Rozprawy doktorskie na temat „Botrytis cinerea”

Latent B. cinerea was detected in nine symptomless wild host species from the families Asteraceae and Brassicaceae, in addition to greenhouse grown lettuce. Conventional testing methods revealed that latent B. cinerea was equally prevalent in the root system as the above ground parts. Incidence of latent infection was moderate in some species (Achillea milleforlium, Arabidopsis thaliana, Centraurea nigra, Cirsium vulgare, Senecio jacobaea, Senecio vulgaris and Taraaxacum agg.) and rare in others (Tussilago farfara and Bellis perennis). In greenhouse lettuce, latent infection was activated by prolonged water stress and artificial inoculation. Despite inoculation, unstressed, vigorously growing lettuce and Arabidopsis plants remained asymptomatic throughout the growing period. Fungicide seed treatment did not significantly affect the amount of latent B. cinerea recovered from the lettuce plants. Introduction of antagonistic micro-organism Trichoderma harzianum T-39 into the soil decreased the amount of latent infection recovered from lettuce leaves but increased it in the stem. A weak negative correlation was found between photosynthesis and the amount of B. cinerea recovered from the leaves. Weight of the plants was reduced due to inoculation of B. cinerea even though latent infection was unaltered. There was no relation between plant weight and total endophytic B. cinerea. A marginal increase of the phenolic contents of the leaf was observed due to inoculation, but no changes to the antioxidant activity, chlorophyll content or carotenoids were found. The high incidence of latent infection found in greenhouse grown lettuce plants with or without successful inoculation may have been due to the presence of several genetically distinct isolates of B. cinerea. Eight different haplotypes were identified among the 32 isolates assessed. A single very common haplotype presumably originated from seed borne infection, because it was rare in plants grown from fungicide treated seed. Latency may be attributed to a mild strain defence response by the presence of several genetically different strains of the pathogen present within the plant as endophytes.

Systemic Botrytis cinerea isolates were collected from non symptomatic fruits of Rubus fruticosus (blackberry) and Fragaria x ananassae (strawberry) and from roots and leaves of wild Primula vulgaris (primrose) and Taraxacum agg. (dandelion) in Brighton, Bath, and Reading separated by 80km or more. Isolates recovered from a Primula x polyantha crop at Reading were also tested. Approximately 300 isolates were genetically characterised using 9 published microsatellite primers and the presence of two transposable elements, boty and flipper. In wild primula and dandelion, incidence of non-symptomatic infection was very variable both geographically and between years. Isolates were usually haploid at all loci, with most haplotypes unique and no detectable linkage disequilibrium between loci.

The study was carried out to clarify the nature of symptomless infection by Botrytis cinerea and to what extent it differs from aggressive necrotic infection in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless plants were produced by dry spore inoculation in plants growing in controlled environmental conditions or in glasshouses. Plating out of surface-disinfected and non-surface-disinfected samples of inoculated, apparently healthy, plants on selective medium revealed that the fungus was spreading from the initial inoculation site to newly developing plant organs both internally and externally. Similar findings were obtained in microscope experiments in which host plants were inoculated with GFP labelled B. cinerea and symptomless spreading was monitored under confocal laser scanning microscope. Spore germination on leaf surface was followed by development of sub-cuticular vesicles and plant cell damage in the infected epidermal cell and a few nearby cells. Sparsely branched long hyphae arose from the vesicles and spread on the leaf surface; spread was mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells. In the late symptomless phase, mycelium arising from single vesicles formed several mycelial networks on leaves. Experiments were carried out to compare the extent of gene expression in symptomless and necrotic infections, using RT-qPCR. Expression of selected genes was quantified in tissue samples based on the amount of mRNA of the respective genes found. In both host species, the mRNA concentration of signalling genes bcg1, bmp1 and calcineurin, and the pathogenicity genes bcsod1 and bcpg1 were similar to or slightly greater in symptomless samples than in necrotic samples. The mRNA of the signalling gene bac and pathogenicity genes bcbot1 and bcnep1, were not detected or detected in lower abundance than in necrosis. In lettuce, the leaves developing distant from the site of inoculation showed similar results to A. thaliana, but in healthy leaves close to the site of inoculation mRNA concentrations of bac and bcnep1 were similar to necrotic samples. Thus, in both host species, the fungus grew along with the plant and moved to newly growing plant parts without producing symptoms; during this growth some pathogenicity genes were less expressed than in necrotic infection.

Il presente elaborato descrive gli aspetti generali di Botrytis cinerea quando si sviluppa come muffa grigia e muffa nobile. La differenza fra queste due diverse forme non è data da una differenza genetica del fungo ma dal microclima presente nell'area di infezione. Infatti l'alternanza di condizioni umide a condizioni secche fa si che il fungo si sviluppi solo all'interno dello strato epidermico (acino infavato) senza sviluppare efflorescenze all'esterno dell'acino (muffa grigia).La presenza della muffa apporta all'acino una maggiore concentrazione di solidi solubili, un lieve innalzamento di pH e un aumento di componenti aromatiche. Queste caratteristiche diventano indispensabili per la produzione di vini bianchi dolci come Sauternes e Tokaj. La comparsa di muffa nobile avviene naturalmente in pochissime aree al mondo. questo perché i fattori di sviluppo sono molto limitanti. L'eccesso e la carenza di umidità o di caldo potrebbero compromettere lo sviluppo della muffa nobile. Questo fa pensare che il riscaldamento globale, comportando un innalzamento della temperatura e una diminuzione delle precipitazioni, possa ostacolare lo sviluppo di muffa nobile nelle uniche zone che presentano questa particolarità.

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

Thesis (MScAgric)--University of Stellenbosch, 2005.
ENGLISH ABSTRACT: Grey mould, caused by Botrytis cinerea, is the most important foliar disease of rooibos seedlings. Although the disease is primarily controlled with applications of fungicides, the improvement of cultural methods of disease management should lessen this dependence on chemical control. Such improvements would, however, not be possible without knowledge of the inoculum sources and dispersal of the pathogen. The aim of this study was to investigate the inoculum ecology of B. cinerea in rooibos nurseries in order to identify primary sources of inoculum and to improve the environmentally friendly management of the disease. The study was conducted in four nurseries over two production seasons (March to July 2003 and 2004). Levels of airborne inoculum of B. cinerea were monitored on a monthly basis inside and around the nurseries with spore traps. Samples of plant material and organic debris were taken in the corresponding areas to determine the incidence of plant material infected by the pathogen and the incidences of grey mould in the nurseries were recorded. Low numbers of B. cinerea colonies were observed on the spore traps. Similar levels of airborne inoculum were observed inside and around the nurseries. The incidence of plant material yielding B. cinerea was higher outside the nurseries than inside, indicating the importance of such materials as potential sources of inoculum. Since patterns of airborne inoculum observed in this study confirmed reports of the local dispersal of B. cinerea, the removal of possible hosts outside the nurseries could aid in the management of grey mould in rooibos nurseries. Resistance to dicarboximide fungicides is a genetically stable trait in B. cinerea, and therefore has the potential to be used as a phenotypic marker. This marker can be used to gain knowledge on the dispersal of B. cinerea inoculum inside and outside rooibos nurseries. Isolates of B. cinerea collected from the air and from plant material in and around four rooibos nurseries were assessed for resistance to iprodione at 1 and 3 μg/ml a.i. Some of the isolates showed resistance to iprodione at 1 μg/ml a.i. However, none of the isolates showed resistance at 3 μg/ml a.i. iprodione. The initial incidence of dicarboximide-resistance at the nurseries was slightly higher than expected. As the season progressed, the incidence of iprodione-resistant isolates decreased towards May, after which an increase was observed towards July. A relatively high percentage of isolates collected outside the nurseries was found to be dicarboximide-resistant. Two of the nurseries had a significant higher incidence of resistant isolates on plant material collected inside, than on plant material collected outside the nursery. However, when looking at resistance levels of airborne isolates, no significant differences were found in the incidence of resistant isolates sampled inside and outside the four nurseries. The data indicated the importance of organic debris and seed-borne infections in the survival and dispersal of dicarboximide-resistant isolates of the pathogen. With the current emphasis on organic agriculture the knowledge gained in this study presents valuable possibilities of improving the cultural management of grey mould in rooibos nurseries.
AFRIKAANSE OPSOMMING: Vaalvrot, veroorsaak deur Botrytis cinerea, is die belangrikste bo-grondse siekte van rooibossaailinge. Alhoewel die beheer van die siekte hoofsaaklik op die gebruik van fungisiede berus, behoort die verbetering van verbouingspraktyke hierdie afhanklikheid van chemiese beheer te verminder. Sulke verbeteringe sal egter slegs moontlik wees indien voldoende kennis van die inokulumbronne en verspreiding van die patogeen beskikbaar is. Die doel van hierdie ondersoek was om die inokulum ekologie van B. cinerea in rooibos kwekerye te ondersoek sodat primêre inokulumbronne opgespoor en omgewingsvriendelike siektebestuurspraktyke verbeter kan word. Die ondersoek is in vier kwekerye oor twee produksie seisoene (Maart tot Julie 2003 en 2004) uitgevoer. Vlakke van luggedraagde inokulum van B. cinerea is op ’n maandelikse basis met behulp van spoorvangers binne en buite die kwekerye gemonitor. Monsters van plantmateriaal en organiese materiaal is in ooreenstemmende areas geneem om die voorkoms van B. cinerea geïnfekteerde plantmateriaal vas te stel en die voorkoms van vaalvrot in die kwekerye is aangeteken. Min B. cinerea kolonies is op die spoorvangers waargeneem. Soortgelyke vlakke van luggedraagde inokulum is binne en buite die kwekerye waargeneem. Die hoër voorkoms van B. cinerea geïnfekteerde plantmateriaal buite die kwekerye as binne, dui op die belang van sulke materiaal as potensiële inokulumbronne. Aangesien die patrone van luggedraagde inokulum, soos waargeneem in hierdie ondersoek, ander berigte van B. cinerea se beperkte verspreidingsvermoë bevestig, kan die verwydering van moontlike alternatiewe gashere buite die kwekerye die bestuur van die siekte binne die kwekerye verbeter. Weerstand teen dikarboksimied fungisiede is ’n geneties-stabiele kenmerk in B. cinerea en het daarom potensiaal om as ’n fenotipiese merker gebruik te word. Hierdie merker kan gebruik word om kennis aangaande die verspreiding van B. cinerea in en om rooibos kwekerye in te samel. Botrytis cinerea isolate in lug en op plantmateriaal in en om vier rooibos kwekerye is gedurende 2003 en 2004 versamel. Die isolate is vir weerstandbiedendheid teen iprodioon by konsentrasies van 1 en 3 μg/ml aktiewe bestandeel (a.b.) getoets. Isolate met weerstand teen 1 μg/ml a.b. iprodioon is waargeneem, maar nie teen 3 μg/ml nie. Die aanvanklike voorkoms van dikarboksimiedweerstand by die kwekerye was hoër as verwag. Hierdie vlak het egter gedaal met die verloop van die seisoen tot in Mei, waarna ’n toename tot in Julie waargeneem is. Die persentasie dikarboksimied-weerstandbiedende isolate buite die kwekerye was relatief hoog. In twee van die kwekerye was die voorkoms van weerstandbiedende isolate op plantmateriaal in die kwekerye betekenisvol hoër as op plantmateriaal buite die kwekerye. Daar was egter geen betekenisvolle verskille in die voorkoms van luggedraagde weerstandbiedende isolate nie, ongeag van die kwekery of posisie. Die data dui op die belang van organiese materiaal en saadgedraagde infeksies in die oorlewing en verspreiding van dikarboksimied-weerstandbiedende isolate van die patogeen. Met die huidige klem op organiese landbou bied die inligting wat in hierdie ondersoek versamel is moontlike praktyke wat geïmplementeer kan word om die beheer van vaalvrot in kwekerye met behulp van verbouingspraktyke te verbeter.

