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Estrategias De Manejo De La Roña Cladosporium Cladosporioides (FRESEN) G.A

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Estrategias De Manejo De La Roña Cladosporium Cladosporioides (FRESEN) G.A Estrategias de Manejo de la Roña Cladosporium cladosporioides (FRESEN) G.A. de VRIES de la Gulupa Passiflora edulis f. edulis Sims. Carlos Fernando Castillo Londoño Universidad Nacional de Colombia Facultad de Ciencias Agropecuarias Maestría en Ciencias Agrarias Palmira, 2014 Estrategias de Manejo de la Roña Cladosporium cladosporioides (FRESEN) G.A. de VRIES de la Gulupa Passiflora edulis f. edulis Sims. Carlos Fernando Castillo Londoño Trabajo de investigación presentado como requisito parcial para optar al título de: Magister en Ciencias Agrarias con énfasis en Fitopatología Director: Ph.D. Carlos German Muñoz Perea Codirector: Ph.D. Elizabeth Álvarez Codirector: Ph.D. Kriss Wichechkus Universidad Nacional de Colombia Facultad de Ciencias Agropecuarias Maestría en Ciencias Agrarias Palmira. 2014 A Dios por darme la inmensa voluntad y capacidad de entrega para la hacer las cosas con amor y dedicación. A mi madre Carmen y a Geovanny por su gran confianza, apoyo, consejos y su constante exigencia para formar la persona que soy. AGRADECIMIENTOS A la Universidad Nacional de Colombia sede Palmira por las oportunidades y facilidades brindadas en el transcurso de mi carrera profesional. A la Universidad Jorge Tadeo Lozano y al Centro Internacional de Agricultura Tropical (CIAT) por brindarme la oportunidad de trabajar en el proyecto. A mi director Carlos German Muñoz, y codirectores Elizabeth Álvarez y Kriss Wichechkus, por sus valiosos aportes y orientación en esta investigación. A la Dra. Martha Cárdenas directora del Centro de Transformación Genética (Riverside), por su valiosa colaboración en la revisión del documento. A mis profesores, a quienes les debo gran parte de mis conocimientos, gracias por prepararnos para un futuro competitivo no solo como los mejores profesionales sino también como mejores personas. Gracias a todas las personas que ayudaron directa e indirectamente en la realización de este proyecto. Contenido 2. LA GULUPA (Passiflora edulis f. edulis Sims) ................................................................ 1 2.1 ORIGEN Y DISTRIBUCIÓN ...................................................................................... 1 2.2 TAXONOMÍA DE Passiflora edulis f. edulis Sims. ................................................... 1 2.3 DESCRIPCIÓN ............................................................................................................ 2 2.4 IMPORTANCIA DEL CULTIVO ............................................................................... 3 2.5 PROPIEDADES DE LA GULUPA ............................................................................. 5 2.5.1 VALOR NUTRICIONAL DE LA GULUPA ........................................................... 5 2.5.2 CONDICIONES Y REQUERIMIENTOS AGRO-ECOLÓGICOS ......................... 5 2.6 GENÉTICA EN MATERIALES COMERCIALES DE GULUPA (Passiflora edulis f. edulis SIMS) EN COLOMBIA .............................................................................................. 7 2.6.1 Variabilidad genética ................................................................................................. 7 2.6.2 Mejoramiento genético y variedades ......................................................................... 9 2.6.3 CARACTERIZACIÓN MOLECULAR EN PASIFLORAS .................................. 11 2.7 ENFERMEDADES EN GULUPA ................................................................................ 12 2.7.1 Antracnosis ocasionada por Glomerella cingulata (Stoneman) Spauld. & H. Schrenk (Anamorfo Colletotrichum gloeosporioides (Penz.) Pen0z. & Sacc. in Penz). 13 2.7.2 Características morfológicas................................................................................... 13 2.7.3 Epidemiología de la Antracnosis de la gulupa. ...................................................... 14 2.7.4 Factores que favorecen el desarrollo de Colletotrichum gloeosporioides. ............. 14 2.7.6. La Roña (Cladosporium cladosporioides) (Fresen.) G. A. de Vries ...................... 18 2.7.6.1. Características morfológicas................................................................................ 19 2.7.6.2 Epidemiología de la Roña de la gulupa. .............................................................. 19 2.7.6.3 Sintomatología ..................................................................................................... 20 2.8 MANEJO DE LA ENFERMEDAD .............................................................................. 20 2.8.1 Semillas (Material genético) .................................................................................... 21 2.8.2 Siembra .................................................................................................................... 21 2.8.3 Podas ........................................................................................................................ 22 2.8.4 Fertilización ............................................................................................................. 23 2.8.5 Manejo de arvenses ................................................................................................. 23 2.8.6 Cosecha .................................................................................................................... 24 2.8.7 Manejo químico ...................................................................................................... 24 2.9 MARCADORES MOLECULARES ............................................................................. 30 2.9.1. ITS (Región espaciadora interna transcrita) ........................................................... 31 2.9.3. Microsatélites RAMS ............................................................................................. 31 3. OBJETIVOS .................................................................................................................... 33 3.1 OBJETIVO GENERAL ............................................................................................ 33 3.2 OBJETIVOS ESPECÍFICOS .................................................................................... 33 4. ASPECTOS METODOLÓGICOS .................................................................................. 34 4.1 Recolección de material vegetal y aislamiento de cepas ........................................... 34 4.2.1 Reactivación de los aislamientos ............................................................................. 34 4.2.3 Aislamientos de cepas de Cladosporium spp .......................................................... 35 4.2.4 Desinfestación del material vegetal ......................................................................... 36 4.2.5 Siembra directa en medio de cultivo ....................................................................... 36 4.2.6 Purificación .............................................................................................................. 36 4.2.7 Obtención de cultivos monospóricos ....................................................................... 37 4.2.8 Pruebas de patogenicidad: Postulados de Koch ..................................................... 38 4.2.9 Preparación de los frutos y del material antes de la inoculación ............................. 38 4.2.10 Ajuste de la metodología de inoculación de frutos de gulupa con Cladosporium spp ..................................................................................................................................... 39 4.2.11. Pruebas de patogenicidad ..................................................................................... 40 4.2.11 Evaluación de la enfermedad ................................................................................. 40 5.0. Pruebas moleculares ...................................................................................................... 41 5.1. Extracción de ADN ................................................................................................... 41 4.5.2. Amplificación de las regiones ITS del ADNr ........................................................ 42 4.5.3. Visualización y análisis de productos amplificados. .............................................. 43 4.5.4. Limpieza de producto PCR para secuenciación .................................................... 43 5.4.5 Análisis de secuencia ............................................................................................... 44 4.5.6. Amplificación aleatoria de microsatélites RAMS. ................................................. 44 4.6. Análisis estadístico ........................................................................................................ 46 4.6.1. Patogenicidad ......................................................................................................... 46 4.6.2. RAMS ..................................................................................................................... 47 4.6.3. Análisis de agrupamiento ....................................................................................... 48 4.6.4 Análisis de correspondencia múltiple ...................................................................... 49 4.6.5 Diversidad genética ................................................................................................. 49 4.6.6. Distancia genética ................................................................................................... 50 5.0. Evaluación in vitro de los extractos vegetales para el control de Cladosporium spp ... 51 5.1. Selección de aislamientos de Cladosporium spp. .....................................................
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