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Pfc5813.Pdf (9.887Mb) UNIVERSIDAD POLITÉCNICA DE CARTAGENA ESCUELA TÉCNICA SUPERIOR DE INGENIERÍA AGRONÓMICA DEPARTAMENTO DE PRODUCCIÓN VEGETAL INGENIERO AGRÓNOMO PROYECTO FIN DE CARRERA: “AISLAMIENTO E IDENTIFICACIÓN DE LOS RIZOBIOS ASOCIADOS A LOS NÓDULOS DE ASTRAGALUS NITIDIFLORUS”. Realizado por: Noelia Real Giménez Dirigido por: María José Vicente Colomer Francisco José Segura Carreras Cartagena, Julio de 2014. ÍNDICE GENERAL 1. Introducción…………………………………………………….…………………………………………………1 1.1. Astragalus nitidiflorus………………………………..…………………………………………………2 1.1.1. Encuadre taxonómico……………………………….…..………………………………………………2 1.1.2. El origen de Astragalus nitidiflorus………………………………………………………………..4 1.1.3. Descripción de la especie………..…………………………………………………………………….5 1.1.4. Biología…………………………………………………………………………………………………………7 1.1.4.1. Ciclo vegetativo………………….……………………………………………………………………7 1.1.4.2. Fenología de la floración……………………………………………………………………….9 1.1.4.3. Sistema de reproducción……………………………………………………………………….10 1.1.4.4. Dispersión de los frutos…………………………………….…………………………………..11 1.1.4.5. Nodulación con Rhizobium…………………………………………………………………….12 1.1.4.6. Diversidad genética……………………………………………………………………………....13 1.1.5. Ecología………………………………………………………………………………………………..…….14 1.1.6. Corología y tamaño poblacional……………………………………………………..…………..15 1.1.7. Protección…………………………………………………………………………………………………..18 1.1.8. Amenazas……………………………………………………………………………………………………19 1.1.8.1. Factores bióticos…………………………………………………………………………………..19 1.1.8.2. Factores abióticos………………………………………………………………………………….20 1.1.8.3. Factores antrópicos………………..…………………………………………………………….21 1.2. Los rizobios…………………………………………………………………………………………………25 1.2.1. Origen del estudio y taxonomía…………………………………………………………………..25 1.2.2. Establecimiento de la relación simbiótica entre la leguminosa y el rizobio…..28 1.2.2.1. Iniciación del nódulo……………………………………………………………………………..28 1.2.2.1.1. Señalización entre la planta y Rhizobium……………………………………………….28 1.2.2.1.2. Invasión y formación del canal de infección…………………………………………..29 1.2.2.1.3. Estructura y desarrollo de los nódulos…………………………………………………..31 1.2.3. La fijación biológica de nitrógeno al desarrollo de inoculantes microbianos………………………………………………………………………………………………..34 1.2.3.1. Información general sobre la fijación del nitrógeno……………………………….34 1.2.3.2. El ciclo del nitrógeno y la fijación de N2 atmosférico………………………………35 1.2.4. Los rizobios que nodulan con “Astragalus”………………………………………………….37 1.2.4.1. Características del género Rhizobium…………………………………………………….37 1.2.4.1.1. Rhizobium giardini…………………………………………………………………………………38 1.2.5. Importancia de la biodiversidad y estudio de los rizobios……………………………38 1.3. Inoculación de plantas en viveros………………………………………………………………..40 1.3.1. Introducción………………………………………………………………………………………………..40 1.3.2. Estudios de biodiversidad y caracterización de rizobios……………………………….42 1.3.2.1. Marcadores moleculares: inter simple sequence repeats (ISSRs)……………43 1.3.2.1.1. Fundamento de la PCR…………………………………………………………………………..45 1.3.2.2. Secuenciación del ARNr 16S………………………………………………………………….46 1.3.3. Marco legal y comercialización de los inoculantes en el mundo………………….52 1.3.4. Marco legal de los inoculantes en España……………………………………………………53 1.3.5. Tendencias en la investigación y el desarrollo de inoculante……………………….5 4 2. Objetivos………………………………………………………………………………………………………….56 3. Material y métodos…………………………………………………………………………………………..59 3.1. Material vegetal………………………………………………………………………………………….60 3.2. Método……………………………………………………………………………………………………….60 3.2.1. Aislamiento…………………………………………………………………………………………………61 3.2.1.1. Desinfección de nódulos………………………………………………………………………..61 3.2.1.2. Crecimiento de colonias en medio EL-MARC………………………………………….62 3.2.1.3. Crecimiento de colonias en medio Mc-Conckey……………………………………..64 3.2.1.4. Crecimiento de colonias en medio Kligler………………………………………………65 3.2.1.5. Crecimiento de colonias en medio LMA + Azul Bromotimol……………………67 3.2.2. Identificación………………………………………………………………………………………………67 3.2.2.1. Extracción ADN bacterial……………………………………………………………………….67 3.2.2.2. Amplificación del fragmento perteneciente a la región 16S, nodA y nodC mediante empleo de la PCR…………………………………………………………………..69 3.2.2.3. Electroforesis del ADN……………………………………………………………………………71 3.2.2.4. Purificación del fragmento amplificado………………………………………………...72 3.2.2.5. Secuenciando del fragmento amplificado………………………………………………73 3.2.2.6. Introducción de la secuencia en la base de datos BLAST…………………………7 4 3.2.3. Inoculación………………………………………………………………………………………………….75 3.2.3.1. Desinfección de semillas………………………………………………………………………..75 3.2.3.2. Escarificación y germinación de semillas………………………………………………..75 3.2.3.3. Desinfección de turba……………………………………………………………………………75 3.2.3.4. Preparación del inóculo………………………………………………………………………..75 