Les protéines hnRNP A1 sont impliquées dans l'épissage alternatif. Un mode d'action proposé implique la formation d'homodimères entre molécules hnRNP A1 causant un réarrangement dans la structure de l'ARN pré-messager. Cette modulation de l'ARN permettrait le rapprochement de sites d'épissage 5' et 3' d'exons situés de par et d'autres d'un exon alternatif. Le domaine riche en résidus glycines est responsable, en grande partie, de l'interaction entre les deux protéines hnRNP A1. Comme la protéine hnRNP H contient aussi un domaine riche en résidus glycines, nous avons postulé que cette dernière pouvait moduler l'épissage alternatif de la même manière que hnRNP A1. Afin de vérifier cette hypothèse, nous avons utilisé un ARN pré-messager constitué de deux sites d'épissage 5' (distal et proximal) en compétition pour un seul site d'épissage 3'. En présence de sites de liaison pour hnRNP H, nous observons que le choix du site d'épissage 5' est déplacé vers le site distal. Nous avons confirmé le rôle des protéines hnRNP H dans la sélection des sites d'épissage 5' in vitro et avons déterminé que le domaine riche en résidus glycines (GRD) est important pour l'activité d'épissage de ce régulateur. Nous avons ensuite exploré la possibilité que des combinaisons de sites de liaison pour hnRNP H et hnRNP A1 puissent activer l'utilisation du site d'épissage 5' distal. Nous avons observé que des combinaisons hétérotypiques peuvent reproduire cette activité d'épissage. Finalement, nous avons utilisé la technologie BRET (« bioluminescence resonance energy transfer ») pour démontrer que des interactions homotypiques entre protéines hnRNP H et hétérotypiques entre molécules hnRNP A1 et hnRNP H peuvent se former dans les cellules vivantes. Notre étude suggère que les portéines hnRNP et hnRNP A1 peuvent changer la conformation de l'ARN pré-messager et affecter le choix du site d'épissage.

Il settore della fragola ha conosciuto diversi mutamenti nel corso del Novecento, per via della sua sempre più larga diffusione, delle nuove tecnologie e dei recenti sviluppi in materia scientifica. Tali mutamenti sono in continua evoluzione, influenzati sia dalle tendenze del mercato, sia dall’affermazione del contributo nutrizionale della fragola, oramai riconosciuto. Tuttavia, esistono molteplici problematiche a carico di questo frutto, che dipendono da territorio, clima, tipologie di coltivazione e conservazione post-raccolta, ma anche e soprattutto, dai patogeni, primo fra tutti, Botrytis cinerea. Questo fungo è responsabile della formazione di muffa grigia nella fragola, problema che si riscontra anche nelle coltivazioni a coltura protetta e nella commercializzazione. Nell’ottica di massimizzare le rese della produzione, per elevare il profitto economico, negli anni sono stati usati tecniche e accorgimenti (come i fungicidi) che oggi risultano, in parte, non pienamente efficaci. Inoltre, sono gli stessi consumatori che, secondo un trend in continua espansione, scelgono sempre più di acquistare prodotti biologici, provenienti da aziende ecosostenibili, pur non volendo rinunciare al gusto e all’immagine estetica della fragola. In ragione di questi fattori, l’obiettivo di questo studio è quello di fornire un quadro generale della situazione della fragola ad oggi, ripercorrendo gli sviluppi più significativi avvenuti nel Novecento, dall’introduzione di nuove varietà ottenute grazie alle ricerche scientifiche, ma anche dall’industria di trasformazione, per provare a determinare quali saranno le tendenze future e quali i potenziali, ulteriori progressi in materia.

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Cucumber plants grown in greenhouse conditions are often infected by Botrytis cinerea, which causes the gray mold. The disease is controlled by successive applications of fungicides. In the perspective of integrated management of gray mold, we studied the effects of the interaction Clonostachys rosea, Silicon (Si) and Botrytis cinerea on disease severity. When Si and C. rosea were applied alone or together, the incubation period of disease was increased. When C. rosea was applied, the incubation period increased about 63h and gray mold severity and electrolyte leakage were significantly reduced. The application of either C. rosea or Si alone increased the activity of poliphenoloxidase, peroxidase, chitinase and β-1,3- glucanase, and reduce disease severity, area under the curve of progress of disease (AUCPD), and rate of gray mold progress. The application of just C. rosea was promoted plant growth, considering the dry mass of root system, stem length and dry mass of stems. The antagonist colonized endophytically cucumber plants; the frequency of colonization was higher in the inoculated with B. cinerea then in the uninoculated plants. We conclude that C. rosea is a potential biological control agent of cucumber gray mold that can be endophytic and can promote the growth of cucumber plants.
Plantas de pepino sob cultivo protegido são frequentemente infectadas por Botrytis cinerea, que causa o mofo cinzento. Controla-se a doença com sucessivas aplicações de fungicidas. No contexto do manejo integrado do mofo cinzento, estudou-se o efeito da interação Clonostahcys rosea, Silício (Si) e B. cinerea na severidade da doença. Quando se aplicaram Si e C. rosea isolados ou em conjunto, aumentou-se o período de incubação da doença. Com a aplicação de C. rosea, o período de incubação aumentou-se em aproximadamente 63 h e se reduziram significativamente a severidade do mofo cinzento e o extravasamento de eletrólitos. Com a aplicação isolada de C. rosea ou de Si aumentou-se a atividade de polifenoloxidase, peroxidade, quitinase e β-1,3-glucanase e reduziram-se a severidade, a área abaixo da curva do progresso da doença (AACPD) e a taxa de progresso do mofo cinzento. Quando se aplicou C. rosea isoladamente, ocorreu promoção do crescimento de plantas, considerando-se a massa seca do sistema radicular, o comprimento da parte aérea, e a massa seca da parte aérea. O antagonista colonizou plantas endofiticamente e a frequência de colonização foi maior nas plantas inoculadas com B. cinerea que nas não inoculadas. Conclui-se que C. rosea é um potencial agente de controle biológico do mofo cinzento do pepino, que pode ser endofítico em plantas de pepino e promover seu crescimento.

Il est connu que les ‘pesticides’ (produits phytopharmaceutiques) utilisés depuis plus de 100 ans en agriculture sont néfastes, autant pour les vivants que pour l’environnement. Il est urgent de développer un remplacement de ces produits toxiques à l’homme et à son entourage. Le but de ce travail est double : d’abord, trouver les micro-organismes non pathogènes dans les vignobles et, ensuite, les mettre en concurrence avec les parasites de la vigne. Nous envisageons de tirer bénéfice sur deux fronts : 1. Lutte biologique contre les maladies cryptogamiques de la vigne, 2. Déclenchement de la résistance chez la vigne contre les maladies. Les micro-organismes utilisés deviennent ainsi, des ‘bio-fongicides’ qui remplacent les produits chimiques. La vigne est attaquée par plusieurs parasites; les plus importants en Bourgogne sont : le mildiou par Plasmopara viticola, l’oïdium par Uncinula necator, et la pourriture grise par Botrytis cinerea. Pour cette thèse nous avons choisi de nous concentrer sur la « pourriture grise » de la vigne et d’autres végétaux, maladie provoquée par le redoutable champignon, Botrytis cinerea. Nous avons isolé quelques espèces de Pythium non pathogènes à partir du sol de vignobles ainsi que d’autres sols agricoles et forestiers. .
The pesticides used in agriculture for controlling phyto-pathogens have hazardous health effects on plants, animals and humans. Massive application of toxic pesticides is a serious problem today in almost all developing countries. Developing safer, environment-friendly biological products may help in overcoming the risks posed by pesticides and may also help in protecting public health, thereby promoting safer means of pest control. This thesis aims at the Biological control of plant diseases by non-phytopathogenic microorganisms, isolated from soil of vineyards and rhizosphere of crop fields. Introduction of these microorganisms leads to a twofold benefit 1. Biological control of the disease by the antagonist effect of the micro-organisms; 2. Enhanced disease resistance in plants to phytopathogens. Hence, these microorganisms could serve as an alternative to chemical control of plant diseases. Grapevine is challenged every year by fungal and viral and bacterial parasites. Among the fungal, the major ones are downy mildew by Plasmopara viticola, powdery mildew by Uncinula necator, black rot by Guignardia bidwellii, phomopsis leaf, cane spot and fruit rot disease by Phomopsis viticola, Eutypa Dieback by Eutypa armeniaceae and grey mould by Botrytis cinerea. Crown gall by bacterial parasite Agrobacterium tumefaciens, and viral diseases includes Peach Rosette mosaic virus disease, tomato ringspot and tobacco ringspot by nematode Xiphinema americanum. .

Includes bibliographical references (leaves 199-253).
This study attempted to characterize at a transcriptional level, the defence responses of Arabidopsis thaliana after infection by Botrytis cinerea, using microarrays. The first microarray experiment focused on profiling Arabidopsis genes induced by B. cinerea over time (temporal) while the second investigated spatial expression of Arabidopsis genes from the point of inoculation. A number of genes were up- and down-regulated specifically at 12 hrs, others at 24 hrs while others were up- and down-regulated at both time points. Similarly, some genes were specifically induced very close to the lesion while others in more distal tissue.

La pourriture grise est une maladie qui affecte de nombreuses cultures dont la vigne. Elle est provoquée par un complexe de deux espèces fongiques, l’espèce majoritaire Botrytis cinerea et l’espèce minoritaire Botrytis pseudocinerea. Les deux espèces se distinguent par leur sensibilité à certains fongicides notamment au fenhexamid, inhibiteur de la 3-cétoréductase des stérols. Ce fongicide a un spectre d’action restreint aux espèces phylogénétiquement proches du genre Botrytis (Sclerotinia et Monilinia fructicola). Son utilisation a conduit à la sélection de souches résistantes parmi lesquelles on distingue trois phénotypes : le phénotype HydR1 correspond à l’espèce B. pseudocinerea naturellement résistante ; les phénotypes HydR2 et HydR3 correspondent à l’espèceB. cinerea ayant acquis la résistance suite à l’introduction du fongicide. L’objet de cette thèse est l’étude des phénotypes HydR1 et HydR2 présents à de faibles, voire très faibles fréquences dans des populations de pourriture grise.Chez B. pseudocinerea (HydR1), nous avons identifié une monooxygénase à cytochrome P450 nommée Cyp684 responsable de la résistance au fenhexamid. Le gène cyp684 montre des polymorphismes (structure et séquence nucléotidique) entre les espèces ainsi qu’une induction par le fenhexamid chez B. pseudocinerea. La comparaison de la séquence du gène cyp684 chez plusieurs espèces de Botrytis et de leurs niveaux de résistance au fenhexamid indique que les acides aminés polymorphes de la protéine Cyp684 sont responsables de la résistance au fenhexamid chez B. pseudocinerea. Le rôle connu des monooxygénase à cytochrome P450 dans la métabolisation des xénobiotiques et la synergie entre le fenhexamid et des inhibiteurs de monooxygénases à P450 suggèrent que Cyp684 soit impliqué dans la métabolisation du fenhexamid. Concernant le phénotypeHydR2 de B. cinerea, le mécanisme de résistance reste à identifier. Les souches HydR2 se distinguent par leur phénotype rose dû à un métabolite secondaire nommé bikavérine. Le génotypage réalisé sur les descendantes d’un croisement entre une souche HydR2 et une souche sensible a mis en évidence un lien physique entre le gène ou l’allèle hydR2 et le cluster bikavérine. Afin d’identifier la niche écologique et de comprendre l'épidémiologie de B. pseudocinerea, nous avons développé une méthode de qPCR espèce spécifique, nommée « B. pseudocinerea allele specific PCR » (BpASP). Cet outil permettra de détecter et de quantifier l’espèce B.pseudocinerea dans les populations de Botrytis, selon la saison, la région géographique et les plantes hôtes
Grey mold is a fungal disease affecting many crops including grapevine. It is generated by a species complex of two fungal species, the major one, Botrytis cinerea, and the minor species, Botrytis pseudocinerea. Both species differ by their sensitivity to several fungicides, in particular to fenhexamid, a potent inhibitor of sterol 3-ketoreductase. This fungicide has a narrow spectrum of activity limited to species closely related to the genus Botrytis (e.g., Sclerotinia and Monilinia fructicola). Fenhexamid applications have led to the selection of resistant strains with three different phenotypes: the HydR1 phenotype corresponds to the naturally resistant species B. pseudocinerea; HydR2 and HydR3 phenotypes correspond to B. cinerea strains that acquired resistance after the introduction of fenhexamid. The topic of this thesis is the study of the phenotypes HydR1 andHydR2 present at low or even very low frequencies in grey mold populations. We identified a cytochrome P450 monooxygenase named Cyp684 responsible for resistance to fenhexamid in B.pseudocinerea (HydR1). The cyp684 gene differs between both species in its gene structure, nucleotide sequence (polymorphisms) and expression. The comparison of cyp684 sequences among different Botrytis species and their resistance levels to fenhexamid indicate the polymorphic amino acids of the Cyp684 protein to be responsible for fenhexamid resistance in B. pseudocinerea. The known involvement of cytochrome P450s in xenobiotic metabolisation and synergy between fenhexamid and P450-inhibitors suggest that Cyp684 could be involved in fenhexamid metabolisation. Concerning the B. cinerea HydR2 phenotype, the resistance mechanism remains to be identified. HydR2 strains have a specific purple pigmentation due to the secondary metabolite bikaverin. Genotyping of progeny derived from a cross between a HydR2 and a sensitive strain revealed aphysical link between the hydR2 gene or allele and the gene cluster involved in bikaverin biosynthesis. In order to identify the ecological niche of B. pseudocinerea and its epidemiological behavior we developed a species-specific qPCR method named “B. pseudocinerea allele specific PCR” (BpASP). This tool will allow detecting and quantifying the species B. pseudocinerea in natural Botrytis populations, collected according to season, geographic origin or plant hosts

