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Detección Y Limpieza De Virus En Orquídeas Cultivadas

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Detección Y Limpieza De Virus En Orquídeas Cultivadas CONSEJO NACIONAL DE CIENCIA Y TECNOLOGIA -CONCYT- SECRETARIA NACIONAL DE CIENCIA Y TECNOLOGIA -SENACYT- FONDO NACIONAL DE CIENCIA Y TECNOLOGIA -FONACYT- UNIVERSIDAD DEL VALLE DE GUATEMALA – UVG - INFORME FINAL EVALUACIÓN DE MÉTODOS PARA LA DETECCIÓN Y LIMPIEZA DE VIRUS EN ORQUÍDEAS CULTIVADAS PROYECTO FODECYT No. 067-2007 MARGARITA PALMIERI Investigador Principal GUATEMALA, JULIO DE 2012. ii EQUIPO DE INVESTIGACIÓN: Licda. Margarita Palmieri Santisteban – Investigadora Principal Licda. María Renée Álvarez – Investigadora Asociada iii AGRADECIMIENTOS: La realización de este trabajo, ha sido posible gracias al apoyo financiero dentro del Fondo Nacional de Ciencia y Tecnología, -FONACYT-, otorgado por La Secretaría Nacional de Ciencia y Tecnología -SENACYT- y al Consejo Nacional de Ciencia y Tecnología -CONCYT-. iv OTROS AGRADECIMIENTOS Este trabajo no habría sido posible sin la colaboración de los dueños de los cultivos comerciales y no comerciales de orquídeas que accedieron a colaborar con el proyecto prestando sus orquídeas para su análisis. También se agradece a la Asociación Guatemalteca de Orquideología (AGO) por permitirnos el acceso a sus reuniones y contactos para poder realizar el proyecto. v INDICE CONTENIDO PÁGINA RESUMEN xi ABSTRACT xii PARTE I I.1. Introducción 1 I.2. Planteamiento del problema 2 I.2.1. Antecedentes en Guatemala 3 I.2.2. Justificación 3 I.3. Objetivos e hipótesis I.3.1. Objetivos 5 I.3.1.1. General 5 I.3.1.2. Específicos 5 I.3.2. Hipótesis 5 I.4. Metodología I.4.1. Localización 6 I.4.2. Las variables 6 I.4.2.1. Las variables dependientes 6 I.4.2.2. Las variables independientes 6 I.4.3. Indicadores 6 I.4.4. Estrategia metodológica 7 I.4.5. El método 7 I.4.6. La técnica estadística 9 PARTE II II.1 Marco teórico II.1.A) La familia Orchidaceae 10 II.1.B) Las Orchidaceae en Guatemala 13 II.1.C) Cultivo de orquídeas 14 II.1.D) Híbridos de orquídeas 14 II.1.E) Enfermedades y plagas de orquídeas 15 II.1.F) Enfermedades virales en orquídeas 16 vi II.1.G) Detección de virus en orquídeas 22 II.1.H) Terapias de tratamiento de virus 24 II.1.I) Limpieza de virus en orquídeas 26 PARTE III III.1. Resultados 27 III.1.A) Diagnóstico de enfermedades virales 27 III.1.B) Terapias de limpieza de virus 31 III.2. Discusión de resultados III.2.A) Diagnóstico de enfermedades virales 34 III.2.B) Terapias de limpieza de virus 36 PARTE IV IV.1. Conclusiones 40 IV.2. Recomendaciones 41 IV.3. Referencias bibliográficas 42 IV.4. Anexos IV.4.1. Metodología de ELISA directo 47 IV.4.2. Metodología de ELISA indirecto 49 IV.4.3. Resultados de presencia de 6 virus en la 180 Orquídeas Analizadas 51 IV.4.4. Fotografías de los síntomas más comunes presentados Por las orquídeas infectadas por virus 54 PARTE V V. 1. Informe Financiero 56 vii LISTADO DE FIGURAS CONTENIDO PÁGINA Figura 1. Esquema de una flor de orquídea 11 Figura 2. Flor de Cattleya vista frontal y sección lateral 11 Figura 3. Cápsulas de tres especies de orquídeas 12 Figura 4. Semilla de orquídea, compuesta por el embrión y la testa 17 Figura 5. Daño provocado en hojas por Cymbidium mosaic virus (CyMV) 17 Figura 6. Daño provocado por Odontoglossum ringspot Tobamovirus (ORSV) en a) una hoja de Cymbidium y b) una hoja de Phalaenopsis. 18 Figura 7. Daño provocado por a) Virus de la mancha anillada del Odontoglosum (ORSV o TMV-O) en una flor de Cymbidium b) la combinación de ORSV y el virus del mosaico del Cymbidium (CymMV) en una flor de Cymbidium y c) ORSV en una flor de Cattleya. 19 Figura 8. a) Cattleya percivaliana con síntomas de CMV, b) hoja de Coelogyne rochussenii con síntoma de CMV, c) hojas de Laelia lobata con ORSV y CMV, d) hojas de Epidendrum con síntomas de ORSV y CMV y e) CMV en Pescatoria wallisii 20 Figura 9. Hoja de Dactylorhiza foliosa con síntomas del virus del mosaico amarillo de la judía (BYMV) (Skelton et al. 2006) 20 Figura 10. Síntomas de infección con Tospovirus en una hoja de Phalaenopsis, b) síntomas de TSWV en una hoja de Phalaenopsis 21 Figura 11. a) síntomas de OFV en hojas, b) síntomas de OFV en bulbos, c) síntomas de OFV en hojas de Dendrobium, d) síntomas de OFV en hoja de Cymbidium y e) sintomatología de OFV en hojas de Laelia 22 Figura 12. Micrografía electrónica de viriones de OFV purificado. Un Aumento del virus se encuentra en el área dentro del rectángulo. 22 Figura 13. Pasos del procedimiento de ELISA directo. I = savia de planta infectada con virus y S = savia de planta sana 23 Figura 14. Brote lateral de Cattleya que puede ser utilizado como explante para el cultivo de meristemos 25 Figura 15. Frecuencia de los diferentes virus encontrados en las muestras de Orquídeas colectadas. 