L’activation des réponses immunitaires des plantes repose sur la reconnaissance de motifs moléculaires associés aux pathogènes (aussi appelés PAMP) par des récepteurs de l’immunité, également nommés PRR (pattern recognition receptors). La chitine, principal composant de la paroi des champignons, est un PAMP bien caractérisé qui induit des réponses de défense aussi bien chez les mammifères que chez les plantes.La première partie de cette étude met en évidence que deux chito-oligosaccharides, la chitine et le chitosan, agissent comme des PAMP chez la vigne (Vitis vinifera) puisqu’ils induisent des évènements précoces de signalisation, l’expression de gènes de défense et une résistance contre des agents pathogènes. Ces résultats suggèrent que des systèmes de perception existent chez la vigne. Une analyse phylogénétique a permis d’identifier trois récepteurs kinases à domaine LysM (LysM-RK ou LYK) chez V. vinifera (VvLYK1-1, -2, -3) appartenant au même clade que le récepteur à la chitine chez Arabidopsis et nommé AtCERK1 (Arabidopsis thaliana Chitin Elicitor Receptor Kinase 1). Leur analyse fonctionnelle a été réalisée par complémentation du mutant d’Arabidopsis Atcerk1, affecté dans la perception de la chitine. Nos résultats montrent que VvLYK1-1 et VvLYK1-2, mais pas VvLYK1-3, complémentent fonctionnellement le mutant Atcerk1 en restaurant l’activation des MAPK (Mitogen-Activated Protein Kinases) et l’expression de gènes de défense induits par les chito-oligosaccharides. De plus, l’expression de VvLYK1-1 chez Atcerk1 restaure la résistance basale à l’agent de l’oïdium de la vigne (Erysiphe necator).La seconde partie du projet s’est focalisée sur les éliciteurs oligosaccharidiques de type « damage-associated molecular patterns (DAMP) ». Ces molécules endogènes peuvent provenir de la dégradation de la paroi lors d’une attaque et sont capables d’activer les réponses immunitaires de la plante. Les DAMP les mieux caractérisés actuellement sont les oligogalacturonates (OG), des fragments de pectine qui induisent des réponses immunitaires chez de nombreuses espèces végétales dont l’activation de MAPK, la production d’H2O2, l’expression de gènes de défense et le dépôt de callose. Nous avons montré dans cette étude que les xyloglucanes (Xh), des fragments d’hémicellulose pariétale purifiés, induisaient l’activation de MAPK et l’expression de gènes de défense chez la vigne et Arabidopsis, afin d’induire une résistance contre le champignon nécrotrophe Botrytis cinerea. Les Xh induisent également la production de resvératrol, une phytoalexine majoritaire chez la vigne, et un dépôt de callose chez Arabidopsis. Par une approche génétique, nous avons identifié certains composants de la signalisation induite par les Xh chez Arabidopsis. L’utilisation de mutants suggère que la résistance induite par les Xh contre B. cinerea est dépendante des voies de la camalexine, de l’acide salicylique, de l’acide jasmonique et de l’éthylène chez Arabidopsis. De manière globale, nos résultats mettent en lumière que les xyloglucanes peuvent être considérés comme de nouveaux éliciteurs de l’immunité chez la vigne et Arabidopsis. / Activation of the plant immune responses requires recognition of common pathogen-associated molecular pattern (PAMP) by their cognate pattern recognition receptors (PRR). Chitin, a major component of fungal cell walls, is a well-known PAMP that triggers defense responses in several mammal and plant species.In the first part of this study, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signaling events, defense gene expression, and resistance against pathogens. These two PAMPs are active in grapevine suggesting that at least one perception system exists. Phylogenetic analysis clearly distinguished three V. vinifera LysM Receptor Kinases (VvLYK1-1, -2, -3) located in the same clade as the Arabidopsis Chitin Elicitor Receptor Kinase 1 (AtCERK1), which mediates chitin-induced immune responses. Their functional characterization was achieved by complementation assays in the Atcerk1 mutant, impaired in chitin perception. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the loss of AtCERK1 function by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator).The second part of this study focused on damaged-associated molecular patterns (DAMP), endogenous molecules that can be released from the plant cell wall during an attack and activate the plant innate immunity. Until now, the best characterized DAMPs are oligogalacturonides (OG) coming from pectin fragments that induce innate immune responses in various plant species, including MAPK activation, H2O2 production, defense gene expression and callose deposition. In this study, we showed that purified xyloglucans (Xh), derived from the plant cell wall hemicellulose, elicit MAPK activation and immune gene expression in grapevine (V. vinifera) and Arabidopsis to trigger induced resistance against the necrotrophic fungus Botrytis cinerea. Xh also elicit the production of resveratrol, the main grapevine phytoalexin, and callose deposition in Arabidopsis. Using a genetic approach, we identified some signaling components of Xh-induced immunity. The use of Arabidopsis mutants suggests that Xh-induced resistance against B. cinerea is dependent on the camalexin, salicylate, jasmonate and ethylene pathways. Taken together, our data highlight that Xh can be considered as new elicitors of grapevine and Arabidopsis immunity.

