Submitted by Helena Bejio (helena.bejio@unioeste.br) on 2018-06-08T11:39:58Z No. of bitstreams: 2 Solivan_Rosanelli_2018.pdf: 719096 bytes, checksum: 0cb5b8474f702cc149a662dcf02b832a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-08T11:39:58Z (GMT). No. of bitstreams: 2 Solivan_Rosanelli_2018.pdf: 719096 bytes, checksum: 0cb5b8474f702cc149a662dcf02b832a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The objective of this work was to evaluate the agronomic behavior of the Niagara Rosada grape vine variety, which was cultivated under plastic cover with different defoliation intensities, and its influence on the Plasmopara viticola fungus. The experiment was carried out in Francisco Beltrão (PR), in a vineyard of Niagara Rosada variety (Vitis labrusca L.) conducted in a trellised system during the 2016/2017 and 2017/2018 harvests. The experimental design was randomized blocks, in a 5 x 2 factorial scheme (0, 10, 20, 30 and 40% of defoliation x with and without cover) with three blocks of three plants per experimental portion. The temperature, air humidity and the incidence of mildew on the curls and leaves were registered. When ripe, the clusters were harvested, duly labeled and transported to the Unioeste post-harvest laboratory, where three clusters per plant of each portion were evaluated, thus determining the fresh biomass, length and width of the clusters, soluble solids content, pH, number of clusters per plant, production and productivity. It was concluded that the plastic cover consorted to the environmental conditions and the phytosanitary management can favor the physical-chemical quality and reduce the severity of mildew in the Niagara Rosada grapes, thus providing greater production and productivity. The use of defoliation does not favor the cultivation of Niagara Rosada in the southwestern region of Paraná. / Objetivou-se com o presente trabalho avaliar o comportamento agronômico de videira Niagara Rosada cultivada sob cobertura plástica, utilizando intensidades de desfolha, e sua influência sobre o fungo Plasmopara viticola. O experimento foi realizado em Francisco Beltrão (PR), num vinhedo de cv. Niagara Rosada (Vitis labrusca L.) conduzido em sistema latada durante as safras 2016/2017 e 2017/2018. O delineamento experimental foi blocos casualizados, em esquema fatorial 5 x 2 (0, 10, 20, 30 e 40% de desfolha x com e sem cobertura) com três blocos de três plantas por parcela experimental. Foram registrados a temperatura, umidade do ar e a incidência de míldio nos cachos e folhas. Quando maduros os cachos foram colhidos, devidamente etiquetados, e transportados até o laboratório de póscolheita da Unioeste, onde foram avaliados três cachos por planta de cada parcela para a determinação da biomassa fresca dos cachos, comprimento e largura dos cachos, teor de sólidos solúveis, pH, número de cachos por planta, produção e produtividade. Conclui-se que a cobertura plástica consorciada às condições ambientais e ao manejo fitossanitário podem favorecer a qualidade físico-química e reduzir a severidade do míldio em uva Niagara Rosada; propiciando maior produção e produtividade. A utilização de desfolha não favorece o cultivo da Niagara Rosada no sudoeste paranaense.

