Pseudoperonospora cubensis (Bert. et Curt) Rost. is the causal agent of cucurbit downy mildew (CDM). It is the most damaging cucumber pathogen on the Eastern Shore of Virginia and eastern parts of the United States. Pseudoperonospora cubensis is an obligate oomycete pathogen, infecting crops within the Cucurbitaceae family. The disease is characterized by angular chlorotic lesions and a downy or felt-like appearance on the abaxial side of the leaf. Control of this pathogen includes use of resistant cucumber cultivars and costly fungicide programs. Continuous use has led to resistance to commonly used fungicides. This has become a major concern and in response, seed companies have developed cucumber cultivars which claim downy mildew resistance.  This study evaluates different cucumber cultivars and assesses their level of resistance to CDM. The results indicate that an integrated management approach of reduced fungicide application and the use of resistant cultivars can suppress levels of CDM and yield a cucumber crop. Additionally, a molecular study was conducted, comparing the relative expression of genes encoding a basic PR-1 protein, a cytosolic ascorbate peroxidase protein and three resistance (R) gene proteins, in nineteen cultivars. All of the selected genes were analyzed using real-time PCR. The relative expression levels of the R-genes varied between cultivars. The basic PR-1 protein decreased expression in the majority of the cultivars, suggesting no involvement in the first twenty-four hours. Cytosolic ascorbate peroxidase relative expression levels suggest an increase in susceptible cultivars and a decrease in tolerant cultivars. / Master of Science

This study is concerned with the resistance of tobacco seedlings to a second attack of downy mildew caused by Peronospora tabacina Adam. Observations were made on the development and duration of resistance following recovery of tobacco seedlings from downy mildew. The duration of the period of resistance is variable and the observations indicate that plants in the greenhouse recovering during the summer remain resistant longer than do plants recovering from an initial attack during the winter. Samples of leaf tissue from recovered (resistant), infected and previously uninfected (susceptible) plants grown in soil in the greenhouse were analyzed for ammonia-nitrogen, nitrate-nitrogen, reducing sugars and total sugars. Similar analyses were made of samples of leaf tissue from tobacco plants grown in crocks of quartz sand and irrigated with nutrient solutions and from plants grown in crocks of sand-peat mixture to which various mixed fertilizers were added. There was no correlation between the ammonia-nitrogen content of tobacco plants, as determined in these experiments, and the response of the tobacco plant and its susceptibility to attack by P. tabacina, but there was a strong indication that such a relationship exists. Recovered plants contained a higher ratio of total sugar to nitrate-nitrogen than did comparable plants which had never been infected. Furthermore the ratio of total sugar to nitrate-nitrogen was lower in plants infected for a second time than it was in plants from the same lot shortly after they had recovered from the initial attack. It seems probable however that the changes in the nitrate-nitrogen and sugar content in recovered tobacco leaves are an indication of recovery and are not responsible for the resistance possessed by such plants. A series of experiments were performed to test the immediate effect of several nutrient salts and of sugar upon the sporulation of the fungus on the leaves of diseased tobacco plants. There was a very definite response in sporulation to some of these treatments, especially to potassium nitrate, calcium nitrate, and sucrose, but it is not known whether the response of sporulation of the fungus to such treatment is related to the type of resistance possessed by recovered plants. Sodium chloride and calcium chloride were dissolved in water and applied to the soil in which young tobacco plants were growing in the greenhouse. Although increased chloride in one of the sand cultural experiments seemed to protect the plants somewhat against downy mildew, application of solutions of these salts to the soil at the rates used (100 lbs. And 138 lbs. Per acre respectively) produced no noticeable effect on the severity of the disease or subsequent recovery of the plants. The leaves of recovered (resistant) tobacco plants are usually reduced in number, the lower ones having been killed by the initial attack of the fungus. Removal of the leaves from healthy tobacco plants in imitation of this condition did not produce resistance to an attack by P. tabacina. The artificial production of necrotic areas on healthy tobacco leaves in simulation of those usually following sporulation of P. tabacina on diseased leaves had no effect either on the susceptibility of the plants to infection or upon sporulation of the fungus on leaves already infected at the time the necrotic areas were induced. Results of attempts to confer immunity from downy mildew upon an entire plant by infection of a single leaf of the plant were inconclusive, since under the conditions of the experiment it was impossible to be sure that the single leaf was infected. However, the indication is that infection of a single leaf will not render the entire plant immune from a second attack by the fungus. / Master of Science

