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Instituto Tecnológico De Costa Rica INSTITUTO TECNOLÓGICO DE COSTA RICA ESCUELA DE BIOLOGÍA INGENIERÍA EN BIOTECNOLOGÍA UNIVERSIDAD DE COSTA RICA Centro de Investigación en Biología Celular y Molecular (CIBCM) Centro de Investigación en Estructuras Microscópicas (CIEMic) Grupo Integrado Simbiosis – Hospedero – Microorganismo (GISHM, UCR) Evaluación de la actividad entomopatógena de diversos aislamientos de hongos y cepas de Bacillus thuringiensis para el potencial desarrollo de un bioformulado contra las hormigas cortadoras de hojas de la especie Atta cephalotes Informe presentado a la Escuela de Biología del Instituto Tecnológico de Costa Rica como requisito parcial para optar por el título de Licenciado en Ingeniería en Biotecnología Esteve Mesén Porras Cartago, Setiembre de 2015 Evaluación de la actividad entomopatógena de diversos aislamientos de hongos y cepas de Bacillus thuringiensis para el potencial desarrollo de un bioformulado contra las hormigas cortadoras de hojas de la especie Atta cephalotes Esteve Mesén Porras1 RESUMEN Las hormigas cortadoras de hojas (Atta y Acromyrmex) son una de las plagas más relevantes del Neotrópico debido a su compleja organización jerárquica en castas, la forma de vida claustral, el comportamiento eusocial para cuidar larvas y proteger su hongo simbionte, y la capacidad de forrajeo diurno y nocturno. Debido a estos aspectos y a su habilidad para reconocer y remover patículas dañinas de su jardín fúngico, el control con agroquímicos ha sido una tarea difícil. Por ello, el objetivo de este proyecto fue reconocer si aislamientos de Bacillus thuringiensis y hongos recuperados de colonias de zompopas podrían ser agentes de biocontrol de obreras de Atta cephalotes. El bioensayo de ingestión con Bt permitió reconocer 3 aislamientos que produjeron mortalidades de 45 a 54%, concentraciones subletales entre 9,97×101 y 5,86×107 µg mL-1 y tiempos de muerte de 5 y 6 días; valores que coincidieron con los reportes de algunos autores que han investigado la entomotoxidad de δ-endotoxinas sobre himenópteros. Los ensayos de patogenicidad y virulencia con microhongos permitieron identificar 6 cepas que exhibieron elevada toxicidad y fueron morfotípicamente similares a Metarhizium, Beauveria, Paecilomyces y Aspergillus, patógenos que conviven en las colonias de hormigas podadoras, según la literatura consultada. Estos entomopatógenos ocasionaron mortalidades entre 81% y 33%, siendo la posible cepa de Metarhizium la que generó mayor virulencia sobre las zompopas en relación con las cepas de 1 5 -1 Aspergillus; y cuyos valores de LD50 y LT50 se ubicaron entre 4,28×10 y 1,00×10 esporas mL y entre 5 y 6 días, respectivamente. Palabras clave: Atta cephalotes, control biológico de plagas, Bacillus thuringiensis, Metarhizium, concentración letal media, tiempo subletal, toxicidad del insecto. 1 Informe presentado para el curso TRABAJO FINAL DE GRADUACIÓN. Escuela de Biología, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica, 2015. ii Evaluation of the entomopathogenic activity from isolation of fungi and Bacillus thuringiensis strains for the potential development of a formulation bioproduct for the biological control of leaf-cutting ants, Atta cephalotes Esteve Mesén Porras2 ABSTRACT Leaf-cutting ants (Atta and Acromyrmex) are one of the most devastating pests in the Neotropical region because of their complex hierarchical organization divided in castes, claustral social life, eusocial behavior to care larvae and protect their symbiont fungus and capacity to forage fresh leaves during the day or night. Considering these factors, and including their hability to recognize and remove potentially harmful particles from their fungus garden, agrochemical control has become a hard task to eradicate this pest. Therefore, the objective of this project was to find out if Bacillus thuringiensis and fungi strains isolated from leaf-cutting ant nests could be applied as bioinfectious agents against Atta cephlotes workers. Ingestion assays adding different Bt lyophilized extracts was a proper method to recognize 3 Bt strains that reached mortality percentages in the interval of 45 and 54%, with subletal concentrations between 9,97×101 and 5,86×107 µg mL-1, as well as subletal times between 5 and 6 days; values that fit with similar reports where δ-endotoxins were used to determine their entomotoxicity on hymenopteran species. Patogenicity and virulence assays adding microfungi enhanced doses allowed to identify 6 strains that exhibited high toxicity, which were morphotypically similar to Metarhizium, Beauveria, Paecilomyces and Aspergillus, common parasites that coexist with leaf-cutting ant colonies according to the consulted literature. Those entomopathogenic strains showed mortalities of 81% - 33%, where the possible Metarhizium strain produced the higher virulence on insects in comparison with the Aspergillus strains, and LD50 1 5 -1 y LT50 values were estimated between 4,28×10 -1,00×10 spore mL and 5-6 days, respectively. Key words: Atta cephalotes, pest biology control, Bacillus thuringiensis, Metarhizium anisopliae, sublethal concentration, sublethal time, insect toxicity. 