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

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

Nos travaux ont été réalisés sur Botrytis cinerea, champignon nécrotrophe ascomycète. La première étape de notre travail a consisté à dresser un état des lieux des métabolites carbonés solubles présents chez les partenaires de l’infection : Botrytis cinerea et le cotylédons de tournesol et de suivre leur évolution au cours de l’infection. Au cours de la colonisation des cotylédons de tournesol, les hexoses d’origine végétale disparaissent, alors que du mannitol d’origine fongique est accumulé. Afin de mettre en évidence les éléments impliqués dans la disparition des hexoses dans la plante, nous avons recherché les gènes potentiellement impliqués dans le transport d’hexoses dans le génome de B. Cinerea. Les analyses bioinformatiques ont permis de caractériser dix-huit séquences comme des protéines transmembranaires impliquées dans le transport des hexoses. Les profils transcriptionnels de ces gènes indiquent une grande flexibilité d’expression aussi bien durant l’infection que pendant le développement in vitro du pathogène. Une fois transportés, les hexoses d’origine végétale sont métabolisés par le champignon. Afin de connaitre le devenir des hexoses, nous avons suivi leur assimilation grâce à l’utilisation de sucres marqués. Cette approche a été couplée à une analyse de la voie de synthèse du mannitol, métabolite carboné prédominant accumulé par B. Cinerea en réponse à l’assimilation des sucres. Il existe deux voies de biosynthèse du mannitol chez les champignons Ascomycètes et les résultats obtenus chez B. Cinerea, proposent de nouvelles perspectives de fonctionnement et permettent de mieux cerner l’importance de chacune des voies dans le métabolisme du mannitol
Our work was completed on Botrytis cinerea, a necrotrophic ascomycete fungus. The first stage of our work consisted in drawing up an inventory of the soluble carbon metabolites present in partners of infection: Botrytis cinerea and sunflower cotyledons and to follow their evolution during the infection. During the colonization of the cotyledons of sunflower, hexoses from plant disappear, whereas mannitol of fungal origin is accumulated. In order to highlight the elements implied in the disappearance of hexoses in the plant, we sought genes potentially implied in the transport of hexoses in the genome of B. Cinerea. The bioinformatic analyses made it possible to characterize eighteen sequences like transmembrane proteins implied in the transport of hexoses. Transcriptional profiles of these genes indicate a great flexibility of expression during infection as during in vitro development. Once transported, plant’s hexoses are metabolized by fungi. In order to know to become to it hexoses, we followed their assimilation thanks to the use of marked sugars. This approach was coupled with an analysis of the way of synthesis of the mannitol, metabolite carbonaceous prevalent accumulated by B. Cinerea in answer to the assimilation of sugars. It exists two mannitol biosynthesis pathways in Ascomycetes fungi and the results obtained with B. Cinerea, propose new prospects of operation and make it possible to better determine the importance of each way in the metabolism of the mannitol

La pourriture grise, maladie tres redoutée des viticulteurs, est due à un champignon, Botrytis cinerea. L'objectif de cette étude est la mise au point de bioessais utilisant des méthodes de culture in vitro et permettant d'évaluer la sensibilité de la vigne à cette maladie. La finalité de ce travail est la sélection de clones résistants ou tolérants en utilisant la variabilité somaclonale introduite par l'embryogenèse somatique. Les bioessais mis au point font appel à différents niveaux d'organisation du végétal : vitroplants, feuilles, cultures de tissus (cals) et protoplastes. Ils ont permis d'établir une bonne corrélation entre le niveau de sensibilité de la vigne à B. Cinerea et celui de la vigne in vitro au filtrat de culture du champignon. L'analyse des filtrats de culture de B. Cinerea a mis en évidence l'activité toxique spécifique d'hétéropolysaccharides. Ce travail apporte les outils indispensables pour une étude approfondie de la relation hôte-parasite (moyens d'attaque du pathogène et mécanisme de défense de la plante). Il permet également d'envisager avec optimisme l'utilisation du filtrat de culture plus ou moins purifié dans une stratégie de sélection.

Botrytis cinerea and Sclerotinia sclerotiorum are the two most serious pathogens on kiwifruit in New Zealand. Because of the pesticide regulations in some of the countries to which New Zealand exports fruit, total protection from Botrytis stem end rot with current dicarboximide fungicides is not possible. The aim of this thesis was to investigate biological control measures for Botrytis stem end rot and Sclerotinia diseases of kiwifruit. More than 1000 microorganisms, isolated from the leaves and flowers of kiwifruit during spring and autumn, and selected from BCAs reported to be effective against B. cinerea and./or S. sclerotiorum, were tested in vitro for their antagonistic ability against B. cinerea and S. sclerotiorum. Successful antagonists were those that, in dual culture on agar plates, produced a zone of inhibition, an area of browning of the pathogens, or grew rapidly over the pathogens and inhibited their growth. The fifty most promising isolates from the initial screen were tested on fruit for their ability to reduce Botrytis and Sclerotinia fruit rots. Mature kiwifruit were artificallv wounded and dual inoculated with a spore suspension of one of the fifty test organisms and either a conidial suspension of B. cinerea or a mycelial suspension of S. sclerotiorum. Following 8-12 weeks incubation in a cool store, fruit were assessed for Botrytis or Sclerotinia induced rot. Isolates of Bacillus spp., Epicoccum purpurascens, Pseudomonas sp. and Trichoderma. spp. reduced, Botrytis fruit rot from 92% (inoculated control) to 0%.Isolates of Alternaria spp., pestalotia sp. and a non-sporulating isolate also reduced the number of fruit rotting to some extent. Similarly, isolates of Bacillus spp., E purpurascens and Trichoderma spp. reduce d Sclerotinia fruit rot from 100% (inoculated control) to 0%. Isolates of Alternaria spp., Myrothecium verrucaria and Pestalotia sp. were also successful at reducing the level of Sclerotinia fruit rot. It was considered undesirable if potential biological control agents (BCAs) were able to colonize kiwifruit that were to be marketed for human consumption. In order to determine if microorganisms, shown to be effective in preventing Botrytis or Sclerotinia fruit rot, were capable of themselves colonizing fruit, isolations were made from fruit dual inoculated with B. cinerea, S. sclerotiorum and/or one of several BCAs. Strains of the BCAs Bacillus spp., Pseudomonas sp. and E. purpurascens were not found to be saprophytic on fruit. Isolates of Alternaria sp., Bacillus sp., E purpurascens, pestalotia sp., Pseudomonas sp. and T. harzianum significantly inhibited germination and germ tube elongation of B. cinerea conidia in vitro in a nutrient solution, over a 24 h period. For example, the presence of Alternaria alternata A6 spores in a nutrient solution reduced germination of B. cinerea conidia from 100% to 20%. The presence of E purpurascens A77 spores inhibited B. cinerea conidial germ tube elongation from >840 pm (in control conidia) to 27 µm. The presence of any one of the BCAs tested prevented germination of B. cinerea conidia in a non-nutrient water solution, in comparision to germination of up to 86% in controls. A spore or cell suspension of each of the isolates Bacillus sp.M60, E. purpurascens A77 and T. harzianum C65 were spray inoculated onto kiwifruit blossoms produced in vivo in the glasshouse, immediately prior to inoculation of the blossoms with a condial suspension of B. cinerea. Application of the BCAs were completely effective in preventing colonization of blossoms by B- cinerea conidia. The effectiveness of each of the isolates E. purpurascens A77,T. harzianum C65 and either Bacillus sp.M60 or M53 to reduce the viability of sclerotia of B. cinerea and S. sclerotiorum was tested in soil punnets. A spore or cell suspension of each respective BCA was applied to the surface of replicated punnets that were seeded with either B. cinerea or S. sclerotiorum. Following 8 weeks incubation, punnets were harvested and viability of sclerotia assessed. T. harzianum C65 and Bacillus sp. M60 significantly reduced the viability of B. cinerea sclerotia from 8 sclerotia/punnet (control) to 4 sclerotia/punnet. T. harzianum C65 and E. purpurascens A77 caused a significant reduction in apothecia production of S. sclerotiorum, from 2.7 apothecia/punnet (control) to 0.7 apothecia/punnet. Bacillus sp.M8 and E purpurascens A77 were tested for their ability to reduce Botrytis stem end rot and Sclerotinia field rot in a kiwifruit orchard. The isolates tested did not successfully reduce either disease. Possible explanations for this are discussed. In order to monitor the survival of particular isolates of BCAs in the field, a technique was developed to distinguish between individual strains of a BCA species. The polymerase chain reaction (PCR) was utilized to identify DNA polymorphisms within the genome of T. harzianum C65, in comparison with other strains of Trichoderma spp.. A sequence of polymorphic DNA was cloned, sequenced and used as a hybridization probe in southern blotting to enable T. harzianum c65 to be distinguished from other strains of Trichoderma spp.. From the results obtained in this study, it was considered that Bacillus M60, E purpurascens 477 and Pseudomonas M30 were the best isolates for the biological control of Botrytis stem end rot on kiwifruit. Further work to enable application of these isolates as postharvest BCAs is discussed. Of the isolates tested in this study, T. harzianum C65 was considered the best isolate for use against Sclerotinia diseases on kiwifruit. Methods of selecting more effective BCAs against S. sclerotiorum are discussed.