In the United States, hops (Humulus lupulus L.) are grown mainly in the Pacific Northwest (PNW). For this reason, most cultural information is based on the growing conditions of the PNW. Growing conditions in Virginia differ drastically and present unique disease and production challenges. Three studies were conducted with the intent of increasing hop cultivation knowledge for Virginia growers. For the first study, 13 cultivars of hops grown at the Virginia Tech hop yard were compared for growth, yield, and quality. Mean cone fresh weight per plant ranged from 12.00 g for Mt. Hood to 1002.87 g for Crystal in 2016 and from 97.98 g for Mt. Hood to 900.33 g for Cascade in 2017. In 2016, only Alpharoma, Cascade, Mt. Rainier, and Southern Cross had alpha acid levels, an indicator of cone quality, within the accepted range. In 2017, Alpharoma, Centennial, Mt. Rainier, and Nugget had alpha acid levels within the expected range. Three cultivars (Cascade, Crystal, and Ultra) were above the expected alpha acid range, which indicates more bittering potential for beer brewers. In a nutrient deficiency study, hop plants were grown in hydroponic solutions, and deficiencies were induced for nitrogen (N), phosphorus (P), and potassium (K). After visual deficiency symptoms had been induced, leaf tissue samples were taken and analyzed for nutrient content. Images were taken at each deficiency stage. For N, incipient deficiency symptoms were observed at a mean of 3.18% dry weight in leaf tissue samples. Visual symptoms included a chlorotic appearance, undersized leaves, and red petioles. Incipient symptoms for P were observed at a mean of 0.307% dry weight in leaf tissue samples. Necrotic spots, leaf cupping, and undersized leaves were apparent with this deficiency. Incipient symptoms for K were observed at a mean of 1.21% dry weight in leaf tissue samples. Symptoms included rounded leaf tips, blue veins, and marginal scorch. In the third study, a whole leaf powdery mildew (Podosphaera macularis) assay was developed and tested using five hop cultivars, Alpharoma, Cascade, Comet, Sorachi Ace, and Tahoma. Leaves were inoculated with powdery mildew (PM) using a settling tower. This method was used to rapidly assess the resistance of cultivars. Leaves were successfully inoculated and PM colonies were allowed to grow for two weeks. Images of the PM colony development on inoculated leaves were compared using ImageJ to determine percentage of coverage. Tahoma was the only cultivar found to produce a significantly different mean percent coverage (19.5%) compared with the resistant cultivars Cascade and Comet (<1%). / Master of Science in Life Sciences / Hops (Humulus lupulus) were first grown in the United States in Massachusetts in the early 1600s. Production of this crop eventually spread throughout the Northeastern US. By the mid-1800s, commercial production spread to southern states such as Virginia. Infestation of pests, e.g., hop aphid and diseases such as downy mildew in eastern states, and prohibition on the production of alcohol caused a significant shift in hop production, which favored the Pacific Northwest (PNW). As a result, in Virginia specific knowledge of hop cultivation is now lacking. Three studies were conducted to increase our knowledge of hop cultivation for the region. For the first study, 13 cultivars grown at the Virginia Tech hop yard were compared for growth, yield, and quality. The cultivars Cascade and Alpharoma had alpha acid levels equal to or higher than expected for the 2016 and 2017 seasons. The alpha acid levels are an indicator of the hop quality from the Cascade and Alpharoma cultivars. Cascade was also a top producer of hop cones with a mean fresh weight of 989.67 g and 900.33 g in 2016 and 2017, respectively. In a nutrient deficiency study, plants were grown in Hoagland hydroponic solutions, which contain all essential nutrients needed for plant growth. This was done to provide photographic documentation to assist local growers with deficiency identification. Deficiencies were induced for nitrogen (N), phosphorus (P), and potassium (K) by removing each of the specific nutrient of interest from each treatment solution. After deficiency symptoms were induced, leaf tissue samples were taken and analyzed for nutrient content. Nutrient deficiencies were documented using photographs. For N, visual symptoms included a chlorotic appearance, undersized leaves, and red petioles. Incipient symptoms for P included necrotic spots, leaf cupping, and undersized leaves. For K, leaf cupping, blue green veins, marginal scorch, and rounded leaf tips. Disease resistance of cultivars is important information for growers and can heavily influence hop yard planning. As such, in another study, an