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(Plum Pox Virus) En Especies Frutales Del Género Prunus

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(Plum Pox Virus) En Especies Frutales Del Género Prunus Centro de Edafología y Biología Aplicada de Segura Tesis Doctoral Resistencia a la sharka (Plum pox virus) en especies frutales del género Prunus Directores Dr. Federico Dicenta López-Higuera Dr. Pedro Martínez Gómez Manuel Rubio Angulo Ingeniero Agrónomo Murcia, 2006 ÍNDICE GENERAL 1. ANTECEDENTES, JUSTIFICACIÓN Y OBJETIVOS..................................... 3 1.1. El cultivo de frutales del género Prunus ................................................. 3 1.1.1. Descripción taxonómica y botánica................................................ 3 1.1.2. Origen y situación actual del cultivo .............................................. 5 1.2. El virus causante de la sharka ............................................................. 10 1.2.1. Taxonomía y descripción del virus............................................... 10 1.2.2. Origen y situación actual de la virosis en España.......................... 14 1.2.3. Detección y localización del virus ................................................ 14 1.2.4. Epidemiología y control de la virosis ............................................ 19 1.3. Susceptibilidad varietal, sintomatología y fuentes de resistencia............. 21 1.4. Justificación y objetivos ...................................................................... 29 2. MATERIAL Y MÉTODOS........................................................................... 33 2.1. Material vegetal.................................................................................. 33 2.1.1. Plantas indicadoras .................................................................... 34 2.1.2. Variedades de frutales del género Prunus .................................... 35 2.1.2.1. Albaricoqueros .................................................................. 35 2.1.2.2. Ciruelos ............................................................................ 38 2.1.2.3. Melocotoneros y nectarinas................................................ 40 2.1.2.4. Almendros ........................................................................ 42 2.1.3. Patrones de frutales del género Prunus ....................................... 45 2.1.4. Cruzamientos inter e intraespecíficos........................................... 48 2.1.4.1. Albaricoquero x albaricoquero ............................................ 49 2.1.4.2. P. mandshurica x albaricoquero.......................................... 50 2.1.4.3. Melocotonero x almendro................................................... 50 2.2. Aislado del Plum pox virus .................................................................. 51 2.3. Evaluación de la resistencia a la sharka................................................ 51 2.3.1. Evaluación en condiciones controladas en invernadero ................. 52 2.3.2. Evaluación en condiciones de campo........................................... 57 2.4. Técnicas de detección del virus ........................................................... 58 2.4.1. ELISA-DASI ............................................................................... 58 2.4.2. Hibridación molecular no radioactiva ........................................... 59 2.4.3. RT-PCR ..................................................................................... 60 2.5. Localización del virus .......................................................................... 62 2.5.1. Inmunoimpresión de la proteína de la cápsida ............................. 62 2.5.2. Impresión del ácido nucleico....................................................... 63 2.6. Clasificación del nivel de resistencia..................................................... 64 3. EVALUACIÓN DE LA RESISTENCIA A LA SHARKA EN ESPECIES DEL GÉNERO PRUNUS .......................................................................................................69 3.1. Introducción ...................................................................................... 69 3.2. Material ensayado .............................................................................. 73 3.3. Metodología ....................................................................................... 74 3.3.1. Comparación de las técnicas de detección del virus ...................... 74 3.3.2. Comparación de las técnicas de localización del virus en los tejidos . 74 3.3.3. Estudio del movimiento del virus a larga distancia ........................ 75 3.3.4. Evaluación de la resistencia en invernadero y en campo ............... 77 3.3.5. Optimización de la evaluación de la resistencia en invernadero...... 77 3.4. Resultados y discusión ........................................................................ 78 3.4.1. Comparación de las técnicas de detección del virus ...................... 78 3.4.2. Comparación de las técnicas de localización del virus en los tejidos . 81 3.4.3. Estudio del movimiento del virus a larga distancia ........................ 86 3.4.4. Comparación de la evaluación en invernadero y campo ................ 89 3.4.5. Optimización de la evaluación de la resistencia en invernadero...... 94 3.5. Conclusiones .................................................................................... 102 4. COMPORTAMIENTO FRENTE A LA SHARKA EN ESPECIES DEL GÉNERO PRUNUS ..................................................................................................... 105 4.1. Introducción .................................................................................... 105 4.2. Material ensayado ............................................................................ 106 4.3. Metodología ..................................................................................... 107 4.4. Resultados y discusión ...................................................................... 108 4.4.1. Variedades de frutales del género Prunus .................................. 108 4.4.1.1. Albaricoqueros ................................................................ 108 4.4.1.2. Ciruelos .......................................................................... 112 4.4.1.3. Melocotoneros y nectarinas .............................................. 114 4.4.1.4. Almendros ...................................................................... 118 4.4.2. Patrones de frutales del género Prunus ..................................... 121 4.5. Conclusiones .................................................................................... 128 5. TRANSMISIÓN DE LA RESISTENCIA A LA SHARKA EN ESPECIES DEL GÉNERO PRUNUS ...................................................................................... 131 5.1. Introducción .................................................................................... 131 5.2. Material ensayado ............................................................................ 134 5.3. Métodología ..................................................................................... 135 5.3.1. Evaluación de la resistencia ...................................................... 135 5.3.2. Hípotesis de herencia y ánalisis estadístico................................. 135 5.4. Resultados y discusión ...................................................................... 136 5.4.1. Herencia de la resistencia en albaricoquero................................ 136 5.4.2. Prunus mandshurica como fuente de resistencia en albaricoquero ..... 142 5.4.3. El almendro como fuente de resistencia en melocotonero ........... 144 5.5. Conclusiones .................................................................................... 148 6. RESUMEN Y CONCLUSIONES GENERALES............................................151 7. BIBLIOGRAFÍA.......................................................................................157 8. ANEXOS ..................................................................................................179 1. Cultivo de plantas en invernadero..........................................................179 2. Técnica ELISA-DASI .............................................................................180 3. Hibridación molecular no radioactiva......................................................182 3.1. Síntesis de la sonda .....................................................................182 3.2. Hibridación molecular no radioactiva .............................................183 4. Extracción de ARN con Kit Qiagen® .......................................................185 5. Técnica RT-PCR ...................................................................................186 6. Inmunoimpresión para la localización de la proteina del virus ..................188 7. Impresión para la localización del ácido nucleico del virus .......................190 8. Tablas optimización de la evaluación de la resistencia en invernadero......192 INDICE DE TABLAS 2. MATERIAL Y MÉTODOS Tabla 2.1. Clasificación botánica y uso agrícola de las especies del género Prunus estudiadas................................................................................................. 33 3. EVALUACIÓN DE LA RESISTENCIA A LA SHARKA EN ESPECIES DEL GÉNERO PRUNUS Tabla 3.1. Comparación de tres métodos diferentes para la detección del PPV: ELISA-DASI, hibridación molecular no radioactiva y RT-PCR ............................. 80 Tabla 3.2. Verificación de la presencia de sharka mediante observación visual de síntomas y la técnica ELISA-DASI ................................................................. 82 Tabla 3.3. Localización del PPV en secciones de tallo y pecíolo en diferentes especies del género Prunus mediante inmunoimpresión
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