La réduction des effets secondaires des produits phytosanitaires sur l’environnement est un enjeux majeur du Grenelle de l’environnement. L’utilisation prolongée du cuivre comme fongicide a entrainé son accumulation dans les sols viticoles. Le cuivre étant phytotoxique,son accumulation dans la plante pourrait modifier la morphologie, la biochimie ou la physiologie des organes végétaux et perturber les interactions vigne-bio-agresseur. Nousavons donc évalué les effets de la contamination cuprique des sols viticoles sur la sensibilitéde la vigne à un cortège de bio-agresseurs par une approche plus intégrée de la culture et deson patho-système.Pour cela, nous avons mesuré les teneurs en cuivre total et disponible des parcelles viticoles.Nous avons exposé au cuivre trois cépages puis nous avons mesuré la distribution du cuivre et ses effets dans la plante. Enfin nous avons évalué l’impact induit pour trois bio-agresseurs de la vigne. Nos résultats montrent que la teneur en cuivre disponible du sol dépend surtout de la teneur total en cuivre (qui résulte de l’historique parcellaire) mais aussi des teneurs en carbone total et en particules fines du sol. L’absorption du cuivre et son transfert vers les parties aériennes varient selon les cépages. Lorsque la contamination cuprique augmente, les interactions vigne-bio-agresseurs sont modifiées : si les effets semblent négatifs pourScaphoideus titanus et Plasmopara viticola, la contamination cuprique semble favorable au développement de Neofusicoccum Parvum. Ces résultats soulignent la nécessité d’avoir une vision la plus intégrée possible du système, et d’intensifier les interactions de recherches entre l’agronomie et l’écologie évolutive. / Following the ‘Grenelle of the environment’ conference, one of the current critical issues in agriculture is reducing the side effects of pesticides on the environment. Prolonged use of copper as a fungicide has led to copper accumulation in vineyard soils. Copper can induce aphytotoxicity. Its accumulation in plants may modify the morphology, biochemistry orphysiology of plant organs and may disturb the plant-pest interactions. The effects of coppercontamination of vineyard soils on the vine sensitivity to the pests received until now a verylimited attention. The present work investigates such effects.To carry out this work, we measured the levels of total and available copper in vineyard soils.We contaminated three grapevine cultivars with copper and measured the copper distributionand its effects on plant. Then, we evaluated the impact induced on three pests of the vine. Ourresults show that the available copper content in the soil is related to the total copper content(which results from the history of the plot), but also to total carbon and fine soil particlescontents. The copper absorption and the copper transferred to the aerial parts of the vine varyaccording to the cultivar. When copper contamination increases, the vine-pests interactionsare modified: a negative effects appears for Scaphoideus titanus and Plasmopara viticola,while important copper contaminations appears to promote the development ofNeofusicoccum parvum.These results underscore the need for a more integrated system approach, and to intensifyinteractions between agronomy and evolutionary ecology research.

La vigne est soumise à la pression de nombreux bioagresseurs dont des parasites obligatoires tels que l’oïdium et le mildiou. La lutte contre les maladies causées par les pathogènes biotrophes nécessite une utilisation souvent intensive de fongicides. Le vignoble consomme à lui seul 16% des fongicides commercialisés chaque année en France. Pour réduire leur impact environnemental qui conduit à l’acquisition de la résistance aux pesticides des pathogène et la présence de résidus dans les vins et dans l’atmosphère, des efforts doivent être entrepris pour développer des stratégies de protection innovante de remplacement ou complémentaire permettant de réduire les intrants pesticides.Les stimulateurs des défenses des plantes permettent de limiter le développement des bioagresseurs en conditions contrôlées. Toutefois, leurs efficacités in natura sont variables et souvent décevantes. Suite au grand nombre de produits potentiellement stimulateurs des défenses des plantes, et à l’intérêt que leur portent les viticulteurs, il est nécessaire de disposer de connaissances et d’outils qui permettent d’évaluer leus efficacités et mieux connaitre leurs potentiels de protection du vignoble. Pour ce faire, une méthode d’évaluation de l’efficacité de produits potentialisateurs ou éliciteurs a été développée au niveau biologique, moléculaire (expression de gènes impliqués dans les défenses) et biochimique (analyses qualitatives et quantitatives des polyphénols), nommée "BioMolChem". Cette méthode a permis d’évaluer l’efficacité de deux phosphonates et d’un analogue de l’acide salicylique, sur différents génotypes et phénotypes de mildiou de la vigne et d’oïdium. Cette approche méthodologique "BioMolChem" a permis d’établir des corrélations entre l’expression de gènes de défense, la présence de certains stilbènes et une efficacité des défenses de Vitis vinifera cv. Cabernet-Sauvignon vis-à-vis de l’oïdium et du mildiou. Les modifications des patrons d’expression des 19 gènes suivis dans les feuilles de vigne et les profils HPLC de polyphénols révèlent des mécanismes de défense multigéniques et complexes. Ainsi, les réactions de défense de la plante sont-elles modulées, en fonction de l’éliciteur considéré, mais aussi en fonction de la diversité phénotypique et génétique des agents pathogènes contre lesquels elle se défend. Ces défenses se caractérisent par une sur-expression d’un ensemble de gènes de défense et une accumulation de composés phénoliques spécifiques.Les marqueurs (gènes et molécules) ainsi identifiés, la méthode "BioMolChem" a été appliquée in natura et a conforté, pour partie, les résultats obtenus au laboratoire. Dans des conditions de fortes pressions parasitaires, il est donc possible de protéger les feuilles et les grappes, à l’aide de SDP et des essais d’association ou d’alternance avec des fongicides conventionnels montrent l’intérêt potentiel de l’emploi des SDP au vignoble. Chemin faisant, dans le cadre d’une viticulture innovante et durable, les