assay for powdery mildew (Podosphaera macularis) was developed that allows for rapid low cost testing of hop cultivars. The assay was tested on the following cultivars: Alpharoma, Cascade, Comet, Sorachi Ace, and Tahoma. Leaves were inoculated with powdery mildew (PM) using a settling tower. Powdery mildew colonies were allowed to grow for two weeks and were then analyzed using ImageJ software to determine percent coverage. At the end of the experiment, Tahoma had significantly greater PM coverage compared to the other cultivars, indicating that Tahoma is less resistant to the specific PM strain. / Hops (Humulus lupulus) were first grown in the United States in Massachusetts in the early 1600s. Production of this crop eventually spread throughout the Northeastern US. By the mid-1800s, commercial production spread to southern states such as Virginia. Infestation of pests, e.g., hop aphid and diseases such as downy mildew in eastern states, and prohibition on the production of alcohol caused a significant shift in hop production, which favored the Pacific Northwest (PNW). As a result, in Virginia specific knowledge of hop cultivation is now lacking. Three studies were conducted to increase our knowledge of hop cultivation for the region. For the first study, 13 cultivars grown at the Virginia Tech hop yard were compared for growth, yield, and quality. The cultivars Cascade and Alpharoma had alpha acid levels equal to or higher than expected for the 2016 and 2017 seasons. The alpha acid levels are an indicator of the hop quality from the Cascade and Alpharoma cultivars. Cascade was also a top producer of hop cones with a mean fresh weight of 989.67 g and 900.33 g in 2016 and 2017, respectively. In a nutrient deficiency study, plants were grown in Hoagland hydroponic solutions, which contain all essential nutrients needed for plant growth. This was done to provide photographic documentation to assist local growers with deficiency identification. Deficiencies were induced for nitrogen (N), phosphorus (P), and potassium (K) by removing each of the specific nutrient of interest from each treatment solution. After deficiency symptoms were induced, leaf tissue samples were taken and analyzed for nutrient content. Nutrient deficiencies were documented using photographs. For N, visual symptoms included a chlorotic appearance, undersized leaves, and red petioles. Incipient symptoms for P included necrotic spots, leaf cupping, and undersized leaves. For K, leaf cupping, blue green veins, marginal scorch, and rounded leaf tips. Disease resistance of cultivars is important information for growers and can heavily influence hop yard planning. As such, in another study, an assay for powdery mildew (Podosphaera macularis) was developed that allows for rapid low cost testing of hop cultivars. The assay was tested on the following cultivars: Alpharoma, Cascade, Comet, Sorachi Ace, and Tahoma. Leaves were inoculated with powdery mildew (PM) using a settling tower. Powdery mildew colonies were allowed to grow for two weeks and were then analyzed using ImageJ software to determine percent coverage. At the end of the experiment, Tahoma had significantly greater PM coverage compared to the other cultivars, indicating that Tahoma is less resistant to the specific PM strain.

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.

Les variétés traditionnelles de vigne nécessitent de très nombreux traitements phytosanitaires pour lutter contre les maladies cryptogamiques qui touchent leurs parties herbacées, comme le mildiou et l’oïdium, causés par Plasmopara viticola et Erysiphe necator respectivement. Ces traitements, coûteux et préjudiciables pour l'environnement, pourraient être réduits par l’emploi de variétés résistantes. La vigne cultivée européenne (Vitis vinifera, 2n=38) est très sensible au mildiou et à l'oïdium. En conséquence, la résistance doit être introduite à partir d’autres Vitaceae ayant un niveau de résistance plus élevé à ces maladies. Plusieurs origines de résistance ont déjà été observées et inventoriées, en particulier chez l’espèce d’origine américaine Muscadinia rotundifolia (2n=40). Ces facteurs sont d'un intérêt majeur pour la sélection de variétés résistantes. Cependant, lors du processus d'introgression, des difficultés à obtenir des pépins viables en F1 ainsi que des anomalies phénotypiques dans les descendances en rétrocroisement ont été constatées. Afin d’optimiser la gestion des résistances provenant de cette espèce dans les programmes d’amélioration variétale, il est nécessaire de comprendre l’organisation génétique et génomique de M. rotundifolia, et de compléter la connaissance des facteurs de résistance issus de cette espèce. Dans ce contexte, les objectifs de la thèse sont : (i) de réaliser une analyse comparative des génomes de V. vinifera et M. rotundifolia et (ii) d’identifier de nouveaux facteurs de résistance chez M. rotundifolia utilisables à terme en sélection. Pour cela, une carte génétique de M. rotundifolia a été développée à partir d’une population de 200 individus issue de l’autofécondation de M. rotundifolia