L'infection des baies de raisin par le champignon Botrytis cinerea, responsable de la pourriture grise, est fréquente et occasionne des dégâts importants. Pourtant, il semble que la baie dispose de moyens de défense parmi lesquels des protéines PR "Pathogenesis-Related". Chez le Pinot Noir, une chitinase (CHI4D) et une thaumatin-like (TL3) s'accumulent naturellement en grande quantité à partir de la véraison et présentent une activité antifongique contre B. cinerea in vitro. L'objectif de ce travail est de comprendre comment B. cinerea peut se développer sur des baies censées disposer de défenses suffisantes. Pour cela, l'expression spatio-temporelle des ARNm et des protéines CHI4D et TL3 a été suivie respectivement par hybridation in situ et immunohistolocalisation dans les baies, au cours de la maturation, à la suite d'un stress abiotique (UV-C) ou d'un stress biotique (B. cinerea). Dans des baies avant véraison (vertes), n'exprimant naturellement que très faiblement CHI4D et TL3, les ARNm et les protéines s'accumulent en grande quantité après application d'un stress abiotique (UV-C) ou biotique (infection artificielle par B. cinerea). Les protéines CHI4D et TL3 sont localisées au niveau des faisceaux conducteurs ainsi qu'au niveau des tissus proches des sites d'exposition aux UV-C (exocarpe) ou au niveau des sites d'inoculation de B. cinerea, suggérant qu'elles sont impliquées dans la défense de la baie avant véraison. Après véraison, les ARNm et les protéines sont naturellement accumulés au niveau de l'exocarpe et des faisceaux conducteurs qui correspondent à des sites potentiels d'entrée ou de propagation des agents pathogènes. Alors que l'application d'un stress UV-C sur ces baies ne provoque qu'un effet mineur sur l'expression de CHI4D et de TL3, au cours de l'infection par B. cinerea, les quantités de transcrits et de protéines diminuent. A un stade précoce d'infection, la diminution de la quantité des deux protéines est observée en avant du front de propagation du champignon, suggérant une dégradation par des protéases sécrétées par B. cinerea. A un stade d'infection plus avancé, cette diminution s'étend à l'ensemble de la baie. La production hétérologue des protéines CHI4D et TL3 nous a permis de confirmer que CHI4D pouvait être dégradée par des protéases aspartiques sécrétées par B. cinerea. La dégradation de TL3 n'a pas pu être reproduite in vitro. Des tests antigerminatifs effectués in vitro avec les protéines hétérologues n'ont pas permis de mettre en évidence d'effet antifongique malgré la présence d'une activité chitinase pour CHI4D et β-1,3,-glucanase pour TL3. Il est donc possible que ces protéines possèdent des fonctions autres que celles impliquées dans la défense. / Grape berries infection by the phytopathogenic fungus Botrytis cinerea, the causal agent of gray mold, is quite common and causes significant damage. However, it seems that berries have a mechanism of defense, among which are pathogenesis related proteins. In Pinot Noir grape berries, a chitinase (CHI4D) and a thaumatin-like (TL3) protein naturally accrue in large amounts from véraison and show in vitro an antifungal effect against B. cinerea. The aim of this work was to understand how B. cinerea can develop on grape berries that seem to have sufficient defense mechanisms. To do so, the spatio-temporal expression of CHI4D and TL3 mRNAs and proteins in berries was studied respectively by in situ hybridization and immunohistolocalization during maturation, after an abiotic stress (UV-C) or a biotic stress (B. cinerea). Before véraison (green berries) while the expression of CHI4D and TL3 is naturally low, mRNAs and proteins have accumulated in large amounts in berries after UV-C exposition or artificial infection with B. cinerea. CHI4D and TL3 proteins have accumulated around vascular bundles as well as near the sites of UV-C exposition (exocarp) or B. cinerea inoculation, suggesting that before véraison these proteins could be involved in the berry defense. After veraison, mRNAs and proteins naturally accumulate in the exocarp and around vascular bundles that correspond to potential sites of penetration or propagation of pathogenic agents. While the application of UV-C stress on these berries causes only a minor effect on the expression of CHI4D and TL3, during infection by B. cinerea, the amounts of mRNA and proteins decreased. At an early stage of infection, the less amounts of both proteins were observed around the fungus propagation area, suggesting that these proteins could be degraded by B. cinerea secreted proteases. At a more advanced stage of infection, the decrease extended to the entire berry.Production of heterologous CHI4D and TL3 proteins allowed us to confirm that CHI4D could be degraded by aspartic proteases secreted by B. cinerea whereas no degradation of TL3 could be observed in vitro. Both heterologous proteins showed no antifungal effect while a chitinase and a β-1,3-glucanase activities were observed respectively for CHI4D and TL3. It is therefore possible that these proteins have other functions than those involved in the defense.

