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Virus De La Marchitez Del Tomate (Tomarv) En El Noroeste De México E Identificación De Hospedantes Alternos” TESIS

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Virus De La Marchitez Del Tomate (Tomarv) En El Noroeste De México E Identificación De Hospedantes Alternos” TESIS INSTITUTO POLITÉCNICO NACIONAL CENTRO INTERDISCIPLINARIO DE INVESTIGACIÓN PARA EL DESARROLLO INTEGRAL REGIONAL UNIDAD SINALOA “Presencia del Virus de la marchitez del tomate (ToMarV) en el Noroeste de México e identificación de hospedantes alternos” TESIS QUE PARA OBTENER EL GRADO DE MAESTRÍA EN RECURSOS NATURALES Y MEDIO AMBIENTE PRESENTA ROGELIO ARMENTA CHÁVEZ GUASAVE, SINALOA; MÉXICO DICIEMBRE, 2012 Agradecimientos a proyectos El trabajo de tesis se desarrolló en el Departamento de Biotecnología Agrícola del Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional (CIIDIR) Unidad Sinaloa del Instituto Politécnico Nacional (IPN). El presente trabajo fue apoyado económicamente por el IPN y recursos autogenerados. El alumno Rogelio Armenta Chávez fue apoyado con una beca CONACYT con clave 366650 y por el IPN a través de la beca PIFI dentro del proyecto Determinación de la importancia de hospedantes alternos en la dispersión de Ca. Liberibacter sp. en el Norte de México (Con número de registro 20120507). DEDICATORIA A mis padres A ellos por haberme dado la vida y haberse preocupado día a día por mi bienestar y futuro, a ellos les dedicó esta tesis, mi vida y mi ser. Gracias por ser mis padres. A mi novia A mi novia Karen quien siempre me ha apoyado en todo, sin condición alguna; por darme su amor y cariño, por eso gracias. A mi tía Soledad Armenta Por estar a mi lado en los momentos difíciles y haberme dado su apoyo y fuerza para salir adelante día con día. Gracias por eso y por todo lo que venga. A mi hermana Quien ha estado conmigo en las buenas y en las malas a lo largo de mi vida; gracias hermana por ese apoyo tan peculiar que me das. A mi tutora Gracias a Erika, quien a pesar de sus regaños y su risa de nervios, estuvo conmigo durante los éxitos y altibajos de este trabajo. AGRADECIMIENTOS A mis padres quienes sin escatimar esfuerzo alguno, han sacrificado gran parte de su vida, para formarme y educarme. A quienes la ilusión de su existencia ha sido verme convertido en una persona de provecho. A ustedes padres GRACIAS. Agradezco a mi familia por su cariño, paciencia, comprensión y apoyo desde siempre, inculcándome que todo lo que sueñe es posible realizarlo. A CONACYT por el apoyo económico brindado durante la realización de este proyecto. A COECYT por el apoyo económico brindado para la finalización de este proyecto. Al CIIDIR-IPN unidad Sinaloa, una institución de enorme calidad, y con un enorme potencial de crecimiento, que me brindó todo el apoyo durante la realización de mi trabajo de investigación. A mi director de tesis, Dr. Jesús Méndez Lozano, por darme la oportunidad de ser parte de su grupo de trabajo. Sus consejos, paciencia, algunas veces regaños y opiniones sirvieron para que me sienta satisfecho de mi participación dentro de este proyecto de investigación. A mi Co-directora, M. en C. Erika Camacho Beltrán, por toda su comprensión, nobleza y dedicación que en todo momento mostro hacia mí, por las enseñanzas, sugerencias y apoyo para que juntos pudiéramos llevar de la mano la realización de este trabajo de investigación, por enseñarme que no importa que tan difícil sea el problema, siempre existe una salida o al menos una risa de nervios, gracias jefa por su gran apoyo, y porque creo que mas que una maestra asociada e investigadora adjunta, encontré una amiga A los miembros del Comité de asesores, Dra. Norma Elena Leyva López, M. en C. Ana Elsi Ulloa Pérez y Dr. Miguel Ángel Apodaca Sánchez por las valiosas contribuciones que hicieron durante la realización de esta investigación, así como por el tiempo que dedicaron para revisar el trabajo final. A mis compañeros del Laboratorio de Virología: Lucy, Natalie, Norma Macías, Tavo, JuanJo, Emanuel, Emmanuel, Betty, Hugo, Marielos, Miriam, Lalo (por un tiempo) que de alguna forma fueron parte de este trabajo. A todos ustedes gracias por ser parte de esto. Agradezco a cada uno de los profesores, quienes participaron en mi desarrollo profesional; sin su ayuda y conocimientos no estaría donde hoy me encuentro. A mis compañeros de generación: Libia Armenta, Meymer, JC, Arely, Luis, Miguel, Elizeth, Magnolia, Lorena, Nachuuu, al Señoron Carlos Eduardo, Héctor Leyva, Wendy, Betito (que estuvo poco pero es un tipaso), Sheila, Alicia, KrlaKo, Roció, Camila. A los compañeros de generaciones pasadas: Nadia, Chaparra, Ale, Alex, Gallo, Abraham, Gerardo, Damián. De igual forma a los nuevos compañeros gracias a todos por su apoyo. INDICE GLOSARIO .............................................................................................................. I INDICE DE FIGURAS .......................................................................................... IV INDICE DE CUADROS ........................................................................................ VII RESUMEN .......................................................................................................... VIII I. INTRODUCCIÓN ............................................................................................. 1 II. ANTECEDENTES ............................................................................................ 3 2.1 Generalidades del tomate (Solanum lycopersicum) ..................................... 3 2.2 Factores que afectan el cultivo de tomate .................................................... 4 2.3 Los virus de plantas ...................................................................................... 8 2.4 Virus que infectan el cultivo de tomate ....................................................... 14 2.5 Factores que inducen la emergencia de enfermedades virales transmitidas por mosca blanca ................................................................................................. 15 2.6 Familia Secoviridae .................................................................................... 16 2.6.1 Género Torradovirus ........................................................................... 17 2.6.1.1 Virus torrado del tomate (ToTV) ........................................................... 17 2.6.2 Virus de la necrosis apical del tomate (ToANV) o Virus de la marchitez del tomate (ToMarV) ......................................................................................... 19 2.6.3 Virus de la mancha del chocolate del tomate (ToChSV) o Virus del chocolate (ToChV) ............................................................................................ 22 III. JUSTIFICACIÓN ........................................................................................ 24 IV. HIPÓTESIS ................................................................................................ 25 V. OBJETIVOS ............................................................................................... 25 VI. MATERIALES Y METODOS ...................................................................... 26 6.1 Área de estudio .......................................................................................... 26 6.2 Colecta de muestras ................................................................................... 26 6.3 Extracción de RNA de tejido foliar .............................................................. 27 6.4 Detección molecular del Virus de la marchitez del tomate (ToMarV) por RT- PCR y PCR anidado. ............................................................................................ 28 6.4.1 Síntesis del cDNA de ToMarV ............................................................. 28 6.4.2 Detección de ToMarV y Control Interno por RT-PCR ......................... 28 6.4.3 Detección de ToMarV por RT-PCR anidado ....................................... 29 6.4.4 Electroforesis ....................................................................................... 31 6.4.5 Purificación del producto de RT-PCR anidado ..................................... 31 6.4.6 Ligación del producto de RT-PCR anidado .......................................... 32 6.4.7 Transformación .................................................................................... 32 6.4.8 Extracción del DNA plasmídico (Miniprep) ........................................... 33 6.4.9 Restricción del DNA plasmídico ........................................................... 34 6.4.10 Purificación del DNA plasmídico para secuenciación .......................... 34 6.4.12 Secuenciación y análisis de la secuencia ............................................ 36 6.5 Infectividad del aislado de ToMarV-Guasave en híbridos de tomate y chile36 6.5.1 Obtención del inóculo de ToMarV-Guasave y transmisión mecánica en plantas de tomate y chile. ................................................................................. 37 6.5.2 Detección de ToMarV en plantas de tomate y chile inoculadas ........... 37 6.5.3 Sintomatología inducida por ToMarV ................................................... 38 6.5.4 Infectividad ........................................................................................... 38 VII. RESULTADOS ........................................................................................... 39 7.1 Presencia del Virus de la marchitez del tomate en el Noroeste de México 39 7.1.1 Colecta de muestras de plantas de tomate .......................................... 39 7.1.3 Extracción de RNA ............................................................................... 47 7.1.4 Detección molecular de ToMarV mediante RT-PCR y RT-PCR anidado en muestras de tomate del Noroeste de México. .............................................. 48
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