cv. Regale. Parallèlement, la même population a été testée pour son niveau de résistance au mildiou et à l’oïdium. Une carte génétique couvrant 950 cM a été réalisée. Elle comprend 191 marqueurs microsatellites répartis sur les 20 chromosomes de M. rotundifolia, et permet de conclure à un niveau de macrosynténie très élevé avec V. vinifera. Le groupe de liaison 20 de M. rotundifolia correspondrait à la partie inférieure du groupe de liaison 7 de V. vinifera. Par ailleurs, un QTL de résistance au mildiou a été détecté sur le groupe de liaison 18 de M. rotundifolia, au niveau d’une région riche en gènes de type NBS-LRR, et un nouveau QTL de résistance majeur à la l’oïdium a été mis en évidence sur le groupe de liaison 14 de M. rotundifolia. Ce QTL, nommé Ren5 pour ‘Resistance to Erysiphe necator 5’, montre une action précoce dans l’arrêt de la croissance du mycélium du pathogène, dès l’établissement des premiers stades de biotrophie du champignon. De plus, le QTL Ren5 a été confronté à deux souches supplémentaires d’E. necator, appartenant aux deux groupes d’oïdium retrouvés dans vignobles européens, contre lesquelles il reste efficace. Les données de cartographie génétique générées pour M. rotundifolia dans ce travail, ainsi que la mise en évidence de Ren5 et de son mode d’action, permettront d’améliorer la gestion des facteurs de résistance issus de cette espèce pour la sélection de variétés résistantes au mildiou et à l’oïdium. / Grapevine requires numerous harmful plant-health treatments, in particular to control downy and powdery mildews, caused by Plasmopara viticola and Erysiphe necator, respectively. One way to reduce the use of fungicides in viticulture is to create resistant grapevine cultivars. European cultivated grapevine (Vitis vinifera, 2n=38) is susceptible to mildews, whereas related species such as Muscadinia rotundifolia (2n=40) exhibit high levels of resistance. In breeding programs, resistance factors from these related species are introduced into the susceptible elite varieties. Nevertheless, difficulties in obtaining viable seeds in the siblings of Vitis x Muscadinia crosses are observed. In order to optimize the management of resistance factors from M. rotundifolia in breeding programs, it is necessary to understand the genomic organisation of this species, and to complete the knowledge of these factors. Thus, the main objectives of this work are : (i) making a comparative analysis of V. vinifera and M. rotundifolia genomes and (ii) identifying new resistance factors from M. rotundifolia. For this purpose a framework genetic map of M. rotundifolia cv. Regale has been created, using a 200 individual S1 population. This population has also been screened for its resistance to downy and powdery mildew. A 950 cM genetic map has been generated, including 191 SSR markers distributed across the 20 chromosomes of M. rotundifolia. A high level of macrosynteny has been observed between the Muscadinia map and the genetic maps available for V. vinifera. Linkage group (LG) 20 of M. rotundifolia matches with the lower part of V. vinifera LG7. Furthermore, a QTL for resistance to downy mildew has been identified on M. rotundifolia LG18, and a major QTL for resistance to powdery mildew has been mapped on LG14. The latest, called Ren5 for ‘Resistance to Erysiphe necator 5’, acts during an early stage to stop E. necator’s mycelium growth, since the first stages of biotrophy have been established for the fungus. Moreover, Ren5 has been confronted to two additional powdery mildew strains, belonging to the two European groups of E. necator, and it remained efficient. Gathering knowledge about the genetic organization of M. rotundifolia, and the mechanism and spectrum of action of newly identified resistance factors such as Ren5, will be useful to optimize the management of M. rotundifolia resistance traits in breeding programs aiming to create new resistant varieties to downy and powdery mildews

La compréhension du processus d’adaptation des populations de parasites à leur plante-hôte est une question fondamentale en écologie évolutive. C’est également un enjeu majeur de recherche finalisée qui a des retombées pour la protection des cultures. L’oomycète Plasmopara viticola, agent causal du mildiou de la vigne, attaque les espèces du genre Vitis. Dans un contexte où l’enjeu principal des programmes d’amélioration est la durabilité des résistances, des connaissances nouvelles sur l’écologie et l’évolution de l'interaction entre le parasite et son hôte sont nécessaires afin d’évaluer le potentiel du mildiou à surmonter ces résistances. Dans ma thèse, je me suis intéressée au rôle de la plante-hôte comme facteur d’évolution des populations de mildiou, en posant cette question à différentes échelles évolutives : (i) dans le bassin d’origine du pathogène (Amérique du Nord), j’ai cherché à évaluer le degré de spécialisation du parasite sur sa gamme d’hôtes sauvages et cultivés; (ii) en Europe, où le mildiou de la vigne a été introduit récemment, j’ai étudié l’évolution des populations de mildiou soumis à la pression de sélection