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Universidad Nacional Agraria La Molina UNIVERSIDAD NACIONAL AGRARIA LA MOLINA FACULTAD DE CIENCIAS “Estudio de la diversidad de cepas de Rhizobium provenientes de nódulos de tres variedades de frijol común (Phaseolus vulgaris L.)” Presentado por: Erika Yovana Gonzales Medina Tesis para Optar el Título Profesional de: BIÓLOGO LIMA-PERÚ 2013 1 DEDICATORIA A mi mami Rosaura, a mi papá Carlos y a mis hermanos Rosa y Carlos por su gran apoyo, consejos y palabras de ánimo para la realización de la tesis. 4 AGRADECIMIENTOS Al equipo de Investigación del Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso, en especial a Dra. Doris Zúñiga, por su constante apoyo, consejos y sugerencias durante la realización del trabajo de investigación. A mis compañeros del Laboratorio Katty Ogata Gutiérrez, Minoru Matsubara Bautista, Juan Mayo Hernández, Stefany Cépeda González, Natalia Kohashikahua Takaezu, con quienes compartíamos conocimientos y experiencias en las labores del laboratorio durante la realización de la tesis. A las autoridades de mi centro de trabajo el Centro de Estudios Preuniversitarios de la Universidad Nacional La Molina (CEPUNALM), al Director el Ing. Huanca Velarde por su apoyo y consejos. Además a mi Jefe Académico, el Mg Sc. Víctor Caro-Sánchez Benites por su gran comprensión. A todos mis compañeros de trabajo en especial al Prof. Santiago Ramirez Rivera, por su apoyo, comprensión, consejos, ánimos y palabras de aliento y al Ps. Inami Shimahukuro por sus recomendaciones y palabras motivadoras. A los miembros del jurado por el tiempo dedicado a la revisión de mi proyecto de tesis y borrador de tesis. Por sus recomendaciones y sugerencias para la realización del trabajo de investigación. Al Consejo Ciencia y Tecnología (CONCYTEC) por la oportunidad de apoyarme con el financiamiento de la tesis. Al Programa de Leguminosas para la obtención de las plántulas muestreadas y la semillas de frijol. 5 RESUMEN En la presente investigación se aislaron 23 cepas bacterianas, catorce cepas pertenecientes al género Rhizobium y nueve a Agrobacterium y/u otros géneros. Las cepas fueron aisladas de nódulos de tres variedades de frijol (Phaseolus vulgaris L.) de suelos de La Molina. En la caracterización fenotípica, el 64.35 % de las cepas crecieron a 37°C; el 57.1% de las cepas toleraron pH4 y el 87% de las cepas fueron afectadas a concentraciones superiores a 0.5% de NaCl. En el ensayo en plantas de frijol, el 84.6% de las cepas, formaron nódulos efectivos e infectivos en la variedad ´Canario Centenario´. La cepa 17, no noduladora, incrementó el peso seco de parte aérea (PSPA) en 38.24 % con respecto al N-, siendo esta diferencia la mayor estadísticamente significativa (p<0.05). En el análisis de agrupamiento integrado, se obtuvieron cuatro grupos: los Grupos I y III cada uno conformado por una cepa, el Grupo II diez cepas y el Grupo IV dos cepas. En el Grupo III, la cepa 17 destacó porque contribuyó significativamente al crecimiento de las plántulas de frijol y en el Grupo IV, las cepas 4 y 5 fueron tolerantes hasta 2% de salinidad. Todas las cepas tuvieron la capacidad de solubilizar fosfato, produjeron AIA y ninguna inhibió el crecimiento del hongo Macrophomina phaseolicola. Éstos resultados nos indican la diversidad de cepas de Rhizobium que nodulan frijol, y permite seleccionar aquellas con mejor potencial PGPR para posterior aplicación como bioinoculantes, que aportan nitrógeno al cultivo y no contaminan los suelos. Palabras clave: Rhizobium, Rizobacterias promotoras del crecimiento de plantas (PGPR), Phaseolus vulgaris 6 ABSTRACT In this investigation were isolated 23 bacterial strains, fourteen strains belonging to the genus Rhizobium and nine strains to Agrobacterium and / or other genres. The strains were isolated from nodules of three varieties of beans (Phaseolus vulgaris L.) in soils of La Molina. The phenotypic characterization, the 64.35% of strains grown at 37 ° C, 57.1% of the strains tolerated pH4 and 87% of the strains were affected at concentrations above 0.5% NaCl. In the test in bean plants, 84.6% of strains formed effective nodules and infective in the variety 'Canario Centenario'. Strain 17, not nodule, increased the dry weight of aerial part (PSPA) at 38.24% compared to the N-, the largest difference being statistically significant (p <0.05). In the integrated cluster analysis, we obtained four groups: Groups I and III each consisting of one strain, ten strains of Group II and Group IV two strains. In Group III, strain 17 out because it contributed significantly to the growth of bean seedlings and Group IV, 4 and 5 strains were tolerant to 2% salinity. All strains were able to solubilize phosphate produced AIA and inhibited the growth of the fungus Macrophomina phaseolicola. These results indicate the diversity of strains of Rhizobium nodulating of beans, and to select those with the best potential for further application as PGPR bioinoculants that add nitrogen to the crop and not contamite the soil. Keywords: Rhizobium, plant growth promoting rhizobacteria (PGPR), Phaseolus vulgaris 7 INDICE GENERAL INTRODUCCIÓN .................................................................................................................................... 