la cepa Azo16M2. INTRODUCCIÓN El aguaymanto, cultivo nativo del Perú, está teniendo mayor demanda en los mercados internacionales, por sus propiedades medicinales y alta producción de vitamina C. La exportación peruana de aguaymanto está en aumento respecto a los años anteriores; del año 2007 (6.8 mil Kg) al año 2012 (57.2 mil Kg) hay un incremento de más de 800 % (SIICEX, 2013). Los microorganismos promotores de crecimiento (PGPR) son ampliamente investigados por su capacidad de inducción de resistencia a patógenos y estimulación del crecimiento en plantas (Barka et al., 2000). Es por esto, que en esta investigación se evalúa la respuesta de la planta bajo inoculación foliar y radicular con estos microorganismos. MATERIAL Y MÉTODOS Ensayos de germinación in vitro en semillas de aguaymanto, descrito por Zúñiga (2012). Se utilizaron 2 inóculos de diazotrofos Da29 y Da 30 y 2 de actinomicetos Aa25 y Aa23. Preparación del inoculo con cepas diazótrofas en medio LMC a 28°C por 48 h y cepas de actinomicetos sembradas en medio ISP1 a 28°C por 120 h (Shirling y Gottlieb, 1966). Instalación del ensayo en campo trasplantando plántulas de aguaymanto (provenientes de almácigos con una altura de 3-4 cm) al campo, considerando 1m de distancia entre plantas. Se utilizó un diseño de bloques completos al azar (DBCA). Se añadieron 100 g de compost a cada hoyo de siembra, siendo luego homogenizado con el suelo. El ensayo se realizó en el Jardín Botánico “Octavio Velarde Nuñez” en la Universidad Nacional Agraria La Molina. RESULTADOS Y DISCUSIÓN Ensayo de germinación in vitro en semillas aguaymanto. A los 7 días de instalado el ensayo se observó que tres de las cepas (Da29, Aa25 y Aa23) incrementaron el % de germinación de semillas de aguaymanto respecto al control sin inocular. También se evaluó la presencia de pubescencia en las radículas de las semillas germinadas, encontrándose que el 79% y el 82% de las plantas inoculadas con las cepas Da29 y Aa25 respectivamente presentaron diferencias significativas en comparación al control sin inocular (48%) (Figura 1). La pubescencia es importante para el crecimiento de la planta, ya que permite una mayor área de toma de nutrientes. 285 Session IV SIV-CP-05 De este modo se demostró la capacidad PGPR a nivel in vitro de las cepas seleccionadas. Efecto de la inoculación en campo. Las semillas fueron inoculadas con las cuatros cepas mencionadas anteriormente, de manera individual y sembradas en almacigo. Al cabo de un mes se realizó la medición de altura y número de hojas de las plántulas, encontrándose un incremento del crecimiento con tres de las cepas: Da29 (3.91cm), Aa25 (3.99cm) y Aa23 (4.18cm), respecto al control (3.82cm). Después de 24 h, las plántulas fueron transplantadas a campo y reinoculadas a nivel radicular con las mismas cepas. A los 15 días se realizó una inoculación foliar con la cepa diazótrofa Azo16M2, a todos los tratamientos a excepción del control, cuyo efecto positivo en el crecimiento de aguaymanto fue demostrado previamente (Ogata y Zúñiga, 2013). Un mes después se realizó la medición de la altura y número de hojas de las plántulas, pudiéndose observar que el tratamiento inoculado con la cepa Aa25 en interacción con Azo16M2 presento un incremento significativo de estos parámetros, respecto al control sin inocular (Tabla 1). Por lo tanto la cepa Aa25 resulta ser promisoria en el cultivo de aguaymanto, desde la germinación hasta el desarrollo del cultivo. Tabla 1. Ensayo a nivel de campo con las cepas DA29, Da30, Aa25 y Aa23 como inoculantes radiculares y la cepa Azo16M2 como inoculante foliar. Tratamiento Altura N° Hojas Da29 2.800 a 3.333 a Da30 4.211 b 4.222 ab Aa23 4.656 b 4.000 ab Aa25 9.556 Control d 6.711 c 7.222 c 4.889 b * Las letras en minúscula corresponden a los grupos de homogenización encontrados mediante la prueba de Rangos Multiples LSD. Figura 1. Efecto de PGPRs en el % de semillas pubescentes. Las letras en las barras corresponden a los grupos de homogenización encontrados mediante la prueba de Rangos Multiples LSD. AGRADECIMIENTOS Proyecto PROCYT-CONCYTEC 325-2011, FDA Biol. 111-UNALM, Lima, Perú. BIBLIOGRAFÍA Barka, E., et. al. (2000). FEMS Microb. Let. 186: 91–95. Ogata, K. y Zúñiga, D. (2013). Informe final. Proyecto Procyt-Concytec 325-2011. Shirling, E., and Gottlieb, D. (1966). Int. J. Syst. Bacteriol. 16: 313-340. SIICEX. (2013). Estadística de Biocomercio. Edición No4. www.siicex.gob.pe Zúñiga D. (2012) Universidad Nacional Agraria La Molina. 1ra Edición. Lima, Perú. pp. 40. 286 Session IV SIV-CP-06 Bacillus sp. y hongos micorrícicos para el control biológico del nemátodo Meloidogyne incognita en el cultivo de Aguaymanto (Physalis peruviana). Isla, F.1*, Carbonell, E.2, Ogata, K.1, Zúñiga, D.1 1 Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso. Dpto.de Biología, Universidad Nacional Agraria La Molina. Lima-Perú. Web: www.lamolina.edu.pe/lmt. 2 Laboratorio de Nematología. Dpto. de Agronomía, Universidad Nacional Agraria La Molina. Lima-Perú. * [email protected] RESUMEN Se evaluó la dosis de nemátodos para determinar el grado de infestación de las raíces en aguaymanto; luego se probó el uso de los hongos micorrícicos y una cepa PGPR de Bacillus sp. como biocontrolador en plantas infectadas. La inoculación de la cepa de Bacillus sp. y doble inoculación de Bacillus sp. y micorrizas incrementó significativamente la altura de plantas y mejoró el número de frutos respecto a la planta infectada con nemátodos. INTRODUCCIÓN El cultivo del aguaymanto (Physalis peruviana) planta nativa del Perú, va adquiriendo importancia en la exportación debido a las propiedades nutricionales y medicinales, en los últimos años se viene intensificando su cultivo ya que ha aumentado la demanda de exportación. En el Perú no existen formas adecuadas en el control de nemátodos, siendo la principal forma de control el uso de plaguicidas químicos costosos, tóxicos para la salud y el medio ambiente. Las especies de Bacillus sp. se han reportado como promotoras de crecimiento en un número amplio de plantas (Kokalis-Burelle et al., 2002); además este género es muy efectivo en el control biológico de nemátodos y se ha demostrado que reduce la eclosión de huevo hasta en un 90% (Nagesh et al., 2005). Por otro lado los hongos micorrícicos mejoran la toma de nutrientes del suelo, principalmente fosfato y protegen a la raíz (Liu et al., 2012). MATERIAL Y MÉTODOS Efecto de la poblacional inicial sobre el grado de severidad radicular. Se sembraron plántulas de aguaymanto en macetas de 2 kg en sustrato estéril 2:1 (suelo: arena) y se inoculó después de 1 semana con diferentes niveles de población de nematodos. Los tratamientos fueron: 0,1000, 3000, 5000, 10.000 y 20.000 nemátodos por kg de sustrato. Luego se determinó el grado de severidad radicular (escala del 0 al 5) según (1985). Preparación de agentes de biocontrol. Se extrajeron las esporas de hongos micorríticos nativos de aguaymanto y se inoculó 100 esporas de hongos formadores de micorrizas por plántula. Se regó con solución de Long Ashton con 15 ppm de P. Luego de una semana se inoculó con Bacillus sp. aislada previamente de suelos de cultivo de papa en zonas altoandinas del Perú (Calvo et al., 2010), esta cepa con capacidad PGPR se sembró en LMC e incubó a 35°C por 72 horas. Se tomo 1 ml de la suspensión (108 UCF/ml). Inóculo de nemátodos. Una semana después de haber transplantado las plantas al invernadero se inoculó con 5000 nemátodos por kilo de sustrato. La extracción de nemátodos fue mediante la técnica de Hussey y Barker (1973). 287 Session IV SIV-CP-06 Diseño experimental. El ensayo consistió de 5 tratamientos: (C) Control, (N) M. incognita, (B+N) Bacillus sp. + M. incognita, (M+N) Micorrizas + M. incognita, (B+M+N) Bacillus sp. + Micorriza + M.incognita y analizado con un diseño completamente al azar (DCA). Se evaluó el crecimiento de la planta y número de frutos en condiciones de invernadero durante 90 días. RESULTADOS Y DISCUSIÓN En el primer ensayo, el índice de infección radicular más alto y significativo lo presentó el tratamiento de 5000 nemátodos por kg de sustrato, con un grado de 5, así también disminuyó significativamente el peso fresco radicular de la planta (Figure 1). En el segundo ensayo, se observó diferencias significativas entre los tratamientos, encontrándose que la inoculación con la cepa de Bacillus sp., micorrizas y la doble inoculación (B+M) disminuyen el daño causado por los nemátodos entre10-14% (Tabla 1), lo cual se evidencia con el aumento significativo en la altura de planta y número de frutos en comparación al tratamiento infestado solo con nemátodos (Tabla 1). Tabla 1. Efecto de la inoculación en el desarrollo del cultivo de Aguaymanto. Figura 1. Efecto de la población inicial de M. incognita en el grado de severidad radicular (GSR) y el peso fresco de las raíces (PFR). Las letras indican la significancia del PFR, letras iguales no son significativamente diferentes (p < 0.05). Tratamientos Altura de las plantas N˚ Frutos (C) 75.93 c 5.88 c Eficiencia de los tratamientos respecto al control (%) 100 (N) 60.00 a 1.13 a 79 (B+N) 70.25 bc 2.63b 92.5 (M+N) 67.88 b 1.25 ab 89.4 (B+M+N) 70.68 bc 2.13 ab 93.1 Los promedios seguidos por letras iguales no son significativamente diferentes (p < 0.05) Con estos resultados se podría aplicar inoculantes como Bacillus sp. y micorrizas en el cultivo de aguaymanto para controlar la infección por nemátodos (M. incognita) y mejorar el crecimiento de la planta, dentro de una agricultura sostenible. AGRADECIMIENTOS PROYECTO PROCYT-CONCYTEC 325-2011, FDA Biol. 111-UNALM, Lima, Perú. BIBLIOGRAFÍA Calvo, P., et al. (2010). Bra. J. Microbiol. 41:899-906. Fassuliotis, G. (1985). The role of nematologist in the development of resistant 1: 233-240. Hussey, S., and Barker, R. (1973). Plant Dis. Repor. 57: 1025-1028. Kokalis-Burell, N., et al. (2002). Plant Soil 238: 257-266. Liu, R., et al. (2012). Mycorrhiza 22: 289-296. Nagesh, M., et al. (2005). J. Biol. Control 19: 65-69. 288 Session IV SIV-CP-07 Identificación de dos bacterias diazótrofas asociadas a una Brassicaceae (Lepidium meyeni Walp.) de suelos altoandinos del Perú. Chumpitaz, C., Ogata, K., Santos, R., Zúñiga, D. * Laboratorio de Ecologia Microbiana y Biotecnologia Marino Tabusso, Dpto. Biología, Universidad Nacional Agraria La Molina. Lima12, Perú. Web: www.lamolina.edu.pe/lmt. * [email protected] RESUMEN Se amplificó el gen nifH de la dinitrogenasa reductasa en dos cepas, LMTZ064-109 y LMTZ064-119, asociadas a la rizósfera de Lepidium meyenii Walp. Los aislados pertenecen a los géneros Stenotrophomonas y Rahnella, respectivamente, siendo ambas gram negativas, cocobacilos y oxidasa negativa. La aparición de un producto de aproximadamente 370 pb perteneciente al gen nifH confirmó la capacidad fijadora de nitrógeno molecular de ambas cepas. INTRODUCCIÓN La maca (L. meyenii Walp.) es una Brassicaceae que habita en regiones altoandinas del Perú entre los 3700-4500 msnm. Es muy apreciada por sus cualidades nutricionales y medicinales (Tovar, 2001). Al ser un cultivo muy extractivo genera un empobrecimiento de los suelos, disminuyendo los rendimientos (Zúñiga, 2009). Los diazótrofos, por otro lado, son un grupo de microorganismos capaces de fijar N 2 a formas más asimilables en diferentes ambientes. El gen nifH de la Fe-proteína de la Nitrogenasa (enzima responsable del proceso de fijación) es usado como un indicador determinante de esta capacidad (Zehr et al., 2003). El objetivo de este estudio fue identificar la capacidad fijadora de N2 de los aislados bacterianos asociados al cultivo de maca. MATERIAL Y MÉTODOS Reactivación y caracterización morfológica y bioquímica de bacterias conservados. Amplificación BOX PCR de acuerdo a Versalovic et al. (1991). Amplificación del gen nifH de acuerdo a Barua et al. (2011). Análisis filogenético por medio del gen ARN ribosomal 16S según Weisburg, et al. (1991), usando la base de datos del Genbank (NCBI). RESULTADOS Y DISCUSIÓN Un total de siete bacterias (LMTZ064-21, LMTZ064-30, LMTZ064-94, LMTZ064-95, LMTZ064-109, LMTZ064-119 y LMTZ064-120) fueron seleccionadas de la colección de bacterias promotoras de crecimiento vegetal (186 cepas) aisladas de suelos de cultivo L. meyenni (Garcia, 2011). Estas cepas fueron caracterizadas morfológicamente de acuerdo a su crecimiento en medio mineral sin nitrógeno (MMN) por 48 horas a 2 8°C. Las colonias tuvieron formas convexas, incoloras, de borde liso, mucosas y diámetro entre 0.5-2 mm. Todas resultaron ser gram negativas, cocobacilos y oxidasa negativa. De acuerdo al análisis BOX PCR se obtuvieron cinco perfiles distintos (Figura 1A), a los cuales se les evaluó su capacidad fijadora de nitrógeno por medio de la amplificación del gen nifH de la dinitrogenasa reductasa (Fe-proteína). Solo dos aislados (LMTZ064-109 y LMTZ064-119) resultaron ser nifH positivos (Figura 1B). 289 Session IV SIV-CP-07 A B A B C C D E E A B C D E nifH 370pb Figura 1. A) Perfiles BOX A1R, A (21), B (30), C (94, 95), D (109) y E (119, 120). M: marcador 1kb (fermentas). B) PCR nifH de perfiles BOX diferentes. Cepas nifH+: LMTZ064-109 y LMTZ064-119. CIAT899 Rizhobium tropici (control+). M: marcador 100 pb (Axygen). Figura 2. Árbol filogenético en base al gen del ARNr 16S mostrando la localización de los aislados LMTZ064-109 y LMTZ064-119. Se realizó un análisis Neighbor-joining con un valor bootstrap calculado para 1,000 réplicas. Escala: 2% de secuencias divergentes De acuerdo al análisis filogenético realizado (Figura 2), se encontró que la cepa LMTZ064-109 está relacionada a S. maltophila ATCC 13637T con 98%. Stenotrophomonas spp. es un habitante común de suelos y plantas (Ryan et al., 2009), especies de este género pueden actuar como promotores de crecimiento vegetal mediante diferentes mecanismos como la fijación de nitrógeno y producción de fitohormonas (Liba et al., 2006). Por otro lado, la cepa LMTZ064-119 tuvo un 100% de similitud con Rahnella aquatilis HX2. Esta especie se reportó por primera vez como una enterobacteria fijadora de nitrógeno presente en rizósferas de cultivos de maíz y trigo (Berge et al., 1991) y su capacidad como PGPR en rizósfera de cultivos de soja y papa (Kim et al., 1997; Kohashikawa, 2010) al producir fitohormonas y solubilizar sales fosfato. En este trabajo se pudo determinar que las dos bacterias aisladas del cultivo de maca fueron capaces de fijar nitrógeno molecular. Este parámetro es importante a tener en cuenta para la producción de inoculantes bacterianos, ya que además de reducir el uso de fertilizantes químicos, promueve el crecimiento de cultivos de importancia económica dentro de una agricultura sustentable; mejorando a su vez la calidad del suelo. AGRADECIMIENTOS Perú biodiverso GTZ-CONCYTEC, 2009. Procyt 309-2009-CONCYTEC. FDA Biol.111-UNALM. BIBLIOGRAFÍA Barua, S., et al. (2012). Microbiol. Res. 167: 95-102. Berge, O., et al. (1991). Can. J. Microbiol. 37: 195-203. Garcia, M.M. (2011). Tesis Biología. Efecto de bacterias diazotróficas en el cultivo orgánico de m aca (Lepidium meyenii Walpers) de San Pedro de Cajas-Junín. Universidad Nacional Agraria La Molina. Li ma, Perú. Kohashikawa, N. (2010). Tesis Biología Comportamiento de diferentes bacterias PGPR sobre el crecim iento del cultivo de papa (Solanum tuberosum), Universidad Nacional Agraria La Molina. Lima, Perú Kim, K.Y., et al. (1997). FEMS Microbiol. Lett. 153: 273-277. Liba, C.M., et al. (2006). J. Appl. Microbiol. 101: 1076–1086. Ryan, P., et al. (2009). Nature Rev. Microbiol. 