des résistances des nouvelles variétés de vigne. Pour comprendre la spécialisation plante-hôte dans ce pathosystème où plusieurs espèces cryptiques ont été identifiées, nous avons réalisé des tests d’inoculations croisées entre espèces hôtes (Vitis spp.) et agent pathogène (P. viticola). Les données phénotypiques et morphologiques apportent les preuves d’une spécialisation plante-hôte au sein des populations de P. viticola : les espèces A et D de mildiou sont spécialisées sur leur plante-hôte, tandis que le processus de spécialisation est en cours pour les espèces B et C. Même si aucune différenciation génétique n’a été montrée au sein de l’espèce C, il existe deux groupes distincts au sein de l’espèce B. Les isolats du compartiment cultivé sont en moyenne plus agressifs que les isolats issus des vignes sauvages, indiquant une adaptation des isolats cultivés sur leur plante hôte. A partir d’un large échantillonnage, nous avons étudié la distribution des espèces de mildiou sur leurs plantes-hôtes sauvages et cultivées. Ce travail a permis d’identifier une nouvelle espèce cryptique et a confirmé la spécialisation plante-hôte. En Europe, nos résultats montrent que le déploiement limité de variétés à résistantes partielles a conduit à des modifications des populations de mildiou: apparition d’isolats virulents (i.e. contournant un QTL majeur de résistance), et augmentation de l’agressivité sur Vitis vinifera. Dans le but de comprendre les mécanismes à l’origine de la spécialisation et du contournement des résistances, nous nous sommes intéressés au répertoire d’effecteurs du parasite. Une centaine d’effecteurs candidats ont été identifiés en utilisant les données disponibles sur le génome de P. viticola. L’analyse du polymorphisme de 32 candidats sur une sélection d’isolats montre que trois d’entre eux évoluent sous sélection positive. Ces résultats soulignent l’importance de la plante-hôte comme facteur de diversification des populations de l’agent pathogène et révèlent que le mildiou s’adapte rapidement aux résistances de la vigne. Il est désormais nécessaire de mieux appréhender le déploiement des résistances de la vigne afin qu’elles puissent être durables. / Understanding the process of adaptation of parasite populations to their host-plant is a key issue in evolutionary ecology. It is also a major subject in applied research that has implications for crop protection. The oomycete Plasmopara viticola, the causal agent of downy mildew, attacks the species of the Vitis genus. In a context where the main concern of the breeding programs is the durability of resistance, new knowledge about the ecology and evolution of the interaction between parasite and host is needed in order to evaluate the potential of downy mildew to overcome the resistance. In my thesis, I addressed the role of the host-plant as an evolutionary factor for downy mildew populations, by asking this question at two different evolutionary scales: (i) in the pathogen region of origin (North America) I assessed the degree of specialization of the parasite on its wild and cultivated host range (ii) in Europe, where downy mildew has been introduced recently, I studied the evolution of downy mildew populations subject to the selection pressure imposed by resistant grapevine varieties. To understand the host-plant specialization in this pathosystem, where several cryptic species have been identified, we performed cross inoculations between different host (Vitis spp.) and pathogen (P. viticola) species. Morphological and phenotypic data provide evidence of host-plant specialization in P. viticola populations: downy mildew species A and D are specialized on their host-plant, while the specialization process is ongoing for species B and C. Although no genetic differentiation has been shown inside species C, there are two distinct groups within species B. Isolates from the cultivated compartment are on average more aggressive than isolates from wild vines, indicating an adaptation of isolates growing on cultivated host-plants. Finally, a large-scale study of the distribution of downy mildew species on both their wild and cultivated host-plants resulted in the identification of a new cryptic species and confirmed the host-plant specialization. In Europe, our results show that the limited deployment of resistant varieties has led to changes in downy mildew populations: emergence of virulent isolates (i.e. breakdown of a major QTL for resistance), and increased aggressiveness on Vitis vinifera. In order to understand the mechanisms at the origin of specialization and resistance breakdown, we examined the parasite’s effector repertoire. Over one hundred effector candidates were identified using available data on the P. viticola genome. The polymorphism of 32 candidate genes revealed that three of them evolve under positive selection. Our results reveal the strong ability of downy mildew to adapt to its host plant and to plant resistance. They should be taken into account when devising strategies for the deployment of grapevine resistances in order to guarantee their durability.