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Irrigation scheduling decisions are based either on the direct measurement of soil water status (SWS) or on physiological measurements like plant water status (PWS). Soil based measurements are quick and easily automated, but the plant response for a particular quantity of soil moisture varies as a complex function of evaporative demand. A plant-based approach measures the plant stress response directly, but is an integration of environmental effects as well. In contrary to soil-based methods, plant based measurements can indicate when to irrigate, but not the quantity. Pre-dawn leaf water potential (ΨPD) is determined mostly by the soil moisture level, and can serve as a measure of static water stress in plants and an index of bulk soil water availability or even as an estimate of soil water potential at the root surface. Therefore it should be possible to establish a link between SWS and PWS, but it is largely unknown how stable the link in a heterogeneous vineyard would be, and how the grapevine vegetative and reproductive response relates to this link. Plant water status plays a large role in determining vigour and yield of the plant. The levels of PWS are influenced by irrigation, but it was mostly affected by the season and vine location in the vineyard. More negative plant water potentials reduced vigour, but had a less pronounced effect on yield, while also reducing overall wine quality. Vigour variability in the vineyard was largely attributed to soil heterogeneity, which seemed to have a strong effect on SWS. SWS measurements were calibrated according to the observed variability, increasing the accuracy of measurements significantly. Soil water content values were used to establish a link between SWS and PWS. This link was determined over time using nine plots, consisting of rain-fed and irrigated regimes, in variable vigour areas. A non-linear relationship was found between ΨPD and percentage extraction of plant available water for rain-fed plots. When irrigation was applied, no correlation could be found. In this study, for Merlot in the Stellenbosch region, PWS differences affected vigour, and to a lesser extent yield, as well as wine quality. More negative plant water potentials reduced vigour more in high vigour areas than in lower vigour areas, which in turn led to unbalanced vegetative: reproductive ratios. This disturbed vine balance may have had a bigger impact on wine quality than PWS levels. Therefore a well-managed and balanced vine is able to withstand more stress, with less detrimental effects. This study also highlights the danger of limiting the assessment of soil and plant water status conditions to point measurements in vineyards with high levels of vigour variability. / AFRIKAANSE OPSOMMING: Besluite rakende die skedulering van besproeiing word gewoonlik gebaseer op die direkte meting van grondwaterstatus (GWS), of op fisiologiese metings soos byvoorbeeld plantwaterstatus (PWS). Grond gebaseerde metings is relatief vinnig en maklik om te outomatiseer, maar die plantrespons vir ʼn spesifieke grondwaterinhoud varieer as ʼn komplekse funksie van dampdruktekorte. ‘n Plantgebaseerde benadering meet die plantstresreaksie direk, maar is ʼn integrasie van omgewingstoestande. In teenstelling met grondgebaseerde metodes, kan plantgebaseerde metodes aandui wanneer om te besproei, maar nie die hoeveelhede wat besproei moet word nie. Voorsonsopkoms blaarwaterpotensiaal (ΨPD) word grootliks deur die grondwaterinhoud bepaal, en kan as ʼn maatstaf van statiese waterspanning in plante en as ʼn indeks van bulk grondwaterbeskikbaarheid dien, of selfs as ʼn benadering van die grondwaterpotensiaal by die worteloppervlak. Dit behoort dus moontlik te wees om ʼn verwantskap te bepaal tussen GWS en PWS, maar dit is grootliks onbekend hoe stabiel hierdie verwantskap sal wees in ʼn heterogene wingerd, asook hoe die wingerdstok se vegetatiewe en reproduktiewe reaksie die verwantskap kan beïnvloed. Plantwaterstatus speel ʼn groot rol in die bepaling van groeikrag en opbrengs in die wingerdstok. Die vlakke van plantwaterstatus word deur besproeiing beïnvloed, maar word skynbaar meesal deur die seisoen en wingerdstok se ligging in die wingerd bepaal. Meer negatiewe plantwaterpotensiaalvlakke het gelei tot laer groeikrag, maar het ʼn minder uitgesproke effek gehad op opbrengs, terwyl dit in die algemeen wynkwaliteit verswak het. Groeikrag variasie in die wingerd kon grootliks aan grond heterogeniteit toegeskryf word, wat skynbaar ʼn sterk invloed op grondwaterstatus gehad het. Grondwaterstatus metings is gekalibreer volgens die variasie wat waargeneem is, wat die akkuraatheid van metings beduidend verhoog het. Grondwaterinhoud waardes is gebruik om ʼn verwantskap aan te dui tussen SWS en PWS. Hierdie verwantskap is oor tyd bepaal vir nege persele, wat bestaan het uit droëland asook besproeide persele, in areas waarvan die groeikrag verskil het. ʼn Nie-liniêre verband is gevind tussen ΨPD en die persentasie onttrekking van plantbeskikbare water vir die droëland persele. Waar besproei was, kon geen verband gevind word nie. In hierdie studie, vir Merlot in die Stellenbosch area, het PWS vlakke groeikrag en tot ʼn mindere mate opbrengs en wynkwaliteit beïnvloed. Meer negatiewe plantwaterpotensiaal vlakke het groeikrag meer beïnvloed in hoër groeikrag areas as in die laer groeikrag areas, wat ook gelei het tot ongebalanseerde vegetatiewe:reproduktiewe verhoudings. Hierdie versteurde balans in die wingerdstokke kon dalk ʼn groter impak op wynkwaliteit gehad het as PWS vlakke. Daar moet dus gepoog word om goed bestuurde en gebalanseerde wingerdstokke te hê, sodat strestoestande beter weerstaan kan word met minder nadelige gevolge. Die studie beklemtoon ook die gevaar verbonde daaraan om die bepaling van grond- en plantwaterstatus te beperk tot puntmetings in wingerde met groot variasie in groeikrag.

Vitis vinifera L. est l'une des premières espèces fruitières à avoir été domestiquées. Sous l'effet de la sélection humaine, cette espèce a suivi une évolution agro-morphologique conduisant à une importante diversité répartie en deux morphotypes principaux selon l'usage des raisins (cuve vs table). L'objectif de cette thèse a été de mieux comprendre la structuration et l'origine de la diversité génétique de la vigne domestique au travers de l'étude de marqueurs moléculaires neutres (nucléaires et chloroplastiques) et de gènes codant pour des caractères d'intérêt agronomique (couleur des baies et architecture des grappes). Une meilleure connaissance des ressources génétiques de la vigne est en effet nécessaire pour leur gestion optimisée et leur utilisation appropriée dans de nouveaux programmes d'amélioration. Une étude de parenté basée sur l'analyse de 20 microsatellites nucléaires a d'abord été menée sur 2344 cultivars de la collection INRA du Domaine de Vassal. Elle a permis de préciser l'ascendance directe de plus de 800 cultivars et de révéler les géniteurs clés. A l'aide de ces mêmes marqueurs, une étude de la structuration génétique du compartiment cultivé a ensuite mis en évidence quatre grands groupes de diversité reliés à l'usage des fruits, la géographie et l'histoire de la viticulture. Ces premiers résultats ont été utilisés pour constituer un échantillon de travail de 595 génotypes comprenant i) des cultivars (subsp. vinifera, syn. sativa) représentatifs de la diversité neutre et de catégories historiques préalablement définies et ii) des représentants du compartiment sauvage (subsp. sylvestris). Les résultats de l'étude de la diversité de l'ADN chloroplastique sont compatibles avec l'existence d'un centre primaire de domestication oriental et de centres secondaires répartis sur le pourtour méditerranéen. Le polymorphisme de séquence (SNP et INDEL) a ensuite été exploré pour trois gènes associés à des caractères d'intérêt agronomique. L'analyse de la diversité des gènes VvMybA1 et VvMybA3, associés à la couleur des baies, a permis de préciser l'histoire de ce trait et sa diversification sous l'effet de la sélection artificielle. L'analyse du polymorphisme du gène VvTFL1A, associé à l'architecture des grappes, a montré une structuration différente principalement en relation avec l'usage des fruits. L'ensemble des résultats a permis de mettre en évidence certaines variétés ou groupes de variétés occupant une position originale dans l'histoire de la vigne cultivée depuis sa domestication. / Vitis vinifera L. is one of the first fruit species ever domesticated. Under human selection, this species underwent a morphological and agronomical evolution leading to an extensive diversity and to two distinct morphotypes according to the use of grapes (wine vs. table). The objective of this PhD thesis was to better understand the structure and origin of cultivated grapevine genetic diversity studying neutral (nuclear and chloroplastidial) molecular markers and genes encoding traits of agronomic interest (berry colour and bunch architecture). A better knowledge of grapevine genetic resources is indeed needed for their optimized management and appropriate use in new breeding programmes. A parentage study based on 20 nuclear microsatellites markers was first performed on 2344 cultivars held in the INRA “Domaine de Vassal” repository. This work allowed us to reveal the direct ascent of more than 800 cultivars and to uncover key genitors. Then, a study of the cultivated pool genetic structure was performed using the same markers. The four diversity groups found are related to use of fruits, geography and viticulture history. These first results were used to build a working sample of 595 genotypes that included i) cultivars (subsp. vinifera, syn. sativa) representative of both neutral markers diversity and previously defined historical categories and ii) representatives of the wild compartment (subsp. sylvestris). The results of chloroplastidial DNA diversity study are consistent with the existence of an eastern primary domestication centre with secondary centres distributed on the periphery of the Mediterranean sea. Sequence polymorphism (SNP and INDEL) was then explored in three genes associated with traits of agronomic interest. Diversity analysis of VvMybA1 and VvMybA3 genes associated with berry colour allowed us to better understand the diversification of this trait under artificial selection. Analysis of VvTFAL1A polymorphism, associated to bunch architecture, showed a different structuration mainly related to the use of fruits. All these results highlighted specific cultivars or groups of cultivars which hold an original position in the history of cultivated grapevine since its domestication.

