Thèse de doctorat--Sciences naturelles--Faculté des sciences de Paris, 1911. N°: 1401. / Notes bibliogr.

Thesis (M.S.)--Oregon State University, 2011. / Printout. Includes bibliographical references (leaves 84-90). Also available on the World Wide Web.

Thesis (Ph. D.) -- Lincoln University, 2007. / Also available via the World Wide Web.

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L’objectif de mes travaux de thèse était d’étudier le métabolisme du tréhalose chez la vigne (Vitis vinifera L. cv. Chardonnay) en réponse à l’exposition à deux stress : le froid" chilling " et l’infection par le champignon pathogène Botrytis cinerea.Pour cela, nous avons tout d’abord optimisé un dosage du tréhalose par fluorimétrie qui nous a permis de caractériser le métabolisme du tréhalose en réponse à ces deux stress.Le métabolisme du tréhalose est activé différemment par le froid dans les organes dela vigne. Les gènes VvTPPA, codant une enzyme de synthèse du tréhalose, et VvTRE, codantla tréhalase, l’enzyme de dégradation, sont respectivement induits et réprimés dans les feuilleset leur expression est corrélée à une augmentation de la concentration en tréhalose. En outre,aucune synthèse de tréhalose n’est mesurée dans les tiges et il n’est pas détectable dans les racines. Enfin, la synthèse de T6P, son précurseur, est précoce dans les feuilles (3 heures aprèsl’exposition au froid). Nos résultats s’accordent avec le modèle actuel faisant du T6P une molécule-signal, corrélée à la concentration en saccharose. Ils excluent, pour le tréhalose, une participation significative à l’osmorégulation en réponse au froid chez la vigne. Par ailleurs,nous avons utilisé des plants de vigne bactérisés par Burkholderia phytofirmans, une bactérie endophyte induisant une tolérance au froid chez cette plante. Nous y avons observé une synthèse de T6P et de tréhalose et nous pensons que cette synthèse pourrait constituer une composante importante de la tolérance induite au froid.Lors de l’infection de feuilles de vitroplants de vigne par B. cinerea, nous avons observé (i) une forte augmentation de la quantité de tréhalose, (ii) l’induction de l’expression du gène VvTRE et (iii) l’augmentation de l’activité tréhalase. En revanche, aucune augmentation de la concentration en T6P n’a été détectée. Nous pensons donc que la synthèsede tréhalose in planta n’est pas favorisée durant l’infection et que le tréhalose détecté est probablement d’origine fongique. Nos résultats sont compatibles avec l’hypothèse selon laquelle l’induction du gène codant la tréhalase et l’augmentation concomitante de son activité interviendraient lors des interactions plantes-agents pathogènes pour éviter toute perturbation du rôle de molécule-signal joué par le T6P.Au final, le métabolisme du tréhalose participe à la réponse aux stress environnementaux chez la vigne et son étude mériterait d’être approfondie, notamment en ce qui concerne les stress biotiques. / The purpose of the present thesis was to investigate grapevine trehalose metabolism upon exposure to 2 stress conditions: chilling and infection by the grey mould fungus Botrytis cinerea.Initially, we had to optimize a fluorimetric based assay to assess trehalose concentration in grapevine. Latter, this method was used to characterize trehalose synthesis in this plant when exposed to chilling or infected by B. cinerea.Upon chilling exposure, trehalose metabolism is differentially activated in grapevine organs. VvTPPA, a gene involved in trehalose synthesis, and VvTRE, encoding the trehalose degrading enzyme (trehalase), were respectively induced and repressed and their expression was correlated with an increase of trehalose concentration in leaves. No trehalose synthesis was observed in stems and the sugar was undetectable in roots. T6P (its precursor)concentration increase was faster in leaves (3 hours after chilling exposure). Our results are in agreement with current status of T6P acting as a signal molecule, correlated with sucrose concentration, and exclude any significant participation of trehalose as a global osmoprotectant under chilling stress in grapevine. Additionally, we have used grapevine plants bacterized by Burkholderia phytofirmans, an endophytic bacterium that confers them chilling tolerance. We have detected T6P and trehalose synthesis in these plants and we believe it might contribute to the induced chilling tolerance.During grapevine leaf infection by B. cinerea, we observed: (i) a strong increase oftrehalose concentration, (ii) the induction of VvTRE and (iii) an increase of trehalase activity.However, no increase in T6P concentration was detected during infection. Our results suggest that trehalose metabolism is not activated upon B. cinerea infection and that trehalose detected is mainly of fungal origin. This is compatible with current hypothesis considering trehalase encoding gene induction and increase in trehalase activity as a plant response to avoid interference with T6P signaling pathway during pathogen infection.Overall, trehalose metabolism is involved in environmental stress responses in grapevine and might be consider for further research especially with focus on biotic stress.