SDP et la méthode "BioMolChem" ont été appliqués sur des génotypes hybrides (Vitis vinifera x Muscadinia rotundifolia). Nous révélons que selon le niveau de résistance intrinsèque des génotypes (plus ou moins résistants à l’oïdium et au mildiou), il est possible d’augmenter le niveau de la résistance exprimée par élicitation. Ainsi, les SDP pourraient-ils s’avérer des alliés d’intérêt pour l’utilisation de variétés partiellement résistantes et limiter potentiellement le contournement des QTL de résistance. L’ensemble de ce travail, à but appliqué, a conduit à l’obtention de résultats qui nous permettent de mieux comprendre comment la vigne réagit aux SDP dans son environnement agronomique. Leur exploitation et leur finalisation devraient nous permettre d’exploiter et de mettre en place une utilisation des éliciteurs mieux adaptée, à des stratégies alternatives ou complémentaires de la gestion des bioagresseurs de la vigne. / Powdery (Erysiphe necator) and downy mildew (Plasmopara viticola) are very important grapevine diseases (Vitis vinifera). These two biotrophic pathogens, which are native to the United States, infect green vine tissues and cause significant economic loss as well as environmental damage through the repetitive applications of fungicides. To reduce their environmental impact efforts should be made to develop strategies to protect innovative alternative or complementary to reduce pesticide inputs.In this study, the efficacy and the role of Benzothiadiazole (BTH), a salicylic acid analogue, and two phosphonate derivatives strengthen plant defence mechanisms against various isolates of downy and powdery mildews (Plasmopara viticola and Erysiphe necator). These compounds showed differences in their efficacy depending on the variability of mildews and highly dependent on plant genetics, environmental conditions and selection pressure. The plant defense stimulation could be an alternative or additional method to traditional pest management in the grapevine.Tools “BioMolChem” were developed to better assess the defence status of the plant defences in vitro and in natura. Transcript kinetics of selected defence-related genes and polyphenol contents profiles, during Vitis vinifera-biotrophic pathogen interaction, were characterized, and the impact of pathogen diversity was investigated in the absence or presence of elicitation. In vineyard, under strong pathogen pressures, it is thus possible to protect leaves and clusters, with SDP and assays of association or alternation with conventional fungicides show the potential interest of the use of these SDP in the vineyard.The grapevine defense mechanisms are complex, depending on the elicitor, leading to the coordinated accumulation of pathogenesis-related proteins (PR), the production of phytoalexins, and the reinforcement of plant cell walls.On the way, within the framework of an innovative and sustainable viticulture, the SDP was applied to hybrid genotypes (V. vinifera x M. rotundifolia). We reveal that according to the level of intrinsic resistance of the genotypes (more or less resistant to powdery and to downy mildew), it is possible to increase the level of the expressed resistance. The SDP could become allies of interest in the use of partially resistant grapevine varieties.The present findings provide insights into the potential use of transcripts and stilbenes as markers of the defense status of grapevine leaves with or without elicitation or infection, which should allow us to exploit and develop a better use of elicitors in alternative or complementary strategies in grapevine pest management.

La presente tesi intende esplorare gli effetti della resistenza parziale sulle epidemie di peronospora della vite (Plasmopara viticola). È stato sviluppato un modello di simulazione teorico che comprende lo sviluppo della pianta ospite e le fasi principali della malattia, dalla mobilizzazione dell’inoculo, alla moltiplicazione della malattia sulle foglie, all’infezione dei grappoli. Attraverso esperimenti (monociclici) di inoculazione è stata studiata la risposta alle infezioni di P. Viticola di 16 varietà parzialmente resistenti, analizzando le seguenti componenti: frequenza d’infezione, durata del periodo di latenza, dimensione delle lesioni, produzione di sporangi, durata del periodo infezioso e infettività degli sporangi prodotti sulle lesioni. Queste componenti di resistenza sono state incorporate nel modello, attraverso cui sono stati studiati i loro effetti sull’epidemia (policiclica) in diversi scenari. Le componenti di resistenza hanno mostrato diversi livelli di efficacia nel sopprimere l’epidemia: l’efficienza di infezione e la produzione di sporangi risultano avere un maggiore impatto nella resistenza espressa a livello di pieno campo. Questo approccio è utile per guidare lo studio fenotipico della resistenza dell’ospite e per anticipare le prestazioni di un genotipo a livello di pieno campo, che risulterebbe difficile e dispendioso considerando la natura perenne della vite. / The present dissertation aims to explore the effects of partial resistance on grapevine downy mildew (Plasmopara viticola) epidemics. A theoretical simulation model was developed including host dynamics and main phases of the disease, from inoculum mobilisation to disease multiplication on foliage, and to infection of clusters. The response to P. Viticola infection was studied for 16 grapevine varieties through (monocyclic) inoculation experiments, by measuring components of partial resistance: infection frequency, duration of latent period, size of lesions, production of sporangia, duration of infectious period, and infectivity of sporangia produced on lesion. Components of partial resistance were incorporated into the model and their effects on the (polycyclic) epidemic were investigated accross different scenarios. Components of partial resistance showed different effectiveness on the suppression of epidemics, infection efficiency and spore production having the strongest impact on the overall field resistance response. This approach is an useful tool for phenotyping studies on host plant resistance and for anticipating the performance of a genotype at the field scale, that otherwise is difficult and time requiring due to the perennial nature of grapevine.