Les anthocyanes sont une famille de polyphénols très répandus chez les végétaux. Chez la vigne, elles sont responsables de la coloration des baies des cépages rouges, et sont impliquées dans les propriétés organoleptiques des vins. Une nutrition azotée faible induit la production des anthocyanes dans les cellules de la pellicule de raisin des cépages rouges via des mécanismes de régulation qui ne sont pas encore totalement élucidés. Dans ce contexte, nous avons étudié les mécanismes moléculaires impliqués dans la réponse de l’accumulation des anthocyanes pour différents niveaux d’apports azotés. Deux matériels biologiques complémentaires ont été utilisés : des suspensions cellulaires de vigne (lignée GT3) et des plants de Cabernet-Sauvignon, cultivés au vignoble.L’augmentation de la synthèse d’anthocyanes en réponse à la diminution de la nutrition azotée a été confirmée dans les baies et les cellules de vigne en culture. Les analyses transcriptomiques globales (génome complet) et ciblées (qPCR) ont mis en lumière des modifications de l’expression génique, notamment de gènes liés au métabolisme des flavonoïdes, en réponse à la nutrition azotée. L’expression de nombreux gènes structuraux impliqués dans la voie de biosynthèse des anthocyanes est induite par une faible nutrition azotée. La variation de l’apport azoté influence également de façon coordonnée l’expression des gènes régulateurs positifs (facteurs de transcription de type MYB) et négatifs (protéine de type Lateral organ Boundary Domain (LBD)) des gènes de la biosynthèse des flavonoïdes chez la Vigne. L’expression de gènes liés à la production d’énergie (NADH, NADPH), est également affectée.En parallèle, une approche intégrative a été développée sur les suspensions cellulaires, en combinant des mesures d’activités enzymatiques, des dosages de métabolites primaires et secondaires, avec un modèle de balance de flux (Flux Balance Analysis, FBA). Les cartes de flux obtenues prédisent que la diminution de l’apport azoté entraîne une augmentation des flux métaboliques dans la voie du shikimate et des phénylpropanoïdes ; ainsi qu’une répression de la majorité des flux dans les différentes voies du métabolisme primaire, à l’exception de la voie des pentoses phosphates, dont le flux est maintenu, et de la voie de synthèse de l’amidon qui est accrue. Les résultats obtenus plaident en faveur d’un lien fort entre synthèse des anthocyanes et statut énergétique (ATP, NADPH) des cellules vigne. / Anthocyanins are polyphenol compounds very abundant in most of the plants. In grapevine, they give color to red berries and they improve red wine quality and increase the organoleptic properties of the wine. Low nitrogen supply stimulates anthocyanin production in berry skin cells of red grape varieties through regulation mechanisms that are far from being fully understood. In this context, we worked on the molecular mechanisms involved in anthocyanin biosynthesis response to nitrogen supply. Two complementary biological materials were used: grapevine cell suspensions (GT3 line) that originate from a teinturier cultivar and produce anthocyanins under normal conditions; and red grape berries of cv. Cabernet-Sauvignon cultivated in a commercial vineyard. Increases of anthocyanins synthesis in response to low nitrogen levels were confirmed in the field-grown berries and the cells suspensions. Both comparative global (microarrays) and targeted (qPCR) transcriptomic analysis showed different regulations on the expression of the genes involved in the secondary (especially the anthocyanin) and nitrogen metabolisms. The expression of most structural genes of the anthocyanin biosynthesis pathway was induced by a low nitrogen supply. Nitrogen controls also the expression of the positive (MYB transcription factors) and negative (Lateral organ Boundary Domain family protein LBD39) regulatory genes of the flavonoid pathway in grapevine. Furthermore, some genes improved in energy production (ATP, NADPH) were affected. In parallel, an integrative approach combining enzymatic activities and primary and secondary metabolites measurements with developing a Flux Balance Analysis (FBA) modeling approach was used on cells suspensions GT3. The flux maps deciphered that low nitrogen increases metabolic fluxes in shikimate and phenylpropanoid pathways and represses the majority metabolic fluxes in different pathways of primary metabolism. The two exceptions included the pentose phosphate pathway, which the flux metabolism was maintained, and the starch synthesis pathway, which was enhanced. The results obtained showed a strong link between anthocyanin synthesis and energy status (ATP, NADPH) in the berry cell suspensions.