Caractérisation d'inhibiteurs de protéases lors de l'interaction entre la vigne et Botrytis cinerea. Il a été montré que lors de l'infection de la baie de raisin par B. cinerea, des protéases fongiques pourraient être à l'origine de la dégradation d'une des protéines PR majoritaires de la baie mûre, la chitinase VvChi4D (Thèse S. Colas, 2012 ; van Sluyter et al., 2013). L'hypothèse émise lors de notre étude est que des inhibiteurs de protéases de la vigne pourraient empêcher la dégradation de cette protéine de défense par les protéases de B. cinerea.L'expression de deux inhibiteurs de protéases, un Potato Inhibitor I (VvPin) et un Kunitz (VvKun), ainsi que celle de trois protéases fongiques, une protéase aspartique (BcAp8), une protéase glutamique (BcAcp) et une protéase à sérine (BcSer), ont été suivies lors de l'infection de la baie de Pinot noir et de la feuille de vitroplants. Les résultats obtenus montrent que l'expression des deux IP est induite en même temps que celle de la protéase à sérine mais après celle des deux protéases acides du champignon. La production en système hétérologue des deux IP ainsi que l'obtention de protéases acides et de protéases à sérine de B. cinerea a permis de montrer que la protéine VvKun est capable d'inhiber les protéases à sérine du champignon. En revanche, aucun des deux IP n'est capable d'inhiber les protéases acides du champignon, protéases responsables de la dégradation de la chitinase VvChi4D. / Characterization of protease inhibitors in the interaction between Vitis vinifera and Botrytis cinerea.It has been shown that upon infection of the grape berry by B. cinerea, fungal proteases may be responsible for the degradation of a PR protein of the mature berry VvChi4D chitinase (Thesis S. Colas, 2012; van Sluyter et al, 2013). The hypothesis of our study is that protease inhibitors could prevent the degradation of this defense protein by proteases of B. cinerea.The expression of two protease inhibitors, a Potato Inhibitor I (VvPin) and a Kunitz (VvKun), and that of three fungal proteases, an aspartic protease (BcAp8), a glutamic acid protease (BcAcp) and a serine protease (BcSer) were followed during infection of Pinot Noir berry and leaf plantlets. The results obtained show that the expression of IP is induced both in the same time as the serine protease, but after that the two fungal acid proteases. The heterologous production of both IPs and the production of acid and serine proteases from B. cinerea secretoms have shown that VvKun is capable to inhibit serine proteases of the fungus. However, neither IP is capable of inhibiting fungal acid proteases, responsible for the degradation of the chitinase VvChi4D.

Th. doct.-ing.--Toulouse, I.N.P., 1979. N°: 72.