L'utilisation des champignons mycorhiziens à arbuscules (CMA) pourrait constituer une alternative potentielle aux traitements fongicides conventionnels pour lutter contre les maladies cryptogamiques des plantes. Notre travail a consisté à étudier l'éventuel effet protecteur de la mycorhization arbusculaire chez le blé tendre (triticum aestivum L.) contre Blumeria graminis f. sp. tritici (Bgt), un champignon biotrophe responsable de l'oïdium, une maladie affectant les parties aériennes de la plante. L'inoculation mycorhizienne du blé avec le CMA Funneliformis mosseae (FM), en conditions controlées et optimisées, nous a permis d'obtenir parallèlement à un taux de mycorhization de 38% des plants de blé, une amélioration significative de la biomasse et un taux de protection contre Bgt estimé à 78%. Ces résultats suggèrent l'induction d'une résistance systémique des réactions de défense du blé par mycorhization (Mycorrhiza-Induced Resistance, MIR). Cette protection serait liée à une accumulation de composés phénoliques et de preoxyde d'hydrogène dans les cellules épidermiques des feuilles de blé mycorhizé, au niveau du site de pénétration de Bgt. Une surexpression des gènes POX, PAL, CH11 et NPR1 codant pour des marqueurs de défense a également été mise en évidence dans les feuilles en absence d'infection par Bgt. Enfin, nos travaux ont également souligné l'intégration de divers paramètres pour optimiser l'utilisation des CMA comme agents de biocontrôle chez le blé. La meilleure protection contre l'oïdium a été obtenue aec un apport en phosphore réduit de 5 fois par rapport à celui préconisé au champ et un inoculum mycorhizien à base de Fm, que ce soit chez un cultivar modérément sensible ou un cultivar plus résistant. / The use of arbuscular mycorrhizal fungi (AMF) could be an innovative alternative to chemicals against fungal plant diseases. Our work aimed at studying the possible protective effect of arbuscular mycorrhization in the bread wheat (Triticum aestivum L.) against Blumeria graminis f. sp. tritici (Bgt), a biotrophic fungi responsible of wheat powdery mildew, a disease affecting the aerial plant organs. Wheat mycorrhizal inoculation by Funneliformis mosseae (Fm), under controlled and optimized conditions, allowed us to obtain concomitantly a micorrhizal rate of 38%, a significant increase of plant biomass and a protection level against Bgt estimated at 78%. These results suggest the induction of systemic wheat defense reactions resulting from mycorrhization (Mycorrhiza-Induced Resistance - MIR). This protection is linked to an accumulation of phenolic compounds and hydrogen peroxide at the Bgt penetration sites in epidermal leaf cells of mycorrhized wheat plants. Up-regulations of POX, PAL, NPR1 and CH11 genes encoding for defense markers were also pointed out in leaves of mycorrhizal wheat in the absence of Bgt infection. Moreover, our study highlighted the importance of taking into account various parameters to optimize the use of AMF as biocontrol agents. The highest protection against powdery mildew was obtained with a 5-fold reduced phosphorus input compared to that recommended in the field and with the mycorrhizal inoculum Fm, in both a moderately susceptible or a more resistant cultuva

O meloeiro é uma cultura de relevância econômica no Brasil, principalmente na região Nordeste, onde grande parte da produção é exportada. Entre os fatores limitantes da cultura está o oídio, que pode ser controlado através do uso de variedades resistentes. No entanto, para que isto seja possível, é necessário que se tenha conhecimento sobre qual é o agente causal em ocorrência na região de produção e quais são as fontes de inóculo desta doença. O presente trabalho teve como objetivos o levantamento das espécies e raças de oídio com ênfase na região Nordeste, a verificação da patogenicidade de isolados provenientes de outras cucurbitáceas em relação ao meloeiro, a avaliação de germoplasma de meloeiro para resistência a P. xanthii raça 2 (francesa), a proposição de um sistema de cultivares de meloeiro para caracterização de raças de P. xanthii e a otimização de um protocolo de extração de DNA de P. xanthii. No total de 65 isolados analisados, observou-se que Podosphaera xanthii foi a única espécie encontrada entre as amostras. Dentre estas, as raças 1 e 2 foram as prevalentes. Também foram observadas as raças 0, 3, 4 e 5 em menor freqüência. As culturas de abóbora, abobrinha, melancia e pepino apresentaram-se como fontes de inóculo de oídio para o meloeiro, uma vez que todos os isolados provenientes destas culturas colonizaram discos foliares de meloeiro. Em geral, os genótipos de melão cultivados comercialmente no Brasil apresentaram-se resistentes a raça 2 (francesa) de P. xanthii. A partir da observação das