Le mildiou est une maladie de la vigne affectant les feuilles et les baies et pouvant entraîner des diminutions importantes de la quantité et de la qualité de la vendange. L'agent responsable, Plasmopara viticola, un oomycète biotrophe obligatoire, provoque différentes altérations physiologiques au niveau de la feuille infectée. D'une part, il y a des accumulations anormales d'amidon au niveau des taches d'huile, symptômes caractéristiques de la maladie. D'autre part, l'infection induit une dérégulation des mouvements stomatiques. Les stomates, ouvertures naturelles permettant les échanges gazeux entre la plante et l'atmosphère, restent anormalement ouverts de nuit et ne se ferment plus en réponse à un stress hydrique ou à un traitement à l'ABA. Les deux parties de cette thèse avaient pour objectif de i) expliquer l'origine et le mécanisme de l'accumulation d'amidon et ii) isoler et identifier le facteur responsable de la dérégulation stomatique. Les modifications du métabolisme de l'amidon au niveau des feuilles infectées ont d'abord été étudiées à l'aide d'une analyse transcriptomique afin d'identifier les gènes codant pour des enzymes impliquées dans ce métabolisme dont l'expression était affectée. Après validation de deux gènes de référence, une étude par qRT-PCR a permis de vérifier les changements d'expression de certains de ces gènes au cours de la cinétique de l'infection, puis les activités des enzymes correspondantes ont été déterminées. Ces approches, complétées par des dosages de l'amidon et des sucres solubles ainsi que la mesure de l'activité photosynthétique, ont permis de corréler différents événements avec l'infection. D'après nos résultats, l'accumulation de l'amidon dans les taches d'huile pourrait résulter d'une augmentation de sa synthèse liée à l'augmentation de l'activité AGPase, combinée à une diminution de sa dégradation par modification de l'activité des amylases. Une diminution de la photosynthèse et une augmentation de l'activité invertase traduisent une transition source-puits au niveau des taches d'huile. La deuxième partie de la thèse a porté sur la recherche d'une molécule soluble d'origine végétale ou pathogène, secrétée dans l'apoplasme au cours de l'infection et responsable du dysfonctionnement stomatique. Sur « épidermes isolés », les fluides apoplastiques extraits de feuilles infectées induisent une ouverture stomatique à l'obscurité et contrecarrent la fermeture induite par l'ABA à la lumière, mimant ainsi la dérégulation observée sur feuille entière. Le composé actif serait une protéine assez stable, de taille >50 kDa et modifiée post-traductionellement par une glycosylation essentielle à son activité sur l'ouverture stomatique. Après séparation par chromatographie d'exclusion des fluides apoplastiques de feuilles infectées, deux fractions ont été retenues : une présentant une forte activité sur l'ouverture stomatique, et une seconde sans activité. Neuf protéines de vigne ont été identi fiées spécifiquement dans la fraction active après une analyse comparative de ces deux échantillons par spectrométrie de masse. Des expérimentations supplémentaires seront nécessaires pour purifier et identifier la protéine recherchée. / The grapevine downy mildew affects leaves and young berries and can affect the harvest quality and quantity. The causal agent is the obligate biotroph oomycete Plasmopara viticola that induces severe physiological alterations in infected leaves. One is the abnormal accumulation of starch in oil spots, the characteristic symptoms of the disease. Another is a deregulation of stomatal movements. Stomata, natural openings on the leaf surface allowing gas exchanges between the plant and the atmosphere, stay abnormally open during the night and do no longer close upon water stress or ABA treatment in infected leaves. This thesis is divided into two chapters that aim to i) explain the origin and mechanism of the abnormal starch accumulation and ii) isolate and identify the compound responsible for the stomatal deregulation. The modifications of starch metabolism in infected leaves were first studied by transcriptomic analysis allowing to identify transcriptional modifications of genes involved in starch metabolism. After the validation of two reference genes, a qRT-PCR analysis was performed in order to verify the expression alterations of some of these genes and the corresponding enzymatic activities were measured. The quantification of soluble sugars and starch, and the measurement of photosynthetic activity were included in the analysis and their alterations could be correlated with the infection. Altogether, the obtained results hint towards an increase of starch synthesis via an increase in AGPase activity, that, combined with a decrease of its degradation by modification of amylase activities, could be responsible for the observed starch accumulation in oil spots. Concurrently, a source-sink transition is apparent in infected leaves by decrease of photosynthetic activity and increase of a cell wall invertase activity. The second part of this thesis dealt with a soluble molecule of plant or pathogen origin that is secreted into the apoplast during infection and could be responsible for the stomatal deregulation. The apoplastic wash fluids from infected leaves increase tha stomatal aperture in the dark in grapevine epidermal peels and counteract the ABA-induced stomatal closure, thus mimicking the deregulation observed on whole leaves. The active compound seems to be a stable protein of > 50kDa with a glycosylation that is essentiel for its activity. After separation of the fluids by size exclusion chromatography, two fractions were compared : one active on stomatal aperture and an inactive one. 9 grapevine proteins could be identified in the active sample by mass spectrometry analysis, but further analyses are needed to purify and identify the one responsible for the stomatal deregulation.

