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Caracterización Biológica Y Molecular De Un Rhabdovirus Causal De Mosaico Estriado Amarillo En Maíz (Zea Mays L.) En Argentina

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Caracterización Biológica Y Molecular De Un Rhabdovirus Causal De Mosaico Estriado Amarillo En Maíz (Zea Mays L.) En Argentina TESIS DE DOCTORADO EN CIENCIAS BIOLÓGICAS Caracterización biológica y molecular de un rhabdovirus causal de mosaico estriado amarillo en maíz (Zea mays L.) en Argentina Bióloga María Fernanda Maurino Directora: Biol. (Dra.) Irma Graciela Laguna Desarrollada en el Instituto de Patología Vegetal, Centro de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria (IPAVE-CIAP-INTA) FACULTAD DE CIENCIAS EXACTAS FÍSICAS Y NATURALES UNIVERSIDAD NACIONAL DE CÓRDOBA Córdoba, Argentina 2016 COMISIÓN ASESORA DE TESIS Dra. Irma Graciela Laguna, Instituto de Patología Vegetal (IPAVE, CIAP, INTA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Dra. María Jimena Nores, Instituto Multidisciplinario de Biología Vegetal (IMBIV, CONICET, UNC) Dr. Guillermo Eyhérabide, Estación Experimental Agropecuaria INTA Pergamino (EEA INTA Pergamino) DEFENSA ORAL Y PÚBLICA Lugar y Fecha: Calificación: TRIBUNAL Firma: ............................................ Aclaración: .............................................. Firma: ............................................ Aclaración: .............................................. Firma: ............................................ Aclaración: .............................................. i Bióloga María Fernanda Maurino DEDICATORIA A mis padres, que siempre apoyaron todas mis decisiones y confiaron en mí incondicionalmente, no sólo en cuanto a lo profesional sino en todos los aspectos de la vida, guiando pero siempre con la libertad necesaria para hacerme sentir que podía lograr cualquier cosa que me propusiese. A la hermosa familia que formamos junto a la persona que elijo todos los días, son el motor y motivo de todas mis acciones. No me alcanzan las palabras para agradecerles por apoyarme y comprenderme en esta etapa, pero sobre todas las cosas por hacerme feliz a cada instante de mi vida. Este logro es para ustedes. ii Bióloga María Fernanda Maurino AGRADECIMIENTOS Quisiera agradecer a la Dra. Irma Graciela Laguna y la Dra. María de la Paz Giménez Pecci por su apoyo, guía y aliento constantes, y por confiar en mí en todo este proceso. Muchas gracias a la Dra. Claudia Nome por su asistencia en la sección de microscopía y al Dr. Fabián Giolitti por su apoyo intelectual en gran parte del proceso y técnico en el desarrollo del reactivo de diagnóstico serológico. A la Dra. Ana María Marino de Remes Lenicov por su asistencia en la identificación de los insectos estudiados y al Dr. Eduardo Virla por proveer de ejemplares de los mismos, para su utilización en esta tesis. Por último, aunque no menor, quisiera también agradecer a todos aquellos que desde uno u otro lugar me brindaron siempre su ayuda desinteresada y sin los cuales me hubiese resultado mucho más difícil esta tarea, compañeros de logros y alegrías pero también de frustraciones y desaciertos. iii Bióloga María Fernanda Maurino PUBLICACIONES DERIVADAS DE LA TESIS 2012. Maurino F., Nome C., Laguna I.G. y Giménez Pecci, M.P. Cap. VIII Virosis emergentes en maíz. En: Enfermedades del maíz producidas por virus y mollicutes en Argentina. Editores: Giménez Pecci M.P., Laguna I.G., Lenardon S.L. Ediciones INTA. Buenos Aires, 2012. Pág. 99-107. 2012. Maurino M.F., Laguna I.G., Giolitti F., Nome C. y Giménez Pecci M.P. First Occurrence of a Rhabdovirus Infecting Maize in Argentina. Plant Disease, Vol. 96 Issue 9, pág. 1383. iv Bióloga María Fernanda Maurino Índice de contenidos Índice de contenidos Índice de contenidos ......................................................................................................... v Índice de tablas y figuras .................................................................................................. 1 Abreviaturas...................................................................................................................... 4 Resumen ........................................................................................................................... 7 Summary ........................................................................................................................... 9 Introducción .................................................................................................................... 11 Hipótesis ......................................................................................................................... 21 Objetivo general ............................................................................................................. 21 Objetivos específicos ...................................................................................................... 21 Capítulo I: Localización y morfología de las partículas del virus en estudio................. 22 Introducción ............................................................................................................. 23 Materiales y Métodos .............................................................................................. 25 1-Colección de muestras .............................................................................. 25 2-Microscopía electrónica ........................................................................... 25 3-Caracterización de partículas virales. ....................................................... 26 Resultados ............................................................................................................... 26 1-Colección de muestras. ............................................................................. 26 3-Caracterización de las partículas virales. ................................................. 26 Conclusiones ........................................................................................................... 30 Capítulo II: Reconstrucción de las relaciones filogenéticas del rhabdovirus causal de estriado amarillo en maíz ................................................................................................ 31 Introducción ............................................................................................................. 32 Materiales y Métodos .............................................................................................. 35 1-Colección de muestras. ............................................................................. 35 v Índice de contenidos 2-Extracción de ARN y RT-PCR. ............................................................... 35 3-Clonado y secuenciación. ......................................................................... 36 4-Análisis de identidades nucleotídicas. ...................................................... 36 5-Análisis filogenético. ................................................................................ 39 Resultados ............................................................................................................... 39 1-Colección de muestras. ............................................................................. 39 2-Extracción de ARN y RT-PCR. ............................................................... 40 4-Análisis de identidades nucleotídicas ....................................................... 41 5-Análisis filogenético del gen de la polimerasa L. .................................... 43 Conclusiones ........................................................................................................... 45 Capítulo III: Evaluación, en condiciones experimentales, de insectos vectores del patógeno y de otros medios de transmisión ................................................................................... 46 Introducción ............................................................................................................. 47 Materiales y Métodos .............................................................................................. 49 1-Recolección de insectos delfácidos para cría. .......................................... 49 2-Transmisión mediante insectos vectores. ................................................. 49 3-Transmisión mediante incisiones vasculares (VPI).................................. 51 Resultados ............................................................................................................... 52 1-Recolección de insectos delfácidos para cría. .......................................... 52 2-Transmisión mediante insectos vectores. ................................................. 52 3-Transmisión mediante incisiones vasculares (VPI).................................. 55 Conclusiones ........................................................................................................... 55 Capítulo IV: Desarrollo de métodos de diagnóstico eficientes ...................................... 56 Introducción ............................................................................................................. 57 Materiales y métodos ............................................................................................... 59 1-Producción de un reactivo de diagnóstico molecular específico para MYSV ..................................................................................................................... 59 vi Índice de contenidos 2-Antisuero de virus parcialmente purificado. ............................................ 59 Resultados ............................................................................................................... 63 1-Producción de un reactivo de diagnóstico molecular específico para MYSV ....................................................................................................................
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