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Tesis De Diploma Facultad de Ciencias Agropecuarias Departamento de Biología Carrera Licenciatura en Biología TESIS DE DIPLOMA Selección de cepas de actinomicetos para el control de Rhizoctonia solani Kühn, Sclerotium rolfsii Sacc., Macrophomina phaseolina (Tassi) Goid. y Sclerotinia sclerotiorum (Lib.) de Bary, en frijol común (Phaseolus vulgaris L.) Autora: Neisy Bárbara García Alvarez Santa Clara 2014 Facultad de Ciencias Agropecuarias Departamento de Biología Carrera Licenciatura en Biología TESIS DE DIPLOMA Selección de cepas de actinomicetos para el control de Rhizoctonia solani Kühn, Sclerotium rolfsii Sacc., Macrophomina phaseolina (Tassi) Goid. y Sclerotinia sclerotiorum (Lib.) de Bary, en frijol común (Phaseolus vulgaris L.) Autor: Neisy Bárbara García Alvarez Tutores: Dr. C. Ricardo Medina Marrero Email: [email protected] Dr. C. Alexander Bernal Cabrera Email: [email protected] Santa Clara 2014 Pensamiento “Durante centenares de miles de años, el hombre luchó para abrirse un lugar en la naturaleza. Por primera vez en la historia de nuestra especie, la situación se ha invertido y hoy es indispensable hacerle un lugar a la naturaleza en el mundo del hombre” Santiago Kovadloff DDeeddiiccaattoorriiaa Dedicatoria A mis abuelos, padres y hermanos AAggrraaddeecciimmiieennttooss Agradecimientos Agradecimientos: Quiero agradecerle a Dios por iluminarme el camino a seguir durante esta larga trayectoria. A mis tutores un inmenso agradecimiento por su orientación, colaboración, paciencia y disposición; por la confianza depositada en mi para el desarrollo de este trabajo y sobre todo por su apoyo. A Cupull, Marlen, Milagro, Micaela y Michel Leiva, gracias por la orientación y ayuda brindada; por enseñarme lo necesario para realizar este trabajo; por sus invaluables aportes y oportunos consejos, además de ofrecerme con humildad todos sus conocimientos siempre con buena disposición, amabilidad y cariño. Le quiero agradecer a mima que aunque ya no se encuentra entre nosotros siempre está presente en nuestros corazones, también a mi abuelito por nunca dejar de estar pendiente de nosotras y por sus sabios consejos. A mami y papacito por darnos la vida, guiarnos por el buen camino y nunca darse por vencidos con nosotras, a mi hermana por estar siempre a mi lado en el transcurso de la vida, a mi hermano por todo su amor y cariño. A C. Javier por sus consejos, ayuda incondicional, por ser mi centro de apoyo, alentarme, darme fuerzas para seguir hacia delante y brindarme todo su amor. A Fran A., Angelita, Nereida, Anay, Roque y Marilin por toda su ayuda brindada y porque estuvieron siempre apoyándome y pendientes de lo que necesitaba. A mis amigos de toda la vida Zahily, Dairy y Ediel que han recorrido toda la trayectoria estudiantil a mi lado brindándome su apoyo y cariño. A todas mis amistades que desde primer año estuvieron a mi lado día a día: Maireby, Beatriz, Heidy, Legnara, Denyer y Daykeni, gracias por compartir Agradecimientos conmigo este viaje, que como montaña rusa tuvo altas y bajas, pero lo importante fue que estábamos juntos en un solo vagón y nuestros esfuerzos unidos lo mantenían siempre en movimiento. De cada uno me llevo un hermoso recuerdo y la enseñanza de que diferentes formas de ser pueden formar un equipo íntegro y exitoso. Gracias por apoyarme, brindarme ánimo, consejos y una amistad que trascenderá por siempre en nuestras vidas. A Marlon y Franko, gracias por su amistad, por hacerme reír todos los días y pasar tantos momentos inolvidables juntos, porque con su alegría hacían que todo fuera más sencillo. A Daylon, Julio, Arturo, Yenisbel, Dania, Riskel, Yasenia y Dayana por siempre tenerme presente y brindarme su amistad. A mis compañeros de aula por enseñarme que en la unión esta la fuerza y por luchar juntos en cada momento de esta etapa inolvidable. A los profesores que con su amor, dedicación y comprensión contribuyeron de una forma u otra en mi formación estudiantil. En fin le quiero agradecer a todas esas bellas personas que me han brindado su amistad, amor y apoyo sincero e incondicional. Neisy Bárbara García Alvarez RReessuummeenn Resumen RESUMEN Una gran limitante en la germinación y desarrollo vegetativo de Phaseolus vulgaris L. es el ataque de hongos fitopatógenos que han inducido el uso desmedido de fungicidas tóxicos, los cuales necesitan ser suplantados por agentes biocontroladores novedosos tales como los actinomicetos. Se seleccionaron 69 cepas al azar, a partir de una colección de 700 cepas de actinomicetos pertenecientes al Departamento de Microbiología del Centro de Bioactivos Químicos, a las cuales se les determinó la actividad antagonista in vitro contra los hongos fitopatógenos del suelo Rhizoctonia solani Kühn, Sclerotium rolfsii