7: 514-25 Tovar, O. (2001). Publicación CONCYTEC. Lima 144 pp. Versalovic, J., et al. (1991). Nucleic Acids Res. 19, 6823-6831. Weisburg, W.G., et al. (1991). J. Bacteriol.173: 697-703 Zehr, J.P., et al. (2003). Environ. Microbiol. 5: 539-554. Zúñiga, D. (2009). Perú biodiverso GTZ-Concytec. Lima, Perú. 290 Session IV SIV-CP-08 Selección de cultivos rizobianos aislados de nódulos de leguminosas de diferentes regiones del Perú con capacidad de promover el crecimiento de lechuga, páprika y tomate. Soriano-Bernilla, B.1*, Prado-Chávarry, G.2, Zavaleta-Verde, D.2, Valdez-Nuñez, R.3 1 Dpto.de Microbiología y Parasitología-Facultad de Ciencias Biológicas, Universidad Nacional de Trujillo, Perú. 2 Universidad César Vallejo, Piura-Perú. 3 Gloria S.A Tarapoto, San Martín-Perú. * [email protected] RESUMEN La selección y evaluación de los cultivos rizobianos aislados de nódulos de leguminosas y posibles PGPR se realizó en laboratorio, determinando la altura de las plantas, longitud de las hojas, peso seco de la parte aérea, raíz y peso seco total de las plántulas de lechuga y páprika, así como la germinación de las semillas de tomate. Se encontró que los cultivos rizobianos Rc-455-02, Rf 188-03, Ra112-01 y Ra 014-02 promueven el crecimiento de no leguminosas considerándose una alternativa biológica frente a fertilizantes químicos que contaminan nuestro ecosistema. INTRODUCCIÓN En años recientes, se ha tomado interés de utilizar rizobacterias promotoras de crecimiento vegetal (PGPR) en la producción de cultivos agrícolas. Esto aplicado a semillas, tubérculos o raíz, y son capaces de colonizar las raíces de las plantas y estimular el crecimiento y rendimiento de cultivo. Dentro de la flora peruana se encuentran especies útiles para el hombre como la papa, tomate, pimientos, tabaco y berenjena, entre otras. Una de las hortalizas también de importancia en el Perú por su producción es la lechuga, se consume cruda, debido a su aporte de calcio, vitamina A y minerales (Ogata et al., 2008). Investigadores evaluaron el efecto de 30 cepas bacterianas en la germinación y el crecimiento de lechuga, obteniendo un incremento en la germinación del 36.5% con respecto al control sin inocular. Asimismo, nuestro país está orientado a la exportación de páprika y actualmente se ha convertido en uno de los líderes del mercado mundial. El páprika presenta un valor nutricional alto en contenido de vitamina C, A, sales minerales, azúcares y oleorresinas (capsaicinas, que otorga el carotenoide colorante) (Santillana et al., 2005; Quintero et al.., 2000). MATERIAL Y MÉTODOS Colección y transporte de los nódulos a partir de leguminosas (frijol común, caupí, arveja y frijo palo) de regiones peruanas (Lambayeque, Cajamarca, La Libertad y Ucayali). Aislamiento de rizobios a partir de los nódulos según metodología de Somasegaran y Hobe (1994); caracterización bioquímica y autenticación de cultivos rizobianos. Preparación y estandarización de los cultivos rizobianos autenticados. Determinación de la capacidad promotora de crecimiento vegetal por cultivos rizobianos mediante pruebas bioquímicas. Tratamiento de las semillas de lechuga, páprika y tomate. Evaluación de la siembra e inoculación de las semillas de lechuga y páprika en muestras de suelo agrícola preparada en bolsas de polietileno. Se determinó la longitud del tallo y raíz, peso seco de la parte aérea y radicular de las plántulas después de 20 días de inoculadas. Se realizó la inoculación rizobiana de las semillas de tomate colocadas en una placa Petri y al quinto día se hizo la medida de radícula e hipocotilo. 291 Session IV SIV-CP-08 RESULTADOS Y DISCUSIÓN De 503 cultivos aislados, 234 se consideraron positivos a las pruebas de purificación y autenticación determinándose así la presencia de cultivos rizobianos. Figura 1. Porcentaje de cultivos rizobianos aislados Figura 2 Autentificación de los cultivos rizobianos en leguminosas Se seleccionaron, mediante pruebas bioquímicas, a 24 cultivos rizobianos aislados de frijol por tener actividad promotora del crecimiento vegetal, y se determinó en base a sus mecanismos de promoción directa o indirecta, en la cual indica que si reaccionan positivamente a una de las pruebas consideradas como promotoras de crecimiento vegetal (producción de indol, AIA, sideróforos y solubilización de fosfatos) sean consideradas como tal y dentro de este grupo se seleccionó a Rc-455-02 y Rf 188-03 para la evaluación en plántulas de lechuga y páprika, después de 20 días de inoculadas. Figura 3. Plántulas de lechuga. Figura 4. Plántulas de páprika. Figura 5. Plántulas de tomate. De los rizobios aislados de nódulos de arveja se seleccionaron a 8 cultivos para la evaluación de la germinación de semillas de tomate y dentro de éstos a Ra 112-01 y Ra 14-02 por ser positivos al menos de una de las pruebas realizadas y presentar diferencia significativa entre la longitud de radícula e hipocotilo con respecto al control en semillas de tomate. BIBLIOGRAFÍA Ogata K., et al. (2008). Zonas Áridas; 12: 137-153. Quintero, I., et al. (2000). Rev. Fac. Agron. (LUZ). 17: 482-491. Santillana, N., et al. (2005). Ecol. Apl. 4: 47-51. Somasegaran, P., and Hoben, H. (1994). Handbook for rhizobia: methods en legume-Rhizobium technology. Springer-Verlag. New York. 292 Session IV SIV-CP-09 Promoção de crescimento de tomate (Solanum lycopersicum L.) estimulado por Bacillus amyloliquefaciens FZB42. Szilagyi-Zecchin, V.J. *, Ruaro, L., Mógor, A.F. Departamento de Fitotecnia e Fitossanitarismo, Universidade Federal do Paraná, Brasil. * [email protected] RESUMO A bactéria B. amyloliquefaciens foi inoculada em sementes de tomate, em diferentes doses, e não alterou o potencial germinativo. Na produção de mudas, a menor dose, proporcionou significativos incrementos em todos os aspectos da parte aérea da planta. INTRODUÇÃO O tomateiro é cultivado no mundo inteiro e possui grande relevância econômica e social (Faostat, 2011). Os microrganismos vêm despertando atenção devido à necessidade de reduzir o uso de produtos químicos, visando um sistema de agricultura sustentável, centrada na proteção ambiental (Vale et al., 2010). Uma das estratégias é explorar os benefícios da ação destes microrganismos na forma de inoculantes (Lucy et al., 2004). Objetivou-se neste estudo verificar a capacidade do B. amyloliquefaciens em promover o crescimento de mudas de tomate. MATERIAL E MÉTODOS A solução de B. amyloliquefaciens FZB42- (Omex® Agrifluids do Brasil Ltda) a 1x1011 UFC/mL foi inoculada nas sementes na proporção de 320 uL/g de semente. Os tratamentos corresponderam a porcentagens em volume aplicado. A cultivar de tomate utilizada foi Santa Clara I-5300 (Isla®). O teste de germinação foi realizado segundo a RAS (2009). Aos dez dias, foram avaliados volume e comprimento das raízes e hipocótilo, no programa Win-Rhizo, acoplado a um scanner LA1600. A produção de mudas foi conduzida de acordo com Koyama et al., (2013). Aos 30 dias após plantio as mesmas variáveis acima foram analisadas, além da determinação de área foliar, massa seca da parte aérea e raiz e altura da planta. RESULTADOS E DISCUSSÃO No teste de germinação, verificou-se que nenhum tratamento alterou negativamente o percentual de germinação. No entanto, também não contribuíram para incrementos na parte aérea ou raiz das plântulas. O tratamento de bactéria a 80% chegou a inibir o volume de raiz e hipocótilo, indicando um provável excesso de dose. Na produção de mudas, a menor dose de bactéria, mostrou-se superior para todos os itens avaliados (exceto clorofila) da parte aérea (Tabela 1). As demais doses não diferiram da testemunha. Uma parte aérea maior indica uma melhor taxa fotossintética, que implica em mais fotoassimilados, que poderão ser translocados para os frutos nos estádios seguintes (Taiz and Zeiger, 2004). Este aumento da parte aérea pode ter uma relação características benéficas já constatadas para esta cepa, tais como, presença de compostos auxínicos (Idris et al., 2004), produção de sideróforos (Chen et al., 2007) e solubilização de fosfato inorgânico (Cuartas et al., 2010). No sistema radicular, as maiores doses tiveram influência negativamente. A menor dose se igualou a testemunha. Neste estádio de desenvolvimento observou-se também um indicativo de excesso de dose nos tratamentos 3 e 4. 293 Session IV SIV-CP-09 Tabela 1. Mudas de tomate inoculadas com B. amyloliquefaciens em diferentes doses, avaliadas aos 3 0 dias após plantio. (DMS) Diferença mínima significativa, (MG) Média Geral, (CV) Coeficiente de Variação. Curitiba, Paraná, UFPR, 2013. Tratamentos 1 - água 2 - bact20 3 - bact40 4 - bact80 MG CV (%) Volume (cm3) 5.33 a 5.11 a 4.85 a 4.61 a 4.98 13.66 MUDAS DE TOMATE 30 DIAS APÓS PLANTIO Raiz Parte aérea Comprimento Massa seca Área Comprimento Volume (cm) (g) * (cm2)* (cm) * (cm3) 2232.39 a 0.1789 a 180.69 b 162.77 c 16.02 b 2156.89 a 0.1734 a 255.16 a 261.32 a 20.02 a 2111.69 a 0.1489 b 202.71 b 201.71 b 16.37 b 2031.23 a 0.1223 c 208.82 b 224.78 b 15.52 b 2133.05 0.15 211.85 212.65 16.99 12.33 9.95 12.80 12.66 18.15 Massa seca (g) * 0.8893 b 1.1586 a 0.9504 b 0.8111 b 0.95 9.79 Clorofila total (mg/g) 6.67 a 8.11 a 6.83 a 8.52 a 7.53 27.53 Teste de Duncan a 5% de probabilidade. * Teste de Duncan a 1% de probabilidade. A inoculação de tomate com B. amyloliquefaciens promoveu incrementos na parte aérea da planta, mostrando-se benéfica a utilização da menor dose na produção de mudas. AGRADECIMENTOS Agradecemos à Omex® Agrifluids do Brasil Ltda, por ceder a cepa da bactéria e à CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) pelo apoio financeiro. REFERÊNCIAS Chen XH. et al. (2007). Nature Biotech. 25: 1007-1014. Cuartas, CAR. (2010). Faculty of Auburn University. 144p. FAOSTAT. (2011). Disponível em: Acessado em 12/07/2012. Idris, EES. et al. (2004). J. Plant Dis. Protect.111: 583-597. Koyama, R., et al. (2013). Amazon. J. Agric. Environ. Sci. 55: 282-287. Lucy, M. et al. (2004). Antonie Leewenhoek 86: 1-25. RAS, Regras para análise de sementes. (2009). 365p. Taiz, L., and Zieger, E. (2004). Fisiología Vegetal. 719 pp. Vale, M. et al. (2010). Plant growth and health promoting bacteria. pp. 21-43. 294 Session IV SIV-CP-10 Efecto de volátiles emitidos por la bacteria bCT34 en la promoción de crecimiento de Arabidopsis thaliana. Parada, M.1*, Quiroz, A.2, Parra, L.2 Mendoza, D.1, Fincheira, P.1, 2 1 Centro Biotecnológicos de Estudios Microbianos (CEBEM), Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera. Chile. 2 Laboratorio de Química Ecológica, Facultad de Ingeniería, Ciencias y Administración. Universidad de La Frontera. Chile. * [email protected] RESUMEN Muchos de los microorganismos presentes en el suelo tienen la capacidad de emitir Compuestos Orgánicos Volátiles (COVs) los que permiten aumentar el crecimiento vegetal, sin embargo, su modo de acción es poco conocido. En este trabajo se aplicaron indirectamente 8µl de inoculante de la cepa bacteriana bCT34 a plántulas de Arabidopsis thaliana en placas Petri divididas y ordenadas en una distribución completamente al azar. La captura de COVs fue realizada mediante el uso de SPME y la identificación por medio de cromatografía gaseosa acoplada a espectrometría de masas. Los resultados indican que plantas de Arabidopsis thaliana inoculadas con bCT34 aumentan su peso fresco total, el número de hojas, la longitud radical y el número de raíces secundarias (p<0,05). Además, se identificaron COVs tales como oximas, terpenos, hidrocarburos aromáticos y aldehídos. Se concluye que la bacteria bCT34 solo a través de la emisión de COVs puede modificar la arquitectura radical en A. thaliana y aumentar el número de hojas. INTRODUCCIÓN Algunos microorganismos de suelo tienen la capacidad de promover el crecimiento vegetal a través de mecanismos directos (fitohormona) e indirectos (antibióticos) (Bhattacharyya and Jha, 2012). Últimamente ha surgido un mecanismo asociado a COVs emitidos por bacterias y definidos como compuestos de bajo peso molecular (<300g/mol-1) que tienen una alta presión de vapor (Ortiz et al., 2009). Ryu et al. (2003) demostraron por primera vez que volátiles bacterianos emitidos por Bacillus subtilis GB03 y Bacillus amyloliquefaciens IN937a son capaces de promover el crecimiento en Arabidopsis thaliana a través de la emisión de acetoína y 2,3-butanodiol. Este reporte se enfoca en determinar los efectos morfológicos en las plántulas tras la inoculación indirecta de la cepa bCT34 y determinar el perfil preliminar de volátiles involucrados en la relación A. thaliana- bCT34 y sus efectos morfológicos en las plántulas tras su inoculación indirecta. MATERIAL Y MÉTODOS La planta modelo utilizada fue Arabidopsis thaliana ecotipo Col0 cultivada en medio básico Murashinge y Skoog (MS 0.5 X) suplementado con 1% sacarosa y 0,8% de agar (pH 5.8). La bacteria bCT34 del cepario del laboratorio del Centro Biotecnológico de Estudios Microbianos (CEBEM) y propagada en el medio universal Plate Count. Las semillas a utilizar se desinfectaron con hipoclorito de sodio por 8 minutos y lavadas con agua destilada estéril. Plántulas con una semana de desarrollo fueron inoculadas indirectamente en medio MS con 8µl de la bacteria bCT34 disuelta en Caldo Nutritivo (Densidad Óptica 0,1 a 600 nm) utilizando placas Petri divididas y con un fotoperiodo 16:8 y a 22˚C. Los efectos morfológicos fueron evaluados a las 2 semanas de inoculadas. La captura e identificación de volátiles fue realizada al mismo tiempo según la metodología descrita por Schalchli et al. (2011). El análisis estadístico de comparación de medias fue realizado en SPSS versión 11.5. 295 Session IV SIV-CP-10 RESULTADOS Y DISCUSIÓN Se observó un aumento significativo del peso fresco total y del número de hojas, así como también se observó diferencias en la arquitectura radical. (A) a (B) a b b (C) (D) P P+I Figura 1. Efecto de la inoculación en el número de raíces laterales, longitud radical y número de hojas de A.thaliana comparadas con plantas no inoculadas. (A) N˚ de raíces secundarias (B) longitud radi cal (C) N˚ de hojas (D) Arquitectura radical. Donde P= A. thaliana y P+I= A. thaliana inoculada con bCT34. Letras diferentes indican diferencias significativas. (Prueba T- Student, ≥ 0.5, n = 4). Preliminarmente se identificaron algunos volátiles emitidos desde la interacción A. thaliana-bCT34. Los compuestos identificados por medio de una comparación de espectros de masas de la biblioteca NIST: fueron acetato de etilo, estireno, metoxi fenil oxima, - y -pineno, limoneno e hidrocarburos aromáticos, entre otros. Probablemente el efecto de los volátiles activó mecanismos que podrían estar involucrados en producir fitohormonas del crecimiento, observado por ejemplo, en el incremento de raíces secundarias, el cual pudiera ser causado por un aumento de la auxina y con ello la regulación de la división y expansión celular. El análisis preliminar de los perfiles de COVs no mostró la presencia de acetoína o 2,3-butanodiol, además los COVs identificados no han sido bioensayados para probar su efecto sobre el crecimiento de A. thaliana. En una segunda etapa se probarán nuevas metodologías de atrapamiento de volátiles y se inducirá la respuesta utilizando compuestos puros y sus respectivas mezclas. El crecimiento de las plantas estimuladas por los COVs de bCT34 podría ser producto de la activación de citocininas implicadas a través de su acción en la división celular y regulación de componentes específicos del ciclo celular (Ryu et al, 2003). Sin embargo, se necesitan más pruebas para establecer el mecanismo de señalización que se ve activado en las plántulas mediante la emisión de volátiles de bCT34. Por otra parte, el aumento del número de hojas, puede estar provocado por una mayor disponibilidad de nutrientes presentes en el medio de cultivo, modificación la arquitectura radical. AGRADECIMIENTOS Proyecto Fondo de Investigación para Bosque Nativo, CONAF 059/2011. Proyecto FONDECYT 1100812. BIBLIOGRAFÍA. Bhattacharyya, P.N., and Jha, D.K. (2012). World J. Microbiol. Biotechnol. 