raças ocorrentes no país e de dados da literatura, propôs-se um sistema de cultivares de meloeiro diferenciadoras para raças de P. xanthii a ser empregado em monitoramentos do patógeno. Um protocolo para extração de DNA de P. xanthii também foi obtido com a finalidade de facilitar estudos genéticos sobre o patógeno com base em marcadores moleculares. / Melon is an economically important crop in Brazil, mainly in the north-east region, where most of the production is exported. Among the limiting factors of the crop is powdery mildew, which can be controled by using resistant varieties. However, to make it possible, it is necessary to know the causal agent in occurrence in the production region and what are the inoculum sources of this disease. The present work had as objectives the survey of the powdery mildew species and races with emphasis in the north-east region; the verification of the strains pathogenicity deriving from others cucurbits in melon; the evaluation of melon germplasm to Podosphaera xanthii race 2 (french) resistance; the proposition of a system of melon cultivars to characterize the P. xanthii races and the optimization of a protocol to P. xanthii DNA extraction. Over all of the 65 strains analysed, it was observed that P. xanthii was the unique specie encountered between the samples. Within that, the races 1 and 2 were prevalents. Also were observed the races 0, 3, 4 and 5 at low frequency. Squash, zucchini, watermelon and cucumber crops showed up like inoculum sources of powdery mildew to melon. Usually, the melon genotypes commercially cultivated in Brazil showed up resistant to P. xanthii race 2 (french). After the observation of races in occurrence and datas from literature, a system of melon cultivars was proposed to differentiate P. xanthii races to be used in surveys of the pathogen. A protocol to extraction P. xanthii DNA also was proposed with the purpose to facility genetics studies about the pathogen basis on molecular markers.

At present, the global population is increasing, while soil and fresh water resources for crop production are declining. It is important to adopt sustainable practices to optimise the use of limited natural resources without compromising the environment, and to enhance continuous production in the long term. The rapid growth of UK strawberry industry has been achieved through the precision use of varieties, nutrients and polythene tunnels. This intensive production has caused significant environmental impacts especially Greenhouse Gas (GHG) emissions from the production. Strawberry powdery mildew (Podosphaera aphanis) is a major fungal disease affecting strawberry production worldwide particularly in polythene tunnels. The disease can result in yield losses of up to 70% of the crop. A ruleQbased system was used in the field trials to predict high risk days of P. aphanis development, taking into account the optimal environmental conditions conducive to conidial germination and disease development. The results (Chapter 3) showed that the use of this prediction system achieved satisfactory control of P. aphanis in commercial strawberry production, with reduced fungicide applications compared with commercial spray programme. The results were consistent in two consecutive years and on different varieties. In addition, it was suggested that the use of the prediction system may also lead to lower GHG emissions associated with fewer fungicide applications, thereby benefit strawberry growers both environmentally and economically. Results from 2014 & 2015 silicon fertigation trials showed that the use of a silicon nutrient via the fertigation system reduced the strawberry susceptibility to P. aphanis and twoQspotted spider mites (Tetranychus urticae Koch) in two consecutive years on different varieties (Chapter 4). In both years, crops received the silicon nutrient only without fungicides had both lower rate of epidemic (r) and lower value of Area Under the Disease Progress Curve (AUDPC) (r = 0.0036, AUDPC = 475 in 2014; r = 0.001, AUDPC = 267 in 2015) compared with the untreated control (r = 0.0042, AUDPC = 662 in 2014; r = 0.0011, AUDPC = 281 in 2015). Silicon also delayed the epidemic buildQup in the silicon nutrient only treatment for approximately two weeks compared with the untreated control. Crops from the silicon nutrient plus fungicides treatment had lower susceptibility (r = 0.0012 in 2014; r = 0.0004 in 2015) than those from the fungicides only treatment (r = 0.0017 in 2014; r = 0.0005 in 2015) suggesting that the silicon nutrient may also enhance fungicides performance in reducing the epidemic buildQup when used together. Moreover, the presence of T. urticae on strawberry leaves was significantly lower (P < 0.001) in plants treated with the silicon nutrient