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.

La vite è in grado di attivare meccanismi di difesa in risposta a stress biotici e abiotici producendo principalmente metaboliti secondari tra i quali composti stilbenici. Questi composti, detti anche fitoalessine, hanno una azione benefica e riconosciuta sulla salute umana. Una delle malattie che apporta più danni in tutto il mondo viticolo è la peronospora, causata dal patogeno Plasmopara viticola L, questa tesi ha lo scopo di confrontare le risposte di diverse varietà e cloni attaccate da questo patogeno. Sono state utilizzate foglie e bacche di 10 cloni di Cabernet Sauvignon per verificare l’andamento dell’infezione e la produzione degli stilbeni nel tempo. Stessa procedura è stata applicata su foglie giovani e vecchie delle varietà di Müller-Thurgau, Chardonnay, Sangiovese, Pinot noir, Cabernet Sauvignon e Solaris. Queste sono state monitorate per verificare l’andamento dell’infezione, la produzione degli stilbeni e l’attivazione della stilbene sintasi. I risultati delle prove condotte hanno evidenziato differenze tra le varietà e tra i cloni e, soprattutto, tra i diversi organi studiati. / The grapevine is able to activate a defence mechanism whenever it interacts with biotic or abiotic stress, mainly by producing defence substances as the stilbenic compounds, phytoalexins known for their health related influence. Combining this fact and the knowledge that the Plasmopara viticola Berk et Curtis, the causal agent of downy mildew, causes considerable damages worldwide to Vitis vinifera L. production during its life cycle, this thesis aims to explore the differences that several grapevine varieties and clones display when confronted with this pathogen. The leaves and small berries of 10 different clones of Cabernet Sauvignon were used to asses infection time course and stilbenic production overtime. Likewise, young and old leaves of the varieties Müller-Thurgau, Chardonnay, Sangiovese, Pinot noir, Cabernet Sauvignon and Solaris, ordered in assumed increasing resistance, were used to assess infection time course, stilbene production on the leaves and the activation of a stilbene synthase gene. All trials conducted highlighted not only the differences within grapevine varieties and grapevine clones but most importantly between the studied grapevine organs.

Downy mildew, caused by the biotrophic Oomycete Plasmopara viticola, is one of the most important diseases of grapevines world wide. It is particularly destructive in temperate viticultural regions that experience warm wet conditions during the vegetative growth of the vine (Wong et al., 2001). The disease is not normally a problem in mediterranean climates where the growing season tends to be hot and dry (Mullins et al., 1992; Sivasithamparam, 1993). Grape downy mildew is however a major disease in Australian viticulture (McLean et al., 1984; Magarey et al., 1991). Grape downy mildew was first reported in Europe in 1878 (Viennot-Bourgin, 1981). In Australia, it was recorded for the first time in 1917 at Rutherglen in Victoria (Vic) (de Castella, 1917). The first recorded outbreak of the disease in Western Australia (WA) occurred in 1997 in a small planting of vines in the far north of the state. In the subsequent year, it was detected in widespread commercial viticulture in the Swan Valley production area, near Perth (McKirdy et al., 1999). The pathogen has since been found in all grape growing regions of WA. Since its introduction into European vineyards in the 1880?s, P. viticola has become one of the world?s most investigated grapevine pathogens. Many aspects its basic biology however remain unknown (Wong et al., 2001). Due to the recent detection of P. viticola in WA, little is known of the nature of strains of the pathogen in the state and their response to local environmental conditions. Much of the research concerning the influence of environmental factors on the development of P. viticola has been conducted in Europe e.g. parts of France and Germany. Due to significant differences in climatic conditions and a shorter selection time on the pathogen in WA, much of the information described in European studies may not be directly applicable to the grape downy mildew disease situation in WA. The focus of this thesis was to examine epidemiological aspects of P. viticola in the mediterranean climate of WA. The environmental conditions that could favour the development of epidemics by strains of the pathogen that have been detected in the state were determined. The existence of P. viticola ecotypes and genetic variation among strains from WA and the Eastern states of Australia was also investigated.