28: 1327-1350. Ortiz, R., et al., (2009). Plant Signal Behav. 4: 701-712. Ryu, C., et al. (2003). Plant Physiol.100: 927-4932. Schalchli, H., et al. (2011). Chemistry and Ecology. 27: 503-513. 296 Session IV SIV-CP-11 Functionality of phosphate solubilizing bacteria isolated from the rhizosphere of papaya plants under conventional and organic farming systems. Melo, J.1*, Azevedo-Junior, R.R. 1, Eutropio, F.J.1, Correira, P.2, Carvalho, L. 2, Meleiro, A.I.2, Teixeira, M.S.1, Carolino, M.2, Ramos, A.C.1, Cruz, C.2 1 Enviromental Microbiology and Biotecnology Lab, University of Vila Velha (UVV). Street Comissário José Dantas de Melo 21, 29102-770, Vila Velha, Espirito Santo State, Brazil. 2 Centre for Environm ental Biology, Faculty of Science, University of Lisbon, Ed. C2,1749-016 Lisbon, Portugal. * [email protected] ABSTRACT In order to determine the effects of agricultural practices on soil microbial communities this study compare phosphate solubilizing bacteria in two different soil managements: a conventional with manure, synthetic fertilizers and pesticides; and as organic system a soil amended with a composting product. 12 bacterial strains were isolated from rhizospheric soil samples of Carica papaya L. from Espírito Santo State – Brasil. All the strains were positive for inorganic and organic phosphorous solubilization, with isolates from organic system having higher P solubilizing capacity. We found microbial functional differences between the rhizosphere of C papaya subjected to distinct managements, and a higher number of P solubilizing bacteria in organic soil. INTRODUCTION Conventional farming systems have been associated with loss of soil fertility, soil erosion, and ground water pollution (Drinkwater et al., 1995). In addition, some conventional agricultural practices inhibit the activity and function of soil microbes. Unlikely, the organic farmers do not use synthetic fertilizers or pesticides and attempt to have a close nutrient cycle on their farms, protect environmental quality, and enhance beneficial biological interactions and processes (Vandermer, 1995). Soils contain enormous numbers of diverse living organisms assembled in complex and varied communities. These organisms play an essential role in the sustainable function of all ecosystems, including nutrient recycling and acquisition, plant health by suppression of undesirable organisms. Currently, phosphate solubilizing microorganisms have attracted the attention of agriculturists as soil inoculums to improve the plant growth and yield. The goal of this work was to evaluate the influence of organic farming in comparison with conventional agriculture on soil microorganism with particular incidence in phosphate solubilizing bacteria. MATERIAL AND METHODS Strains Isolation. Bacterial strains were isolated from the rizosphere of Carica papaya L. according to Nautiyal, (1999). Viable Phosphate Solubilizing Bacteria. Plate counts of solubilizing bacteria were determined in solid medium (NBRIP) of serial dilutions of each soil samples. Phosphate Solubilization Index. Bacterial strains were inoculated in NBRIP medium using different inorganic and organic phosphate sources (e.g. Phytate, Tricalcicum phosphate). Results were expressed as a solubilizing index (SI) representing the diameter of halo solubilization per colony diameter. According to Silva Filho & Vidor (2000) P solubilization can be classified as low (IS < 2); medium (2< IS < 3) and high (IS > 3). Phosphate solubilizing quantification. Bacterial strains were grown in liquids media (NBRIP) using different inorganic and organic phosphate sources (e.g. Phytate, 297 Session IV SIV-CP-11 Tricalcium phosphate). Solubilized phosphate was quantified according to a modified assay of D’Angelo et al.(2001). Pairwise interactions. The isolated strains were tested for their pairwise interactions in Nutrient Broth and their growth rate and P solubilization quantification were determined. RESULTS AND DISCUSSION The colony formation unit did not differ in both systems (data not shown), but abundance of P solubilizing bacteria was significantly higher (P<0.05) in organic soil than on conventional agriculture (Figure 1A). All strains are classified as low solubilizers of tricalcium phosphate as the index was lower than 2 units, nevertheless strains from organic soils had a higher SI (Figure 1B). All strains are classified as medium solubilizers of organic phosphate with a SI higher than 2 and the strains isolated from organic agriculture have the highest solubilization index (Figure 1B). Pi quantification tend to be higher in organic soil compared with conventional practice (Figure 1C). Pairwise interactions between the isolated strains have shown that combined strains from different origin (organic and conventional) can positively interact and enhanced their growth (data not shown). Although the number of total viable bacteria is about the same in both soil systems the number and activity of solubilizing bacteria differ in organic and conventional farming, which led us to conclude that different agriculture practices influence microbial functionality. We suggest that organic management or introducing microbial consortia with bacteria isolated from organically managed fields into conventional fields are potential strategies to improve phosphorus use efficiency. A Solubilization Index 50000 40000 30000 20000 Organic Pi Quantification 300,00 Tricalcium 250,00 2,0 1,5 200,00 150,00 1,0 100,00 0,5 50,00 0,00 0,0 Conventional 350,00 Phytate 2,5 10000 0 C B mg Pi /L 60000 CFU g -1 3,0 Solubilizing Phosphate Bacteria Solubiliziation Index 70000 Conventional Organic Conventional Organic Figure 1. Phosphate solubilization bacteria in conventional versus organic agriculture. A. Total o f viable phosphate solubilizing bacteria in NBRIP medium. B. Solubilization index of bacterial strains. C. Quantification of solubilized phosphate using Ca3PO4 as P source. ACKNOWLEGMENTS Acknowledgments: This work is supported by CNPq/475436/2010-5 and FAPES/54687985-2011 grants. FAPES also conceded fellowships to FJE and JM. REFERENCES D’Angelo, E., et al. (2001). J. Environ. Qual. 30:2206-2209. Drinkwater, L.E., et al. (1995). Ecol. Appl. 5: 1098-1112. Nautiyal, C.S. (1999). FEMS Microbiol. Lett. 170: 265-270 Silva Filho, G.N., et al. (2000). Rev. Bra. Ciên. Solo 24:311-319. Vandermer,, J. (1995). Annu. Rev. Ecol. Syst. 26: 201-224. 298 Session IV SIV-CP-12 Plant-growth promoting Sphingomonas sp. associated with annual ryegrass. Dourado, A.C.1, Castanheira, N.2, 3, Cortés Pallero, A.3, Delgado Rodriguéz, A.I.3, Alves, P.I.L.1 ,2, Barreto Crespo, M.T.1, 2, Fareleira, P.3* 1 Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal. 2 Instituto de Tecnologia Quím ica e Biológica/UNL, Oeiras, Portugal. 3 Instituto Nacional de Investigação Agrária e Veterinária, Oeira s, Portugal. * [email protected] ABSTRACT Bacterial strains previously isolated from annual ryegrass were found to be closely related to Sphingomonas paucimobilis and to contain a nifH gene sequence similar to Rhizobium spp. Although all isolates were able to grow in N-free medium, no acetylene reduction activity could be detected. Plant inoculation assays showed increases in the biomass of plants grown in either N-free or N-supplemented medium. INTRODUCTION The genus Sphingomonas includes about 80 species of strictly aerobic, chemoheterotrophic Proteobac teria that contain glycosphingolipids as cell envelope components (Takeuchi et al., 2001). Sphingomonas spp. are widely distributed in natural environments, such as soils, aquatic systems and plants, but the only species described to date as capable of nitrogen fixation is S. azotifigens, originally isolated from the rhizosphere and roots of rice (Xie and Yokota, 2006). Some species, including S. paucimobilis, are opportunistic pathogens and have been isolated from human clinical specimens and hospital environments (Ryan and Adley, 2010). Annual ryegrass (Lolium multiflorum Lam.) is a forage crop that is extensively used in poorproduct ive areas in southern Portugal dedicated to the exploitation of cork and holm oaks (“montado” or “dehesa”). While searching for diazotrophic bacteria associated with this crop, we have recently obtained a collection of isolates originated from both the root external environment and surface-sterilized plant tissues. The collection included a group of Sphingomonas sp. that was able to grow well in nitrogen-free culture medium (work to be published). Here we describe the characterization of these isolates and the evaluation of their ability to promote plant growth. MATERIAL AND METHODS Twenty-one bacterial isolates from annual ryegrass were used. Near full length 16S rDNA (1.5-kb) was amplified from genomic DNA and sequenced. A fragment of the recA gene (468-bp) was obtained according to Chen et al. (2012) and sequenced. A fragment of the nifH gene (725-bp) was amplified as described by Xie and Yokota (2006); an inner segment (432-bp) was re-amplified (Rösch et al. 2002) and used for sequencing. The most similar sequences in GenBank were selected and phylogenetic trees were constructed by the neighbor-joining method (Saitou and Nei, 1987) using the Kimura’s two-parameter substitution model (Kimura, 1980) in MEGA version 5.0. The acetylene reduction activity was assessed by gas chromatography in semi-solid NFb cultures (Mascarua-Esparza et al., 1988). Auxin production was evaluated according to Ashgar et al. (2002) in 16h-grown cultures in TY medium supplement with 100 µg/ml tryptophan. Plant inoculation assays were performed in Evans medium; 3-5 days old seedlings were inoculated with 108 cells, incubated for 4 weeks in a controlledenvironment growth chamber, and afterwards removed and dried until constant weight. 299 Session IV SIV-CP-12 RESULTS AND DISCUSSION A group of 21 bacterial strains previously isolated from annual ryegrass using an N-free semi-solid medium (NFb) was addressed in this work. Sequence analyses of the 16S rDNA and the housekeeping gene recA revealed that the isolates were phylogenetically related to S. paucimobilis (respectively 99.8% and 100% similarity with the type strain DSM 1098), which is not known as a diazotroph, and more distant from the nitrogenfixer S. azotifig ens (Figure 1A). Despite their ability to grow in N-free medium, no acetylene reduction activity could be detected in any of the isolates. The search for a nifH gene fragment revealed a distinct sequence from S. azotifigens, and more related to Rhizobium sp. and Sinorhizobium (now Ensifer) sp. sequences (Figure 1B). Interestingly, the same sequence was found in the type strain S. paucimobilis DSM 1098, indicating the presence of a nitrogen-fixing gene in this species. Figure 1. Neighbor-joining phylogenetic trees based on (A) 16S rDNA and (B) nifH gene sequences. The numbers at the nodes indicate bootstrap values based on 1,000 replicates (>70%). Sequences in boldface were obtained in this work. GenBank accession numbers are enclosed in parentheses. Plant inoculation assays revealed that most Sphingomonas isolates could enhance the growth of annual ryegrass plants in N-free Evans medium, with average increases of 50% and 25% respectively in the roots and shoots biomass, relative to non-inoculated controls. These results suggested that, regardless the negative results in the ARA assay, nitrogen fixation could be an important process in the association of these bacteria with the host plant. However, subsequent inoculation assays with isolate G2Ac10 indicated that increases in the roots biomass could also occur when nitrogen was present in the culture medium, suggesting the involvement of mechanisms other than nitrogen fixation. IAA production among isolates varied between 6.6 and 24.4 µg/ml culture. Five isolates produced above 20 µg IAA/ml culture, but there was no obvious relationship with major increases in plants biomass. Preliminary tests with the nonpathogenic S. a zotifigens also resulted in enhanced roots biomass, revealing a good potential as an alternative inoculant for annual ryegrass. ACKNOWLEGMENTS Work financed with National Funds by Fundação para a Ciência e a Tecnologia (FCT), Portugal, throu gh Project PTDC/AGR-AAM/100577/2008 and grant # PEst-OE/EQB/LA0004/2011. A. Cortés Pallero and A.I. Delgado Rodríguez were granted by Fundecyt, España. N. Castanheira is granted by FCT, Portuga l. A.C. Dourado and N. Castanheira contributed equally to this work. REFERENCES Asghar, H.N., et al. (2002). Biol. Fertil. Soils 35: 231-237. Chen, H. (2012). Dissertation at the Faculty of Biology Ludwig-Maximilians-University Munich. Kimura, M. (1980). J. Mol. Evol. 16: 111-120. Mascarua-Esparza, M.A., et al. (1988). Plant Soil 106:91-95. Rösch, C., et al. (2002). Appl .Environ. Microbiol. 68: 3818-3829. Ryan, M.P., and Adley, C.C. (2010). J. Hosp. Infect. 75: 153-157. Saitou, N., and Nei, M. (1987). Mol. Biol. Evol. 4: 406-425. Takeuchi, M., et al. (2001). Int. J. Syst. Evol. Microbiol. 51: 1405-1417. Xie, C.-H., and Yokota, A. (2006). Int. J. Syst. Evol. Microbiol. 56: 889-893. 300 Session IV SIV-CP-13 Paenibacillus species isolated from Pisum sativum nodules in Lanzarote present several in vitro plant growth promotion mechanisms. Ramírez-Bahena, M.H.1, 2, Flores-Félix, J.D.3, Tejedor, C.3, León-Barrios, M.4, Peix, A.1, 2*, Velázquez, E.2, 3 1 Instituto de Recursos Naturales y Agrobiología, IRNASA-CSIC, Salamanca, Spain. 2 Unidad Asociada Grupo de Interacción Planta-Microorganismo Universidad de Salamanca-IRNASA-CSIC. 3 Departamento de Microbiología y Genética, Universidad de Salamanca, Spain. 4 Departamento de Microbiología y Biología Celular. Universidad de la Laguna. Tenerife. Spain. * [email protected] ABSTRACT In the present work we explored the diversity of Gram positive endophytes isolated from nodules of Pisum sativum growing in three Lanzarote soils through 16S rRNA gene sequencing. The results showed the presence of six species from genus Paenibacillus and two putative new species of this genus close to Paenibacillus xinjiangensis, Paenibacillus peoriae, Paenibacillus borealis and Paenibacillus taohuashanense. All strains isolated were able to produce siderophores an indole acetic acid that are included in the in vitro mechanisms involved in plant growth promotion abilities. The data obtained suggested that these endophytes can play a role in the formation and functioning of Pisum sativum nodules and then we are carrying out microcosms experiments in order to analyze their effect in plant when coinoculated with Rhizobium strains isolated from the same nodules in Lanzarote soils. INTRODUCTION Lanzarote is the most northern and oriental island of Canary archipelago included in the Biosphere Reserves since 1993. The particular characteristics of the soils present in this volcanic islands make them interesting ecosystems to study the diversity of legume nodules. In this way we showed that Phaseolus vulgaris is nodulated in Lanzarote soils by Ensifer meliloti strains that are not able to nodulate Medicago sativa (Zurdo-Piñeiro et al., 2009). Nevertheless, there are no data about the endosymbionts of other legumes cultivated in small orchards in this island neither about the endophytes present in these nodules. In the last years the study of legume nodular endophytes is increasing with special emphasis on their ability to have in vitro plant growth promotion mechanisms that has been found in some nodule endophytic strains including sporulated Gram positive genera. The most abundant genera of this bacterial group found in legume nodules are Bacillus and Paenibacillus and some of their species are able to produce indole acetic acid an siderophores (Selvakumar et al., 2007, Li et al., 2011). Therefore, the objective of this work was the isolation and identification of the endophytic species isolated from nodules of Pisum sativum used as trap plant in three sites of Lanzarote (Canary Islands) as well as the analysis of the in vitro plant growth promotion mechanisms presented by the endophytes isolated. MATERIAL AND METHODS The isolation of endophytes and rhizobia was performed following the method of Vincent (1970). The Pisum sativum plants were harvested at flowering in two soils subjected to integrated production in Valladolid (Spain). Amplification and sequencing of the 16S rRNA gene were performed according to Rivas et al. (2007). The sequences obtained were compared with those from EzTaxon-e server (Kim et al., 2012). 