Com o avanço no desenvolvimento industrial sustentável estão sendo exploradas novas aplicações de uso dos subprodutos agroindustriais. O bagaço de uva obtido a partir da vinificação ou produção de sucos mantém importantes quantidades de polifenóis em suas cascas, sementes e engaço após o processamento, úteis para proteção da pele contra os raios ultravioleta. Neste contexto, o objetivo do nosso estudo foi caracterizar quimicamente o extrato do bagaço de uva Cabernet Sauvignon e avaliar a sua segurança e eficácia clínica em fotoprotetor tópico. Nesta dissertação, o extrato hidroetanólico (70% v/v) do bagaço de uva total, incluindo cascas, sementes e engaços de Vitis vinifera L., foi obtido por percolação seguido de liofilização. Os ensaios divididos em duas etapas: i) In vitro: determinação dos fenólicos totais (FT) usando Folin-Ciocalteu e expresso em equivalentes de ácido gálico (GAE); flavonoides por meio da reação com tricloreto de alumínio e expresso em quercetina; atividade antioxidante do extrato e das formulações através da desativação de radicais livres de DPPH• expressa em equivalentes de Trolox (TE); identificação dos principais compostos fenólicos do extrato bruto por espectrometria de massa (HPLC/ESI-MS/MS); desenvolvimento de nove formulações usando o delineamento experimental; medição dos valores de transmitância das formulações referentes ao fator de proteção solar (FPS), fotoestabilidade e fator de proteção UVA (FP-UVA) através do equipamento Labsphere UV-2000S® e ii) Em seres humanos, apenas duas formulações foram avaliadas clinicamente, C sem extrato e com filtros UVA e UVB: butilmetoxidibenzoilmetano, etil-hexilmetoxicinamato e etil-hexildimetil PABA (11,5% p/p) e D com extrato (bagaço de uva 10% p/p) e filtros UV. Inicialmente, a segurança dermatológica, irritabilidade e sensibilização; fototoxicidade e fotossensibilização foram avaliadas pelo Teste de Contato Repetitivo em Humanos (HRIPT). Posteriormente, a eficácia fotoprotetora clínica foi testada na pele de dez voluntários. O teor de FT encontrado no extrato de bagaço de uva foi de 141,11 ± 3,38 mg GAE.g-1 e de flavonoides 3,01 ± 0,14 mg QE.g-1. A eficácia antioxidante das soluções metanólicas com extrato e fotoprotetor na concentração de 1 mg.mL-1 em peso seco em pH 5 e 7 foram aproximadamente: 707 µmol TE.g-1 e 520 µmol TE.g-1, respectivamente. Isto significa uma queda em torno de 26,45% da capacidade antioxidante do extrato quando incorporado à formulação. Foram identificadas por espectrometria de massa dezenove substâncias fenólicas, entre elas dímeros e trímeros de procianidinas, di-hidroflavonol, flavonóis e antocianidinas. Os resultados demonstraram efeito sinérgico entre os filtros UV e extrato vegetal no FPS e efeito antagônico com a variação do tempo de irradiação sobre as respostas do FPS, atividade antioxidante, comprimento de onda crítico, proteção UVA, proteção UVB e razão UVA/UVB. A melhor combinação eficiência-resposta foi detectada na formulação D com um aumento do FPS in vitro em cerca de 210%. Os testes de segurança não induziram qualquer reação adversa. A eficácia fotoprotetora clínica de D foi 21% (10,20 - 12,30) superior em relação à formulação C, demonstrando maior eficiência na proteção UVA. Sendo assim, C e D foram consideradas seguras para uso tópico e D estatisticamente superior tanto na proteção UVA quanto UVB comparado à amostra só associada de filtros. Logo, o bagaço de uva é um promissor ingrediente sustentável multifuncional útil para o uso em protetor solar. / With the advance in sustainable industrial development, new applications of agro-industrial by-products are being explored. Grape pomace obtained from winemaking or juice production keeps important amounts of polyphenols in their skins, seeds and stalks after processing that are useful for protection of skin against ultraviolet rays. In this context, the objective of our study was to chemically characterize Cabernet Sauvignon pomace extract and to evaluate its safety and clinical efficacy in topical photoprotector. The hydroethanolic extract (70% v/v) of the Vitis vinifera L. whole grape pomace, including seeds, skins and stalks, was obtained by percolation after lyophilization. The tests were divided into two stages: i) In vitro: total phenols (TF) using Folin-Ciocalteu and expressed in Gallic Acid Equivalent (GAE); flavonoids by reaction with aluminum trichloride and expressed as quercetin; antioxidant activity of the extract and formulations by inhibition by DPPH• expressed as Trolox equivalents (TE); identification of the main phenolic compounds of the crude extract by mass spectrometry (HPLC/ESI-MS/MS); development of nine formulations using experimental design and evaluated the SPF, photostability and UVA protection factor (UVA-PF) through the Labsphere UV-2000S® equipment; ii) In humans, only two formulations were clinically evaluated, C without extract and with UVA and UVB filters: butilmetoxidibenzoilmetano, etil-hexilmetoxicinamato and etil-hexildimetil PABA (11.5% w/w) and D with extract (10% w/w grape pomace) and filters UV. Initially, the dermatological safety, irritability and sensitization; phototoxicity and photosensitization were evaluated by the Human Repeat Insult Patch Test (HRIPT). After, the clinical photoprotective efficacy was tested on the skin of ten volunteers. The FT content found in the dried extract was 141.11 ± 3.38 mg GAE.g-1 and flavonoids 3.01 ± 0.14 mg QE.g-1. The antioxidant potential of the methanolic solutions with extracts and photoprotectors at the concentration of 1 mg.ml-1 in dry weight and at pH 5 and 7 were around: 707 µmol TE.g-1 and 520 µmol TE.g-1, respectively. This means a drop of about 26.45% of the antioxidant capacity of the extract when incorporated into the formulation. Mass spectrometry identified nineteen phenolic substances, among them procyanidins dimers and trimmers, di-hydroflavonol, flavonols and anthocyanin. The results showed a synergistic effect between the UV filters and the herbal extract in the SPF and the antagonistic effect with the variance of the irradiation time in the SPF responses, antioxidant activity, critical wavelength, UVA protection, UVB protection and UVA/UVB ratio. The best efficiency-response combination was detected in the D formulation with an in vitro SPF increase of about 210%. Safety tests did not induce any adverse reaction. The clinical efficiency of D photoprotection was 21% (10.20 - 12.30) higher than the C formulation, demonstrating high efficiency in UVA protection. Thus, C and D were considered safe for topical use and D statistically superior in both UVA and UVB protection to the only combined sample of filters. Therefore, grape pomace is a promising multifunctional sustainable ingredient qualified to be used in sunscreen industry.