This study was undertaken to determine the sensitivity of grape downy mildew (DM, Plasmopara viticola) and powdery mildew (PM, Erysiphe necator) to commonly used single-site fungicides in Virginia and nearby states. DM and PM isolates were collected from 2005 to 2007. In grape leaf disc bioassays, 92% of the DM isolates were QoI (azoxystrobin)-resistant, but none were resistant to mefenoxam. Eighty-two percent of the PM isolates were QoI-resistant, but none were resistant to boscalid and quinoxyfen. The frequency of the G143A point mutation, which confers high levels of QoI resistance, was quantified in DM and PM isolates by real-time PCR. Most of the QoI-resistant DM and PM isolates contained >95% of the 143A allele. QoI-sensitive DM isolates contained less than 1% of 143A. One out of 145 and 14 out of 154 QoI-resistant DM and PM isolates (able to grow on azoxystrobin concentration ï ³ 1 µg/ml), respectively, contained less than 1% 143A. Most PM isolates exhibited reduced sensitivity to five DMI fungicides when compared to a sensitive subgroup (n=9) and compared to published reports for unexposed populations; the resistance factor (median EC50 of the entire isolate collection divided by median EC50 of sensitive subgroup) was highest for tebuconazole (360) and myclobutanil (350), followed by triflumizole (79), triadimefon (61), and fenarimol (53). Sensitivities to all five DMI fungicides, but also azoxystrobin, were moderately to strongly correlated (pairwise r-values ranging from 0.60 to 0.88). / Master of Science in Life Sciences

Development of fungicide resistance in fungal and oomycete pathogens is a serious problem in grape production. Quinoxyfen is a fungicide widely used against grape powdery mildew (Erysiphe necator). In 2013, E. necator isolates with reduced quinoxyfen sensitivity (designated as quinoxyfen lab resistance or QLR) were detected in Virginia. Field trials were conducted in 2014, 2015, and 2016 at the affected vineyard to determine to what extent quinoxyfen might still contribute to disease control. Powdery mildew control by quinoxyfen was good, similar to, or only slightly less, than that provided by myclobutanil and boscalid in all three years. The frequency of QLR in vines not treated with quinoxyfen declined only slowly over the three years, from 65% to 46%. Information about the mode of action of quinoxyfen is limited; previous research suggests that quinoxyfen interferes with the signal transduction process. We profiled the transcriptomes of QLR and sensitive isolates in response to quinoxyfen treatment, providing support for this hypothesis. Additional transcriptional targets of quinoxyfen were revealed to be involved in the positive regulation of the MAPK signaling cascade, pathogenesis, and sporulation activity. Grape downy mildew (Plasmopara viticola), another important grape pathogen, is commonly controlled by phosphite fungicides. A field trial and laboratory bioassays were conducted to determine whether P. viticola isolates from vineyards with suspected control failures showed reduced sensitivity against phosphite fungicides. Prophyt applied at 14-day intervals under high disease pressure provided poor downy mildew control in the field. Next-generation sequencing technologies were utilized to identify 391,930 single nucleotide polymorphisms (SNPs) and generated a draft P. viticola genome assembly at ~130 megabase (Mb). Finally, field isolates of P. viticola collected from a Virginia vineyard with suspected mandipropamid control failure were bioassayed. The EC50 values of the isolates were >240 μg.ml-1 for mandipropamid, well above the field rate. The PvCesA3 gene of two resistant isolates was sequenced revealing that these isolates had a GGC-to-AGC substitution at codon 1105, the same mutation that has been found associated with CAA resistance elsewhere. / PHD / Powdery and downy mildew are two diseases of grapes that can cause large yield losses, and are usually controlled by regular fungicide applications. Development of fungicide resistance has been a growing challenge. Quinoxyfen is a protectant fungicide commonly used against powdery mildews. Unusual grape powdery mildew isolates that grew well on quinoxyfen-treated plants in the laboratory (designated as quinoxyfen lab resistance or QLR) were detected in a Virginia vineyard. In 2014, the first year of this study, 65% of powdery mildew isolates from parts of this vineyard that received no further quinoxyfen treatments had the QLR type of resistance, and this declined only slowly to 46% by the third year. Field trials were conducted in 2014, 2015, and 2016 to determine the efficacy of quinoxyfen in the presence of QLR. Powdery mildew control by