301 Session IV SIV-CP-13 The phosphate solubilization, siderophores production, indole acetic acid production and nitrogen fixation were analyzed as previously described (García-Fraile et al., 2012). RESULTS AND DISCUSSION Pisum sativum plants nodulated in the three soil samples analyzed from Guatiza and Mala, one of them recovered from a volcano soil. The nodules were effective and typical of rhizobia, nevertheless most of the isolated colonies on YMA plates despite their mucoid aspect and white colour did not correspond to rhizobia but to Paenibacillus species. Only a strain from species Rhizobium leguminosarum was recovered from the Mala soil. The Paenibacillus species were identified as Paenibacillus validus, Paenibacillus xinjiangensis, Paenibacillus peoriae, Paenibacillus borealis, Paenibacillus taohuashanense and Paenibacillus polymyxa with more than 99.5% identity in the 16S rRNA gene. Some strains belonged to undescribed species from genus Paenibacillus related to Paenibacillus xinjiangensis, Paenibacillus peoriae, Paenibacillus borealis and Paenibacillus taohuashanense. All the strains presented at least one of the four in vitro mechanisms of plant growth promotion studied. Siderophores were produced by all strains and particularly by the strains of Paenibacillus validus. ACKNOWLEDGMENTS This work was supported by MINECO (AGL2010-17380). MHRB is recipient of a JAE-Doc researcher contract from CSIC, cofinanced by ERDF. REFERENCES García-Fraile, P., et al. (2012). PLoS One. 7: e38122. Kim, O.S., et al. (2012). Int. J. Syst. Evol. Microbiol. 62: 716-721. Li, L., et al. (2011). FEMS Microbiol. Ecol. 79: 46-68. Rivas, R., et al. (2007). Lett. Appl. Microbiol. 44: 181-187. Selvakumar, G., et al. (2007). Curr. Microbiol. 56: 134-139. Vincent J.M. (1970). A Manual for the Practical Study of Root-Nodule. pp. 1-13. Edited by J.M. Vin cent. Oxford : Blackwell Scientific Publications. Zurdo-Piñeiro, J. L., et al. (2009). Appl. Environ. Microbiol. 75: 2354-2359. 302 Session IV SIV-CP-14 Efecto de rizobacterias en el enraizamiento de miniestacas en dos clones híbridos de Eucalyptus spp. González-Candia, P.1*, Sossa, K.2, Rodríguez, F.3, Sanfuentes, E.1 1 Laboratorio de Patología Forestal, Facultad de Ciencias Forestales, Universidad de Concepción. Concepción, Chile. 2 Laboratorio de Biopelículas y Microbiología Ambiental, Centro de Biotecnologí a, Universidad de Concepción. Concepción, Chile. 3 Forestal Mininco S.A. * [email protected] RESUMEN Mediante tres ensayos se evaluaron rizobacterias en el enraizamiento de miniestacas de dos clones híbridos de Eucalyptus nitens x Eucalyptus globulus. Los resultados de esta investigación confirmaron el positivo efecto de las rizobacterias en promover el enraizamiento, confirmando el potencial medioambiental y de bajo costo que esta estrategia implicaría para la industria forestal. Además la identificación mostró a Bacillus y Pseudomonas como las bacterias con mayor efecto. INTRODUCCIÓN Eucalyptus globulus y sus híbridos son relevantes para industria forestal en Chile, por lo que mucho esfuerzo se ha destinado a mejorar la producción de esta especie en condiciones de vivero. En este sentido la reproducción asexual es promisoria, sin embargo, los problemas en el enraizamiento de algunos clones puede limitar su implementación (Teixeira et al., 2007). Para mejorar esto, se ha comenzado a utilizar rizobacterias en virtud de su positivo efecto en el enraizamiento, de su bajo impacto ambiental y menor costo (Adesemoye et al., 2009). Estas bacterias captan carbono y nitrógeno desde los exudados de las raíces (Benizri et al., 2001), influenciado el enraizamiento de la planta (Compant et al., 2010). A pesar de estas ventajas, el efecto de estas bacterias ha sido poco estudiado en especies forestales (Chanway, 1997). Este estudio evaluó el efecto de rizobacterias en el enraizamiento de miniestacas de dos clones híbridos recalcitrantes de Eucalyptus. MATERIAL Y MÉTODOS Se aislaron 106 cepas bacterianas desde la rizósfera de cinco clones de Eucalyptus spp. (Díaz et al., 2009). Fueron realizados tres ensayos (E1, E2, E3) en vivero de la empresa Forestal Mininco S.A, en Los Ángeles, Chile. Las rizobacterias fueron aplicadas al substrato por aspersión, y en la base de las miniestacas de dos clones E. nitens x E. globulus A y B mediante inmersión (108ufc/ml). En E1 se utilizó solo un clon híbrido mientras que en E2 y E3 se consideraron los dos clones. Como control se utilizó agua destilada. El diseño experimental fue en bloques completos al azar, con 20 repeticiones para E1 y E2, y 30 para E3. Luego de 45 días se evaluó el enraizamiento (%). Los resultados fueron analizados mediante test paramétricos o no paramétricos en función de la distribución de los residuos (SAS 9.2). Posteriormente las cepas con mejores resultados fueron identificadas mediante extracción, purificación y secuenciación de ADNr 16S. RESULTADOS Y DISCUSIÓN En el primer ensayo 32 cepas aumentaron significativamente (p>0,05) el enraizamiento de las miniestacas, con un enraizamiento máximo que fue de 75%; 2,6 veces mayor al control (28%). Estos resultados implican rendimientos comparables a los obtenidos por Teixeira et al. (2007), mediante inoculación de cepas de rizobacterias en estacas de E. grandis. En el segundo ensayo 9 y 7 cepas incrementaron significativamente el 303 Session IV SIV-CP-14 enraizamiento en los clones A y B, respectivamente. En el tercer ensayo 12 cepas aumentaron el enraizamiento en ambos clones. Estos resultados permiten concluir que rizobacterias seleccionadas pueden incrementar el enraizamiento en miniestacas de clones E. nitens x E. globulus, con diferencias clon-específicas. Los resultados de la identificación indicaron que las cepas bacterianas pertenecieron principalmente al género Bacillus sp. y Pseudomonas sp. Otras cepas fueron identificadas como Musilaginibacter sp., Flavobacterium sp. y Rhodococcus sp, géneros de bacterias ambientales indicadas como bacterias con efecto en el enraizamiento de plantas (Belimov et al., 2005, Madhaiyan et al., 2010). Tabla 1. Enraizamiento de miniestacas en los híbridos E. nitens x E. globulus clones A y B. Se muestran aquellas cepas con enraizamiento significativamente mayor al control, que además estuvieron presenten en los resultados de E1, E2 y E3. Cepa Control 14 121 46 63 18 49 43 116 56 59 119 Enraizamiento (%) Clon A 7 29 25 28 32 37 28 25 29 27 27 40 Enraizamiento (%) Clon B 54 74 75 74 79 86 78 78 68 67 68 77 AGRADECIMIENTOS CONICYT, Forestal Mininco S.A. BIBLIOGRAFÍA Adesemoye, A., et al. (2009). Microbiol. Ecol. 58: 921-929. Belimov, A., et al. ( 2005). Soil Biol. Biochem. 37: 241-250 Benizri, E., et al. (2001). Biocontrol Sci and Techn. 11: 557-574. Chanway, C., (1997). For. Sci. 43 99-112. Compant, S., et al. (2010). Soil Biol. Biochem. 42: 669-678. Díaz, K., et al. (2009). World J. Microbiol. Biotechnol. 25: 867-873. Madhaiyan, M., et al. (2010).IJSEM. 60: 2451-2457. Teixeira, D., et al. (2007). Braz. J. Microbiol. 38: 118-123. 304 Session IV SIV-CP-15 Azoarcus sp. CIB, un nuevo endófito del arroz que degrada anaeróbicamente hidrocarburos aromáticos. Fernández, H.1, Prandoni, N.1, Fernández-Pascual, M.2, Morcillo, C.2, Fajardo, S.2, Díaz, E.1, Carmona, M.1* 1 Departamento Biología Ambiental. Centro de Investigaciones Biológicas-CSIC. Protección Vegetal. Instituto de Ciencias Agrarias-CSIC. * [email protected] 2 Departamento de RESUMEN En nuestro laboratorio hemos demostrado que Azoarcus sp. CIB, una bacteria modelo en el estudio del catabolismo anaeróbico de compuestos aromáticos, es capaz de invadir la raíz del arroz y de vivir como endófito. Dado que Azoarcus sp. CIB degrada diversos contaminantes, podría ser empleada como herramienta biotecnológica en tareas de endofitorremediación. INTRODUCCIÓN Azoarcus sp. CIB es una -Proteobacteria desnitrificante con la capacidad de degradar, tanto aeróbica como anaeróbicamente, un elevado número de compuestos aromáticos, incluyendo contaminantes como el tolueno y m-xileno (López-Barragán et al., 2004). Otras bacterias del género Azoarcus, e.g., Azoarcus sp. BH72 y A. communis, son capaces de invadir plantas y vivir como endófitos (Reinhold-Hurek and Hurek, 2000). Hasta la fecha, no se había descrito ningún Azoarcus que degrade anaeróbicamente compuestos aromáticos y que sea endófito, llegándose a sugerir la existencia de un nuevo género, Aromatoleum, para los Azoarcus no endófitos degradadores (ReinholdHurek and Hurek, 2 000). En este trabajo presentamos evidencias experimentales de que Azoarcus sp. CIB es capaz de invadir la raíz del arroz. MATERIAL Y MÉTODOS Recuento del número de bacterias dentro de la raíz del arroz. Las semillas de arroz fueron desinfectadas e inoculadas con bacterias tal y como se ha descrito anteriormente (Compant et al., 2005). Tras 5 días de crecimiento se determinó el número de viables en el interior de la raíz siguiendo protocolos ya establecidos (Compant et al., 2005). Técnicas microscópicas. Se empleó un microscopio confocal Leica TCS-SP5-AOBS y un microscopio electrónico (TEM) STEM LEO 910. Las muestras para confocal fueron preparadas según (Reinhold-Hurek and Hurek, 2011). El procesamiento e inmunolocalización para TEM se llevo a cabo según de María et al. (2005) utilizando un anticuerpo anti-GFP comercial. RESULTADOS Y DISCUSIÓN Identificación de genes necesarios para la endofitosis en el genoma de Azoarcus sp. CIB. Análisis genómicos en bacterias endofíticas ha permitido identificar una serie de genes esenciales para establecer un estilo de vida endófito (Krause et al., 2006; ReinholdHurek and Hure k, 2011). Recientemente hemos secuenciado el genoma de Azoarcus sp. CIB, y se han identificado genes ortólogos a los relacionados en otras bacterias con la asociación como endófito: i) genes que codifican el flagelo, ii) genes de fimbrias tipo IV, iii) genes implicados en la síntesis de exopolisacáridos, iv) genes implicados en la producción de análogos de hormonas vegetales. Estos resultados nos permitieron sugerir que Azoarcus sp. CIB podría ser el primer degradador anaeróbico de hidrocarburos aromáticos que se describe capaz de establecerse también como un endófito de plantas. 305 Session IV SIV-CP-15 Localización de Azoarcus sp. CIB en el interior de la raíz del arroz. Dado que el arroz es una planta que colonizan otras bacterias del género Azoarcus (Reinhold-Hurek and Hurek, 2011), se utilizó como modelo de planta huésped para estudiar la capacidad endofítica de la cepa CIB. Se inocularon, de forma independiente, plántulas previamente desinfectadas con Azoarcus sp. CIB, Azoarcus communis (control positivo) y Escherichia coli (control negativo). El recuento de bacterias presentes en el interior de las raíces reveló que por gramo de raíz había 10 5 células de A. communis, 5x104 de Azoarcus sp. CIB y ninguna de E. coli. Estos resultados sugieren que Azoarcus sp. CIB es capaz de colonizar la raíz del arroz tan eficientemente como A. communis. Observación de Azoarcus sp. CIB en el interior de la raíz del arroz. Azoarcus sp. CIB-GFP, una cepa que expresa la “green fluorescence protein”, fue detectada en el interior de estructuras de la raíz del arroz tanto mediante observación con microscopía de fluorescencia (Figura 1A), como con microscopía confocal (Figura 1B). La microscopía electrónica permitió corroborar estos resultados, localizando a Azoarcus sp. CIB en los espacios intercelulares de la exodermis radicular (Figura 1C). B A C PC Az EI Az Az 10 m 50m 0,2µm Figura 1. Observación microscópica de la interacción de Azoarcus sp. CIB con la raíz del arroz. (A) Microscopía de fluorescencia de un pelo radicular donde se observa un grupo de Azoarcus sp. CI BGFP. (B) Microscopía confocal en la que se observan agregados de Azoarcus sp. CIB-GFP en las zona s internas de la raíz. (C) Inmunolocalización con anti GFP de Azoarcus sp. CIB-GFP en un espacio intercelular de la exodermis radicular de arroz. PC, pared celular; EI, espacio intercelular, Az,A zoarcus. Este trabajo constituye la primera descripción de un organismo capaz de degradar anaeróbicamente compuestos aromáticos y de vivir como endófito en plantas, abriendo la posibilidad de utilizar endofitorrediación de ambientes contaminados con hidrocarburos aromáticos. AGRADECIMIENTOS Este trabajo ha sido financiado por los proyectos BIO2009-10438 y BIO2012-39501 del Ministerio de Economía y Competitividad y la Comunidad de Castilla-La Mancha proyecto POII10-0211-5015. BIBLIOGRAFÍA Compant, S., et al. (2005). Appl. Environ. Microbiol. 71: 1685-1693. de María, N. et al. (2005). Plant Physiol. Biochem. 43: 985-996. Krause, A., et al., (2006). Nat. Biotech. 24: 1385-1391. López-Barragán, M.J., et al. (2004). J. Bacteriol. 186: 5762-5774. Reinhold-Hurek, B., and Hurek, T. (2000). Int. J. Syst. Evol. Microbiol. 50: 649-659. Reinhold-Hurek, B. and Hurek, T. (2011). Curr. Op. Plant Biol. 14: 435-443. 306 Session IV SIV-CP-16 Impacts of rice-bean intercropping on soil microbial activity and the development of rice plants. Razakatiana, A.T.E.1, 2*, Henintsoa, M.3 Becquer, T.4, Ramanankierana, H.2, Baohanta, H.R.2, Raherimandimby, M.1, Rabeharisoa, L.3, Duponnois, R.5 1 Laboratoire de Biotechnologie et de Microbiologie, Faculté des Sciences, Université d’Antananarivo , BP 906, Antananarivo 101, Madagascar. 2Laboratoire de Microbiologie de l’Environnement (LME), Centre National de Recherches sur l’Environnement (CNRE), BP 1739, Antananarivo 101, Madagascar. 3 Laboratoire des Radioisotopes (LRI), Université d’Antananarivo, BP 3383, Antananarivo 101, Madagascar. 4IRD, UMR 210 Eco&Sols, CIRAD – INRA – SupAgro, 2 place Viala, Bâtiment 12, 34060 Montpellier Cedex , France. 