than those without. In addition, initial results suggested that silicon may play a positive role in raising °Brix of strawberry leaf petiole, improving pollen viability, and influencing the length of flower receptacle and stamens. Maltmas Farm has a wide range of semiQnatural habitats that provide food and nesting resources for wild pollinators. Hoverflies, bumblebees and solitary bees were found to be the main wild pollinators that pollinate commercial strawberries at Maltmas Farm (Chapter 5). The number of pollinators in tunnels or open fields significantly correlated with the abundance of strawberry flowers (P < 0.05). Pollinator presence also differed between groups throughout the day and over the seasons. Hoverflies appeared early in the day and were abundant in summer months; bumblebees and solitary bees were present most of the day and throughout the season, whereas honeybees were only active in sunny days. Temperatures, relative humidity and cloud coverage also affected pollinator presence. In addition, pollinator activity was not significantly (P > 0.05) affected by the application of the silicon nutrient via the fertigation system. The integrated use of the prediction system (to reduce fungicide applications and subsequent GHG emissions), the silicon nutrient (to reduce crop susceptibility to P. aphanis and T. urticae), and sustainable farmland management (to encourage the presence of wild pollinators) could help strawberry growers to achieve a more sustainable production.

A cultura do meloeiro é de grande importância econômica no Brasil, sendo a região Nordeste a principal produtora e exportadora do fruto. A baixa resistência aos principais patógenos é um dos fatores limitantes para a competitividade brasileira da cultura no mercado internacional. Uma das doenças mais importantes é o oídio, causado por Podosphaera xanthii. Geralmente, o controle de P. xanthii é obtido com o uso de fungicidas. Porém, é necessário empregar medidas mais econômicas e que não agridam o ambiente, como a utilização de cultivares resistentes. O objetivo deste trabalho foi identificar marcadores moleculares de vários tipos ligados ao gene Pm-1 de resistência à raça 1 de P. xanthii com o intuito de auxiliar programas de melhoramento genético. Para tanto foram utilizadas duas linhagens quase-isogênicas (LQI) de melão AF426pm1 (P1) e AF426Pm1 (P2), ambas pertencentes à variedade inodorus, que são contrastantes para ausência e presença, respectivamente, do gene Pm-1. Para a análise de co-segregação entre gene e marcadores candidatos, foi utilizada uma população de retrocruzamento RC1F1, fenotipada para resistência a raça 1 de oídio, obtida a partir do cruzamento entre linhagens. As técnicas de LM-PCR e AFLP resultaram em marcadores polimórficos, porém somente os encontrados com a técnica de AFLP puderam ser utilizados como marcadores no teste de co-segregação. Foram encontrados dois marcadores AFLP. No entanto, somente um, denominado HF155 mostrou-se ligado a uma distância de 4,9 cM do gene Pm-1. Devido à proximidade deste marcador ao gene este pode ser utilizado em programas de seleção assistida por marcadores, visando o melhoramento de linhagens com resistência ao P. xanthii. / Melon crop is of great economical importance in Brazil being the Northeast region the main producer and exporter of the fruit. The low levels of resistance to pathogens is one of the restricting factors for the Brazilian competitiveness worldwide. One of the most important diseases is powdery mildew caused by Podosphaera xanthii. Generally, the control of P. xanthii is achieved with the use of fungicides. However it is necessary to use less expensive control methods with a minimum environmental impact such as resistant cultivars. The objective of this work was to identify molecular markers linked to the Pm-1 gene which confers resistance to race 1 of P. xanthii with the purpose of assisting boding program. Two near isogenic lines (LQIs) of melon Agro AF426pm1 (P1) and AF426Pm1 (P2), both belonging to the inodorus variety, which are respectivally contrasting for abscence and presence of Pm-1 gene were analyzed. For the cosegregation analyses between gene and candidate markers, it was used the BC1F1 population was used screened for resistance to powdery mildew and obtained from a cross between the LQIs. The LM-PCR and AFLP techniques were efficient in the polymorphisms detection, however only those ones which were found using AFLP technique could be used as markers in the co-segregation test. It was found two markers with this technique but just the HF155 is located 4.9 cM of the Pm-1 gene. Due the proximity of the HF155 marker to the Pm-1 gene this one can be used in markerassisted selection aiming to develop melon cultivars resistant to P. xanthii.