Submitted by Eunice Novais (enovais@uepg.br) on 2018-02-07T12:08:16Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Isabela Leticia Pessenti.pdf: 2816974 bytes, checksum: 7ddb844219627cfd4008623f5e811191 (MD5) / Made available in DSpace on 2018-02-07T12:08:17Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Isabela Leticia Pessenti.pdf: 2816974 bytes, checksum: 7ddb844219627cfd4008623f5e811191 (MD5) Previous issue date: 2017-07-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As condições climáticas apresentam forte influência na cultura da videira. O alto índice de precipitação e a baixa amplitude térmica, por exemplo, podem fazer com que as uvas não alcancem índices adequados de maturação. Com o objetivo de verificar o efeito da desfolha, da aplicação do ácido abscisico e de extratos vegetais na qualidade de uvas, dois experimentos foram conduzidos em vinhedo comercial com as cvs. Primitivo e Malbec, respectivamente, em Água Doce–SC. Os tratamentos foram os seguintes para o experimento 1: 1) testemunha (sem tratamento); 2) desfolha manual no início da maturação (estádio “pintor”); 3) desfolha manual 15 dias após a primeira desfolha; 4) ácido abscísico 200 mg L-1; 5) ácido abscísico 400 mg L-1; 6) ácido abscísico 600 mg L-1. Para o experimento 2 foram incluídos dois tratamentos adicionais: 7) extrato de gervão 100 g L-1, 8) extrato de capim limão 100 g L-1. Foram realizadas as seguintes avaliações: massa e diâmetro de bagas, diâmetro e comprimento de ramos, massa de cachos, teor de sólidos solúveis, acidez titulável, relação sólidos solúveis/acidez, pH, teor de antocianinas, polifenóis totais, teor de flavonoides, intensidade de cor, radiação fotossinteticamente ativa, índice de clorofila, índice de desfolha e de clorose, luminosidade, croma, tonalidade, índice de coloração para bagas vermelhas e teor de álcool para o vinho. A desfolha e a aplicação de S-ABA não apresentou efeitos sobre as características químicas dos cachos e bagas nas duas cultivares. Houve maior radiação fotossinteticamente ativa nos tratamentos de desfolha e S-ABA. A aplicação de S-ABA proporcionou clorose nas folhas, seguido de baixo teor de clorofila, ocasionando a senescência, seguido de desfolha. A desfolha e a aplicação de ácido abscísico proporcionaram níveis aumentados de polifenóis totais, antocianinas, flavonoides e intensidade de cor, tanto na uva Primitivo, quanto na Malbec e no vinho. Os extratos vegetais aplicados não influenciaram nas análises físico-químicas, no teor de antocianinas e polifenóis totais, apenas no teor de flavonoides e intensidade de cor na cv Malbec. / Climatic conditions have a strong influence on grapevine cultivation. The high rate of rainfall and low temperature range, for example, can cause the grapes do not reach adequate maturation indexes. In order to check the effect of defoliation, the application of abscisic acid and application of plant extracts on the quality of grapes, two experiments were conducted in commercial vineyard with the cvs. Primitivo and Malbec, respectively, in Água Doce – SC. The treatments were as follows for experiment 1: 1) control (no treatment); 2) manual defoliation in early maturation (‘veraison’); 3) defoliation 15 days after the first manual defoliation; 4) abscisic acid 200 mg L-1; 5) abscisic acid 400 mg L-1; 6) abscisic acid 600 mg L-1. For experiment 2 were included two additional treatments: 7) gervão extract 100 g L-1, 8) extract lemon grass 100 g L-1. The following reviews were carried out: mass and berry diameter, diameter and length of branches, number and mass of branches, soluble solids, titratable acidity, soluble solids/acidity, pH, anthocyanins, total polyphenols, flavonoids, color intensity, photosynthetically active radiation, chlorophyll index, index of defoliation and chlorosis, lightness, chroma, hue, color index for red berries and alcohol content for wine. Defoliation and application of S-ABA showed no effects on the chemical characteristics of the bunches and berries in the two cultivars. There was a higher photosynthetically active radiation treatments of defoliation and S-ABA. The application of S-ABA provided chlorosis in leaves, followed by low chlorophyll content, causing the senescence, followed by defoliation. Defoliation and application of abscisic acid have provided increased levels of total polyphenols, anthocyanins, flavonoids and color intensity, both in Primitivo grape as the Malbec and wine. The plant extracts applied did not influence on the physical and chemical analyses, the anthocyanins and total polyphenols, flavonoids content only and intensity of color in cv Malbec.

Com o avanço no desenvolvimento industrial sustentável estão sendo exploradas novas aplicações de uso dos subprodutos agroindustriais. O bagaço de uva obtido a partir da vinificação ou produção de sucos mantém importantes quantidades de polifenóis em suas cascas, sementes e engaço após o processamento, úteis para proteção da pele contra os raios ultravioleta. Neste contexto, o objetivo do nosso estudo foi caracterizar quimicamente o extrato do bagaço de uva Cabernet Sauvignon e avaliar a sua segurança e eficácia clínica em fotoprotetor tópico. Nesta dissertação, o extrato hidroetanólico (70% v/v) do bagaço de uva total, incluindo cascas, sementes e engaços de Vitis vinifera L., foi obtido por percolação seguido de liofilização. Os ensaios divididos em duas etapas: i) In vitro: determinação dos fenólicos totais (FT) usando Folin-Ciocalteu e expresso em equivalentes de ácido gálico (GAE); flavonoides por meio da reação com tricloreto de alumínio e expresso em quercetina; atividade antioxidante do extrato e das formulações através da desativação de radicais livres de DPPH• expressa em equivalentes de Trolox (TE); identificação dos principais compostos fenólicos do extrato bruto por espectrometria de massa (HPLC/ESI-MS/MS); desenvolvimento de nove formulações usando o delineamento experimental; medição dos valores de transmitância das formulações referentes ao fator de proteção solar (FPS), fotoestabilidade e fator de proteção UVA (FP-UVA) através do equipamento Labsphere UV-2000S® e ii) Em seres humanos, apenas duas formulações foram avaliadas clinicamente, C sem extrato e com filtros UVA e UVB: butilmetoxidibenzoilmetano, etil-hexilmetoxicinamato e etil-hexildimetil PABA (11,5% p/p) e D com extrato (bagaço de uva 10% p/p) e filtros UV. Inicialmente, a segurança dermatológica, irritabilidade e sensibilização; fototoxicidade e fotossensibilização foram avaliadas pelo Teste de Contato Repetitivo em Humanos (HRIPT). Posteriormente, a eficácia fotoprotetora clínica foi testada na pele de dez voluntários. O teor de FT encontrado no extrato de bagaço de uva foi de 141,11 ± 3,38 mg GAE.g-1 e de flavonoides 3,01 ± 0,14 mg QE.g-1. A eficácia antioxidante das soluções metanólicas com extrato e fotoprotetor na concentração de 1 mg.mL-1 em peso seco em pH 5 e 7 foram aproximadamente: 707 µmol TE.g-1 e 520 µmol TE.g-1, respectivamente. Isto significa uma queda em torno de 26,45% da capacidade antioxidante do extrato