quinoxyfen on both grape clusters and leaves was similar to, or only slightly less, than that provided by the standard anti-powdery mildew fungicides myclobutanil and boscalid in all three years. In order to gain a better understanding of the mode(s) of action and resistance mechanism(s) of quinoxyfen, gene expression of QLR and sensitive isolates, both in the presence and absence of quinoxyfen, was analyzed by nucleic acid sequencing. This study confirms previous research suggesting that quinoxyfen interferes with the important biological process signal transduction, and revealed additional gene targets of quinoxyfen. The phosphites are a group of fungicides commonly used to control grape downy mildew. Control failures after phosphite application have occasionally been suspected, and downy mildew isolates from vineyards with and without suspected control failures were tested in laboratory bioassays to determine if any level of resistance could be demonstrated. There was a limited range of sensitivity, and none of the isolates showed a notable loss of sensitivity. A field trial was conducted to determine the efficacy of one phosphite fungicide, Prophyt, applied at 14-day intervals under conditions favorable for disease development. Prophyt provided poor downy mildew control, suggesting that it has to be applied more frequently. Next-generation sequencing technologies were utilized to identify genetic markers for clade identification and generated a draft genome assembly of grape downy mildew, which improves the understanding of grape downy mildew genome. Grape downy mildew isolates collected from a vineyard in Virginia where mandipropamid provided poor control of downy mildew were bioassayed. The isolates tolerated mandipropamid rates well above the field rate, showing that they were indeed resistant. The mutation that confers mandipropamid resistance on other continents was found in the PvCesA3 gene of two resistant isolates.

Maîtriser l’impact des maladies sur les cultures est un défi majeur de l’agriculture moderne. Cette préoccupation est un aspect important de l’optimisation de la productivité, notamment en viticulture. En France, le mildiou de la vigne causé par Plasmopara viticola est une des maladies cryptogamiques responsable des épidémies les plus dévastatrices et les plus redoutées. Les traitements reposent sur l’utilisation préventive, systématique et onéreuse de composés chimiques antifongiques dont l’utilisation massive constitue un risque à la fois pour l’homme et l’environnement. La réduction de l’utilisation de fongicide implique le développement d’outils de diagnostic au champ, qui requiert la compréhension des interactions entre la plante et les agents pathogènes. Les travaux de cette thèse pluridisciplinaire ont porté sur le pathosystème Plasmopara viticola - Vitis vinifera, notamment pour répondre à l’intérêt croissant pour un outil de diagnostic en temps réel de la maladie utilisable au vignoble. Les stilbènes sont des phytoalexines impliqués dans la défense de certaines plantes supérieures vis-à-vis de stress biotiques et abiotiques. L’autofluorescence de ces composés phénoliques, dont la biosynthèse est induite dans les feuilles de vigne par P. viticola, en fait un potentiel marqueur naturel de l’infection. En effet, la faible autofluorescence bleu-verte des feuilles de vigne saines est considérablement renforcée par l’autofluorescence violet-bleue des stilbènes à la surface de feuilles de vigne infectée par P. viticola. Cette étude a montré que quelque soit le niveau de résistance du génotype, l’autofluorescence violet-bleue des stilbènes induit par l’infection est présente au niveau des parois des cellules de l’épiderme. En dehors de la concentration, la viscosité s’est révélé être la principale variable physico-chimique influençant l’intensité de l’autofluorescence des stilbènes dans les différents compartiments cellulaires des feuilles de vigne. Ceci explique la fluorescence intense des parois, particulièrement rigides, des cellules de garde (stomates) des feuilles infectées. Le suivi cinétique journalier a révélé la nature transitoire de l’autofluorescence des stilbènes lors de l’infection. La robustesse et l’intérêt de ce signal a également été validée par la mesure à différentes échelles (de la cellule à la feuille entière) et avec différentes méthodes fluorimétriques. Les résultats de ce travail ont permis des avancées sur la connaissance du rôle de composés phénoliques induits et constitutifs dans la défense contre P. viticola. En plus de la localisation de l’autofluorescence des stilbènes en surface des feuilles, la microscopie confocale couplée à la microspectrofluorimetrie a révélé différentes