Thesis (MScAgric)--University of Stellenbosch
ENGLISH ABSTRACT: An understanding of the infection pathways of Botrytis cinerea in grape bunches will help to combat this devastating pathogen of grape. Many studies have been done to determine the possible infection pathways of B. cinerea. Most of these studies made use of artificial inoculations that deposit groups of conidia on the plant surface. The deposition of clusters of conidia is not a common phenomenon in nature. The aim of this study was to investigate the infection pathways of (i) naturally- as well as (ii) artificially inoculated B. cinerea conidia during all the phenological stages of three wine grape cultivars, and to compare the (iii) pathogenicity and virulence, on grape and nectarine fruit, of isolates obtained from different host plants. In the natural infection study the occurrence of Botrytis cinerea and subsequent disease expression at different positions in bunches of wine grapes (cultivars Chenin Blanc, Shiraz and Chardonnay) was determined from 1999 to 2001. Different techniques were used to detect viable inoculum at different positions (rachises, laterals, pedicels, and the peicel end, cheek and style end of berries) in bunches. Isolations were made on Kerssies' B. cinerea selective medium, or bunches were used untreated, or treated with paraquat. Paraquat was used to terminate host resistance and to promote the development of the pathogen from the tissues. The material was used untreated to detect the pathogen on the surface, or were surface-sterilized to detect mycelia (latent infection) in the tissue. In the artificial inoculation study, bunches of wine grapes (cultivars Chenin Blanc, Chardonnay and Shiraz) at pea size, bunch closure, and harvest were dusted with dry conidia of Botrytis cinerea in a settling tower and incubated for 24 h at high relative humidity (±93%). Following incubation, the bunches were divided in two groups. The one group was surface-sterilised in 70% ethanol for 5 s, the other group was left untreated. Bunches of the sterile group, and from the untreated group were used for isolation. From each bunch rachis segments, laterals, pedicels and berry skin segments (from the pedicel-end and cheek) were removed. The sections were placed in Petri dishes on Kerssies' B. cinerea selective medium and on a water agar medium supplemented with paraquat, and incubated at 22°C under diurnal light. Occupation by the pathogen was positively identified by the formation of sporulating colonies of B. cinerea on the different tissues. Lastly, in the virulence and pathogenicity experiment on grape and nectarine fruit Botrytis cinerea isolates, which were obtained from different host plants, were compared by simulating natural infection. Cold-stored fruit, considered highly susceptible to B. cinerea were therefore inoculated with single, airborne conidia of the pathogen. Different tests were conducted to assess surface penetration and lesion formation. Isolations were made from fruit skins on Kerssies' B. cinerea selective medium. Nectarine fruit were treated with paraquat, and grape berries were frozen for 1 h at -12°C. Paraquat and freezing were used to terminate host resistance and to promote the development of the pathogen from the tissues. In the natural infection studies B. cinerea occurred in a consistent pattern in bunches of the three cultivars. B. cinerea consistently developed from the tissue of the rachis, laterals, pedicel and pedicel-end, but not from the berry cheek. The rachis, lateral and pedicel contained much higher levels of B. cinerea than any position on the berry. Furthermore, the pathogen consistenly occurred at relatively high levels on the rachises throughout the season. Collectively, the data showed that in the Western Cape province, B. cinerea occured more regularly in wine grape bunches during the early part of the season, than later in the season. The data of the artificial studies confirmed the findings made with the natural infection studies. In these experiments the pathogen resided more often on the structural bunch parts than on the berries. Overall, the isolation studies revealed that conidia occurred predominantly on the rachis. The incidence of B. cinerea was furthermore constantly high in the inner bunch after each inoculation, and in bunches of different maturities. The data therefore indicated that, when available, conidia penetrated loose and tight clustered bunches in a similar way. Finally, in the virulence and pathogenicity experiments the results showed clearly that no host specialisation exists in the B. cinerea isolates used in this study. From these studies it is clear that in the Western Cape province B. cinerea occurs more readily in the inner structural parts of the bunches and more so during the earlier parts of the season. These findings should be considered when planning and implementing disease control programmes.
AFRIKAANSE OPSOMMING: INFEKSIEWEË VAN BOTRYTIS CINEREA OP GESELEKTEERDE WYNDRUIF KULTIVARS Indiepte kennis van die infeksieweë van Botrytis cinerea op druiwetrosse word benodig vir die beheer van dié vernietigende patogeen van druiwe. Vele studies is al gedoen om die moontlike infeksieweë van die swam op druiwe trosse te ondersoek. Die meeste van die studies het gebruik gemaak van kunsmatige inokulasie tegnieke waar die konidia van die swam in groepe op die korreloppervlak gedeponeer is. In die natuur is dit 'n rare verskynsel dat konidia in groepe op die korreloppervlak land. Die doel van die studie was om die infeksieweë van B. cinerea op drie wyndruif kultivars te ondersoek wat (i) natuurlik- en (ii) kunsmatig geïnokuleer is met konidia gedurende al die fenologiese stadia, en om die (iii) virulensie en patogenisisteit van isolate wat van verskillende gashere verkry is, op druiwe en nektariens te vergelyk. In die natuurlik-geïnokuleerde druiwe is die voorkoms van B. cinerea en die gevolglike siektevoorkoms op verkillende posisies in trosse van wyndruiwe (Chenin Blanc, Chardonnay, Shiraz) gedurende 1999 tot 2001 bepaal. Verskillende tegnieke is gebruik om lewensvatbare inokulum by verskillende posisies (ragis, lateraal, pedisel en pedisel-end van die korrel) in die tros waar te neem. Isolasies is op Kerssies' B. cinerea selektiewe medium gemaak, of trosse is onbehandeld gebruik, of behandel met paraquat. Paraquat is gebruik om die gasheer se natuurlike weerstand te verlaag en om die ontwikkeling van die patogeen te bevorder. Die plantmateriaal is onbehandeld gelaat om die patogeen op die oppervlak waar te neem, of die oppervlak is gesteriliseer om die latente myselium in die weefsel waar te neem. In die kunsmatige inokulasiestudies is trosse, van wyndruiwe (Chenin Blanc, Chardonnay, Shiraz), geïnokuleer met droë spore, van B. cinerea, in 'n inokulasietoring en die plantmateriaal is dan geinkubeer vir 24 h by 'n hoë relatiewe humiditeit (93%). Na die inkubasie proses is die trosse in twee groepe verdeel. Die een groep druiwe het oppervlak sterilisasie ondergaan in 70% etanol vir 5 s, en die ander groep was onbehandeld gelaat. Trosse van die onbehandelde en gesteriliseerde groep druiwe is gebruik vir isolasies. Vanuit elke tros is daar segmente van die ragis, laterale, pediselle en korrels (van die pedisel-end en wang gedeeltes) geïsoleer. Die segmente is in Petri bakkies met Kerssies' B. cinerea selektiewe medium en op water agar medium, wat paraquat bevat het, geïsoleer en geïnkubeer onder 'n 12 h dagligperiode teen 22°C. Die patogeen is positief geïdentifiseer deur sporuierende kolonies op die onderskeie weefseltipes. Laastens, in die virulensie- en patogenisiteitsproewe op druiwe en nektariens is verskillende isolate van B. cinerea, verkry vanaf verskillende gasheerplante, vergelyk deur natuurlike inokulasie toestande na te boots. Koue opgebergde vrugte, wat beskou word as hoogs vatbaar vir die infeksie van B. cinerea, is geïnokuleer met droë, enkel luggedraagde spore van die patogeen. Verskillende toetse is gedoen om die oppervlak penetrerende en letselvormende vermoëns van die onderskeie isolate te toets. Isolasies is van die skille van die vrugte gemaak en op Kerssies' B. cinerea selektiewe medium geplaas. Die nektarienvrugte is met paraquat behandel en die druifkorrels is gevries vir 1 h teen -12°C. Paraquat en bevriesing is gebruik om die gasheer se weerstand te verlaag en om die ontwikkeling van die patogeen te bevorder. In die natuurlik-geïnokuleerde studies het B. cinerea 'n konstante patroon getoon in die trosse van die drie verskillende wyndruif kultivars. B. cinerea het konstant ontwikkel uit die ragis, laterale, pedisel en pedisel-end, maar selde uit die korrelwang. Die ragis, lateral en pedisel dele het baie hoër vlakke van van die swam bevat as enige deel op die korrel. Die patogeen het ook konstant volop deur die hele seisoen op die ragis voorgekom. Gesamentlik wys die data dat, B. cinerea in wyndruiwe, in die Wes Kaap provinsie, meer geredelik vroeër in die seisoen voorkom, eerder as later. Data van die kunsmatige inokulasiestudies het die bevindinge van die natuurlike inokulasiestudies tot 'n groot mate bevestig. In dié studies het die patogeen meer geredelik die strukturele dele van die tros, eerder as op die korrels, bewoon. Oor die algemeen het die isolasieproewe gewys dat die konidia meer op die ragis voorkom as op enige ander deel. Die voorkoms van B. cinerea was ook oor die algemeen baie hoër in die strukturele dele van die tros, as op die korrel self. Die verskynsel het onder trosse van verskillende ontwikkelingsvlakke voorgekom. Die data het dus ook gewys dat konidia, wanner dit beskikbaar is, minder- sowel as meer kompakte trosse op 'n soortgelyke manier penetreer. Laastens, in die virulensie en patogenisiteitseksperimente het die resultate duidelik gewys dat daar geen gasheer spesifieke gedrag onder B. cinerea isolate is nie. In die studies het dit duidelik na vore gekom dat, B. cinerea meer geredelik in die strukturele binne dele van die wyndruif tros, in die Wes Kaap provinsie voorkom. En so ook eerder aan die begin van die seisoen, as later in die seisoen. Dié kennis moet in aanmerking geneem word by die beplanning en implementering van siektebeheerprogramme.

El fitopatógeno Botrytis cinerea afecta el botón floral del cultivo de rosa bajo condiciones de campo y poscosecha. En la región florícola del Estado de México, el manejo se basa en el uso de fungicidas entre los que se encuentra el tiabendazol, al cual ha perdido sensibilidad y en algunos casos ha desarrollado resistencia. Por lo anterior, el objetivo de la presente investigación fue determinar la DL50 y la DL95 bajo condiciones in vitro de tiabendazol frente a B. cinerea. Se utilizaron las cepas VBc1 y TBc2 provenientes de los municipios de Villa Guerrero y Tenancingo, Estado de México, respectivamente. Se realizaron bioensayos para calcular la ventana biológica; es decir, los extremos del intervalo con efecto tóxico del fungicida (0 y 100 %) y a partir de estos resultados se calcularon dosis logarítmicas. La mortalidad se transformó a unidades Probit y las dosis a logaritmo, los datos se sometieron a una regresión lineal simple y a partir de la ecuación se determinó la DL50 y la DL95. En el caso de la cepa TBc2 la DL50 y la DL95 correspondieron a 0.0394 y 0.8673 g de i. a L-1, respectivamente; mientras que para la cepa VBc1 fueron de 0.0576 y la 6.047 g de i. a. L-1, respectivamente.

Includes bibliographical references (leaves 63-86).
Disease resistance in plants has been extensively studied for the past century with many new and exciting results being discovered each year. A plant utilises both preformed and induced defence responses to resist pathogen attack but researchers have focused on dissecting the induced defence response pathway. The complex signal transduction pathway underlying the establishment of resistance to a wide range of pathogen attack is currently being dissected using Arabidopsis thaliana as a model organism. Arabidopsis mutants displaying altered disease resistance response to pathogen infections can help us to get a beUer understanding of the genetiC and molecular basis of the disease resistance pathway. Extensive research has shown that accumulation of 3 signalling molecules are vitally important for establishing a resistance response, as aberrant signalling or accumulation of salicylic acid , ethylene or jasmonic acid `leads to an altered resistance response. Researchers continue to isolate and characterise defence-related mutants to piece together the intricate puzzle of defence-signalling components. A dominant Arabidopsis mutant, constitutive induced resistance 3 (cir3), had been isolated from an ethylmethane sulfonate (EMS) mutagenised transgenic line expressing luciferase under the control of the PR-1 promoter (PR-1

The objective of this thesis was to investigate BiOWiSHTM-Aqua, a commercial dry solid formulation containing a consortium of bacteria and yeast, as a biopesticide for treatment of Botrytis cinerea, a gray mold that affects strawberries. BiOWiSHTM-Aqua was compared with another commercial product specifically used as a fungicide and bacteriocide, Serenade® Garden Disease Control Spray (concentrated Bacillus subtilis strain QST 713). Both laboratory tests as well as in vivo lab tests were conducted. BiOWiSHTM-Aqua results varied widely from plate to plate, regardless of experimental conditions. In some of these plates, inhibition zones were observed around colonies from BiOWiSHTM-Aqua, indicating efficacy. The organism responsible for the inhibition zones of B. cinerea growth was isolated from BiOWiSHTM-Aqua, and 16s rRNA analysis identified this culture as a strain of B. subtilis. This strain was designated as B. subtilis ssp. KLB. The B. subtilis KLB concentration required to completely inhibit B. cinerea was 9.1x104 CFU/mL when B. subtilis KLB was inoculated 48 hours before B. cinerea, 1.3x105 CFU/mL at 24 hours, and 3.2x106 CFU/mL when both were inoculated at the same time. Various preliminary experiments using B. subtilis KLB were also conducted to investigate its economic feasibility, to characterize the organism, and to test its post-harvest in vivo viability. B. subtilis KLB cell concentration was 1.6x109 CFU/mL in a bioreactor with LB at the end of the log growth phase. B. subtilis KLB achieved cell concentrations as high as 5x109 CFU/mL in shake flasks with food-grade tapioca as a carbon source. Inoculation of B. subtilis KLB on post-harvest strawberries did not have an effect on Botrytis infection rates compared to the negative control. These various experiments were the first step in research to potentially produce B. subtilis KLB on a commercial scale.

