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Fire Blight (Erwinia Amylovora) Of FIRE BLIGHT (ERWINIA AMYLOVORA) OF ROSACEOUS PLANTS. PATHOGEN VIRULENCE AND SELECTION AND CHARACTERIZATION OF BIOLOGICAL CONTROL AGENTS Jordi CABREFIGA OLAMENDI ISBN: 84-689-0992-0 Dipòsit legal: GI-142-2005 Universitat de Girona Departament d’Enginyeria Química, Agrària i Tecnologia Agroalimentària Institut de Tecnologia Agroalimentària TESI DOCTORAL FIRE BLIGHT (Erwinia amylovora) OF ROSACEOUS PLANTS. PATHOGEN VIRULENCE AND SELECTION AND CHARACTERIZATION OF BIOLOGICAL CONTROL AGENTS. Memòria presentada per optar al Grau de Doctor per la Universitat de Girona, per Jordi Cabrefiga i Olamendi 2004 Emili Montesinos Seguí i Anna Bonaterra Carreras, Catedràtic i Professor Titular de l’Àrea de Producció Vegetal, membres del Departament d’Enginyeria Química, Agrària i Tecnologia Agroalimentària i del Institut de Tecnologia Agroalimentària de la Universitat de Girona, CERTIFIQUEN: Que el llicenciat en Biologia Jordi Cabrefiga Olamendi ha dut a terme, sota la seva direcció, el treball amb el títol “Fire blight (Erwinia amylovora) of rosaceous plants. Pathogen virulence and selection and characterization of biological control agents”, que presenta en aquesta memòria la qual constitueix la seva Tesi per a optar al Grau de Doctor per la Universitat de Girona. I per a què consti als efectes oportuns, signen la present a Girona, el 14 d’abril de 2004. Vist-i-plau directors de la Tesi Dr. Emili Montesinos Seguí Dra. Anna Bonaterra Carreras Emili Montesinos Seguí, Catedràtic de Producció Vegetal de la Universitat de Girona, i director dels projectes de recerca ‘Estudios epidemiológicos y evaluación de estrategias de control integrado de fuego bacteriano (Erwinia amylovora) en España’ (Ref. AGF98-0402- C03-01) i ‘Optimización de los métodos de detección, prevención y control del fuego bacteriano de las rosáceas en España’ (Ref. AGL2001-2349-C03-01) de la Comisión Interministerial de Ciencia y Tecnologia, en els que es circumscriu la tesi doctoral titulada ‘Fire blight (Erwinia amylovora) of rosaceous plants. Pathogen virulence and selection and characterization of biological control agents’ amb el número de registre 307 de 5 de maig de 2000 realitzada per Jordi Cabrefiga Olamendi, DECLARA QUE: Aquesta tesi està sotmesa a la propietat intel·lectual compartida amb els investigadors del grup de Patologia Vegetal i del Institut de Tecnologia Agroalimentària de la Universitat de Girona que participen en els esmentats projectes (Article 2. apartat 2, RD 1326/2003 de 24-10-2003; Llei de la Propietat Intel·lectual, RD 1/1996 de 12-04-1996). I per a què consti als efectes oportuns, signen la present a Girona, el 14 d’abril de 2004. Vist-i-plau Dr. Emili Montesinos Seguí Jordi Cabrefiga Olamendi AGRAÏMENTS Són moltes les persones que d’una o altra manera han contribuït a la realització d’aquest treball, des d’ajudar a la realització de la part experimental fins a la de crear un bon ambient de treball. Són moltes hores, i alguns anys que dura i són moltes les paraules necessàries per expressar tots aquests agraïments. Arribat aquest punt són poques les ganes que tinc d’enrotllar-me i per tant aniré al gra. En primer lloc voldria agrair al Dr. Emili Montesinos per haver-me donat la oportunitat de formar part del seu grup d’investigació i haver-me contagiat la seva fascinació per la fitopatologia. Als meus directors de tesi, la Dra. Anna Bonaterra i el Dr. Emili Montesinos, per la direcció i correcció de la memòria, per tota la formació que m’han donat a tots el nivells, i sobretot per la seva paciència, dedicació i disponibilitat. Als meus companys, especialment a l’Anna, que a més de ser directora de tesi és una gran amiga, a l’Esther per fer-me de guia en els meus inicis, a en Jesús, el meu inseparable company de despatx que no hi ha maneres de treure-me’l de sobre, a la Lídia, per tantes coses (les que te apuntades i les que no), a la Lusi i a l’Olga per la quantitat de favors, a la Cun i a l’Isidre per donar-me bons consells sempre que els he necessitat, a en Pere i en Josep per la seva ajuda quan ha fet falta, a la Sussi, a la Carme i a la Lourdes per la seva ajuda en els aspectes administratius. També als nouvinguts que contribueixen a alegrar l’ambient del laboratori, la Mari, la Marta, la Patrícia, la Rosalia, la Lourdes, l’Albert, i a les que ja no hi són, la Roser i la Carme. A les meves “cosines” de Tecnologia d’Aliments, l’Anna Maria, l’Elena, la Mònica, la Dolors i la Lucero, i als nouvinguts, l’Eduard, la Nuri, la Maria, en David, les Annes, l’Ariadna i en Batista. I no voldria oblidar les companyes de l’EQATA, la Nuri i la Gemma, ni en Jaume, pels esmorzars, els pica-piques, les sortides nocturnes i les xerrades que hem compartit. De forma especial a tots als meus amics, per compartir tants moments, per fer