quando incorporado à formulação. Foram identificadas por espectrometria de massa dezenove substâncias fenólicas, entre elas dímeros e trímeros de procianidinas, di-hidroflavonol, flavonóis e antocianidinas. Os resultados demonstraram efeito sinérgico entre os filtros UV e extrato vegetal no FPS e efeito antagônico com a variação do tempo de irradiação sobre as respostas do FPS, atividade antioxidante, comprimento de onda crítico, proteção UVA, proteção UVB e razão UVA/UVB. A melhor combinação eficiência-resposta foi detectada na formulação D com um aumento do FPS in vitro em cerca de 210%. Os testes de segurança não induziram qualquer reação adversa. A eficácia fotoprotetora clínica de D foi 21% (10,20 - 12,30) superior em relação à formulação C, demonstrando maior eficiência na proteção UVA. Sendo assim, C e D foram consideradas seguras para uso tópico e D estatisticamente superior tanto na proteção UVA quanto UVB comparado à amostra só associada de filtros. Logo, o bagaço de uva é um promissor ingrediente sustentável multifuncional útil para o uso em protetor solar. / With the advance in sustainable industrial development, new applications of agro-industrial by-products are being explored. Grape pomace obtained from winemaking or juice production keeps important amounts of polyphenols in their skins, seeds and stalks after processing that are useful for protection of skin against ultraviolet rays. In this context, the objective of our study was to chemically characterize Cabernet Sauvignon pomace extract and to evaluate its safety and clinical efficacy in topical photoprotector. The hydroethanolic extract (70% v/v) of the Vitis vinifera L. whole grape pomace, including seeds, skins and stalks, was obtained by percolation after lyophilization. The tests were divided into two stages: i) In vitro: total phenols (TF) using Folin-Ciocalteu and expressed in Gallic Acid Equivalent (GAE); flavonoids by reaction with aluminum trichloride and expressed as quercetin; antioxidant activity of the extract and formulations by inhibition by DPPH• expressed as Trolox equivalents (TE); identification of the main phenolic compounds of the crude extract by mass spectrometry (HPLC/ESI-MS/MS); development of nine formulations using experimental design and evaluated the SPF, photostability and UVA protection factor (UVA-PF) through the Labsphere UV-2000S® equipment; ii) In humans, only two formulations were clinically evaluated, C without extract and with UVA and UVB filters: butilmetoxidibenzoilmetano, etil-hexilmetoxicinamato and etil-hexildimetil PABA (11.5% w/w) and D with extract (10% w/w grape pomace) and filters UV. Initially, the dermatological safety, irritability and sensitization; phototoxicity and photosensitization were evaluated by the Human Repeat Insult Patch Test (HRIPT). After, the clinical photoprotective efficacy was tested on the skin of ten volunteers. The FT content found in the dried extract was 141.11 ± 3.38 mg GAE.g-1 and flavonoids 3.01 ± 0.14 mg QE.g-1. The antioxidant potential of the methanolic solutions with extracts and photoprotectors at the concentration of 1 mg.ml-1 in dry weight and at pH 5 and 7 were around: 707 µmol TE.g-1 and 520 µmol TE.g-1, respectively. This means a drop of about 26.45% of the antioxidant capacity of the extract when incorporated into the formulation. Mass spectrometry identified nineteen phenolic substances, among them procyanidins dimers and trimmers, di-hydroflavonol, flavonols and anthocyanin. The results showed a synergistic effect between the UV filters and the herbal extract in the SPF and the antagonistic effect with the variance of the irradiation time in the SPF responses, antioxidant activity, critical wavelength, UVA protection, UVB protection and UVA/UVB ratio. The best efficiency-response combination was detected in the D formulation with an in vitro SPF increase of about 210%. Safety tests did not induce any adverse reaction. The clinical efficiency of D photoprotection was 21% (10.20 - 12.30) higher than the C formulation, demonstrating high efficiency in UVA protection. Thus, C and D were considered safe for topical use and D statistically superior in both UVA and UVB protection to the only combined sample of filters. Therefore, grape pomace is a promising multifunctional sustainable ingredient qualified to be used in sunscreen industry.

Made available in DSpace on 2017-07-10T17:37:44Z (GMT). No. of bitstreams: 1 Tatiana_Werle.pdf: 502997 bytes, checksum: a8ed05a8a76d34c68e008dc33707334a (MD5) Previous issue date: 2011-08-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In an attempt to induce the grapevines buds sprouting in mild winter regions, characterized by mild temperatures during the hibernal season, it becomes necessary the use of sprouting inducers. Considering the grapevines cultivation expansion in Western Paraná, the current study had as its aims to determine the optimal concentration of hydrogenated cyanamide, ethephon and Stimulate® bio-stimulant for the induction and uniformity of sprouting in Niagara Rosada cultivar, as well to indicate the most effective product in the uniformity sprouting inducing and which promotes improved productivity and fruits quality. The experiment was conducted in a commercial vineyard located in the municipality of Quatro Pontes, West of Paraná State. The experiment was carried out in outlined blocks in factorial scheme 3 X 5, totaling 15 treatments with four repetitions, and each plant was considered an experimental unit. The treatments consisted of the following concentrations: 0; 10 mL L-1; 20 mL L-1; 30 mL L-1 and 40 mL L-1 of hydrogenated cyanamide (H2CN2); 0 ; 2 L ha-1; 4 L ha-1; 6 L ha-1 and 8 L ha-1 of ethephon; 0; 5 mL L-1; 10 mL L-1; 15 mL L-1 and 20 mL L-1 Stimulate® bio-stimulant. The products application was made immediately after pruning, by spraying with backpack sprayer. After pruning, it was assessed the number of buds per plant, time to sprouting onset after application, number and percentage of sprouting per plant, production per plant, expected productivity, average weight of berries, number of berries per cluster, width of shoulder and berries, berries length, soluble solids content, titration total acidity, pH and ratio. Considering the conditions under which the experiment was conducted, one can conclude that: The concentration of hydrogenated cyanamide which showed higher percentage of 'Niagara Rosada' grapevine sprouted buds was the 20mL L-1; the ethephon concentration which provided the highest percentage of Niagara Rosada grapevine sprouted buds was the 8 L ha-1; the bio-stimulant concentration which provided the highest percentage of Niagara Rosada grapevine sprouted buds was the 5 mL L-1; the hydrogenated cyanamide provided the anticipation and uniformity of the Niagara Rosada grapevine sprouting. The buds spraying with hydrogenated cyanamide, ethephon and bio-stimulant resulted in no effect on the grapevine productivity and quality, cv. Niagara Rosada / Na tentativa de induzir a brotação das gemas de videiras em regiões