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Colletotrichum Coccodes (Antracnosis Del Tomate Y Chile)

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Colletotrichum Coccodes (Antracnosis Del Tomate Y Chile) DIRECCIÓN GENERAL DE SANIDAD VEGETAL CENTRO NACIONAL DE REFERENCIA FITOSANITARIA Área de Diagnóstico Fitosanitario Laboratorio de Micología Protocolo de Diagnóstico: Colletotrichum coccodes (Antracnosis del tomate y chile) Tecámac, Estado de México, julio 2019 Senasica, agricultura sana para el bienestar Aviso El presente protocolo de diagnóstico fitosanitario fue desarrollado en las instalaciones de la Dirección del Centro Nacional de Referencia Fitosanitaria (CNRF), de la Dirección General de Sanidad Vegetal (DGSV) del Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria (SENASICA), con el objetivo de diagnosticar específicamente la presencia o ausencia de Colletotrichum coccodes. La metodología descrita, tiene un sustento científico que respalda los resultados obtenidos al aplicarlo. La incorrecta implementación o variaciones en la metodología especificada en este documento de referencia pueden derivar en resultados no esperados, por lo que es responsabilidad del usuario seguir y aplicar el protocolo de forma correcta. La presente versión podrá ser mejorada y/o actualizada quedando el registro en el historial de cambios. I. ÍNDICE 1. OBJETIVO Y ALCANCE DEL PROTOCOLO .............................................................................................. 1 2. INTRODUCCIÓN ............................................................................................................................................... 1 2.1 Información sobre la plaga .............................................................................................................................. 1 2.2 Información taxonómica .................................................................................................................................. 2 2.3 Flujo de trabajo ................................................................................................................................................ 4 3. DETECCIÓN E IDENTIFICACIÓN ................................................................................................................ 5 3.1 Identificación morfométrica ............................................................................................................................ 5 3.1.1 Observación directa y cortes histológicos .................................................................................................. 5 3.1.2 Incubación en papel secante ....................................................................................................................... 6 3.1.3 Aislamiento en medios de cultivo .............................................................................................................. 7 3.2 Descripción morfométrica ............................................................................................................................... 8 3.2.1 Descripción colonial .................................................................................................................................. 8 3.2.2 Descripción morfométrica ....................................................................................................................... 10 3.3 Identificación del patógeno ........................................................................................................................... 11 4. REGISTROS ...................................................................................................................................................... 12 5. CONTACTO PARA INFORMACIÓN ADICIONAL ................................................................................... 12 6. RECONOCIMIENTO....................................................................................................................................... 12 7. REFERENCIAS ................................................................................................................................................ 13 8. ANEXOS ............................................................................................................................................................ 16 8.1 Signos y síntomas .......................................................................................................................................... 16 8.2 Ciclo de la enfermedad .................................................................................................................................. 17 8.3 Elaboración de montajes................................................................................................................................ 18 8.3.1 Preparaciones temporales con cubreobjetos ............................................................................................. 18 8.3.2 Preparaciones temporales con cinta adhesiva .......................................................................................... 18 8.3.3 Preparaciones permanentes ...................................................................................................................... 18 8.4 Medios de montaje ........................................................................................................................................ 19 8.5 Medios de cultivo .......................................................................................................................................... 19 8.6 Corroboración molecular ............................................................................................................................... 20 8.6.1 Extracción de DNA .................................................................................................................................. 20 8.6.2 Verificación de la calidad del DNA ......................................................................................................... 21 8.6.3 PCR punto final ....................................................................................................................................... 21 8.6.4 Interpretación de resultados para los ensayos de PCR punto final ........................................................... 23 8.6.5 Corroboración mediante Filogenia Molecular ......................................................................................... 23 II. ÍNDICE DE FIGURAS Figura 1. Crecimiento colonial de Colletotrichum coccodes en medio de cultivo EMA, con concentración de microesclerocios al centro de la colonia y arreglo radial de microesclerocios hacia la periferia. .................................. 8 Figura 2. Diferenciación colonial de especies de Colletotricum, incubación a 25 ºC durante 8 días. .......................... 9 Figura 3. Apariencia colonial de especies de Colletotrichum sobre PDA................................................................... 9 Figura 4. Estructuras de reproducción de Colletotrichum coccodes. .......................................................................... 10 Figura 5. Estructuras de Colletotrichum coccodes. ..................................................................................................... 11 Figura 6. Síntomas causados por Colletotrichum coccodes en tomate y chile. ........................................................... 16 Figura 7. Ciclo de vida de Colletotrichum coccodes en tomate (Solanum lycopersicum). ......................................... 17 Figura 8. Ciclo de vida de Colletrichum coccodes en chile (Capsicum annum L.). ................................................... 17 Figura 9. Ensayo de control endógeno con el par de primers ITS-1 e ITS-4 .............................................................. 23 Figura 10. Alineamiento de secuencias utilizando Nucleotide BLAST. ..................................................................... 24 Figura 11. Alineamiento múltiple de secuencias. ....................................................................................................... 26 Figura 12. Filogenia molecular. .................................................................................................................................. 26 III. ÍNDICE DE CUADROS Cuadro 1. Medios de montaje recomendados ............................................................................................................. 19 Cuadro 2. Medios de cultivo recomendados ............................................................................................................... 19 Cuadro 3. Primers utilizados en el ensayo de PCR punto final para le detección del gen endógeno ribosomal ......... 21 Cuadro 4. Preparación de la mezcla de reacción para el ensayo de PCR punto final del control endógeno ............... 22 Cuadro 5. Programa de termociclaje para la detección del control endógeno ............................................................ 22 1. OBJETIVO Y ALCANCE DEL PROTOCOLO Describir la metodología aplicada en el Laboratorio de Micología del Centro Nacional de Referencia Fitosanitaria para la detección e identificación de Colletotrichum coccodes mediante caracterización morfométrica y técnicas moleculares a partir de semillas y material vegetal de tomate y chile, bajo las condiciones de uso de reactivos que aquí se detallan. Este método es apropiado para tejido vegetal de tomate (Solanum lycopersicum) y de chile (Capsicum sp.) que presente signos y síntomas característicos, como tallos o frutos, así como a partir de semilla de ambos cultivos. 2. INTRODUCCIÓN 2.1 Información sobre la plaga C. coccodes es el agente etiológico de la enfermedad conocida como antracnosis o pudrición negra en solanáceas. Afecta
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