5Laboratoire des Symbioses Tropicales Méditerranéennes LSTM UMR CIRAD/ IRD /SupAgro/UM2 USC INRA TA A-82/ J Campus International de Baillarguet 34398 Montpellier Cedex 5, France. * [email protected] ABSTRACT The impact of the intercropping system with common bean and rice under different treatments such as organic fertilizer, mineral fertiliser, cropping system with no-tillage or conventional tillage on plant development and on microbial activity was assessed. INTRODUCTION Soil tillage, organic amendment type or amount of phosphorus (P) fertilizer could influence both plant development, production yield and soil microbial activities (Asensio et al, 2010). The main objective of the study was to evaluate the effects of different cropping system on plant development and soil microbial activities. This will provide new way to reduce the use of chemical fertilizer in farming system. MATERIAL AND METHODS The study site is a field experiment located in Lazaina, a rural area in the suburb of Antananarivo, Madagascar (18°46’53.56’’South and 47°32’05.03’’East). Different ricebean intercropp ing system corresponding to a combination of two soil tillage (conventional (CT) and no-tillage (NT)), two organic treatments corresponding to an input of 20 kg P ha-1 as manure (M20) and as green manure of stylosanthes (GM20) and two levels of, triple superphosphate (TSP) chemical fertilizer (20 (TSP20) and 50 (TSP50) kg P ha-1), were compared to a control (CT without organic and mineral fertilizers). The shoot production and the grain yields of rice and bean were determined. Soil microbial enzymatic activities (rhizospheric soils of rice and bean) were assessed with an emphasis on phosphatasic activities and FDA (Fluorescein diacetate) hydrolysing activities. Phosphatasic activities were conducted in acid and alkaline media. The paraNitrophenyl Phosphate (pNPP) substrate in contact with the soil was hydrolyzed to pNitrophenol by phosphatasi c enzymes (Feller et al.,1994) and FDA in contact with the soil was hydrolyzed by different enzymes producing there fluoresceine (Gaël et al., 2002; Alef K.,1998; Schnurer, J. and Rosswall. T.,1982). RESULTS AND DISCUSSION Compared to the control, the organic manure mixed with mineral fertilizer in moderate doses promotes plant growth and yield of both rice and beans (Fig 1). However, the green manure promoted a decrease of the shoot biomass productions and grain yields, probably due to the poor nitrogen content of the stylosanthes shoots that we used. 307 Session IV SIV-CP-16 Quantity of pnitrophenol (µg/h/g of soil) (a) (a) (ab) (ab) (ab) Production yield (t.ha-1) 200 3 (a) (b) (a) (a) (ab) (a) 0 50 40 30 20 10 0 (b) (a) (a) (a) (a) (a) (a) (a) (a) c (abc) (bc) (abc)(a) (abc) (ab) (ab) (abc) (abc) (a) Acid phosphatase Quantity of p-nitrophenol (µg/h/g of soil) Production yield(kg.ha-) 400 50 40 30 20 10 0 2 (b) (b) (b) (b) (ab) (ab) (ab) (a) 1 (a) (ab) (a) 0 c (a)(a) (ab) (ab) (ab) (b) Acid phosphatase Alcaline phosphatase (d ) (b) (a) (b) (ab) (ab) (ab) (a) (ab) (ab) (ab) (ab) (ab) (ab) Alcaline phosphatase Figure1. Comparison of yield of bean (a), yield of rice (b) and soil phosphatase activity of rice soil (c) and bean soil (d) on the field expériment in Lazaina Table 1. Global microbial activity Treatment CT-Co CT-TSP20 CT-TSP50 CT-M20 CT-TSP20-M20 CT-TSP50-M20 CT-TSP20-GM20 CT-TSP50-GM20 NT-TSP20-M20 NT-TSP20-GM20 Fluorecsein diacetat (mg/h/g) of rice soil 5,53 (abc) 2,55 (a) 3,61 (abc) 2,32 (a) 3,03 (ab) 2,63 (abc) 7,05 (bcd) 9,80 (d) 4,20 (abc) 6,95 (cd) Fluorecsein diacetat (mg/h/g) of bean soil 3,92(a) 2,21 (a) 4,18(a) 2 (a) 6,68 (a) 4,04 (a) 11,76 (b) 10,78 (b) 3,29 (a) 2,61 (a) Using stylosanthes as green manure significantly stimulated the global microbial activity of soil microorganisms evaluated by the fluorescein diacetate hydrolysis assay, in rice and bean soils (Table1), as observed also by Acosta-Martinez et al. (2007) and Gaël et al. (2002). Soil phosphatase activity texted by the p-nitrophenyl phosphate (pNPP) hydrolysis assay (Feller et al.,1994) was also significantly higher in tillage treatments using manure alone or mixed with mineral fertilizer. Thus, our results showed that organic fertilizers on rice-bean intercropping system positively influence soil microbial activities. ACKNOWLEGMENTS This work was supported by the project Fabatropimed supported by Agropolis Foundation under the reference ID 1001-009. REFERENCES Acosta-Martínez, V., et al. (2007). Appl. Soil. Ecol. 37: 41–52. Alef, K. (1998). Methods in applied soil microbiology and biochemestry. Academic Press, London, pp . 232-233. Asensio, R. (2010). Les Cahiers du PRAM 8: 25-29. Feller, C., et al. (1994). Can. J. Soil Sci. 73: 121-129. Gaël, A., et al. (2002). Activités biologiques et fertilité des sols. ITAB, Paris. 27p. Schnurer, J., and Rosswall, T. (1982). Appl. Environ. Microbiol. 43:1256-1261. 308 Session IV SIV-CP-17 Efeito de bactérias PRPG com indução de ácido salicílico em plântulas de duas cultivares de palma forrageira (Opuntia e Nopalea). Lyra, M.C.C.P.1*, Pérez-Montaño, F.2, Jiménez-Guerrero, I.2, Madinabeitia, N.2, DiazOlivares, I.M. 2, Gutiérrez-Alcántara, R.2, Ollero, F.J.2 1 Agronômico de Pernambuco-IPA. Laboratório de Genoma-Avenida General San Martin, 1371, Bongi, CEP. 52761000 Recife-Pernambuco. Brasil. 2 Departamento de Microbiología. Facultad de Biología. Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012 Sevilla-España. * [email protected] ABSTRACT Prickly Pear Cactus has great economic interest in Brazil due to its ability to withstand long periods of drought. The use of PRPG bacteria induced with AS can have an effect on plant growth. This study aimed to evaluate this interaction. We observed that the Nopalea did most amount of AS was detected by luminescence. The AM07 isolated was inhibited by the presence of AS. The growth parameters of the plants only showed significant differences in the variety Miúda due to its homogeneity of germination and consequently the same physiological stage. INTRODUÇÃO A palma forrageira é uma cultura muito utilizada no nordeste brasileiro por apresentar uma grande capacidade de adaptação às condições de seca. Os mecanismos pelo qual as rizobactérias promotoras de crescimento de plantas (PGPR) atuam podem ser diretos ou indiretos e estes mecanismos podem ser ativados em diferentes estágios do crescimento da planta. Algumas enzimas e compostos fenólicos estão envolvidas nestes diferentes mecanismos de defesa na planta quando inoculadas com bactérias PGPR (Figueiredo et al, 2010). Um destes compostos é o acido salicílico (AS) que deriva do aminoácido fenilalanina. A importância do SA como regulador do crescimento em plantas está reduzida a poucos processos mas sua presença afetam a síntese de outros reguladores de crescimento. O AS reduz a síntese de etileno e em algumas espécies pode causar atraso na senescência de flores ou indução da floração. Ryals et al. (1996) observaram que os níveis de SA aumenta no tecido da planta quando estas sofrem algum ataque de patógeno e a aplicação de SA exógeno aumenta a resistência da planta para grande gama de patógenos.Este trabalho teve como objetivo observar o efeito de dois isolados endofíticos com e sem indução de acido salicílico em plântulas de palma forrageira . MATERIAL E MÉTODOS As sementes de Opuntia fícus indica (var. Orelha de Elefante Africana) e de Nopalea Cochenillifera (var. Miúda) da família Cactaceae foram oriundas do Agreste Pernambucano (Banco de Germoplasma de Arcoverde-Estação Experimental do Instituto Agronômico de Pernambuco-IPA-Brasil). Oito tratamentos foram realizados: T1: Controle, T2: G. diazotrophicus (AM07), T3: H. seropedicae (AM37), T4: AM07 + 1 mM (10-4M) de ácido salicílico (AS), T5: AM37 + 1 mM AS, T6: AM07 + AM37, T7: AM07+ AM37 + 1mM AS, e T8: 1 mM AS. Foram estudados: Peso Fresco da Planta (PFP, g), Comprimento da planta (CP, cm) e Largura do Cladódio (LC, cm – realizada exatamente na metade da folha). O delineamento foi inteiramente ao acaso, com três repetições. A inoculação com os isolados foi realizada cinco dias após o transplante onde as mesmas foram cultivadas em meio DYGs por 24 hs. O SA foi dissolvido em água segundo Bordiec et al. (2011). A extração e determinação do AS foi realizada de acordo com Huang et al. (2005) com algumas modificações, onde macerou-se 0,2 g do tecido da planta, sonicação e centrifugação resultando em um extrato cru contendo AS. O extrato cru foi misturado com Acinetobacter sp. strain ADP1 (Metzgar et al, 2004) e 309 Session IV SIV-CP-17 colocada em uma placa e incubada a 37°C por 1 h em três réplicas. A quantificação do AS (Luminescência) foi realizado no equipamento Synergy HT Biotek com Programa Gen51.11. RESULTADOS E DISCUSSÃO A variedade Orelha de Elefante Africana (Opuntia fícus-indica), não apresentou diferenças significativas para os parâmetros estudados talvez em função do alto coeficiente de variação (CV%=40,53). O tratamento T6 com os isolados AM07 e AM37 foi quem obteve o maior percentual em relação ao controle absoluto para a PFP (129%), vindo logo após os tratamentos T2 (93%) e T3 (81%). Ao adicionar o AS juntamente com os isolados observamos uma redução da PFP de 17% negativo em relação ao controle negativo e de 35% menos em relação ao T6. O mesmo comportamento foi observado para as outras duas variáveis (CP e LC). Resultado não observado para o parâmetro LC, que foi negativo. Resultados contrários, foram observado por Ryals et al, (1996) onde observaram que os níveis de SA aumentava no tecido da planta quando estas sofriam algum ataque de patógeno. Na variedade N. Cochenillifera (var. Miúda) observamos diferenças significativas em que o melhor tratamento foi T2 seguido dos tratamentos T3, T7, T5 e T4, respectivamente. Resultados semelhantes foram observados com o uso simultâneo do AS com os isolados influenciando negativamente nos tratamentos em que o T8 foi quem apresentou os piores resultados chegando a obter um percentual de -17% (PFP), 10% (CP) e 9% (LC). O maior valor de luminescência usando a bactéria Acinetobacter sp. ADP1, foi observado com a Nopalea var. Miúda nos tratamentos T8 (59,87 ng) e T5 (31,2 ng), os demais tratamentos variaram entre 9 e 12,3 ng. No caso da planta Opuntia var. Orelha de Elefante, o maior valor de AS foi obtido no T8 (29,92 ng). 100% a mais de AS na planta Nopalea. Se analisarmos estes dados comparando com as variáveis estudadas de PFP, CP e LC, podemos afirmar que quanto mais AS na planta mais afeta o crescimento da mesma. O isolado AM07 foi o mais afetado com a adição de AS nas duas variedades. Podemos afirmar que como a palma forrageira possui um metabolismo fotossintético do tipo CAM, que o caracteriza por terem um crescimento lento, estas plantas competem de forma desvantajosa com outras espécies de plantas quando as condições ambientais são extremas. O uso de AS inoculado com PRPG em cactáceas deve ser melhor estudado. CONCLUSÕES A variedade Nopalea foi quem mais quantidade de AS foi detectada por luminescência. A bactéria AM07 foi inibida pela presença de AS. Os parâmetros de crescimento das plantas apresentaram diferenças significativas apenas na variedade Miúda devido a sua homogeneidade na germinação e consequentemente um mesmo estagio fisiológico. Por serem de metabolismo lento, resultados satisfatórios poderiam ser observados em um período de tempo maior, bem como, determinar a melhor dose de AS para estas plantas. REFERÊNCIAS Bordiec, S., et al. (2011). J. Exp. Bot. 62: 595-603. Figueiredo, M.V.B.F., et al. (2010). Plant Growth Promoting Rhizobacteria: Fundamentals and Applications. Huang, W.E., et al. (2005). Environ. Microbiol. 7: 1339-1348. Metzgar, M.L., et al. (2004). Nucleic Acids Res.32: 1792-1797 Ryals, J.A., et al (1996). Plant Cell 8: 1809-1819. 310 Session IV SIV-CP-18 Análisis cualitativo y cuantitativo de vitamina C en fresas, en presencia de microorganismos endófitos. Aranda-Alonso, C.1, Serrano, A.M.1 2* , Rodríguez-Carvajal, M.A.1, Ollero, F.J.2, Megías, M.3, Gil1 Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla. Sevilla, España. 2 Departamento de Microbiología Vegetal y Biología Molecular, Facultad de Biología, Universidad de Sevilla. Sevilla, España. 3 Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla. Sevilla, España. * [email protected] RESUMEN El contenido en ácido L-ascórbico o vitamina C en fresas ha sido determinado mediante HPLC-PDA en fase reversa. Fueron analizadas ocho tesis inoculadas con diferentes microorganismos endófitos y en tres de ellas el aumento en ácido L-ascórbico fue muy significativo, siendo la cepa Pantoea vagans LMG 24199 (T) (NTABE-P310) + Micorriza la que presentó un efecto más acusado como inductor de la biosíntesis de vitamina C. INTRODUCCIÓN La vitamina C está formada en un 90% por ácido L-ascórbico (AA), que es la forma predominantemente activa, si bien también se puede encontrar en la forma oxidada: ácido L-deshidroascórbico (DHA), que también presenta actividad biológica (Campos et al., 2009). Aunque para cuantificar de forma precisa el contenido en vitamina C de la matriz biológica en cuestión es importante determinar el contenido de ambos, se puede considerar únicamente el porcentaje de ácido L-ascórbico en una determinación rápida y aproximada. En este trabajo se presenta un método de análisis HPLC-PDA en fase reversa, que no compromete la integridad del ácido ascórbico y que a la vez proporciona unos resultados fiables, siendo rápido y de bajo coste. MATERIAL Y MÉTODOS Preparación de las muestras y extracción del ácido ascórbico. Se parte de unos 10 g de fresas trituradas. La rotura mecánica de las células se lleva a cabo con nitrógeno líquido en mortero y, a continuación, se liofiliza la muestra. El liofilizado se trata con 10 ml de una disolución de H3PO4 (3%) y AcOH (8%) (Frenich et al., 2005), se centrifuga a 15000 rpm durante 10 min a 5 °C y se recoge el sobrenadante, que se diluye con agua hasta 50 ml. El extracto resultante se pasa a través de filtros de nylon de 0,45 µm para su posterior inyección en el HPLC-PAD (Pallauf et al., 2012). Condiciones cromatográficas. Se ha utilizado un sistema de HPLC Waters-Alliance 2695 acoplado a un detector PDA Waters 2996 (Photo Diode Array), con una pre-columna SecurityGuard Cartridge C18 4 x 3,0 mm ID y una columna Hyperclone ODS C18 - 5µm x 4,0 mm x 250 mm (Phenomenex, EEUU). La fase móvil empleada ha sido H 2SO4 0,01% (Burini, 2007). El volumen de inyección para cada ensayo fue de 100 µl y cada cromatograma se ha registrado durante 30 minutos. Debido a la inestabilidad térmica observada en las muestras y en los patrones, éstos se prepararon frescos para cada análisis y manteniendo en hielo los viales justo antes de introducirlos en el carrusel del equipo. 311 Session IV SIV-CP-18 RESULTADOS Y DISCUSIÓN Antes de establecer el método de cuantificación, se hicieron ensayos para detectar el posible efecto matriz intrínseco en muestras biológicas. La variación en las respuestas instrumentales fue considerable, por lo que se corroboró la presencia del efecto matriz y se decidió aplicar la técnica de las adiciones estándar (Miller, 2002) para subsanar este efecto en la cuantificación. Los resultados de dicha cuantificación fueron los siguientes: El incremento que se produce a causa de los microrganismos endófitos es más que notable. Este resultado plantea la necesidad de realizar ensayos futuros más exhaustivos, para confirmar estas bacterias y hongos como agentes inductores de la biosíntesis de vitamina C. AGRADECIMIENTOS Agradecemos la financiación al Ministerio de Educación y Ciencia (AGL2009-13487-C04-02). BIBLIOGRAFÍA Burini, G., et al. (2007). J. Chromatogr. 1154: 97-102. Campos, F. M., et al. (2009) Quim. Nova 32: 87-91. Frenich, A. G., et al. (2005). J. Agric. Food Chem. 53: 7371-7376. Miller, J., (2002). Estadística y Quimiometría para Química Analítica. p 296. Pallauf, K., et al. (2012). J. Food Compos. Anal. 21: 273-281. 312 Session IV SIV-CP-19 Occurrence of rhizobacteria with PGPR activities in different ecosystems and agro-ecosystems of northern, central and southern Chile. Barra, P., Jorquera, M., Acuña, J. *, Lagos, L., Marileo, L., Inostroza, N., Mora, M.L. Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile. * [email protected] ABSTRACT The occurrence of rhizobacteria with PGPR activities in different ecosystems and agroecosystems of northern, central and southern Chile was studied. Around 200 isolates showed the ability to growth in culture media supplemented with phytate and 1aminocyclopropane-1-c arboxylate (ACC) as sole source of phosphorus and carbonnitrogen, respectively. Genes involved in phytate (-propeller phytase gene) and ACC (acdS gene) degradation were detected by PCR in isolates from both ecosystems and agro-ecosystems. Indole acetic acid (IAA) production (9-67 µg mL-1) was also observed in selected isolates. INTRODUCTION Soil bacteria play an essential role in nutrient cycling and plant productivity. In terrestrial ecosystems, bacterial communities of the plant rhizosphere carry out functions that are essential to plant growth. Thus, plant-growth promoting rhizobacteria (PGPR) have been isolated from agro-ecosystems and commonly proposed as inoculants to contribute to nutrition (e.g. nitrogen and phosphorus) and stress tolerance of plants. However, all of our knowledge of rhizosphere bacterial communities for different plants so far has come from studies on agricultural crops and forest tree species, and almost nothing is known yet about microbial communities in soil under natural vegetation. In comparison to managed ecosystems, the long term selection and co-evolution of microbial communities under natural vegetation provides a unique opportunity to examine how certain rhizobacteria may help plants acquire nutrients and survive in poor soils. Under this scenario, molecular tools, such polymerase chain reaction (PCR), now offer great promise for exploring the genetic diversity of rhizobacteria and understanding how bacterial communities function to support plant growth and tolerance to environmental stress. The main goal of this study was to explore the occurrence of rhizobacteria with PGPR activities in different ecosystems and agro-ecosystems of northern, central and southern Chile. The PGPR activities and related genes evaluated were: phytate-degrading activity (-propeller phytase [BPP] gene); 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity (acdS gene) and indole acetic acid (IAA) production. MATERIAL AND METHODS Rhizosphere samples were collected from plants grown in different ecosystems (Atacama Desert [22°S; 68°W], Conguillio National Park [38°S; 71°W], Patagonia [53°S; 70°W]) and agro-ecosystems (avocado orchards [32°S; 70°W], cereal crops and pastures [38°S; 72°W]) from northern, central and southern Chile. Rhizosphere samples were enriched in diverse minimal culture media supplemented with phytate (PSM media) and ACC (DF media) as sole source of phosphorus and carbon-nitrogen, respectively. Isolates grown in PSM and DF media were screened by PCR for putative genes involved in phytate (BPP gene) and ACC (acdS gene) degrading activities as described by Jorquera et al. (2012). Production of phytohormone, indole acetic acid 313 Session IV SIV-CP-19 (IAA), was also evaluated in 25 isolates showing PCR positive isolates for BPP and acdS genes. RESULTS AND DISCUSSION Count of culturable bacteria revealed higher bacterial loads rhizosphere soils of Conguillio National Park, cereal crops and pastures (1-10×106 CFU g-1) compared with Atacama Desert, avocado orchards and Patagonia (1-5×105 CFU g-1). Around 220 from 800 isolates grown in media enriched with phytate and ACC were selected for PCR screening based on different phenotypes. Amplification of BPP and acdS genes was observed in both ecosystems and agro-ecosystems. By using PSM media, 7-29% and 222% of isolates sh owed positive PCR amplification for BBP gene in ecosystems and agro-ecosystems, respectively. In relation to acdS gene, 9-27% and 12-32% of isolates showed positive PCR amplification in ecosystems and agro-ecosystems, respectively. By using DF media, 0-26% and 0-18% of isolates showed positive PCR amplification for BBP gene in ecosystems and agro-ecosystems, respectively. Respect to acdS gene, 21-29% and 6-28% of isolates showed positive PCR amplification in ecosystems and agro-ecosystems, respectively. Our results also showed higher IAA productions in isolates from agro-ecosystems (23-67 µg mL-1) compared with ecosystems (9-35 µg mL1 ). This study revealed the occurrence of rhizobacteria with PGR activities in diverse managed and undisturbed ecosystems. Rhizobacteria with PGR activities obtained from natural vegetation may be taken in account to help plants to acquire nutrients in agroecosystems w ith lower rates of fertilization and improve the tolerance of plants to stressful conditions (e.g. drought, salinity or frost). ACKNOWLEGMENTS This work was supported by the CONICYT Doctoral Scholarships 21110473 and FONDECYT Project no. 1120505. REFERENCES Jorquera, M.A., et al. (2012). Microbial Ecol. 64: 1008-1017. 314 Session IV SIV-CP-20 Efecto de plaguicidas sobre la actividad biológica de la cubierta vegetal (L. perenne) de un sistema de biopurificación denominado lecho biológico. Diez, M.C.1*, Gallardo, F.2 1 Departamento. de Ingeniería Química, Universidad de La Frontera, Temuco, Chile. 2 Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Temuco, Chile. * [email protected], [email protected] RESUMEN Se evaluó el efecto de repetidas aplicaciones de una mezcla de plaguicidas (atrazina, clorpirifos e iprodiona) sobre la actividad biológica en la cubierta vegetal de L. perenne de un sistema de biopurificación denominado lecho biológico. En general, las actividades biológicas en la biomezcla no variaron significativamente entre el control y los tratamientos en relación a la concentración de plaguicida adicionado. El ácido succínico se encontró en los exudados radicales en la mayor concentración, mientras que el menor encontrado fue el ácido oxálico. INTRODUCCIÓN El sistema de biopurificación denominado lecho biológico está diseñado para retener y degradar plaguicidas en elevadas concentraciones. La adsorción y degradación suceden en una biomezcla conformada por suelo, paja y turba en proporción volumétrica de 1:2:1. El suelo aporta microorganismos y capacidad de adsorción, la paja propicia el crecimiento de hongos con actividad ligninolítica y la turba adsorbe contaminantes y mantiene la humedad necesaria para la actividad biológica en la biomezcla. Sobre esta biomezcla, se instala una cubierta vegetal de césped que propicia una rizósfera que colabora con la degradación de los contaminantes a través de los exudados radicales y por la absorción de contaminantes; además de favorecer la evapotranspiración en el sistema de biopurificación (Castillo et al., 2008). MATERIAL Y MÉTODOS Para evaluar el efecto de los plaguicidas sobre la actividad biológica de la cubierta vegetal de L. perenne (ballica) de un lecho biológico, se instaló un ensayo en modalidad destructiva durante 90 días. Se utilizaron vasos de plumavit (300 ml) con 100 g de biomezcla c/u y cuya característica se presenta en la Tabla 1. Se germinaron las semillas por dos semanas bajo cámara de crecimiento con temperatura de 20 ±1 ºC y un foto período de 17 h/día. Luego se raleó dejando 6 plántulas con características fenotípicas semejantes por vaso. Luego al día 15 se contaminan los vasos con 4 niveles de una mezcla de atrazina (ATZ), clorpirifos (CHL) e iprodiona (IPR) (0, 1, 5, 10 mg/l) dejando 3 vasos sin contaminar (blancos) para cada tiempo de muestreo. Al día 16 se tomaran 12 vasos de los cuales 3 son blanco (vasos sin contaminar) y 9 contaminados con la respectiva concentración de los plaguicidas y se realizan los análisis correspondientes. Se re-contaminan luego cada 15 días y se realiza toma de muestra antes y después de cada contaminación. Antes y después de cada aplicación, se realizan los análisis de fluoresceína diacetato (FDA), ureasa, peroxidasa, fosfatasa ácida, plaguicida residual y ácidos orgánicos exudados. 315 Session IV SIV-CP-20 Tabla 1. Características fisicoquímicas de la biomezcla preparada con suelo trumao (Andisol): Biomezcla pH CO (%) NTK (%) C/N 4,76 19,6 0,60 32,7 CO: carbono orgánico; NTK: nitrógeno total Kjeldahl RESULTADOS Y DISCUSIÓN La actividad ureasa no presentó diferencias significativas entre el control y las dosis de plaguicidas adicionados. Luego de la primera adición de los plaguicidas, la actividad ureasa disminuyó drásticamente, incrementando luego su actividad a partir del día 31 y hasta el día 59, luego de lo cual disminuyó nuevamente en todos los tratamientos utilizados. La actividad fosfatasa ácida no presentó diferencias significativas entre el control y las dosis de plaguicidas adicionados. La fosfatasa duplicó su actividad entre la primera y segunda contaminación, manteniéndose prácticamente sin modificar hasta el final del ensayo con valores entre 15 y 20 µg PNP/g, a pesar de las sucesivas contaminaciones con los plaguicidas. Respecto de la actividad de la FDA, ésta presentó un comportamiento oscilatorio en el tiempo, sin mostrar una directa con la concentración ni con la degradación de los plaguicidas y similar al observado en estudios anteriores (Tortella et al., 2012) lo cual puede deberse a que la FDA incluye un grupo de enzimas hidrolíticas las cuales son producidas por varios tipos de microorganismos. La actividad peroxidasa se mantiene similar hasta el día 44 del ensayo en valores de 0,4 a 0,6 U/g y si mostrar diferencias significativas entre los tratamientos luego de lo cual incrementa hasta valores de 1 a 1,2 U/g, debido a la mayor actividad de los hongos que se desarrollan favorecidos por la presencia de la paja. El ácido orgánico que se encontró en mayor concentración en la biomezcla corresponde a ácido succínico (5,689 mg/l), seguido de ácido cítrico (1,624 mg/l). Los ácidos málico y oxálico se encontraron en menor concentración (0,536 y 0,197, respectivamente). A modo general podemos señalar que los pesticidas extraídos en los exudados radicales incrementan durante el tiempo debido a las re-contaminaciones a las cuales fueron siendo sometidas las plantas y a la acumulación de pesticidas en la raíz de las plantas. En general, la iprodiona en las 3 concentraciones adicionadas (1, 5 y 10 mg/l) se presenta en mayor concentración en los exudados indicando su menor degradación. Estos resultados son concordantes con los resultados obtenidos de la degradación de iprodiona en la biomezcla sin la cubierta vegetal (Diez et al., 2013). Por otro lado, atrazina y clorpirifos presentan un nivel elevado de remoción. AGRADECIMIENTOS Este trabajo ha sido financiado por el proyecto Fondecyt 1120963. BIBLIOGRAFÍA Castillo M.d.P. et al. (2008). J. Agric. Food Chem. 56: 6206-6219. Diez, M.C. et al. (2013). J. Biobased Mat. and Bioenergy (en prensa). Tortella, G.R. et al. (2012). Biodegradation DOI 10.1007/s10532-013-9619-4. 316 Session V Phisiology and biochemistry of beneficial microorganisms and associated plants Session V SV-P-1 Fijación de nitrógeno en leguminosas en entornos variables: el bacteroide, el nódulo y la planta. Arrese Igor, C.* Universidad Pública de Navarra. Pamplona, España. * [email protected] 318 Session V SV-P-2 Molecular physiology of nickel and cobalt homeostasis in Rhizobium leguminosarum. Palacios, J.M.1*, Rubio-Sanz, L.2, Prieto, R.I.1, Menéndez-Cerón, M.1, Albareda, M.1, Clavijo, C.1, Imperial, J.1, 3, Mandrand-Berthelot, M.A.4, Rodrigue, A.4, Cacho, C.5, RuizArgüeso, T.1, Brito, B.1 1 CBGP and Departamento de Biotecnología, E.T.S. Ingenieros Agrónomos, Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón, 28223 Madrid, Spain. 2 Departamento de Química y Análisis Agrícola, E.U. Ingeniería Técnica Agrícola, Ciudad Universitaria s/n 28040. 3 C .S.I.C. Madrid. 4 Université Lyon-INSA-CNRS UMR5240, Lyon, France. 5 IHCP JCR, Ispra, Italy. * [email protected] INTRODUCTION Transition metals such as Fe, Cu, Mn, Ni, or Co are essential nutrients, as they are constitutive elements of a significant fraction of cell proteins. Such metals are present in the active site of many enzymes, and also participate as structural elements in different proteins. From a chemical point of view, metals have a defined order of affinity for binding, designated as the Irving-Williams series (Irving and Williams, 1948) Mg2+ < Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+>Zn2+ Since cells contain a high number of different proteins harbouring different metal ions, a simplistic model in which proteins are synthesized and metals imported into a “cytoplasmic soup” cannot explain the final product that we find in the cell. Instead we need to envisage a complex model in which specific ligands are present in definite amounts to leave the right amounts of available metals and protein binding sites, so specific pairs can bind appropriately. A critical control on the amount of ligands and metal present is exerted through specific metal-responsive regulators able to induce the synthesis of the right amount of ligands (essentially metal binding proteins), import and efflux proteins. These systems are adapted to establish the metal-protein equilibria compatible with the formation of the right metalloprotein complexes. Understanding this complex network of interactions is central to the understanding of metal metabolism for the synthesis of metalloenzymes, a key topic in the Rhizobium-legume symbiosis. In the case of the Rhizobium leguminosarum bv viciae (Rlv) UPM791 Pisum sativum symbiot ic system, the concentration of nickel in the plant nutrient solution is a limiting factor for hydrogenase expression, and provision of high amounts of this element to the plant nutrient solution is required to ensure optimal levels of enzyme synthesis (Brito et al., 1994). Usually, transition metals are present at low levels in natural habitats, and specific, high affinity transport systems have been developed by bacteria to import them from the soil solution. At the same time, these elements are toxic even at moderate concentrations, since they can catalyze the production of free radicals, or compete with other metals for the active site of metalloenzymes. In this talk we will present our recent work on the mechanisms involved in the homeostasis of transition metals nickel and cobalt in free living and symbiotic cells of the endosymbiotic bacterium Rhizobium leguminosarum. 