2 Report presented to accomplish the licenciature course FINAL GRADUATION PROJECT. Departament of Biology, Technology Institute of Costa Rica, Cartago, Costa Rica, 2015. iii ACREDITACIÓN Evaluación de la actividad entomopatógena de diversos aislamientos de hongos y cepas de Bacillus thuringiensis para el potencial desarrollo de un bioformulado contra las hormigas cortadoras de hojas de la especie Atta cephalotes Informe presentado a la Escuela de Biología del Instituto Tecnológico de Costa Rica como requisito parcial para optar por el título de Licenciatura en Ingeniería en Biotecnología Miembros del tribunal Ph.D. Adrián Pinto Tomás Lic. Fabiola Jiménez Rodríguez M.Sc. Catalina Murillo Cruz iv DEDICATORIA A mis abuelos maternos Elia y Bernardo, y a mi padre Amancio Ronny; personas que me dieron muchas enseñanzas de vida y fueron mi ejemplo a seguir para impulsar mi carrera y tratara de dar mi mejor esfuerzo en cada trabajo a realizar. A Dios, quien siempre me ha concedió muchas bendiciones mientras realizaba mis estudios universitarios. v AGRADECIMIENTOS Me gustaría dar un sentido agradecimiento al Ph.D. Adrián Pinto Tomás, quien me introdujo en este mundo de la investigación, ha tratado de hacer su mayor esfuerzo por posicionarme en el área de la biotecnología vinculada con el control biológico de plagas y me ha incentivado a a pensar de manera minuciosa sobre la manera más práctica para estudiar el comportamiento ecológico de un insecto de importancia agronómica, con el fin de buscar soluciones biológicas para su control. Seguidamente, quisiera agradecer el apoyo incondicional y el seguimiento en mi crecimiento personal y profesional que me ha brindado la Lic. Fabiola Jiménez Rodríguez en todos los proyectos que he desarrollado, quien además ha sido una persona facilitadora, amable, atenta y consejera en todo momento para llevar a cabo el trabajo de laboratorio de la mejor manera posible. De la misma forma, también me gustaría agradecer a la M.Sc. Catalina Murillo Cruz, quien es una pieza fundamental dentro del grupo de investigación Simbiosis-Hospedero-Microorganismo y es la persona que me proporcionó el apoyo logístico y de organización desde el inicio del proyecto, además fue una gran colaboradora porque mostró un gran interés por la oportuna presentación de avances, pronta compra y obtención de los materiales que necesité en el transcurso de este trabajo. Para el desarrollo exitoso de este trabajo de graduación, siempre conté con el trabajo en equipo de los colegas de los grupos Integrado Simbiosis-Hospedero-Microorganismo y de Control Biológico, así como dos compañeros extranjeros provenientes de Perú y Alemania, quienes aunque participaron de manera temporal o transitoria durante la realización de este trabajo; su colaboración prestada fue clave en labores de mantenimiento de las colonias de zompopas, giras de campo, sugerencias para mejorar algunos protocolos, selección de los aislamientos de hongos o cepas de Bacillus thuringiensis, y el establecimiento y registro de datos de algunos de los protocolos o bioensayos que realicé durante el periodo en que se llevó a cabo este proyecto de investigación. Me gustaría agradecer a las instituciones y a la amabilidad de las personas que brindaron su apoyo durante las giras de campo en las que asistí para la colecta de colonias de zompopas, en especial a Danilo Brenes Madrigal, Bernal Matarrita Carranza y a la atención brindada por el personal de La Estación Biológica La Selva; a Arturo Ramírez, quien fue un asesor y colaborador en las colectas del Centro Agronómico Tropical de Investigación y Enseñanza; y a German Alvarado, quien nos recibió y ayudó a reconocer colonias en La Estación Experimental Agrícola Fabio Baudrit Moreno. vi Asimismo, quisiera dar un agradecimiento por la colaboración y amabilidad de todo el personal del Centro de Investigación en Biología Celular y Molecular (CIBCM), así como las personas que trabajan en el Centro de Investigación en Estructuras Microscópicas (CIEMic); quienes me facilitaron y ubicaron en los lugares físicos para desempeñar el trabajo de laboratorio, me brindaron su amistad, materiales de gira y fueron muy atentos en el préstamo y uso de los equipos de laboratorio. También me gustaría reconocer la ayuda a nivel de financiamiento que proporcionó la empresa agroquímica industrial RIMAC S.A., así como la Fundación de la Universidad de Costa Rica para la Investigación (FUNDEVI, UCR)
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