La pourriture grise, causée par le champignon Botrytis cinerea, est l'une des principales maladies aériennes fongiques sur diverses cultures d'importance agronomique. La diversité génétique de B. cinerea est très forte et la capacité rapide d'adaptation de ce champignon à une pression sélective est également avérée. Ce champignon est ainsi capable de développer des résistances à une grande variété de composés fongicides de synthèse ou d'origine naturelle. Des méthodes alternatives de lutte ont de ce fait été développées ces dernières années : divers agents de lutte biologique (ALB) présentant différents modes d'actions ont été identifiés et pour certains d'entre eux commercialisés pour contrôler B. cinerea. Cependant la durabilité de la lutte biologique est un domaine encore très peu étudié. La perte d'efficacité d'un ALB pourrait résulter de la préexistence d'isolats moins sensibles de pathogènes dans les populations naturelles et/ou de la capacité de l'agent pathogène à produire, sous une pression de sélection continue exercée par l'ALB, des mutants ayant une sensibilité réduite. L'objectif global de la présente étude est d'évaluer le risque potentiel de perte d'efficacité de la lutte biologique vis-à-vis de B. cinerea. Dans cette étude, les efforts ont été concentrés sur la pyrrolnitrine, un antibiotique produit par divers ALBs, dont certains sont efficaces contre B. cinerea. Les objectifs spécifiques de l'étude étaient (i) d'évaluer la diversité de la sensibilité à la pyrrolnitrine au sein de la population naturelle de B. cinerea, (ii) d'estimer le risque de perte d'efficacité des ALBs produisant la pyrrolnitrine due à la pression de sélection exercée par la pyrrolnitrine et (iii) d'étudier le mécanisme de résistance à la pyrrolnitrine chez B. cinerea. Parmi 204 isolats de B. cinerea, une gamme importante de sensibilité à la pyrrolnitrine a été observée, avec un facteur de résistance de 8,4 entre l'isolat le plus sensible et l'isolat le moins sensible. La production de 20 générations successives pour 4 isolats de B. cinerea, sur des doses croissantes de pyrrolnitrine, a abouti au développement de mutants avec des niveaux élevés de résistance à l'antibiotique, et à une réduction in vitro de la sensibilité à la bactérie productrice de pyrrolnitrine Pseudomonas chlororaphis PhZ24. La comparaison entre les mutants résistants à la pyrrolnitrine et leurs parents sensibles pour la croissance mycélienne, la sporulation et l'agressivité sur plantes a révélé que la résistance à la pyrrolnitrine est associée à un fort coût adaptatif. Des observations cytohistologiques sur tomates ont confirmé que l'isolat sensible à la pyrrolnitrine attaque le pétiole rapidement et envahit la tige, alors que le mutant résistant à la pyrrolnitrine ne s'étend pas au-delà du pétiole. De plus, ce dernier mutant forme un mycélium anormal et des cellules ressemblant à des chlamydospores. Les résultats ont d'autre part révélé que les mutants de B. cinerea résistants à la pyrrolnitrine sont résistants au fongicide iprodione, suggérant ainsi qu'une pression exercée par la pyrrolnitrine sur le champignon conduit à une résistance au fongicide. Réciproquement, la production de générations successives sur iprodione conduit à une résistance à l'antibiotique. Afin d'étudier les déterminants moléculaires de la résistance de B. cinerea à la pyrrolnitrine, le gène histidine kinase Bos1, impliqué entre autres dans la résistance aux fongicides chez B. cinerea a été séquencé chez les souches sensibles et les mutants résistants. La comparaison des séquences a mis en évidence des mutations ponctuelles différentes chez les mutants de B. cinerea obtenus sur la pyrrolnitrine et ceux obtenus sur l'iprodione. De plus, les résistances à la pyrrolnitrine et à l'iprodione ne sont pas systématiquement associées à une mutation ponctuelle dans le gène Bos1. Enfin, aucune modification n'a été détectée dans la taille des allèles de neuf locus microsatellites quelle que soit la pression sélective exercée et quelle que soit le phénotype du mutant produit. Cette étude montre qu'un champignon pathogène des plantes est capable de développer progressivement une moindre sensibilité à un agent de lutte biologique mais que cette moindre sensibilité est associée à une forte perte de fitness

There is no published research regarding postharvest infection of freesia flowers by Botrytis cinerea. Although, infection problems have concerned freesia growers and wholesalers in recent years. The overall objectives of this study were firstly to evaluate the factors affecting B. cinerea postharvest disease establishment and secondly to evaluate a range of novel potential treatments to reduce postharvest freesia infection. These treatment options include plant activators such as acibenzolar-S-methyl and methyl jasmonate and biotic (Aureobasidium pullulans) and abiotic (UV-C irradiation) biological/elicitors agents. Research was undertaken in an attempt to explain the variation in B. cinerea incidence on cut freesia flowers as noted by the UK importer Zwetsloots & Sons Ltd. in 2000. Higher monthly rejections of freesia flower stems throughout 2000 due to B. cinerea infection were recorded during spring (April-May), early summer (June) and autumn (October). Comparatively higher proportions of rejected freesia stems were associated with glasshouse temperatures ranging from 13-17°C. In the presence of B. cinerea inoculum on freesia petal surface, temperature was not a limiting factor for disease establishment. Incubation of artificially inoculated freesia flowers at 12°C resulted in overall higher disease severity and lesion numbers compared to flowers incubated at 5 or 20°C. In contrast, relative humidity was the most important factor for postharvest infection by B. cinerea. Elicitor based strategies for IPM using the potent activator acibenzolar provided limited protection of freesia flowers against B. cinerea when applied postharvest. Acibenzolar significantly reduced disease severity, lesion numbers and lesion diameters compared to the untreated control when applied at 0.15 g A. 1. U1. Methyl jasmonate (MeJA) applied as gas, pulse and spray generally suppressed B. cinerea disease on cut freesia flowers. Disease severity, lesion numbers and lesion diameters of flowers gassed with 0.1 μL MeJA L"' were reduced by 56,43 and 37%, respectively compared to untreated control flowers. Gaseous MeJA treated freesia flowers at 0.1 μL L"1 increased PPO activity by 57% compared to untreated controls 24h after MeJA treatment. After 36h of incubation at 20°C, disease severity, lesion numbers and lesion diameters of gaseous MeJA treated flowers were reduced by 68,56 and 50%, respectively, compared to the untreated controls. However, PAL activity in MeJA treated freesia flowers did not decrease significantly over time compared to untreated control 12h post-inoculation and thereafter. These findings suggest that MeJA treatment might suppress the action of PAL in the phenylpropanoid pathway and consequently block SA production. UV-C irradiation might be used in an integrated postharvest disease management program for freesia flowers. UV-C irradiation after artificial inoculation resulted in markedly reduced B. cinerea disease severity scores and lesion numbers. In detail, UV-C irradiation of cut freesia flowers with 0.5,1,2.5 and 5 kJ m''' reduced disease severity by up to 44,70,74 and 59% and lesion numbers by up to 37,62,68 and 60%, respectively. UV-C irradiation at 1 kJ M-2 before artificial inoculation slightly reduced disease severity and lesion numbers possibly by inducing defence responses. However, the limited disease suppression suggested that apparently B. cinerea could overcome the UV-C induced effect. The effect of preharvest treatments on freesia crops with acibenzolar was investigated in glasshouse trials in view to suppress postharvest B. cinerea infection via SAR induction. Acibenzolar was effective in selected treatments and conditions. Disease pressure varied over the 3 years and over varieties tested. However, it was unclear whether acibenzolar induced systemic and/or local defence responses. The latter was supported by biochemical investigations in 2001 which suggested that acibenzolar did not induce PAL activity. In contrast, preharvest MeJA treatment resulted in markedly systemic protection of treated flowers compared to untreated ones. MeJA efficacy was dependent on variety and on postharvest incubation temperatures. Disease severity, lesion numbers and lesion diameters on MeJA treated freesia var. 'Dukaat' flowers incubated at 20°C were reduced by 56,61, and 49% compared to controls, respectively. Also, disease severity, lesion numbers and lesion diameters on MeJA treated 'Cote d'Azur' flowers incubated at 20°C were reduced by 36,26, and 49% compared to controls, respectively.

Botrytis cinerea was systemically transferred into lettuce seeds by dry inoculation of the flower/bud with fungal spores. Plants grown from seeds were found to be systemically infected. Plants grown from infected seeds were used to investigate the behaviour of this systemic pathogen under different soil water saturations and the interaction of the pathogen with organisms at higher trophic levels. Variation in soil water saturation stressed the host plants resulting in a lower rate of photosynthesis, chlorophyll fluorescence and root weight. When the lettuce plants were infested with aphids (Myzus persicae) the rate of population growth of the aphids was slower on infected lettuce plants than uninfected plants. Numbers of lesions of B. cinerea were higher in aphid-free plants than on infested plants. Although the rate of chlorophyll fluorescence was not significantly affected by systemic B. cinerea and infestation with aphids, the effects of stress were evident in lettuce plants as the presence of aphids and 8. cinerea significantly affected the rate of photosynthesis, shoot and root weight of the plants. This confirms the interaction between two economically important pests of lettuce, the aphid Myzus persicae and the fungal pathogen, Botrytis cinerea. When the infested plants were attacked by the parasitoid Aphidius colemani, a greater number of M. persicae and their parasitoids were reared on lettuce plants free from infection by Botrytis cinerea. However, parasitoids attacked proportionally more aphids on uninfected plants, and both aphids and emerging parasitoids were significantly smaller when reared on infected plants. There was no difference in parasitoid sex ratio, with a 50:50 sex ratio found with parasitoids emerging from hosts reared on both infected and un infected plants. The results also revealed that the aphid hosts are larger when reared on plants free from B. cinerea infection than infected ones. In aphid hosts body size is a measure of host quality. The low number of parasitoid mummies recorded in aphids reared on B. cinerea infected plants indicated the negative effects of B. cinerea on both the preference and performance of the parasitoids. Experiments showed that learning has an influence on parasitoid host choice and that this is influenced by host plant infection status. These results show that the aphid M. persicae reared on uninfected plants when given a choice shows preference for uninfected plants while aphids grown on infected plants when given choice do not show a preference to either host. However, Aphidius colemani reared on aphids grown on uninfected plants but allowed to gain experience attacking aphids reared on infected plants showed a significant preference to the aphid hosts reared on infected plants when given a choice. While Aphidius colemani which emerged from aphids reared on infected plants but allowed to gain experience on hosts reared on uninfected plants showed a significant preference for the aphid hosts reared on uninfected plants when given a choice. Therefore, the behaviour of both host and parasitoid is affected by experience, and with the parasitoids, learning can alter host preference behaviour. The result from the field study confirmed the results of experiments done in a controlled environment room. Numbers of parasitoids were greater on exposed uninfected plants. Together, these results suggest that systemic infection by Botrytis cinerea may have considerable effects on ecological interactions at higher trophic levels.