319 Session V SV-P-2 Bacteroid Ni HupE Ni-Malate Ni Ni HupE2 Ni Ni-Citrate Ni ? NiFe Hydrogenase Ni Ni Ni-X Ni Ni dmeRF ? ? Ni Ni Ni Figure 1. Nickel uptake and efflux systems involved in nickel homeostasis in the Rhizobium leguminosarum-Pisum sativum symbiosis. Nickel complexes in the nodule cytosol. A first stage to be considered in the supply of metals to the bacteroid in legume nodules is the chemical species present in the nodule cytosol surrounding the symbiosome. Analysis of the chemical nature of nickel complexes has been made possible through the development of a new analytical methodology for the direct identification and quantification of nickel complexes in extracts from nodule cytosol (Cacho et al., 2010), a useful tool to study the mechanism involved in the nickel provision for endosymbiotic bacterial cells. The use of this methodology has revealed that nickel species vary in different legume plants. Pea nodules contain similar levels of nickel malate and nickel citrate, whereas most nickel is chelated with citrate in lentil nodules. Other legumes such as bean or soybean contain different balances of nickel chelates. The binding affinity of the metal ions for the different organic ligands might influence the capacity of the metal to be detached from the chelate, or to be transported as a chelate across the different membranes. Nickel complexes found in plant cytoplasm has been used as substrates for experiments of hydrogenase induction of wild-type and hupE/hupE2 mutants to ascertain the actual role of such compounds in the provision of this metal for metalloenzyme synthesis under controlled conditions. At this point, further characterization of potential metal- or chelate- transporters through the plantderived per ibacteroid membrane is required to ascertain the actual roles of metal complexes in the provision of nickel to the bacteroid. Nickel transport through bacterial outer membrane. Once inside the symbiosome, and before crossing the cytoplasmic membrane, metals must cross the bacterial outer membrane (OM). Whereas small molecules (<600 Da) can cross this membrane by passive diffusion through porins when they are moderately concentrated in the environment, larger molecules and molecules present at low concentration require energy-dependent systems. The OM does not maintain a proton gradient nor ATP synthesis, and these energy requirements can be fulfilled by TonBdependent transp ort systems, in which a protein complex (TonB-ExbB-ExbD) spanning the periplasmic space is able to transduce proton-motive-force from cytoplasmic membrane to power up transport in the outer membrane (Postle and Larsen, 2007). Although most TonB-dependent transporter (TBDT)-like proteins are predicted to transport iron compounds (siderophores or heme) or cobalamine, recent reports indicate that this kind of receptors are also involved in the uptake of other metals, such as nickel. In the case of endosymbiotic bacteria, a TonB-dependent transporter (TBDT) involved in nickel uptake has been found adjacent to hydrogenase genes in the genome of B. 320 Session V SV-P-2 japonicum (Schauer et al., 2008). This gene has been cloned and transferred into R. leguminosarum, and its role on the import of nickel ions for hydrogenase synthesis in this bacterial species is being analyzed. Nickel uptake through cytoplasmic membrane. Once in the periplasm, metal ions must cross the cytoplasmic membrane, the main permeability barrier for metal uptake. Two proteins (HupE and HupE2) of the HupE/UreJ family are the main nickel transporters through cytoplasmic membrane in Rlv UPM791 (Brito et al., 2010). These transporters are integral membrane proteins with six predicted transmembrane domains that work as single-component permeases. Mutant analysis demonstrated the essentiality of these proteins for the synthesis of active R. leguminosarum NiFe hydrogenase under nickel limitation both in free-living culture and in symbiosis (Brito et al. 2010). Hydrogenase activity determinations and Ni63 transport assays demonstrated that HupE is a nickel transporter that provides this metal for the synthesis of hydrogenase in Rlv UPM791 free-living cells and lentil bacteroids. Site directed mutagenesis has allowed the identification of two histidine-rich domains essential for Ni2+ transport. The characterization of kinetic and functional properties of Rlv UPM791 HupE is being carried out through heterologous expression of HupE in a nik-deficient Escherichia coli background. The specificity of this transporter for Ni2+ is being determined in competition assays with other divalent metal ions. A HupE variant lacking the N-terminal periplasmic region of the mature protein has been constructed to ascertain the potential role of this domain in ion scavenging and concentration around the transporter. The relevance of other residues conserved in members of the HupE/UreJ family is being tested through the analysis of the corresponding site-specific mutants. The results of the on-going work will be presented and discussed. Figure 2. Topological model proposed for the R. leguminosarum nickel transporter HupE. Histidine residues are highlighted, and histidine-rich motifs are indicated on the top (adapted from Brito et al., 2010). Metal efflux systems. The commonest mechanism for metal resistance in living cells is metal efflux. Efflux systems described in different bacteria include members of the Resistance-Nodulationcell Division (RND) protein family, P-type ATPases, cation diffussion facilitators, and other resistance factors (Nies, 2003). In order to have an idea of the metal resistance systems present in endosymbiotic bacteria we have carried out g enomic and physiological analyses of R. leguminosarum strains isolated from ultramafic soils. 321 Session V SV-P-2 Transposon mutagenesis of the highly resistant R. leguminosarum strain UPM1137 has led to the identification of potential metal efflux determinants conferring resistance to high metal levels under free-living and symbiotic conditions. A metal-inducible resistance mechanism involving a member of the cation diffusion facilitator (CDF) family has been studied in detail. Phenotypic characterization of a deletion mutant has shown that this protein acts as a key component for cobalt detoxification in R. leguminosarum. The gene encoding this CDF forms an operon with a nickel-and cobaltresponsive metal loregulator of the RcnR-type. Highly similar operons have been identified in other endosymbiotic bacteria, suggesting that metal-inducible, RcnRregulated CDF sys tems constitute a widespread strategy for transition metal detoxification in this group of bacteria. The characterization of such resistance systems will allow a better understanding on how endosymbiotic bacteria regulate intracellular metal levels, a central aspect of metal homeostasis in this relevant group of bacteria. ACKNOWLEDGEMENTS This work has been funded by Spain’s MICINN (project BIO2010-15301 to J.P.), Comunidad Autónoma de Madrid (Microambiente-CM S2009/AMB-1511 to T.R.A), Universidad Politécnica de Madrid (AL11P(I+D )-4 to B.B.), and by France’s PICS project (SYMBIONI n°5992 to A.R. and MA. M-B.). REFERENCES Brito, B., et al. (1994). J. Bacteriol. 176: 5297-5303. Brito, B., et al. (2010). J. Bacteriol. 192: 925-935. Cacho, C., et al. (2010). Talanta 83: 78-83. Irving, H., and Williams, R.J.P. (1948). Nature 162: 746-747. Nies, D.H. (2003). FEMS Microbiol. Rev. 27: 313-339. Postle, K., and Larsen, R.A. (2007). Biometals 20:453-465. Schauer, K., et al. (2008). Trends Biochem. Sci. 33: 330-338. 322 Session V SV-CO-1 Engineering a hydrogen biosensor in the nitrogen-fixing strain Rhodobacter capsulatus. Gónzalez de Heredia, E.M.*, Barahona, E., Jiménez-Vicente, E., Echavarri-Erasun, C., Rubio, L.M. Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón 28223, Madrid. * [email protected] ABSTRACT R.capsulatus has a system to regulate the expression of its uptake hydrogenase in response to H2. We are studying this system and using some of its elements to generate a H2 biosensor: a R. capsulatus strain able to detect presence of H2 yielding a response of our choice. INTRODUCTION Numerous microorganisms can produce H2 by reactions related to their metabolism and most of them are also able to consume it. Hydrogenase enzymes (metalloproteins that contain iron-sulfur clusters and Fe-Ni or Fe-Fe binuclear active sites) catalyze the reaction: 2H+ + 2e- Ö H2. [NiFe]-H2ases have been thoroughly studied in Bacteria and, in some cases, their expression is regulated by the so-called soluble sensor hydrogenases. In addition, nitrogenase enzymes produce H 2 when reducing N2 into NH3. The nitrogen-fixing bacterium R. capsulatus carries nitrogenase and hydrogenase enzymes able to produce H2. It also carries a system to detect H2 that is composed of three proteins: a H2-sensor hydrogenase (HupUV), a histidine kinase (HupT) and a response regulator (NtrC-like transcription factor, HupR) (Vignais et al., 2005). In the presence of H2, this sensor triggers expression of hydrogenase structural and biosynthetic genes. The aim of this work is to construct a tool relying on this complex regulatory signal cascade that will transduce H2 levels into a signal of our choice. A reporter gene has been introduced in a wild-type strain of R. capsulatus, so that it is expressed in presence of H2. This biosensor will allow us to screen or select for randomly generated nitrogenase mutants with increased H2-producing activity. MATERIAL AND METHODS The strain used in this study is R. capsulatus SB1003. This strain was cultured under aerobic (synthetic air) or anaerobic conditions (N 2) and in the presence or absence of 6.5% H2 in the gas phase. Chromosomal mutations were generated by simple or double recombination using pVIK112 (Kalogeraki and Winans, 1997) or pK18mobsac plasmids (Kirchner and Tauch, 2003) -galactosidase activity assays were carried out as described by Miller (1972). RNA from cultures of exponentially growing cells (OD of 0.8) was extracted by using TRizol and chloroform. cDNA was obtained using High Capacity cDNA Reverse Transcription kit using above RNA as template (Applied Biosystem). Expression analyses were performed on cDNA samples by RT-qPCR. RESULTS AND DICUSSION Detection and analysis of hupS promoter. The expression of structural genes for the uptake hydrogenase is activated when the phosphorylated form of the transcriptional regulator HupR binds to the promoter of hupS (small subunit of uptake hydrogenase). A putative promoter sequence upstream of hupS was cloned into the lacZ-containing vector pMP220 (Spaink et al., 1987), and this construct was introduced into R. capsulatus to generate R. capsulatus pMP220::PhupS 323 Session V SV-CO-1 strain. Both strains, R. capsulatus pMP220 (wild type) and R. capsulatuspMP220::PhupS were cultured in PY medium under aerobic conditions and in absence or the presence of 6.5 % H2. In absence of externally-added H2, galactosidase activity was higher in R. capsulatus pMP220::PhupS than in wild type (800 vs. 17.5 Miller units, respectively). Furthermore, in presence of 6.5 % H 2 strain pMP220::PhupS exhibited 1450 Miller units compared to 56 units in the wild-type strain. This result shows that the PhupS region is indeed a promoter and that it is activated by H2. Constructing and testing the “hydrogen sensor”. The regulatory effect of H2 in the expression of hydrogenase structural genes was analyzed by RT-qPCR. R. capsulatus was cultured in the absence or the presence of 6.5 % H2. Total RNA was extracted, and hupR and hupS expression was analyzed by RTqPCR. hupR and hupS expression was ca. 10 times higher in the presence of H 2 than in its absence. The lacZ expressing H2 sensor was constructed into the chromosome of R. capsulatus. A recombinant suicide plasmid, fusing PhupS to the lacZ reporter (pVIK112::PhupS::lacZ), was constructed and introduced into R. capsulatus wild-type strain by biparental matting to generate strain PhupS::lacZ, which contains the lacZ reporter gen inserted into the chromosome. R. capsulatus wild-type and PhupS::lacZ strains were cultured under the same conditions for 7 hours and their -galactosidase activities determined. In the PhupS::lacZ strain activities were found to be higher in the presence of H2, confirming functionality of our H2 sensor tool in a stable chromosomal location. The H2 sensor strain will be used to screen or select clones expressing randomly generated nitrogenase variants able to produce high levels of H 2 into the cell (see Barahona E. et al. communication: Towards the optimization of hydrogen production by nitrogenase: In vitro directed evolution of Rhodobacter capsulatus molybdenum nitrogenase). REFERENCES Adams, M.W.W., et al. (1981). Biochim. Biophys. Acta. 594:105-176. Vignais, P.M., et al. (2005). Biochem. Soc. Trans. 33: 28-32 Kalogeraki, V.S., and Winans, S.C. (1997). Gene. 188: 69-75. Kirchner, O., and Tauch, A. (2003). J. Biotechnol. 104: 287-299 Miller, J.H. (1972). Cold Spring Harbor, N.Y. Spaink, H.P., et al. (1987). Plant Mol. Biol. 9: 27-39. 324 Session V SV-CO-2 MtNramp1 mediates iron import in rhizobia-infected Medicago truncatula cells. Rodríguez-Haas, B.1, Finney, L.2, Kryvoruchko, I.3, González-Melendi, P.1, Udvardi, M.3, Imperial, J.1, 4, González-Guerrero, M.1* 1 Centro de Biotecnología y Genómica de Plantas (CBGP) Universidad Politécnica de Madrid. Campus de Montegancedo, Pozuelo de Alarcón, 28223 Madrid, España. 2 Biosciences Division. Advanced Photon Source, Argonne National Laboratory Argonne, Illinois, Estados Unidos de América. 3 The Samuel Roberts Noble Foundation, Plant Biology Division, Ardmore, Oklahoma, 73401, USA. 4 Consejo Superior de Investigaciones Científicas (CSIC). Madrid, España. * [email protected] ABSTRACT Symbiotic nitrogen fixation is a process that requires relatively high quantities of iron provided by the host legume. Using synchrotron-based X-ray fluorescence, we have determined that this iron is released from the vasculature into the apoplast of zone II of M. truncatula nodules. This overlaps with the distribution of MtNramp1, a plasma membrane iron importer. The importance of MtNramp1 in iron transport for nitrogen fixation is indicated by the 60% reduction of nitrogenase activity observed in knockdown lines, mo st likely due to deficient incorporation of this essential metal cofactor at the necessary levels. INTRODUCTION Iron is an essential growth-limiting micronutrient for plants (Grotz and Guerinot, 2002). Most of the proteins (nitrogenase, leghemoglobin, etc…) involved in symbiotic nitrogen fixation require iron cofactors (Udvardi and Day, 1997). In spite of this very little is known about how iron reaches the nodule and how it is incorporated by rhizobiacontaining cells. He re we show that iron is released in the infection/maturation areas of indeterminate nodules, and that a Nramp transporter introduces it into the infected cells. MATERIAL AND METHODS Synchrotron-based X-ray fluorescence X-ray fluorescence assays were performed as described by Rodríguez-Haas et al (2013). Plant transformation. M. truncatula plants were transformed with Agrobacterium rhizogenes as indicated (Boisson-Dernier et al., 2001). MtNramp1 localization. Promoter:GUS and confocal immunofluorescence studies were performed as described (Limpens et al., 2009). Generation of knock-down plants The first 450 bp of MtNramp1 cDNA sequence were cloned in pENTR1A and transferred using Gateway Technology to pFRN. Nitrogenase activity measurements. Nitrogenase activity was measured as described (Ruíz-Argüeso et al., 1979). RESULTS AND DISCUSSION There are three possible mechanisms for iron transfer to the nodule: i) increased uptake from the epidermis, ii) use of accumulated stores from the nodule primordia, and iii) long-distance transport from the vasculature. These possibilities would present different iron distribution patterns, and these could be determined by means of synchrotron-based 325 Session V SV-CO-2 X-ray fluorescence. The data showed that iron is transported by the vasculature and accumulates in the apoplast of the zone II of the nodule (Figure 1A). This also indicates that plasma membrane iron importers must be present in these cells to introduce apoplastic iron into the cytosol. In a previous microarray analysis we identified MtNramp1 as a metal transporter gene induced more than 7-fold by nodulation. This result was verified by qPCR. MtNramp1 was able to complement the phenotype of the fet3/fet4 yeast mutant that has a reduced capability of incorporating iron from the medium. Promoter:GUS studies of MtNramp indicated that it is expressed in the apical region of the nodule, coincidental with zone II, where iron is being released into the apoplast. This was verified with immunolocalization of HA-tagged MtNramp1 using confocal microscopy (Figure 1B). It appeared that MtNramp1 is localized in the plasma membrane of cells of nodule zone II. All these results suggest that MtNramp1 could be the transporter that introduces apoplastic iron into the infected cells that would be used for nitrogen fixation. This was tested by obtaining knock-down lines and determining the nitrogenase activity of their nodules, since nitrogenase is one of the most abundant iron proteins in the nodule. On average, a 90 % reduction of expression levels was achieved that caused a 60 % reduction of nitrogenase activity (Figure 1C), supporting a role of MtNramp1 in symbiotic iron transport. B Zone III C 100 % Nitrogenase activity A Zone II Zone I 80 60 40 20 WT Knock-down Figure 1. Iron transport in M. truncatula nodules. (A) Elemental distribution in Zone II. Iron is in green, zinc in blue and calcium in red. (B) Immunolocalization of MtNramp1. Blue is DAPI stained DNA, gre en indicates GFP expressing rhizobia and red is MtNramp1-HA labelled with anti-HA mouse antibody conjugated to Alexa594. (C) Nitrogenase activity in wild type and MtNramp1 knock-down nodules. ACKNOWLEDGMENTS This work was supported by the Ramón y Cajal Fellowship RYC-2010-06363, and the Marie Curie International Reintegration Grant MENOMED (all to MGG). Use of the Advanced Photon Source was supported by the U.S. Department of Energy under contract number DE-AC02-06CH11357. REFERENCES Grotz, N., and Guerinot, M.L. (2002). Curr. Opin. Plant Biol. 5: 158-163. Udvardi, M.K., and Day, D.A. (1997). Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 493-523. Rodríguez-Haas, B., et al. (2013). Metallomics doi 10.1039/c3mt00060e Boisson-Dernier, A., et al. (2001). Mol. Plant Microbe Interact. 14: 695-700. Limpens, E., et al. (2009). Plant Cell 21: 2811-2828. Ruíz-Argüeso, T., et al. (1979). Biochem. Biophys. Res. Comm. 86: 259-264. 326 Session V SV-CO-3 Selección de líneas de garbanzo tolerantes a la salinidad empleando caracteres de producción de biomasa y funcionamiento nodular como indicadores. Gómez, L.A.1*, Vadez, V.2, Vaillhe, H.3, Pernot, C.3, Drevon, J.J.3 1 Instituto de Suelos. Autopista, Costa-Costa y Antigua Carretera de Vento, Capdevila, Boyeros. La Habana, Cuba. 2 Instituto Internacional de Investigaciones de Cultivo para el Trópico Semiárido. Patancheru, Hyderabad 502 324, Andhra Pradesh, India. 3 Instituto Nacional de Investigaciones Agronómicas, INRA, Place Viala F-34060, Montpellier, France. * [email protected] RESUMEN Se estudió la tolerancia a la salinidad de 30 líneas cultivadas en solución nutritiva, evaluando nodulación, conductancia nodular al O2 y biomasa. En condiciones no salinas las plantas mostraron alta nodulación y crecimiento, mientras que la salinidad (25 mM NaCl), disminuyó significativamente estos parámetros. La sensibilidad a la sal determinada como crecimiento foliar, y nodular relativo y eficiencia de uso de la simbiosis con Mesorhizobium mostró importantes diferencias entre líneas, las que se agruparon en 4 clases. Se concluyó que la tolerancia a la salinidad está relacionada con alto potencial simbiótico y elevada y estable conductancia nodular al O2. INTRODUCCIÓN. La salinidad afecta cerca de 80 millones de ha de suelos cultivable en todo el mundo un factor que ejerce un impacto negativo sobre la fijación simbiótica del nitrógeno (FSN) y el rendimiento de grano de plantas de garbanzo (Cicer arietinum L.) en interacción con Mesorhizobium, dado la particular sensibilidad de esta simbiosis a la salinidad (Flowers et al., 2010). Una alternativa para resolver este problema pudiera ser la selección de líneas resistentes. El presente trabajo evalúa la tolerancia a la salinidad de 30 líneas inoculadas con la cepa de Mesorhizobium ciceri UPMCa 7. MATERIAL Y MÉTODOS Se evaluaron 30 líneas (ICCV2; ICCV10; ICC67; ICC867; ICC1431; ICC1915; ICC2580; ICC3946; ICC4495; ICC4593; ICC5003; ICC5337; ICC6263; ICC6306; ICC8058; ICC8522; ICC8950; ICC9942; ICC10885; ICC11121; ICC12155; ICC13357; ICC15518; ICC15610; ICC96029; JG11; CSG8962; ICC4973 (L550); INRAT 93.1 y Amdoun, Vadez et al., 2007), cultivadas en solución nutritiva (Drevon et al., 1988), a dos niveles de salinidad (0 y 25 mM NaCl). Se evaluó la conductancia nodular al O2 (CNO), biomasa foliar (BF), número de nódulos (NN), peso individual de los nódulos (PIN), biomasa de nódulos (BN), relaciones entre órganos, Crecimiento Relativo foliar y nodular relativo y Eficiencia en la Utilización de la Simbiosis con Mesorhizobium (EUSM), según L’Taief et al., 2007. Los datos se procesaron para componentes principales (ACP) y dendograma (XLSTAT, Addinsoft SARL, Paris, Francia, 2010). RESULTADOS Y DISCUSIÓN En condiciones no salinas las plantas exhibieron alta nodulación y crecimiento (Tabla 1), mientras bajo salinidad se afecto la BN (11 – 89 %); el NN (12 – 83 %), y la BF (2589 %), las raíces se afectaron solo en el 50 % de las líneas. El ACP en el tratamiento no salino evidencio que F1, F2 y F3 fueron vectores de “distribución de biomasa”, “nodulación y crecimiento” y “crecimiento individual de los nódulos” respectivamente, en cambio en condiciones salinas F1, F2 y F3 fueron considerados vectores de “EUSM”, “nodulación” y “crecimiento Foliar y Nodular relativo” respectivamente. Los índices CFR, CNR y EUSM variaron considerablemente entre genotipos los que se agruparon en 4 clases y 8 sub-clases de acuerdo a su tolerancia a la salinidad (Tabla 1). 327 Session V SV-CO-3 A 0 mM NaCl las líneas representativas de las clases 1, 2, 3 y 4 fueron ICC4593, ICC13352, ICC10885 e ICC8522 respectivamente, las clases 3 y 4 agruparon las líneas con más alta nodulación y crecimiento (Tabla 1). En condiciones salinas las líneas representativas de las clases/ sub-clases 1A, 1B, 1C, 2A, 2B, 2C, 3 y 4 fueron CSG 8962, ICC6306, ICC12155, ICC15610, ICC3946, INRAT 93-1, ICC8950 y Amdoun respectivamente, la clase 3 agrupó las líneas más tolerantes (Tabla 1). Las líneas ICC8950, ICC1431, ICC4495, Amdoun, ICC4973 e ICC5337 fueron consideradas tolerantes, mientras ICCV10, ICC13357 e ICC1915 sensibles. Tabla 1. Clases/sub-clases resultado del análisis de dendograma para disimilitudes fenotípicas líneas cultivadas a 0 and 25 mM de NaCl. Clases BF BN NN PIN BR CFR CNR /sub-classes g planta-1 g planta-1 Numero x planta-1 mg nodulo-1 g planta-1 0 mM 1 3.60 0.355 212 1.86 0.46 2 3.20 0.606 458 1.44 0.51 3 5.85 0.529 646 0.82 0.81 4 7.37 0.785 984 0.79 0.91 25 mM NaCl 1A 0.62 0.058 56 1.04 0.14 30.1 39.7 1B 1.55 0.186 132 1.51 0.34 36.4 43.6 1C 1.24 0.120 108 1.38 0.20 92.4 48.0 2A 2.11 0.239 212 1.21 0.45 59.6 72.7 2B 2.62 0.210 225 0.93 0.52 89.4 34.4 2C 1.81 0.154 196 0.85 0.36 37.0 26.4 3 2.03 0.201 138 1.50 0.27 91.9 122.0 4 3.36 0.366 322 1.17 0.56 54.9 49.5 en 30 EUSM 3.12 5.74 6.7 7.1 11.4 8.7 8.3 8.3 La pO2 crítica asociada al metabolismo nodular mostró en el tratamiento no salino un valor medio de 40 % y no varió por influencia de la sal. La pendiente de la relación consumo de O2 y pO2 multiplicado por el área nodular (cm2 g-1 nódulo) que representa la conductancia nodular al O2 (CN0) incremento significativamente (p<0.001) solo en las líneas sensibles, por lo tanto el incremento en la CNO pudiera ser un mecanismo de adaptación en respuesta a la demanda de N de la planta y en compensación a la disminución de la nodulación por estrés salino, pero también por el alto costo de la respiración de la FSN (Jebara y Drevon, 2001). Se concluyó que la tolerancia del garbanzo a la salinidad está relacionada en parte a la capacidad de mantener alto potencial simbiótico bajo condiciones salinas, el cultivar ideal parece ser el que exhibe un gran número de pequeños nódulos y una elevada y estable CNO. BIBLIOGRAFÍA Drevon, J.J., et al. (1988). Plant Physiol. Bioch. 26: 73-78. Flowers, T.J., et al. (2010). Plant. Cell. Environ. 33: 490-509. L´Taief, B., et al. (2007). J. Plant Physiol. 164: 1028-1036. Vadez, V., et al. (2007). Field. Crop. Res. 104: 123-129. 328 Session V SV-CO-4 Estrés abitotico induce cambios en las señales de comunicación y en la interacción temprana entre maní y rizobacterias. Cesari, A., Paulucci, N., García, M., Dardanelli, M.S. * Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Argentina. * [email protected] ABSTRACT En este trabajo mostramos como el estrés hídrico (PEG6000-0,28MPa) es capaz de inducir cambios en la composición de lípidos de rizobacterias y en rizodeposiciones de maní, importantes para la señalización rizosférica, en la movilidad bacteriana y de relevancia en los eventos tempranos de interacción. INTRODUCCIÓN La diversidad estructural de moléculas producidas por las leguminosas al igual que las rizobacterias con capacidad de asociarse con ellas, es vasta. Proponemos examinar cómo el perfil de lípidos de rizobacterias y de rizodeposiciones de maní (señal rizosférica), así como la movilidad de rizobacterias (evento temprano de interacción) que se asocian a maní, cambia frente estrés hídrico (PEG6000 -0,28MPa). Es relevante el papel que proteínas, lípidos, flavonoides, ácidos orgánicos, entre otros, juegan en la tolerancia a condiciones de estrés abiótico y cambios ambientales pueden comprometer la eficacia del diálogo molecular entre ambos socios, alterándose consecuentemente el desarrollo de diversos eventos como por ejemplo la movilidad, la adhesión, la colonización y la nodulación (Dardanelli et al., 2009; Guasch-Vidal et al., 2013). MATERIAL Y MÉTODOS Recolección y análisis de rizodeposiciones: plantas de 7 días de crecimiento en solución Hoagland (PEG6000 -0,28MPa) fueron cosechadas y las rizodeposiciones fueron empleadas en los estudios (Dardanelli et al., 2008). Composición de ácidos grasos (AG) de rizodeposiciones y de rizobacterias: el análisis de los esteres metílico se realizó a partir de los lípidos totales, por cromatografia gaseosa (GC) según Paulucci et al. (2013). Identificación y cuantificación de fosfolípidos de rizobacterias: mediante TLC, empleando Acetato de sodio [1-14C], agregado al medio de cultivo al momento de la inoculación se realizó el estudio de estas moléculas (Paulucci et al., 2013). Estudios de movilidad swarming y swimming de rizobacterias: fue llevado a cabo tal como se describe en Albareda et al. (2006) y Medeot et al. (2010). RESULTADOS Y DISCUSIÓN Efecto del estrés hídrico sobre lípidos de rizodeposiciones de Arachis hypogaea Las plantas de maní fueron sometidas a un gradiente de PEG6000, determinando que el efecto significativo ocurre a un potencial de -0,28MPa (estrés moderado). La composición de AG de las rizodeposiciones muestra que existen cambios con disminución de ácidos palmítico, palmitoleico y esteárico, mientras que se observó un incremento de oleico y otras moléculas no identificadas (NI) de más de 20 carbonos. Efecto del estrés hídrico sobre lípidos de las rizobacterias asociadas a Arachis hypogaea Estudios realizados con PEG6000 sobre las rizobacterias Azospirillum brasilense AZ39 y Bradyrhizobium sp. SEMIA6144 indicaron una mayor tolerancia por parte de los 329 Session V SV-CO-4 microorganismos en comparación con el vegetal. Sin embargo dentro de las bacterias empleadas, AZ39 mostró ser mayor tolerante al efecto de PEG6000 que SEMIA6144. En la condición de estrés inducido con PEG6000 -0,28MPa (15mM), AZ39 presentó una disminución de la biomasa húmeda en un 34% con respecto al control cuando alcanza la fase estacionaria, mientras que SEMIA 6144 disminuyó un 48%. El perfil de AG de AZ39, se modificó mostrando incrementos significativos a nivel de palmítico, esteárico y otros NI de mas de 20C, y una disminución del AG oleico, mientras que SEMIA6144 incrementó el AG cis-vaccenico, y disminuyó el palmítico, palmitoleico y esteárico frente al estrés hídrico. Estudios de TLC revelaron que los principales fosfolípidos en AZ 39 son fosfatidilcolina (PC), fosfatidiletanolamina (PE) y fosfatidilglicerol (PG) y cardiolipina (Cd) y en estrés se incrementó PC y disminuyó PE y Cd. Efecto del estrés hídrico sobre la movilidad de rizobacterias que se asocian a maní Con respecto a la movilidad, se evaluó swarming y swimming de bacterias crecidas en condiciones de estrés hídrico, observando un incremento de los dos tipos de movilidad cuando las bacterias estuvieron sometidas a dicha condición experimental (Figura 1 paneles A y B). Estos son los primeros reportes de movilidad en estas condiciones experimentales para rizobacterias. A B Figura 1. Swarming (Agar-agua 0.5%) y Swimming (Agar-agua 0.3%) en condición control y estrés PEG6000 -0,28 MPa (15mM). Paneles A AZ39 y B SEMIA 6144. AGRADECIMIENTOS Este trabajo fue financiado por Proyecto PIP CONICET y SECyT UNRC. BIBLIOGRAFÍA Albareda, M., et al. (2006). FEMS Microbiol. Lett. 259: 67-73. Dardanelli, M., et al. (2008). Soil Biol. Biochem. 40: 2713-2721. Dardanelli, M., et al. (2009). Symbiosis 40: 175-180. Guasch-Vidal, B,. et al. (2013). Mol. Plant Microbe. Interact. 26: 451-460. Medeot, D., et al. (2010). FEMS Microbiol. Lett. 303: 123-131. Pauluci, N., et al. (2013). J Appl. Microbiol. 114: 1457-1467. 330 Session V SV-CP-01 Movilidad en cepas de rizobios nodulantes de maní. Vicario, J.C.*, Dardanelli, M.S., Giordano, W.F. Departamento de Biología Molecular, FCEFQyN, Universidad de Río Cuarto. Córdoba. Argentina. * [email protected] RESUMEN La movilidad es una característica de los rizobios que puede contribuir en el éxito de la competitividad simbiótica de estas bacterias (Ames et al., 1981). En este trabajo se evaluó la capacidad de movilidad de cepas nodulantes de maní en diferentes condiciones y se realizaron estudios de morfología por SEM. El comportamiento mostró no ser homogéneo en las condiciones ensayadas y se observó un aumento significativo en el largo de las células swarmer en comparación con las vegetativas. INTRODUCCIÓN Se han identificado en bacterias seis categorías de movilidad: swimming, swarming, twitching, gliding, sliding y darting (Henrichsen, 1972). Los movimientos de mayor importancia en el suelo son los de tipo swimming, el cual puede también ocurrir en medio de cultivo con baja concentración de agar (0,2-0,4%). En contraste al movimiento bacteriano individual que ocurre en el swimming, el swarming es considerado un comportamiento bacteriano grupal asociado con migración en superficie semisólidas (Williams y Schwarzhoff, 1972). Un mayor número de flagelos son requeridos para el swarming, lo cual puede deberse a una mayor fricción de la colonia con el medio (Harshey, 2003). Cuando las células son inoculadas en la superficie de un medio agar, se forman colonias regulares en el punto de inoculación. Luego las células inician un proceso de diferenciación en células largas multinucleadas e hiper flageladas llamadas células swarm. OBJETIVO Evaluar la movilidad bacteriana de cepas simbiontes de maní, determinándose si la misma puede ser el condicionante para una respuesta efectiva cuando la bacteria se inocula en la semilla. MATERIAL Y MÉTODOS Se realizaron ensayos de movilidad para las cepas recomendadas de Bradyrhizobium C145, SEMIA6144, USDA4438 y las nativas 15A y Pc34 aisladas de nódulos radicales de maní cultivados en el sur de Córdoba (Bogino et al., 2010) La concentración de agar para todos los ensayos de swimming fue de 0,3% y para todos los ensayos de swarming fue de 0,5%. La movilidad fue determinada midiendo el diámetro del halo. Los ensayos anteriormente descriptos, fueron modificados para realizar un estudio de movilidad en un gradiente de agar, fuentes