14 II.OBJETIVOS ......................................................................................................................................... 16 2.1 OBJETIVO GENERAL......................................................................................................................... 16 2.2 OBJETIVOS ESPECÍFICOS ................................................................................................................... 16 III. REVISIÓN BIBLIOGRÁFICA ........................................................................................................ 17 3.1 EL CULTIVO DE FRIJOL ............................................................................................................... 17 3.1.1 TAXONOMÍA .................................................................................................................................. 17 3.1.2 ORIGEN .......................................................................................................................................... 17 3.1.3 VARIEDADES DE PHASEOLUS VULGARIS .......................................................................................... 18 3.1.4 DISTRIBUCIÓN............................................................................................................................... 18 3.1.5 EXIGENCIAS AMBIENTALES............................................................................................................ 18 3.1.6 PRODUCCIÓN ................................................................................................................................. 19 3.1.7 FERTILIZACIÓN DEL FRIJOL ............................................................................................................ 19 3.2. EL NITRÓGENO ............................................................................................................................. 20 3.2.1 DEMANDA DEL NITRÓGENO, CONTAMINACIÓN Y EFECTOS TÓXICOS EN EL HOMBRE ...................... 20 3.2.2 FIJACIÓN BIOLÓGICA DE NITRÓGENO (FBN) .............................................................................. 21 3.2.3 ASOCIACIONES SIMBIÓTICAS RHIZOBIUM LEGUMINOSAS Y NO LEGUMINOSAS ............................... 22 3.2.4 FUNCIONAMIENTO DEL NÓDULO .................................................................................................... 23 3.3 TAXONOMÍA DE RHIZOBIUM ..................................................................................................... 24 3.4 CARACTERÍSTICAS DE RHIZOBIUM ........................................................................................ 29 3.5.1 MÉTODOS CULTURALES......................................................................................................... 30 3.5.2 CRECIMIENTO DE RHIZOBIUM A DIFERENTES CONDICIONES AMBIENTALES ......... 31 3.6 RIZOBACTERIAS PROMOTORAS DE CRECIMIENTO VEGETAL (PGPR) ...................... 34 3.6.1 RIZOBIOS COMO PROMOTORES DE CRECIMIENTO VEGETAL ........................................................... 34 3.6.2 FITOHORMONAS ............................................................................................................................. 36 3.6.3 AUXINAS ....................................................................................................................................... 36 3.6.4 EL FÓSFORO .............................................................................................................................. 39 3.6.5 RHIZOBIUM COMO CONTROLADORES BIOLÓGICOS DE PATÓGENOS ........................... 42 3.7 INVESTIGACIONES DE RHIZOBIUM REALIZADAS EN PERÚ ............................................ 43 IV. MATERIALES Y MÉTODOS .......................................................................................................... 44 4.1 LUGAR ............................................................................................................................................... 44 4.2 MATERIAL BIOLÓGICO ............................................................................................................... 44 4.2.1 MATERIAL VEGETAL ...................................................................................................................... 44 4.2.2 MICROORGANISMOS .....................................................................................................................
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