28 viii Figura 16. Número de plantas de orquídeas con infecciones mixtas por 2 y 3 virus simultáneamente. 28 Figura 17. Gráfica de la distribución de la presencia de virus en Orquídeas infectadas. 29 Figura 18. Movimiento del virus del Mosaico del Cymbidium (CyMV) En híbridos de Dendrobium infectadas. 37 Figura 19. Efecto de la termoterapia para la eliminación de PVY 39 ix LISTADO DE CUADROS CONTENIDO PÁGINA Cuadro 1: Número de plantas positivas para los virus analizados. 27 Cuadro 2. Orquídeas infectadas trasladadas al invernadero de la UVG 30 Cuadro 3. Orquídeas tratadas con hidroterapia, termoterapia y cultivo de meristemos 30 Cuadro 4. Resultados de la prueba de ELISA en las orquídeas tratadas con hidroterapia. 31 Cuadro 5. Resultados de la prueba de ELISA en las orquídeas tratadas con termoterapia. 32 Cuadro 6. Resultados de la prueba de ELISA en las orquídeas tratadas con cultivo de meristemos. 32 Cuadro 7. Resultados de la prueba de ELISA en las orquídeas tratadas con termoterapia a 42°C luego del cultivo de meristemos. 33 x RESUMEN La familia Orchidaceae es una de las más grandes del reino vegetal, en Guatemala representa aproximadamente el 10% de la flora. La belleza de sus flores ha hecho que sean muy apreciadas ornamentalmente, por lo cual su cultivo ha aumentado en los últimos años. Las condiciones ambientales en que se cultivan las hacen susceptibles a la infecciones por virus; siendo los más comunes: virus de la mancha anillada del Odontoglossum (ORSV), virus del mosaico del Cymbidium (CyMV), mosaico del pepino (CMV), virus del bronceado del tomate (TSWV), virus del mosaico del tabaco (TMV) y potyvirus. En este trabajo se realizó un diagnóstico de la presencia de los 6 virus por medio de pruebas ELISA en 6 cultivares. Además se implementaron terapias para la eliminación de virus en las orquídeas infectadas. Éstas fueron: hidroterapia a 45°C durante 1 hora, termoterapia a 42°C durante 24 horas, cultivo de meristemos y cultivo de meristemos seguido de termoterapia a 42°C y 8, 16, 24 y 48 horas. Se encontró que el 19.89% (36 plantas infectadas) de todas las plantas analizadas estaban infectadas con uno o más virus. Se observó la presencia de 3 virus: ORSV, CyMV y TMV. La sintomatología observada fue: líneas cloróticas en presencia de CyMV y manchas cloróticas en presencia del virus de ORSV. Ninguna de las terapias realizadas fue efectiva para la limpieza de virus en orquídeas. Únicamente una planta mostró eliminación de los virus por medio de hidroterapia. Palabras clave: Orquídeas, virus, pruebas ELISA, terapias de eliminación, cultivo de meristemos xi ABSTRACT The Orchidaceae family is one of the largest in the plant kingdom, in Guatemala, representing approximately 10% of the flora. The beauty of their flowers has made them highly prized ornamentals, for which cultivation has increased in recent years. The environmental conditions in which they are grown make them susceptible to virus infections, the most common being: Odontoglossum ringspot virus (ORSV), Cymbidium mosaic virus (CyMV), cucumber mosaic virus (CMV), tomato spotted wilt virus (TSWV), tobacco mosaic virus (TMV) and potyvirus. In this work we carried out an assessment of the presence of the 6 viruses using ELISA in 6 cultivars. In addition, therapies were implemented to eliminate virus from infected orchids. Those were: hydrotherapy at 45 ° C for 1 hour, thermotherapy at 42 ° C for 24 hours, meristem culture and meristem culture followed by thermotherapy at 42 ° C for 8, 16, 24 and 48 hours. We found that 19.89% of all plants tested were infected with one or more viruses. We observed the presence of 3 viruses: ORSV, CyMV and TMV. The symptoms observed were: chlorotic lines in the presence of CyMV and chlorotic spots in the presence of ORSV. None of the therapies performed were effective in cleaning viruses in orchids. Only one plant was cleaned by hydrotherapy. Keywords: Orchids, virus, ELISA, elimination therapies, meristem culture xii PARTE I I.1. INTRODUCCIÓN Las orquídeas son plantas monocotiledóneas que se caracterizan por la belleza de sus flores. Muchas de éstas han sido cultivadas e hibridizadas para su comercialización. Su cultivo ha ido creciendo en todas partes del mundo, incluyendo Guatemala. El cultivo de orquídeas es una producción intensiva en el que se posee un importante capital en muy poco espacio. Un vivero mediano cuenta con alrededor de quince mil plantas, las cuales necesitan muy buena calefacción y alto porcentaje de humedad. Estas condiciones son propicias para el desarrollo de diversas plagas y enfermedades. Es por ello que es necesario tener un exhaustivo control en las colecciones. Las plagas y enfermedades en orquídeas son consecuencia de un riego incorrecto, mala ventilación, hábitos de fertilización incorrectos y otros errores de cultivo.
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