localisations de ces phytoalexines dans la profondeur des tissus en corrélation avec le niveau de résistance des génotypes. L’utilisation de l’autofluorescence des stilbènes comme marqueur de l’infection a permis de mettre en évidence : 1) le fait que les flavonols constitutifs des feuilles de V. vinifera retardent le développement de l’infection par P. viticola; et 2) le fait que les acides hydroxycinnamiques constitutifs ne semble pas participer à la défense contre P. viticola. Enfin, une nouvelle méthode de diagnostic non-destructive du mildiou sur feuille basée sur l’autofluorescence des stilbènes a été développée. Elle a montré une détection pré-symptomatique du mildiou sur les feuilles de vigne entières dès le premier jour après l’infection sur la face abaxiale et le troisième jour sur la face adaxiale. Cette méthode de diagnostic du mildiou a été validée au laboratoire notamment grâce à un prototype de capteur proximal développé en collaboration avec la société Force-A. La validation de la méthode au vignoble dans le cadre d’infection naturelle est la prochaine étape pour une utilisation de ce capteur optique dans le cadre de l’agriculture durable et de la sélection variétale. / Controlling the impact of diseases on crops is a major challenge of modern agriculture. This concern is an important aspect of optimizing productivity, notably in viticulture. In France, downy mildew caused by Plasmopara viticola is a fungal disease responsible for the most devastating epidemics. The preventive and systematic treatments are expensive, while the massive use of antifungal chemicals is a risk to both humans and the environment. Reducing the use of fungicide involves the development of diagnostic tools in the field, which requires understanding the interactions between plants and pathogens. The work of this multidisciplinary thesis focused on the pathosystem Plasmopara viticola - Vitis vinifera, especially to meet the growing interest in a real-time diagnostic tool of disease applicable in the vineyard. Stilbenes are phytoalexins involved in the defense of certain higher plants against biotic and abiotic stresses. The autofluorescence of these phenolic compounds, whose biosynthesis is induced in grapevine leaves by P. viticola, makes it a potential marker of natural infection. Indeed, the low blue-green autofluorescence of grapevine leaves is greatly enhanced by the violet-blue autofluorescence of stilbenes on the surface of leaves infected by P. viticola. This study showed that whatever the level of resistance in various genotypes, violet-blue autofluorescence induced by stilbene is present in the walls of epidermal cells. In addition to their concentration, viscosity proved the main physico-chemical variable affecting the intensity of the autofluorescence of stilbenes in different compartments of vine leaves. This explains the intense fluorescence of the walls, particularly rigid, of guard cells (stomata) of infected leaves. Daily monitoring revealed a kinetic with a transient rise of the autofluorescence of stilbenes during infection. The robustness and value of this signal was also validated by measuring at different levels (cellular to whole leaf) and with various fluorimetric methods (imaging, spectroscopy, proximal sensing). These results advance our understanding of the role of constitutive and induced phenolic compounds in plant defence against P. viticola. In addition to a common location of the autofluorescence of stilbenes on the leaf surface, confocal microscopy coupled with microspectrofluorometry revealed distinctive localizations of these phytoalexins in the deep tissue correlated with the level of resistance in genotypes. This aspect no doubt needs broader testing. The use of autofluorescence of stilbene as a marker of infection allowed us to ascertain that: 1) constitutive flavonols of the leaves of V. vinifera retard the development of infection by P. viticola and 2) the constitutive hydroxycinnamic acids do not seem to participate in the defence against P. viticola. Finally, a new method for the non-destructive diagnosis of leaf infection based on the autofluorescence of stilbenes has been developed. We have demonstrated a pre-symptomatic detection of downy mildew on whole grape leaves from the first day after infection on the abaxial surface and from the third day on the adaxial surface. This method of diagnosis has been validated in the laboratory thanks to a proximal sensor prototype developed in collaboration with the company Force-A. The validation of the method in the vineyard in a context of natural infections is the next step for use of this optical sensor as a tool for sustainable agriculture and for genetic screening.