Orientador: Wagner Bettiol / Banca: Edson Luis Furtado / Banca: Antonio Carlos Maringoni / Banca: Marcelo Augusto Boechat Morandi / Banca: Gilberto Ubida Leite Braga / Resumo: A incidência de radiação ultravioleta (UV 100 a 400 nm) na terra , em especial a radiação UV - B (280 - 320 nm), por ser filtrada exclusivamente pela camada de ozônio e apresentar grande efetividade biológica , quando comparada com os outros espectros da radiação UV , está sendo alterad a com as mudanças climáticas . Sendo a radiação solar um importante componente climático durante o desenvolvimento de um microrganismo no ambiente, se fez necessário avaliar a tolerância de fitopatógenos, bem como de agentes de biocont role à radiação UV - B . Assim , o presente trabalho teve como objetivo s estudar alguns aspectos d a interação morangueiro × Botrytis cinerea × Clonostachys rosea × radiação UV - B. Nos estudos foram observadas diferença s significativa s entre os 13 isolados de B. cinerea em relação a germinação de esporos e esporulação em discos de folhas de morango após irradiação com UV - B de 2, 9 a 8, 9 KJ m - 2 . A germinação relativa variou de 75% a 9 5% e a esporulação variou em mais do que 100% entre os isolados de B. cinerea após exposição à radiação UV - B de 6,4 KJ m - 2 . O isolado LQC - 150 de B. cinerea apresentou maior germinação e esporulação em discos de folhas após irradiação e foi selecionado como o mais tolerante. O isolado LQC - 150 de B. cinerea apresentou LD 50 de 6,2KJ m - 2 . A esporulação de ambos os fungos em discos de folhas de morangueiro , quando inoculados individualmente, foi inversamente proporcional ... / Abstract: The incidence of ultraviolet (UV 100 to 400 nm) in the earth , especially UV - B radiation (280 - 320 nm) is being altered with climate change. The solar radiation is an import ant component for the development of microorganism in the environment, thus is important evaluate the tolerance of plant pathogens as well as the biocontrol agents to UV - B radiation. T he present study aimed to study the interaction of strawberry x Botrytis cinerea x Clonostachys rosea x UV - B radiation. There were significantly differences among the thirteen B. cinerea strains in relation to spore germination and sporulation on leaf disks after irradiation ranging from 2.9 to 8.9 KJ m - 2 . The relative germina tion ranged from 95 to 75% and the sporulation varied more than 100% among B. cinerea strains after exposure to 4 radiation of 6.4 KJ m - 2 . The LQC - 150 strain showed high germination and sporulation on leaf disk after irradiation and was selected as a toleran t strain. Survival curve of B. cinerea strain LQC - 150 showed lethal dose 50 (LD 50 ) of 6.2 KJ m - 2 . The sporulation of both fungi on leaf disks was inversely proportional to the dose of UV - B radiation, while inoculated alone. When confronted in the same leaf disk and not irradiated, C. rosea reduced the incidence of the pathogen and its sporulation in about 50% and 80%, respectively. However, the ability of C. rosea to control B. cinerea on leaf disks was gradually reduced with the increase of UV - B radiation, reaching 20% and 50%, respectively for pathogen incidence and sporulation, on higher UV - B doses. When the bioagent was applied in the morning, the development was lower than when applied afternoon. The effect of PABA in the induction of resistante in plan ts of Nicotiana benthamiana against B. cinerea was evaluated and it was found that plants treated with PABA were more resistant to the pathogen. The evaluations of size of plants and leaves ... / Doutor