3.2.3.5. Inoculación……………………………………………………………………………………………76 4. Resultados………………………………………………………………………………………………………..77 4.1. Aislamiento………………………………………………………………………………………………..78 4.1.1. Crecimiento de colonias en medio EL-MARC……………………………………………….78 4.1.2. Crecimiento de colonias en medio McConkey……………………………………………..79 4.1.3. Crecimiento de colonias en medio Kligler…………………………………………………….80 4.1.4. Crecimiento de colonias en medio LMA+Azul Bromotimol…………………………..81 4.2. Identificación………………………………………………………………………………………………83 4.3. Inoculación………………………………………………………………………………………………….87 5. Conclusión………………………………………………………………………………………………………..88 6. Referencia bibliográficas………………………………………………………………………………….90 Índice de imágenes Imagen 1. (Izq.)Planta de Astragalus nitidifloru sen periodo de floración; (Der.) Planta de Astragalus nitidiflorus, de 30-40 cm de diámetro, en su segundo año de vida. A los cuatro años puede llegar a alcanzar hasta un metro de diámetro………………………………..4 Imagen 2. Las hojas de Astragalus nitidiflorus son imparipinnadas, con 8-12 pares de folíolos vilosos por ambas caras……………………………………………………………………………………5 Imagen 3. Inflorescencia de Astragalus nitidiflorus en la que puede observarse la característica corola papilionácea de las Fabáceas con 5 pétalos libres; 1 superior muy desarrollado, denominado estandarte, 2 laterales o alas y 2 inferiores que pueden estar soldados y forman la quilla………………………………………………………………………………….6 Imagen 4. (Izq.) Frutos de Astragalus nitidiflorus; y (Der.) Semilla reniforme de Astragalus nitidiflorus………………………………………………………………………………………………….7 Imagen 5. Detalle de frutos y semillas de Astragalus nitidiflorus. Los frutos de esta especie contienen por término medio unas doce semillas……………………………………………7 Imagen 6. Tras el reposo estival, se produce el rebrote de las yemas situadas en la base de los tallos, a ras del suelo. De esta forma comienza la planta un nuevo periodo de actividad vegetativa……………………………………………………………………………………………………..8 Imagen 7. Esquema del ciclo de Astragalus nitidiflorus durante tres años, aunque se ha comprobado que puede vivir al menos hasta cinco años en el medio natural………………8 Imagen 8. En su primer año de vida las plántulas de Astragalus nitidiflorus desarrollan tan sólo 4-5 hojas. En su segundo año de vida y con mayor desarrollo vegetativo florecen por primera vez, dando lugar por término medio a 100 flores por planta………9 Imagen 9. Durante el periodo de floración, las inflorescencias se van sucediendo en los tallos conforme estos van creciendo. El número de inflorescencias producidas varía en función de la edad de la planta…………………………………………………………………………………….9 Imagen 10. Tras la fecundación de las flores se desprenden los pétalos marchitos y comienza el cuajado de los frutos………………………………………………………………………………10 Imagen 11. Las flores de Astragalus nitidiflorus son polinizadas por abejas del género Osmia, también conocidas como “abejas albañiles” por construir sus nidos y celdas con barro………………………………………………………………………………………………………………………….10 Imagen 12. Detalle de una abeja albañil posada sobre un piroclasto originado en el afloramiento volcánico de los Pérez Bajos………………………………………………………………….11 Imagen 13. Los frutos al madurar en verano se desprenden y quedan debajo de los tallos secos de la planta madre. Cuando la planta rebrota los frutos quedan bajo su sombra……………………………………………………………………………………………………………………….11 Imagen 14. Germinación de semillas de Astragalus nitidiflorus sobre papel de filtro en placas de Petri……………………………………………………………………………………………………………12 Imagen 15. Detalle de la germinación de semillas de Astragalus nitidiflorus donde se observa la emergencia de la radícula………………………………………………………………………….12 Imagen 16. Nódulos de Rhizobium giardinii que se han desarrollado en plantas de Astragalus nitidiflorus cultivadas en maceta con mezcla de turba y suelo del hábitat............................................................................................................................13 Imagen 17. Hábitat natural de Astragalus nitidiflorus. Las plantas del Garbancillo se encuentran sobre terrenos volcánicos de naturaleza basáltica, alterados por la actividad humana……………………………………………………………………………………………………….14 Imagen 18. Ejemplar de Astragalus nitidiflorus sobre basaltos alcalinos volcánicos……14 Imagen 19. Cabezo Negro de Tallante, volcán extinguido que constituye el último episodio de vulcanismo del sureste español hace 2,6 millones de años……………………..15 Imagen 20. Localización de las subpoblaciones existentes de Astragalus nitidiflorus en el complejo de Cabezos del Pericón……………………………………………………………………………17 Imagen 21. Hembra de Bruchophagus astragali…………………………………………………………20 Imagen 22. La proporción de flores que se convierten en frutos es muy baja, apenas alcanza el 20%, lo que da lugar a una media de 4-5 frutos por inflorescencia…………….21
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