Despite the significant progress achieved in molecular biology in the last few years, our knowledge about the mechanisms of plant resistance against necrotrophic and non-host pathogens is still rudimentary. Here, we report the isolation and characterization of three Arabidopsis mutants selected for altered resistance against B. cinerea. Mutations in the Increased Botrytis Resistance (IBRJ) gene resulted in significant resistance against B. cinerea and A. brassicicola, another necrotrophic fungus. Interestingly, ibri plants also exhibited enhanced susceptibility to the host and non-host bacterial pathogens P. syringae pv. tomato and P. fluorescens, respectively. Conversely, resistance against B. gram mis f. sp. tritici and E. cichoracearum was not affected, suggesting JBRJ is required for resistance against some, but not all pathogens. By TAIL-PCR IBRJ gene was found to be allelic to asymetric leaves 1 (as1). AS1 belongs to the R2R3 subfamily of the MYB-domain and has been extensively studied because of its central function in leaf development. Interestingly, neither as2 or erecta mutants or the conditional line KNJ are affected in resistance against B. cinerea or PstDC3 000. These results suggest that as1-mediated disease resistance and susceptibility is independent from the AS1 -dependent pathway regulating leaf development. Moreover, we show that AS1 function in disease resistance & susceptibility is conserved in at least three evolutionary divergent plant species, suggesting AS1 function in disease resistance is relatively ancient. The analysis of a substantial series of as1 double mutants in Arab idopsis revealed that both the production and perception of jasmonate (JA) and ethylene (ET) are required for as1-mediated disease resistance against necrotrophic pathogens. Moreover, the expression of JA/ET-dependent defence genes was shown to be accelerated in as1 plants in response to B. cinerea challenge. While as1 resistance against necrotrophic pathogens seems to be associated with enhanced expression of JA/ET - dependent genes, susceptibility against the different strains of host and non-host bacterial pathogens occurred in the presence of normal PR-1 expression and salicylic acid accumulation, suggesting susceptibility towards bacterial pathogens is not associated with defects in the activation of SA signalling pathway. In sum, our findings indicate that AS1 is a negative regulator of resistance against necrotrophic fungi and a positive regulator of nonhost resistance and basal protection against bacterial pathogens in both Arabidopsis and other plant species. In contrast to ibr1, the ibr2 and ebsl (Enhanced Botrytis susceptible1) mutants seem to specifically affect resistance against B. cinerea. The ibr2 was mapped to a short interval on chromosome five. Despite ibr2 cloning is not accomplished, we speculated that the mutation is in an alpha-tubulin gene because ibr2 plant morphology resembles the phenotype of the previously characterized lefty1 and lefty2 mutants, which also encode alpha-tubulins. Our speculation is supported by the presence of two alpha-tubulins in the mapped region on chromosome 5, close to the predicted location of ibr2. The Botrytis susceptible line ebs1 co-segregates with basta resistance, indicating that the mutated gene is tagged. However, the T-DNA inserted contains additional sequences on its left border that prevented successful cloning. The ebs1 plants show impaired resistance against B. cinerea, but unaffected resistance against virulent and avirulent strains of PstDC3 000, suggetsing EBS1 plays a more specific role in resistance against necrotrophic pathogens. Further characterization of ibr2 and ebs1 is required in order to elucidate their role in plant disease resistance.

Les cascades de signalisation fongiques du type Hog1 sont impliquées dans diverses fonctions telles l'adaptation aux stress, la résistance aux fongicides, le développement et, dans certains cas, la virulence. Nous avons caractérisé la cascade homologue chez l'ascomycète phytopathogène Botrytis cinerea via l'inactivation de l'histidine-kinase senseur Bos1, sa relation par rapport à la MAPK Sak1 et la régulation de gènes cibles. Les analyses de phosphorylation montrent que Bos1 inhibe la phosphorylation de Sak1 en l'absence de stimulus externe. En conditions de stress, cette inhibition est levée conduisant à la phosphorylation de Sak1, laquelle est impliquée dans l'adaptation aux stress ionique et peroxide, le développement des macroconidies, la pénétration et le développement des nécroses. Par un test d'épistasie, nous montrons que Bos1 régule certaines fonctions cellulaires indépendamment de Sak1 (sensibilité au superoxide; adaptation et conidiation en milieu hyperosmotique neutre; sensibilité aux fongicides de la classe des dicarboximides, phénylpyrroles et aromatiques; production de la mélanine). Deux cascades parallèles sous le contrôle de Bos1 régulent l'intégrité de la paroi et le développement des appressoria et des sclérotes. Afin d'identifier des gènes régulés par la cascade Bos1-Sak1, nous avons réalisé des analyses de RT-qPCR sur des gènes sélectionnés en fonction des phénotypes mutants. Certains des phénotypes peuvent ainsi être corrélés à des expressions différentielles des gènes chez les mutants. Les profils d'expression, en conditions standard, de la plupart des gènes co-régulés par Bos1 et Sak1 corroborent le contrôle négatif de la phosphorylation de Sak1
Hog1-like fungal signal transduction cascades are involved in diverse cellular functions, such as adaptation to various stresses, fungicide resistance, development and, in some cases, virulence. In this work we characterized the homologous pathway of the plant pathogenic ascomycete Botrytis cinerea via the inactivation of the sensor histidine kinase Bos1, its relationship to the downstream MAP kinase (MAPK) Sak1, and the regulation of target genes. Phosphorylation assays show that, without any external stimulus, Bos1 inhibits Sak1 phosphorylation. Under stress conditions, this inhibition is released, leading to Sak1 phosphorylation, which is involved in the adaptation to high ionic and peroxide stress, macroconidia development, plant penetration and necrosis development. Through an epistasis test, we demonstrate that Bos1 regulates certain functions, independently of Sak1. They include superoxide tolerance, adaptation and conidiation on high neutral osmolarity, and susceptibility to three families of fungicides (dicarboximides, phenylpyrroles and aromatic hydrocarbons) as well as melanin production. Cell wall integrity, appressoria- and sclerotia development are probably controlled by two parallel signalling cascades both regulated by the Bos1 HK. To identify the downstream genes regulated by the Bos1-Sak1 cascade, real-time RT-PCR analysis was conducted on selected sets of genes based on the different mutant phenotypes. Some but not all phenotypes can be related to differential gene expression. Expression pattern of most Bos1-Sak1 controlled genes under standard conditions corroborates the negative control of Sak1 phosphorylation

Gray mold of strawberry, caused by Botrytis cinerea, is a very destructive pre- and post-harvest fruit rot. Outside of California, fungicide resistance in B. cinerea has been reported to every site-specific chemical class labeled for use against gray mold. One objective of this study was to characterize the resistance of 888 isolates of B. cinerea from California strawberry fields to ten active ingredients. Isolates were collected from the same planting block in 47 fields during the early-season (0 to 8 fungicide applications) and late-season (16 to 26 fungicide applications) of 2016. Sensitivity of each isolate was determined using the following active ingredients at a discriminatory dosage (μg/ml): boscalid (75), cyprodinil (4), fenhexamid (50), fludioxonil (0.5), fluopyram (10), iprodione (10), isofetamid (5), penthiopyrad (5), pyraclostrobin (10), and thiophanate-methyl (100). Resistance to each active ingredient was observed at varied frequencies (early-season %, late-season %): boscalid (12, 35), cyprodinil (12, 46), fenhexamid (53, 91), fludioxonil (1, 4), fluopyram (2, 7), iprodione (25, 8), isofetamid (0, 1), penthiopyrad (8, 25), pyraclostrobin (77, 98), and thiophanate-methyl (81, 96). Captan, boscalid, cyprodinil, fenhexamid, and fludioxonil were the most commonly used fungicides in surveyed strawberry fields. A selection of 100 isolates was identified to the species level. All isolates were B. cinerea, excluding one isolate of Botrytis mali. A fungicide resistance trial was conducted v to observe resistance responses in populations of B. cinerea. Frequencies of resistance to boscalid and fludioxonil remained unchanged despite consecutive applications of these fungicides. Frequency of resistance to fenhexamid increased when this fungicide was applied and decreased when it was not. This occurred in fungicide treatments including fungicide rotation, tank-mixing with captan, and consecutive applications of fenhexamid. Multi-fungicide resistance was widespread in California strawberries; isolates resistant to fenhexamid, thiophanate-methyl and pyraclostrobin were the most common phenotype. The frequency of resistance increased from the early-season to late-season for multiple active ingredients tested. This within-season change in frequency of resistance was tested and confirmed in a field trial, where common resistance management strategies failed to prevent the buildup of fenhexamid resistance. New and improved methods of resistance management may need to be enacted to ensure the future efficacy of site-specific fungicides.

The circadian clock is an endogenous mechanism that provides a wide variety of organisms with the ability to anticipate daily environmental changes. It was shown that under cyclic and constant light growth conditions Arabidopsis exhibits rhythmicity in Botrytis cinerea resistance, with maximal resistance observed when leaves were inoculated at dawn. Crucially, this mechanism was confirmed to be under circadian clock regulation. To understand how the circadian clock was driving an effective defence response, genes that were more rapidly induced or repressed after inoculation at dawn compared to night were identified. This indicated a complex interaction between the circadian clock and the defence regulatory network. Phytohormone defence signalling, in particular, jasmonate (JA) and ethylene responses, was shown to contribute to the observed rhythmic variation in resistance. This was further confirmed by the identification of a JA signalling mutant (jaz6), which displayed no difference in resistance to B. cinerea following inoculation at dawn or night under cyclic or constant light conditions. Given the central role of JAZ6 in the circadian defence response against B. cinerea, it was likely transcription factors (TFs) bound by JAZ6 were potential links between the plant circadian clock and the defence response. Elucidating the TFs that interacted with JAZ6 revealed JAZ6 to be able to interact with a TF shown to be crucial to the B. cinerea defence response, EIN3. Moreover, JAZ6 was also able to interact with a central regulator of circadian clock, FHY3. Both TF interactors indicate JAZ6 is a linking protein between the circadian clock and the defence response against B. cinerea. To further understand how the circadian clock was mediating the plant defence response against B. cinerea genome-wide chromatin accessibility data was generated using ATAC-Seq. This aimed to enable the comparison of chromatin accessibility as well as TF binding in regions surrounding genes related to B. cinerea defences between the two time-points. This protocol was not optimized for plant tissue. Steps within in! laboratory-based ATAC-Seq protocol were therefore pinpointed for tissue specific optimization. Thus, a protocol specific to ATAC-Seq data analysis was proposed.