Magíster en Bioquímica área de Especialización en Bioquímica ambiental y Memoria para optar al Título de Bioquímico / Autor no autoriza el acceso a texto completo de su documento hasta enero 2018 / El mercado de la uva de mesa en Chile es una importante fuente de ingreso económico, siendo nuestro país catalogado como líder exportador de esta fruta. Sin embargo, el principal problema de índole fitopatológico que afecta la uva de mesa son las infecciones producidas por el hongo Botrytis cinerea, que se caracterizan por generar pudriciones o atizonamientos de las partes florales, brotes y bayas, causando importantes pérdidas económicas. Su control incluye un manejo integrado del cultivo, además de un tratamiento químico preventivo mediante el uso de fungicidas. No obstante, aunque se realicen todas estas prácticas el control total no resulta satisfactorio, debido al aumento de cepas fúngicas resistentes a la acción de los fungicidas. Frente a esta problemática, se han estudiado otras formas de control dentro de las que se incluye la utilización de microorganismos capaces de impedir la infección de B. cinerea sobre cultivos susceptibles, entre los cuales podemos destacar el biocontrolador comercial Serenade, que posee como ingrediente activo la cepa bacteriana Bacillus subtilis QST 713. El objetivo principal de esta tesis fue caracterizar cepas bacterianas silvestres con actividad antifúngica y determinar la capacidad protectora de las bacterias contra el ataque del hongo sobre el vegetal. Se seleccionaron cepas bacterianas silvestres que al ser caracterizadas a nivel filogenético, bioquímico y ultraestructural, fueron clasificadas en el género Bacillus y en las especies B. subtilis y B. pumilus. Luego de evaluar su capacidad antifúngica mediante bioensayos in vitro e in vivo, se seleccionaron las cepas B. subtilis NB y B. pumilus G2, las que fueron capaces de proteger al tejido vegetal de forma más eficiente que el producto comercial Serenade. Posteriormente, se realizó extracción orgánica de las moléculas secretadas por estas bacterias, determinándose preliminarmente su naturaleza y estructura química, utilizando las técnicas TLC, IR, HPLC, LC-MS/MS y GC-MS. Finalmente, con el uso de estas metodologías se pudo demostrar que B. subtilis NB y B. pumilus G2 secretan enzimas que degradan la pared celular fúngica, dipéptidos cíclicos ciclo-(Pro-Leu) y ciclo- (Pro-Phe) y lipopéptidos cíclicos (iturinas, fengicinas y surfactinas). Estas bacterias poseen un gran potencial para controlar infecciones producidas por B. cinerea en las variedades de uva de mesa afectadas por este hongo. Sin embargo, se requieren estudios posteriores que permitan confirmar y ampliar los resultados de esta tesis, antes que se pueda desarrollar un producto comercial / Chile is a leading exporter of table grapes, and this market is an important source of income for the country. However, infection with the fungus Botrytis cinerea, characterized by the rotting of grape flowers, buds, and berries, causes important economic losses. Fungus control includes integrated crop management and preventive chemical fungicide treatments. Despite these practices, overall control is unsatisfactory as fungal strains become increasingly resistant to fungicides. Alternative control methods have been studied, including the use of microorganisms capable of preventing B. cinerea infection on susceptible cultivars. Among these is the commercial biocontrol Serenade, which contains the Bacillus subtilis QST 713 bacterial strain as the main active ingredient. The main objectives of this thesis were to characterize wild bacterial strains with antifungal activity and determine the protective ability of these strains for table grape vines challenged by B. cinerea. Wild bacterial strains were characterized at the phylogenetic, biochemical, and ultrastructural levels and classified in the genus Bacillus and species B. subtilis and B. pumilus. After evaluating antifungal capacities through in vitro and in vivo bioassays, the B. subtilis NB and B. pumilus G2 strains were selected. These strains were able to more efficiently protect plant tissue than the commercial product Serenade. Subsequently, the molecules secreted by these bacteria were organically extracted, and their chemical structures and natures were determined using the techniques TLC, IR, HPLC, LC-MS/MS, and GC-MS. These methodologies revealed that B. subtilis NB and B. pumilus G2 secreted enzymes that degraded the fungal cell wall, the cyclic dipeptides cyclo-(Pro-Leu) and cyclo-(Pro-Phe), and cyclic lipopeptides (iturins, fengycins, and surfactins). These bacteria show promising potential in controlling B. cinerea infections among affected Chilean table grape species. However, further studies are required to expand upon the present results before a product can be developed for commercial use / Proyecto DGT-2014AC

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