de inverno ameno, caracterizado por temperaturas suaves durante a estação hibernal, torna-se necessário o emprego de indutores de brotação. Considerando a expansão do cultivo de videiras no Oeste do Paraná, o presente trabalho teve como objetivos verificar a concentração ideal de cianamida hidrogenada, ethephon e do bioestimulante Stimulate® para a indução e uniformização de brotação na cultivar Niagara Rosada, bem como indicar o produto mais eficiente na indução de uniformização de brotação e que promove melhor produtividade e qualidade de frutos. O experimento foi conduzido em vinhedo comercial localizado no município de Quatro Pontes, Oeste do Estado do Paraná. O experimento foi implantado em delineamento de blocos em esquema fatorial 3 X 5, totalizando 15 tratamentos com quatro repetições, sendo que cada planta foi considerada uma parcela experimental. Os tratamentos foram compostos pelas seguintes concentrações: 0; 10 mL L-1; 20 mL L-1; 30 mL L-1 e 40 mL L-1 de cianamida hidrogenada (H2CN2); 0; 2 L ha-1; 4 L ha-1; 6 L ha-1 e 8 L ha-1 de ethephon; 0; 5 mL L-1; 10 mL L-1; 15 mL L-1 e 20 mL L-1 de bioestimulante Stimulate®. A aplicação dos produtos foi feita imediatamente após a poda, através de pulverização com pulverizador costal. Após a poda avaliou-se o número de gemas por planta, tempo para início da brotação após a aplicação, número e porcentagem de brotações por planta, produção por planta, produtividade esperada, massa média das bagas, número de bagas por cacho, largura do ombro e das bagas, comprimento das bagas, teor de sólidos solúveis, acidez total titulável, pH e ratio. Considerando as condições em que foi conduzido o experimento pode-se concluir que: A concentração de cianamida hidrogenada que proporcionou maior porcentagem de gemas brotadas de videira Niagara Rosada foi a de 20 mL L-1; A concentração de ethephon que proporcionou maior porcentagem de gemas brotadas de videira Niagara Rosada foi a de 8 L ha-1; A concentração de bioestimulante que proporcionou maior porcentagem de gemas brotadas de videira Niagara Rosada foi a de 5 mL L-1; A cianamida hidrogenada proporcionou a antecipação e uniformização das brotações de videira Niagara Rosada. A pulverização das gemas com cianamida hidrogenada, ethephon e bioestimulante não resultou em efeito sobre a produtividade e qualidade de videira, cv. Niagara Rosada

Vitis vinifera L. représente l’une des cultures les plus répandues dans les pays producteurs de vin, laquelle est soumise à de nombreuses contraintes environnementales pouvant favoriser l’émergence des maladies de dépérissement du bois (MDB). La famille des Botryosphaeriaceae est responsable du Botryosphaeria dieback, provoquant des chancres et des nécroses qui conduisent à une dépréciation de la qualité du vin, voire à la mort des ceps. Les méthodes de lutte sont peu efficaces. Sept espèces sont retrouvées dans le vignoble français dont B. dothidea, D. intermedia, D. mutila, D. seriata, Do. viticola, N. parvum et L. viticola. Nous avons étudié différents traits d’histoire de vie de ces agents pathogènes : (i) leur agressivité in planta, (ii) leur adaptation à des contraintes environnementales (e.g. température, fongicides), (iii) la présence de mycovirus, pour acquérir des connaissances sur leur pouvoir adaptatif face aux contraintes environnementales et expliquer la variabilité de leur agressivité. En complément, l’évaluation de la sensibilité de cultivars de Vitis face à une infection par N. parvum et D. seriata a été réalisée, et le potentiel de défenses de différents cépages (Ugni Blanc, Cabernet-Sauvignon et Merlot) a été étudié. L’ensemble des travaux menés ont permis de révéler des espèces très agressives telles que N. parvum et L. viticola par rapport à D. seriata, lors d’inoculations en serre sur des boutures. Les températures optimales de croissance déterminées montrent que certaines espèces (ex. Lasidiplodia spp.) sont mieux adaptées à des températures élevées (33°C). Par ailleurs, la sensibilité de 65 souches et génotypes a été testée pour 9 fongicides avec des modes d’action différents (inhibiteurs de la respiration mitochondriale, de la biosynthèse des stérols, du cytosquelette, multi-sites, etc.). De nombreuses espèces sont peu ou pas sensibles à certains de ces fongicides et des souches résistantes ont été trouvées avec un facteur de résistance pouvant atteindre plus de 1000. D’autre part, la détection de mycovirus au sein des 65 isolats a permis d’identifier la présence de 6 mycovirus, dont Neofusicoccum luteum mitovirus 1 et Neofusicoccum luteum fusavirus 1. L’évaluation du potentiel de défense des trois cultivars Vitis face à une infection par N. parvum et D. seriata a montré des réponses différentes entre cépages et en fonction de l’agent pathogène. In fine, des analyses croisant les différents traits d’histoire de vie et les interactions plante-pathogènes ont été faites, et l’ensemble des résultats nous a fourni de nouvelles pistes d’étude pour lutter contre ces agents pathogènes. / Vitis vinifera L. is largely cultivated in countries producing wine but an increase in grapevine trunk diseases (GTDs) have been observed due to the attack of several fungal pathogens including those belonging to the Botryosphaeriaceae family (Botryosphaeria dieback). Seven species were isolated in the French vineyards B. dothidea, D. intermedia, D. mutila, D. seriata, Do. viticola, N. parvum and L. viticola. Nowadays, no efficient products are available to control these diseases. Studying, different life traits of 65 strains of different genotypes of the Botryosphaeriaceae’s family, known to have members displaying different aggressiveness, would lead to a better understanding of these wood pathogens. Their in planta aggressiveness, their adaptation towards environmental pressures (temperature, fungicides), and the detection of mycoviruses were carried out. In order to have a better comprehension of the interaction within the plant, the expression analysis of genes involved in the plant defense were assayed upon 3 cultivars (Cabernet Sauvignon, Merlot, and Ugni-Blanc) but also on two species (N. parvum and D. seriata). L. viticola, and N. parvum, were shown to be more aggressive than D. seriata and. L. viticola is more adapted to higher temperature (33°C). Moreover, the strains tested with 9 different fungicides (mitochondrial respiratory, sterol biosynthesis, cytoskeleton or multisite inhibitors, etc.) showed lower sensitivity within some species, (with a resistance factor reaching a 1000). In addition, at least 6 mycoviruses were characterized. Amongst them, two mycoviruses were isolated from a N. luteum strain and were fully sequenced (Neofusicoccum luteum mitovirus 1 and Neofusicoccum luteum fusavirus 1). The three cultivars infected with either N. parvum or D. seriata showed different gene responses between themselves but also between the different strains inoculated. These different studies are giving us more information upon these Botryospheriaceae fungi, to find out new efficient or complementary methods in order to control GTDs