Botrytis cinerea est un champignon nécrotrophe et polyphage capable de provoquer la pourriture grise sur plusieurs centaines d’espèces végétales. Les pertes engendrées par cette maladie sont importantes à travers le monde notamment sur des espèces économiquement importantes comme la tomate, la fraise ou encore la vigne. Parmi les facteurs de virulence identifiés chez B. cinerea se trouvent deux toxines non-hôte spécifiques. Il s'agit du sesquiterpène botrydial et du polycétide acide botcinique. Même si leur rôle redondant dans la nécrotrophie a été démontré, les mécanismes qui gouvernent l’expression des clusters responsables de leur synthèse (respectivement BOT et BOA) restent inconnus. Dans ce contexte, l’objectif de mon projet de thèse était de caractériser les différents mécanismes qui régulent le métabolisme secondaire chez B. cinerea. Je me suis particulièrement intéressé aux clusters BOT et BOA ainsi qu’à un troisième cluster (PKS7) qui, d’après le phénotype d’un mutant d’insertion (ADN-T), pourrait être impliqué dans la nécrotrophie. Grâce à la disponibilité du nouvel assemblage du génome de la souche modèle B05.10, un gène candidat codant un facteur de transcription (FT) putatif a pu être identifié à proximité du cluster BOT. La caractérisation de ce gène a permis de démontrer le rôle majeur de la protéine (BcBot6) dans l’activation des gènes Bcbot et la production subséquente de botrydial. De même, la caractérisation de Bcboa13, un gène codant un FT putatif présent au sein du cluster BOA, a permis de démontrer le rôle de régulateur positif de BcBoa13 envers les gènes Bcboa. A la différence des clusters BOT et BOA, le cluster PKS7 ne contient pas de gène codant pour un potentiel FT spécifique. Afin de confirmer le rôle du métabolite putatif produit par ce cluster et d’identifier sa structure chimique, l’inactivation du gène clé codant une PKS-NRPS (Bcpks7) a été réalisée et des analyses métaboliques ont été initiées. Finalement, la présence au sein des clusters BOT et BOA de nombreux transposons ayant subi des mutations de type RIP (Repeat-Induced Point mutations) ainsi que la position sub-télomérique des clusters BOA et PKS7 nous a amené à nous intéresser au rôle de la structure chromatinienne dans la régulation de ces clusters. Dans ce cadre, trois mutants délétés dans des gènes codant des modificateurs chromatiniens putatifs (les histones méthyltransférases BcDim-5 et BcKmt6 et la protéine hétérochromatinienne BcHp1) ont été générés. L’expression des gènes clés des clusters BOT, BOA et PKS7 chez les mutants Bcdim-5, Bchp1 et Bckmt6 suggère que différents mécanismes chromatiniens interviennent pour le contrôle des clusters BOT et BOA d’une part et du cluster PKS7 d’autre part. L’ensemble des résultats obtenus pendant cette thèse apporte une contribution majeure à la compréhension des mécanismes de régulation spécifiques mais aussi de ceux en lien avec la structure chromatinienne de la production de métabolites phytotoxiques impliqués dans la nécrotrophie chez B. cinerea
Botrytis cinerea is a necrotrophic polyphagous fungus able to induce the gray mold disease on hundreds of plant species. The resulting losses are important worldwide notably on economically important crops such as tomato, strawberry or grapevine. Among the virulence factors identified in B. cinerea stand two non-host specific toxins: the sesquiterpene botrydial and the polyketide botcinic acid. Although their redundant role in necrotrophy has been shown, the mechanisms governing the clusters responsible for their synthesis (respectively BOT and BOA) remain unknown. In this context, the aim of my PhD project was to characterize the different mechanisms that regulate secondary metabolism in B. cinerea. I particularly focused on BOT and BOA clusters as well as on a third one (PKS7) which, according to the phenotype of an insertion-based mutant (T-DNA), could be involved in necrotrophy. Thanks to the newly assembled genome of the B05.10 wild type strain, a candidate gene encoding a putative transcription factor (TF) could be identified near the BOT cluster. The characterization of this gene allowed pointing out the major role of the protein (BcBot6) in the activation of Bcbot genes and in the subsequent botrydial production. Similarly, the characterization of Bcboa13, a putative TF-encoding gene present into the BOA cluster, allowed demonstrating the positive regulatory role of BcBoa13 on Bcboa genes. Unlike the BOT and BOA clusters, the PKS7 one does not contain any putative TF-encoding gene. In order to confirm the role of the putative metabolite produced by this cluster and to identify its chemical structure, the inactivation of the PKS-NRPS key enzyme-encoding gene (Bcpks7) was conducted and metabolic analyses were initiated. Finally, the presence of many RIP(Repeat-Induced Point mutations)-inactivated transposons within BOT and BOA clusters as well as the subtelomeric location of BOA and PKS7 clusters raised our interest about the role of chromatin structure on those clusters regulation. In this context, three mutants inactivated into putative chromatin modifiers encoding-genes (the histone methyltransferases BcDim-5 and BcKmt6 and the heterochromatin protein BcHp1) were generated. The expression analysis of the key genes of the BOT, BOA and PKS clusters suggests different chromatin-based mechanisms that intervene for the BOT and BOA cluster on one side and on the PKS7 cluster on another. Altogether, the results generated during this PhD project are a major contribution to the comprehension of pathway-specific as well as chromatin-based mechanisms that regulate the production of necrotrophy-involved phytotoxins by B. cinerea

Botrytis cinerea in contact with mature grape berries encounters an environment particularly rich in polyphenols and PR proteins, where the stilbenic phytoalexin trans-resveratrol may accumulate. To mimic conditions similar to those found in grape berries, B. cinerea was grown in vitro with grape PR proteins and polyphenols extracted from mature grapes and with trans-resveratrol. Results showed that in the presence of highly toxic amounts of trans-resveratrol, grape polyphenols allowed total recovery of fungal growth, and proteins allowed partial recovery. These resveratrol-polyphenol or resveratrol-protein combinations also induced a strong release into the medium of laccase activity, which is likely to be involved in trans-resveratrol detoxification. The grape protein pattern changed during fungal growth; most grape proteins quickly disappeared from the culture when polyphenols and trans-resveratrol were present together. Similar protein patterns were obtained in vitro by incubating grape proteins with grape polyphenols and/or trans-resveratrol with a purified B. cinerea laccase. Under these conditions, most proteins became insoluble. The grape protein pattern obtained from grape berries infected by B. cinerea strongly resembled that obtained in vitro by incubating grape proteins and polyphenols with fungal laccase. It seems that B. cinerea, through laccase secretion and activity and by exploiting the berry polyphenols, easily neutralizes the toxicity of grape stilbenic phytoalexins and makes the grape pathogenesis-related proteins insoluble. The effect of laccase, resveratrol and polyphenols on fungal spore germination was also studied. Results showed that resveratrol alone initially does not inhibit the spore germination. But the inhibition was completely relieved by the presence of grape polyphenols. Instead, the pre-incubation of resveratrol with laccase completely inhibited the spore germination. In addition, we investigate the involvement of B. cinerea proteases in the degradation of grape PR proteins. An aspartyl and a tripeptidyl protease were purified from B. cinerea in vitro culture. The purified proteases activities partially degraded PR proteins. The expression analysis of tripeptidyl and aspartic protease gene families revealed that several members of these families are expressed in the presence of grape PR proteins. In conclusion, our results support that in a grape berry environment characterized by an abundance of polyphenols, B. cinerea laccase not only detoxify the trans-resveratrol but also modifies the solubility of grape proteins and this environment may facilitate the fungal protease to degrade grape PR proteins.
Durante l’infezione dell’uva il fungo fitopatogeno Botrytis cinerea incontra tessuti particolarmente ricchi di polifenoli e proteine PR e dove si accumula la fitoalessina trans-resveratrolo. Per simulare condizioni simili a quelli trovati negli acini d'uva, B. cinerea è stato coltivato in vitro con proteine e polifenoli estratti da uve mature, e con trans-resveratrolo. I risultati hanno dimostrato che in presenza di livelli tossici di trans-resveratrolo, i polifenoli dell'uva favoriscono una normale crescita del patogeno mentre le proteine consentono un parziale recupero della crescita. Le combinazioni polifenoli-resveratrolo o resveratrolo-proteina inducevano il rilascio di una forte attività laccasica nel mezzo, che sembra essere coinvolta nella disintossicazione del trans-resveratrolo. I risultati hanno di mostrato anche che il pattern delle proteine dell’uva era alterato durante la crescita del fungo. Infatti, le proteine dell’uva scomparivano rapidamente dalla coltura nella quale polifenoli e trans-resveratrolo erano presenti simultaneamente. Profili proteici simili sono stati ottenuti in vitro, incubando proteine con polifenoli dell'uva e /o trans-resveratrolo con le laccasi purificata di B. cinerea. In queste condizioni, la maggior parte delle proteine diventava insolubile. Questo pattern era molto simile a quello osservato negli acini infettati da B. cinerea. Pertanto, B. cinerea, attraverso la secrezione di attività laccasica e sfruttando i polifenoli, neutralizza facilmente la tossicità delle fitoalessine stilbeniche e rende le proteine PR insolubili. L'effetto di laccasi è stato studiato anche sulla germinazione delle spore di B. cinerea. Il resveratrolo da solo inizialmente non inibiva la germinazione delle spore, invece, la sua pre-incubazione con le laccasi, inducendo la formazione di trans-ε-viniferina, ne inibiva la germinazione. Invece se nel mezzo erano presenti anche i polifenoli non si osservava alcuna inibizione della germinazione. Successivamente è stato indagato il coinvolgimento delle proteasi di B. cinerea nella degradazione delle proteine d’uva. Del mezzo coltura è stata purificata una aspartyl e una tripeptidyl proteasi. Queste proteasi sono state in grado di degradare parzialmente le proteine d’uva. Un’analisi di espressione dei geni delle famiglie di tripeptidyl e aspartyl proteasi ha dimostrato anche altri membri di queste famiglie erano espresse in presenza di proteine PR dell’uva. I risultati permettono di concludere che nell’acino d’uva, caratterizzato d’una grande varietà di polifenoli, la laccasi non solo anulla la tossicità del trans-resveratrolo, ma modifica anche la solubilità delle proteine dell'uva. Questo effetto potrebbe facilitare l’azione proteasica del fungo verso le proteine PR d’uva.

The ecological significance of mycoviruses is becoming increasingly recognised, not just for their potential as biocontrol agents but also as driving forces in the evolution and diversification of fungi. Therefore, it is important to understand how mycoviruses and fungi interact on the molecular and biochemical level. To this end the interaction between Botrytis cinerea and the mycoviruses Botrytis virus F and Botrytis virus X was studied. Relative and absolute real time PCR protocols were developed for monitoring the titres of BVX and BVF during transfection studies to monitor changes in virus titre in relation to phenotypic and metabolic changes in the fungal host. Phenotypic changes included severe phenotypical alterations, which were associated with extreme up regulation of carbohydrate, amino acid and lipid metabolism, and induction of stress responses (vacuolisation/cell lysis, increased pigmentation). To study the location and distribution of BVX in infected Botrytis the BVX coat protein was recombinantly expressed in E. coli, BVX specific polyclonal antibodies produced, and protocols developed for the serological detection and visualisation of BVX. Immuno-fluorescence microscopy was used to studying the distribution of BVX within growing Botrytis cultures indicated that the virus is present in aggregates located attached to the cell membrane, the septum, in spores, and in hyphal tips. A combination of light and electron microscopy showed that BVX is often closely associated with cell walls, suggesting that the virus may be moving across the cell wall by altering cell wall composition. If this is shown to be the case then it provides an alternative method to transmission via hyphal anastomosis, which is currently considered to be the only method of horizontal transfer.

Les champignons ont développé diverses interactions avec les végétaux. Ces interactions peuvent être bénéfiques pour la plante dans le cas des champignons établissant des symbioses mutualistes ou néfastes si le champignon est pathogène. Elles reposent sur des mécanismes moléculaires mal élucidés. Des études réalisées sur le champignon mutualiste Hebeloma cylindrosporum associé au pin Pinus pinaster ont permis de créer une collection de mutants affectés dans leur capacité à interagir avec les plantes et à former l’organe mixte de la symbiose, l’ectomycorhize. L’objectif de ma thèse a été d’étudier un mutant affecté dans le gène codant une alpha-tubuline Hctubα2. Les tubulines sont des protéines présentes chez tous les Eucaryotes et permettent la formation des microtubules, des éléments clés du cytosquelette. Chez les champignons, on trouve une ou deux alpha tubuline(s). H. cylindrosporum en possède deux. J’ai étudié l’expression de ces deux tubulines lors l’établissement de l’interaction avec les racines de l’hôte. Les résultats indiquent que ces deux gènes sont différentiellement exprimés lors de l’interaction. J’ai étudié au niveau protéomique l’impact de la mutation en comparant les protéomes intracellulaires des deux souches. On retrouve deux alpha-tubulines chez certains champignons phytopathogènes comme Botrytis cinerea. L’hypothèse de l’implication de l’alpha-tubuline 2 dans l’établissement de la pathogénie a été émise. J’ai donc construit des mutants de Botrytis cinerea dans lesquels ce gène a été inactivé. J’ai également tenté de localiser à l’aide de fusions traductionnelles chacune des alpha-tubulines chez le champignon mycorhizien et chez le pathogène
In all terrestrial ecosystems, plants live in close interaction with numerous fungi. The interaction has a negative or positive effect on host plant depending upon the pathogenic or symbiotic status of the fungus. The establishment of these interactions is based on a tightly regulated molecular dialog between symbiotic partners. Previous studies on the ectomycorrhizal fungi, Hebeloma cylindrosporum associated with maritime pine (Pinus pinaster), created a collection of mutants affected in their mycorrhizal abilitiy. The aim of my thesis was to characterize one of these mutants affected in a gene, Hctubα2, encoding an alpha tubulin. Tubulins are eukaryotic cytoskeletal proteins involved in microtubules formation. Fungi have one or two alpha-tubulin. For example, H.cylindrosporum has two alpha-tubulin. The site of mutagenic DNA insertion in fungal genome was characterized. I studied the expression of both alpha-tubulins during the establishement of mycorrhizal interaction. Results showed that the two genes are differentially expressed during the interaction with host plant. At proteomic level, I studied the impact of the mutation comparing the two strains using 2D gel electrophoresis and sequencing differentially accumulated spots. Pathogenic fungi also bear two alpha-tubulins, as Botrytis cinerea. The hypothesis of the involvement of the alpha-tubulin 2 in pathogenesis was investigated. I created Botrytis cinerea mutants deleted for this gene. I also created translational fusions in order to visualize both alpha-tubulins in Hebeloma cylindrosporum and in Botrytis cinerea