Sacc., Sclerotinia sclerotiorum (Lib.) de Bary y Macrophomina phaseolina (Tassi) Goid. que afectan a Phaseolus vulgaris L.. Luego se realizó una selección de las cepas con mejor actividad para la evaluación de la actividad enzimática extracelular, capacidad solubilizadora de fosfatos, producción de ácido indolacético (AIA) y caracterización molecular. De las 69 cepas evaluadas para determinar la actividad antagonista, se obtuvo que 26 cepas presentaron antagonismo contra S. sclerotiorum, 2 contra S. rolfsii, 39 contra M. phaseolina y 11 contra R. solani. Las cepas que presentaron los mayores halos de inhibición contra S. sclerotiorum y R. solani fueron J11, J4 y EA2, y contra M. phaseolina la cepa EBa21. Todas las cepas seleccionadas presentaron actividad enzimática extracelular; destacándose la cepa EBa21 en la producción de quitinasas, las cepas EA2, J4 y L2 en la de celulasas y las cepas EA2 y B8 de proteasas. En la solubilización de fosfato sobresalieron las cepas B8 y EBa21 mientras que en la producción de AIA las cepas EBe3, EA2 y EBa21. La identificación de las cepas mediante la secuenciación del gen del ARNr 16S arrojó que todas tienen una homología del 99% con diferentes especies de Streptomyces. Por tanto, todas estas cepas que presentan las mejores propiedades son candidatas para el control de los hongos fitopatógenos y promover el crecimiento de Phaseolus vulgaris L. Palabras clave: ácido indolacético (AIA), actividad enzimática, antagonismo, fitopatógenos, solubilización de fosfatos. AAbbssttrraacctt Abstract ABSTRACT Phaseolus vulgaris L. germination and vegetative development is usually hampered by phytopathogenic fungi´s attack, which has induced the overuse of toxic fungicides. Consequently, these chemicals should be replaced by novel biocontrol agents such as Actinomycetes. A random selection of 69 strains from a 700 Actinomycetes strains culture collection of the Department of Microbiology of the Chemical Bioactive Center were used to determine the antagonistic activity against the soil phytopathogenic fungi Rhizoctonia solani, Sclerotium rolfsii Sacc., Sclerotinia sclerotiorum (Lib.) of Bary and Macrophomina phaseolina (Tassi) Goid., which impair Phaseolus vulgaris L. culture. Actinomycetes strains displaying the best antimicrobial activity were further selected to assess their extracellular enzymatic activities, phosphate solubilizing capacities, indolacetic acid (IAA) productions as well their molecular characterizations. From all tested strains, 26 had activity against S. sclerotiorum, 2 against S. rolfsii, 39 against M. phaseolina and 11 against R. solani. Strains J11, J4 and EA2 were the best against S. sclerotiorum and R. solani, while strain EBa21 was the best against M. phaseolina. All selected strains exhibited extracellular enzymatic activities with EBa21 being the best chitinase producing strain; EA2, J4 and L2 the best cellulase producing strains and EA2 and B8 the best protease producing strains. Likewise, strains B8 and EBa21 showed the best phosphate solubilizing features and strains EBe3, EA2 and EBa21 the best IAA producing capacities. 16S rRNA gene sequencing showed that all strains had a 99% homology to different Streptomyces species. Consequently, all these strains having the best properties are good candidates to control phytopathogenic fungi and promote the growth of Phaseolus vulgaris L. Key words: Indolacetic acid (IAA), enzymatic activity, antagonism, phytopathogenic fungi, phosphate solubilization. ÍÍnnddiiccee Índice Índice de Contenido 1 INTRODUCCIÓN ....................................................................................................... 1 2 REVISIÓN BIBLIOGRÁFICA .................................................................................... 4 2.1 CULTIVO DEL FRIJOL COMÚN (PHASEOLUS VULGARIS L.) ............................. 4 2.1.1 CARACTERÍSTICAS GENERALES DEL FRIJOL ............................................................... 4 2.1.2 PRINCIPALES AGENTES FITOPATÓGENOS QUE INCIDEN EN EL CULTIVO EN CUBA .......... 4 2.2 HONGOS DEL SUELO ........................................................................................... 5 2.2.1 PRINCIPALES HONGOS DEL SUELO QUE INCIDEN EN EL CULTIVO DEL FRIJOL ................. 5 2.2.2 CARACTERÍSTICAS GENERALES DE RHIZOCTONIA SOLANI KÜHN, SCLEROTIUM ROLFSII SACC., MACROPHOMINA PHASEOLINA (TASSI) GOID. Y SCLEROTINIA SCLEROTIORUM (LIB.) DE BARY .............................................................................................................................. 5 2.2.3 MICROORGANISMOS BIOCONTROLADORES DE HONGOS DEL SUELO ........................... 10 2.3 ACTINOMICETOS ...............................................................................................
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