que cada dia sigui especial i hagi valgut la pena viure’l. A l’Instituto Valenciano de Investigaciones Agrarias, a la Universidad Pública de Navarra, a la Unitat de Sanitat Vegetal del laboratori de Sanitat del DARP, i a la Unidad de Sanidad Vegetal de la Diputación General de Aragón, per haver cedit part de les soques de bacteris utilitzades en aquest treball. Al Dr. Jean Pierre Paulin, i al seu equip de la Station de Pathologie Végétale de l’INRA d’Angers per haver-me acollit durant un mes al seu centre. Al servei de Microscòpia de la Universitat de Girona, per la seva ajuda i assessorament en les observacions al microscopi electrònic i en la preparació de les mostres. i ii Agraïments Al Departament d’Enginyeria Química, Agrària i Tecnologia Agroalimentària (EQATA) de l’Escola Politècnica Superior de la Universitat de Girona, a l’Institut de Tecnologia Agroalimentària (INTEA) i al Centre de Innovació i Desenvolupament en Sanitat Vegetal (CIDSAV) de la mateix universitat per facilitar la infrastructura necessària per realitzar els experiments. A la Generalitat de Catalunya per haver-me concedit una Beca CIRIT Pre-Doctoral per a la formació de personal investigador (FI/FIAP). Finalment, un record per a la família que m’han donat suport, moltes vegades sense saber que estava fent, i especialment a la Rosa per estar amb mi i aguantar-me quan les coses no han anat del tot bé. RESUM El foc bacterià, causat pel bacteri E. amylovora, és una malaltia molt important a nivell comercial i econòmic perquè afecta a les plantes de la família de les rosàcies i és especialment agressiva en pomera (Pyrus malus), perera (Pyrus communis) i plantes ornamentals (Crataegus, Cotoneaster o Pyracantha). Aquesta malaltia està distribuïda per tot el món en zones climàtiques temperades d’Amèrica del Nord, Nova Zelanda, Japó, Israel, Turquia i Europa. A Espanya, el foc bacterià va ser detectat per primera vegada el 1995 al nord del País (Euzkadi) i més tard, en nous focus apareguts en altres zones. L’objectiu principal d’aquesta tesi doctoral va ser la selecció d’agents de biocontrol del foc bacterià i per aquesta raó es van seleccionar un grup reduït de soques d’E. amylovora i es van posar a punt diversos patosistemes ex vivo i in planta per ser utilitzats en el procés de selecció d’agents de biocontrol del foc bacterià. També es va estudiar l’existència de trets o patrons específics dels antagonistes associats a la inhibició de la infecció causada per E. amylovora i els possibles mecanismes d’acció involucrats en el biocontrol d’E. amylovora per una de les millors soques. En el present treball, es van caracteritzar 53 soques d’E. amylovora de diferents orígens (localització geogràfica i planta hoste) en relació al tipus de colònia en diferents medis de cultiu, detecció per PCR i ELISA, perfils metabòlics utilitzant els sistemes API i Biolog GN, patogenicitat en fruits immadurs de pera i diversitat genètica mitjançant RFLPs del gen ams i de l’ADN genòmic. Les soques d’E. amylovora es van mostrar molt homogènies per a totes aquestes característiques. Tot i això, es van observar diferències principalment en els perfils metabòlics, MRFLPs de l’ADN genòmic i patogenicitat en peres immadures. La variació en els MRFLPs de l’ADN genòmic es va relacionar amb la distribució geogràfica, mentre que la variació en els perfils d’utilització de fonts de carboni semblaven estar relacionats amb la planta hoste per la tendència a agrupar les soques en dos grups, un composat principalment per soques aïllades de fruiters i l’altre per soques aïllades de plantes ornamentals. Es va observar major diversitat entre soques a nivell de virulència en peres immadures. Estudis basats en la relació dosi-malaltia i en corbes de progressió de la malaltia van permetre la classificació de les soques en funció de la seva agressivitat (dosi mitjana efectiva, incidència màxima de la malaltia, taxa de progressió de la malaltia), infectivitat (dosi infectiva mínima, moment d’inici de la infecció) o de la seva virulència en termes generals (índex de virulència compost, àrea sota la corba de progressió de la malaltia). Les soques que van presentar la major virulència van ser EPS101, UPN513, UPN544, USV1043, USV4512 i USV4576. D’acord amb aquests resultats, les soques CUCM273 i EPS101 d’E. amylovora van ser elegides per realitzar la selecció d’agents de biocontrol. La soca EPS101 va ser seleccionada per haver estat aïllada de la nostra zona d’influència, a Lleida, i perquè presentava les característiques comunes de la majoria de soques d’E. amylovora. Per contra, la soca Resum CUCM273 va ser aïllada als Estats Units i es va seleccionar per ser probablement una de les soques més divergents, ja que, difereix de la resta en el serotip i en el tipus de colònia (blanca, convexe, brillant i mucosa).
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