Nous avons exprimé dans différents systèmes hétérologues l'ADNc de la xylanase (XYNB) du champignon filamenteux Penicillium funiculosum ainsi que celle du phytopathogène Botrytis cinerea (XynBc) appartenant toutes deux à la famille des GH11 des glycosylhydrolases. Les propriétés physico-chimiques des enzymes recombinantes ont été déterminées. XYNB et XynBc montrent les mêmes spectres d'inhibitions en présence d'inhibiteurs protéiques probablement impliqués dans des mécanismes de défenses des plantes et issus du blé ; les activités sont fortement inhibées par XIP-I et TAXI-I tandis que TAXI-II n'a aucun effet. Des essais de RT-PCR semi-quantitatives ont montré la présence des ARNm au cours des 72 premières heures de l'infection de feuilles de tabacs par B. Cinerea
The phytopathogen fungus Botrytis cinerea and the filamentous fungus Penicillium funiculosum produce various glycosidases with xylanase activity. In the present study, we report the heterologous expression, purification and characterization of two family 11 xylanase produced by these fungi. These enzymes present similar activities on different substrates and identical suceptibilities to different proteinaceous inhibitors from wheat. These xylanases were sensitive to XIP-I, TAXI-I but not to TAXI-II. The inhibition was competitive and the inhibition constant were in the nanomolar range indicting a great affinity between these xylanases and the wheat inhibitors. Moreover, we have shown that the presence of glycosylation or of a carbohydrate binding module in the xylanase, did not affect the interaction of the enzyme with the inhibitor

Le Botrytis cinerea, anamorphe du Botryotinia fuckeliana est l'agent de la pourriture grise de nombreuses plantes et de la vigne en particulier. Le cycle biologique de cet organisme implique une multiplication directe par conidies et un processus sexué dans lequel interviennent des sclérotes sur lesquels se forment des apothécies et des microconidies ou des spermaties assurant la fécondation. Un recensement des connaissances acquises quant à ce cycle montre l'existence de grandes disparités entre les résultats rapportés. Nous avons essayé de définir les conditions optimales d'obtention des différentes phases du cycle afin de mieux connaître la biologie de ce parasite
L'ouvrage que nous présentons comporte essentiellement trois parties : 1) dans la première partie, nous avons étudié différents milieux de culture sur lesquels le Botrytis cinerea est capable de se développer. Nous avons ensuite défini les modifications de la composition de ces milieux qui permettent d'obtenir préférentiellement du mycélium, des conidies, des sclérotes ou des microconidies. Il existe des souches produisant préférentiellement des microconidies ou des sclérotes, nous les avons utilisées spécifiquement afin d'étudier les processus d'initiation et de développement des apothécies ; 2) dans la 2ème partie du travail, nous avons effectué en microscopie photonique et microscopie à balayage une étude cytologique approfondie des différentes étapes de la phase sexuée. Elle a permis d'établir le mode de formation des microconidies et de mettre en évidence la présence de l'ascogone à l'intérieur d'un sclérote ; 3) la 3ème partie consiste en une étude approfondie de la reproduction sexuée du B. Fuckeliana. Dans cette partie, nous avons étudie la nécessité de la spermatisation et d'une vernalisation pour induire la formation des apothécies et leur développement. Des expériences ont été tentées avec différentes fractions de l'organisme susceptibles de fournir des noyaux pour la fécondation des ascogones ; elles ont montré que seules les spermaties étaient capables d’assurer la féondation. L’ensemble de l’ouvrage présenté correspond à une mise à jour et une homogénéisation des connaissances relatives au cycle sexué de Botryotinia fuckeliana

Lors de l'interaction entre un champignon phytopathogène et une plante hôte, des molécules toxiques peuvent être générées par l'agresseur mais aussi par les réactions de défense de la plante. Par ailleurs, les champignons phytopathogènes sont exposés aux traitements chimiques utilisés au champ à des fins antifongiques. Mieux comprendre les mécanismes impliqués dans la tolérance de ces microorganismes à ces molécules toxiques constitue ainsi un point important tant au point de vue des interactions plante-pathogène que de la tolérance aux produits fongicides. Les transporteurs à efflux sont décrits comme participant activement au phénomène de tolérance en exportant les molécules toxiques hors de la cellule. Chez l'agent de la pourriture grise B. cinerea, différentes approches génétiques et bio-informatiques ont conduit à l'existence de 53 transporteurs ABC, dont quatre seulement ont été caractérisés et dont le rôle dans le processus infectieux du champignon est limité. Au cours de ce travail de thèse, la mesure de la réponse transcriptionnelle de l'ensemble des gènes ABC a montré une forte mobilisation des transporteurs à efflux au cours du processus infectieux de B. cinerea, et suggère un rôle global de cette super-famille de gènes dans l'interaction. Jusqu'à aujourd'hui, les études de tolérance des champignons phytopathogènes aux fongicides rapportées dans la littérature ont été conduites in vitro. Ici, l'étude a été conduite in planta et a révélé cinq gènes codant pour des transporteurs ABC sur-exprimés en réponse au traitement fongicide. Leur implication dans la détoxication a été étudiée par une approche de mutagenèse dirigée. Cette méthode n'a cependant pas permis de conclure quant au rôle des transporteurs délétés dans la tolérance au fongicide et/ou dans la pathogénie. Enfin, des résultats contradictoires co-existent actuellement sur le lien qu'il pourrait exister entre les tolérances des champignons aux antifongiques et l'activation de la transcription des gènes codant pour les transporteurs ABC impliqués dans l'efflux de ces derniers. Dans ce travail, ce point a été exploré à l'échelle de tous les gènes ABC de B. cinerea. Leur comportement transcriptionnel a été étudié en réponse à deux fongicides de mode d'action différents, dans des conditions de culture in vitro, mais également à différents stades d'une infection. L'analyse des séquences promotrices des gènes co-régulés par ces deux produits in vitro a permis l'identification de deux motifs putatifs de régulation et suggère l'implication potentielle de plusieurs facteurs de transcription dans la réponse aux fongicides

"Botrytis cinerea" est un champignon phytopathogène qui infecte les grappes de raisins et plus de 200 plantes hôtes, grâce à ses facteurs de virulence. Leurs sécrétions sont régulées par le pH environnant. Ce champignon semble bien adapté pour croître à pH 3, pH rencontré dans la baie de raisin. La baie de "Vitis vinifera" L. Cv. Pinot Noir possède des protéines de défense (PR-protein). Bien que certaines d'entre elles se soient avérées efficaces contre le développement de B. Cinerea, la baie reste très sensible. Ces protéines de défense, chitinase de classe IV (CHV5) et thaumatin-like (TL) sont à une concentration maximale dans la baie mûre. L'infection des grappes de raisin par "B. Cinerea" s'effectue en foyers qui croissent régulièrement. Dans les baies exprimant des symptômes d'infection croissants, les concentrations en protéines, y compris CHV5 et TL, diminuent. L'étude des ARNm dans les baies montre que les gènes de CHV5 et de TL ne sont pas induits par "B. Cinerea"
"Botrytis cinerea" the causal agent of grey mould un grape berries is able to infect more than 200 host plants leading to severe losses. This fungus secretes virulence factors which play a key role during the infection process. At the start of this work, little was known about the regulation of these factors and the regulation of grapevine defense mechanisms. We showed that "B. Cinerea" virulence factors are regulated by environmental pH. PH 3. 0, typically found in grape berries, is optimal for this growth. Berries express defenses responses including defense proteins that help to stop pathogen infection. Among them, the main proteins, abundant in ripe and healthy berries, of "Vitis vinifera" L. Cv. Pinot noir are class IV chitinases (CHV5) and a thaumatin - like (TL). During grape colonisation by "B. Cinerea", infection clusters are regularly spreading followed by a decrease in plant protein concentration. Studies at the mRNA level showed that CHV5 and TL genes are not induced by "B. Cinerea"

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Rio Grande do Sul (RS), the most important state of grape production in Brazil, harvests, approximately, 780 thousand tons annually. One of the biggest constraint factors to the obtainment of higher production numbers is the incidence of late season diseases in vineyards, being botrytis bunch rot, caused by Botrytis cinerea, one of the major contributors for field and post-harvest losses. Red grapes can show more resistance against this fungus, due to higher levels of phenolic compounds. The use of biological control agents (BCAs) emerges as a promising alternative to control botrytis disease. For this reason, the objectives of this study were to test isolates from this fungal pathogen, originated from the two main grape production regions in the state of RS, trough in vitro direct confront test against BCAs fungal isolates (Trichoderma spp. and Gliocladium sp.) and to test the same pathogen isolates on semi in vivo biological control, against the BCAs isolates which obtained the highest biological activity during the in vitro test, on the post-harvest storage of table grapes, on a red and a white cultivar. The B. cinerea isolates used were UFSM SG01, UFSM SG02, from serra (mountain range) region, UFSM CM01, and UFSM CM02, from campanha meridional (meridional pampas) region. The BCAs isolates used in this study were Tricoderma spp., UFSM T20, UFSM T17, UFSM T15.1 (obtained from soil), UFSM TSG, UFSM TCM (obtained from the same grape bunches where B. cinerea was isolated, representing each region), and Gliocladium sp., UFSM G4DB (obtained from soil). UFSM TSG, UFSM TCM and UFSM T15.1 showed the highest biocontrol activity (B. A.) in vitro, in general over 50% against all B. cinerea isolates. The same BCAs were selected to be used at the semi in vivo test with detached berries for three inoculation periods: B+T, pathogen and BCA inoculated at the same time; B+24hT, pathogen inoculated first and BCA 24 h later, T+24hB, BCA inoculated first and pathogen 24 h later. A higher control, considered as the lower damage level, on T+24hB period, showed the importance of preventive treatment. Cracks on berries played a more important role than the color of the skin for botrytis infection.
O Rio Grande do Sul, o estado produtor de uvas mais importante no Brasil, colhe em média 780 mil toneladas por ano. Um dos maiores entraves para a obtenção de maiores médias de produção é a incidência de doenças de final de ciclo (DFC), sendo Botrytis cinerea, agente causador da podridão-cinzenta, um dos maiores responsáveis por perdas de produção no campo e na pós-colheita. Uvas tintas podem apresentar maior resistência à podridão-cinzenta, devido à maior concentração de compostos fenólicos. O uso de agentes de controle biológico (BCAs) é uma alternativa promissora no controle da podridão de botrytis. Dessa maneira, os objetivos deste estudo foram testar isolados do patógeno coletados das duas principais regiões vitivinícolas do RS em confronto direto in vitro com isolados de agentes antagonistas (Trichoderma spp e Gliocladium sp.) e testar os mesmos isolados do patógeno em controle biológico semi in vivo com os isolados antagonistas que obtiveram as maiores médias de atividade de biocontrole no teste in vitro, na pós-colheita de uvas de mesa, em cultivares branca e tinta. Os isolados de B. cinerea empregados foram UFSM SG01, UFSM SG02, UFSM SG 03, oriundos da serra, e UFSM CM01 e UFSM CM02, oriundos da campanha meridional. Os isolados antagonistas empregados no teste in vitro foram UFSM T20, UFSM T17, UFSM T15.1 (oriundos de solo), UFSM TSG, UFSM TCM (oriundos de cachos de uva coletados nas mesmas regiões de coleta do patógeno), de Trichoderma spp., e UFSM G4DB, de Gliocladium sp.. Os isolados UFSM TSG, UFSM TCM e UFSM T15.1 foram os três que obtiveram as maiores médias de atividade de biocontrole, em geral acima de 50%, para todos os isolados de B. cinerea, testados em confronto direto in vitro. Esses isolados BCAs foram selecionados para o teste de controle biológico semi in vivo em bagas destacadas, em três períodos de inoculação: B+T, antagonista e patógeno aplicados ao mesmo tempo; B+24hT, patógeno inoculado primeiro e antagonista 24h após, T+24hB, antagonista aplicado primeiro e patógeno 24h após. O maior controle, assumido a partir do menor grau de dano, no período T+24hB, evidenciou a importância do tratamento preventivo. Rachaduras em bagas tiveram maior influência do que a coloração da casca na ocorrência da podridãocinzenta.