Les recherches sur la lutte biologique (ou biocontrôle) par utilisation de micro-organismes connaissent un essor remarquable, les applications au champ étant cependant encore limitées en raison des variations d’efficacité dans la protection des plantes. Celles-ci sont souvent imputées à la non persistance des agents de biocontrôle dans la rhizosphère ou sur le végétal qu’ils sont censés protéger. Afin de réduire ce risque, une solution consiste à utiliser des micro-organismes isolés du végétal que l’on souhaite protéger. Dans le cadre de cette thèse, Pythium oligandrum, un oomycète colonisateur de la rhizosphère de nombreuses plantes dont la vigne, a été étudié pour lutter contre l’esca, une maladie du bois de la vigne pour laquelle il n’existe actuellement aucune méthode de lutte disponible. Des souches de P. oligandrum ont été isolées de la rhizosphère de ceps cultivés dans 3 régions viticoles (12 vignobles) du Bordelais présentant des sols variés : argilo-calcaire, sable-graveleux et graveleux. Les analyses des communautés fongiques et bactériennes obtenues par empreinte moléculaire (Single Strand Conformation Polymorphism) ont montré que, contrairement aux bactéries, les espèces fongiques différaient selon les régions. Des Pythium spp. aux oospores échinulées ont été isolées à partir des racines des ceps échantillonnés, avec une prédominance de P. oligandrum (séquençage de la région ITS). L’analyse des séquences des gènes codant pour le cytochrome oxydase I et une tubuline a permis de constituer 3 groupes de souches. Le séquençage d’autres gènes codant pour des protéines « élicitines-like » a indiqué que chaque souche présentait au moins un gène codant pour chacun des 2 types d’éliciteurs de P. oligandrum : l’oligandrine et les protéines de la paroi cellulaire (CWPs). Il apparaît que le type de sol et la microflore associée à la rhizosphère n’exerceraient pas une influence suffisante pour que la structure génétique des populations de P. oligandrum soient associées à un contexte tellurique particulier. En revanche, le type de porte-greffe et la méthode de désherbage (chimique ou mécanique) pourraient avoir une incidence sur la colonisation racinaire par P. oligandrum. Les relations entre P. oligandrum et les racines de la vigne ont été étudiées par analyse transcriptomique (microarray Vitis vinifera de 29 549 gènes). Les résultats obtenus montrent que de jeunes plants de vigne ont répondu à la colonisation racinaire par P. oligandrum en modifiant l’expression de gènes intervenant dans plusieurs voies métaboliques. Deux aspects a priori opposés ont été observés : P. oligandrum serait perçu comme (1) un agresseur contre lequel la plante a mis en place des réactions de défense mais en même temps, comme (2) un micro-organisme symbiotique car un certain nombre de modifications transcriptionnelles étaient similaires à celles reportées dans les interactions rhyzosphèriques symbiotiques (e.g. forte stimulation de gènes codant pour des subtilases). Un essai visant à induire chez la vigne une protection contre un champignon pathogène impliqué dans l’esca, Phaeomoniella chlamydospora, grâce à P. oligandrum, a été réalisé. La colonisation des racines par P. oligandrum a été associée à une réduction de la longueur des nécroses dues à P. chlamydospora. En adéquation avec ce résultat, l’analyse transcriptomique par RT-PCRq et microarrays a montré une surexpression de la voie de l’éthylène. Plusieurs gènes spécifiquement induits constitueraient des marqueurs de résistance qu’il conviendra de valider lors de prochaines expérimentations. / Biocontrol research based on the use of microorganisms is expanding very rapidly. However, the use of such bioncontrol agents is still too inconsistent to effectively protect plants in field applications. This phenomenon is often attributed to the non-persistence of biocontrol agents in the rhizosphere or on the plants. In order to reduce the risk of this happening, one solution consists in using microorganisms that are isolated from the plants needing protection. In this thesis, an oomycete called Pythium oligandrum, which colonizes the rhizosphere of many plants, including grapevine, was assessed for the control of esca, a grapevine trunk disease for which no control method is currently available. P. oligandrum strains have been isolated from the rhizosphere of vines cultivated in 3 wine-growing regions (12 grapevines) of Bordeaux with different types of soil: stony-sandy, silty and stony. Analyses of fungal and bacterial communities using a molecular fingerprinting method (Single Strand Conformation Polymorphism) showed that, unlike bacteria, the fungal species varied according to the sampling region. Roots of all the vines sampled were colonized by echinulated-oospore Pythium spp., with P. oligandrum strains predominating. Phylogenetic analyses based on the genes encoding the cytochrome oxidase I and one tubulin allowed these strains to be clustered into three groups. The sequencing of the elicitin-like genes, whose proteins are key components in inducing systemic resistance in plants, showed that each strain held at least one gene encoding for each of the two kinds of P. oligandrum elicitors (i.e. oligandrin and Cell Wall Proteins). Sequencing and molecular fingerprinting analyses showed thus that the type of soil and the rhizosphere microbiota did not shape the population structure of P. oligandrum. However, other factors such as the different kinds of rootstock and weeding management can also have an influence on the root colonization by P. oligandrum. The relationship between P. oligandrum and grapevine was studied using a transcriptomic approach (microarray Vitis vinifera, 29 549 genes). The results highlighted the modifications induced by young vines in response to P. oligandrum root colonization, in the genetic expression of several genes belonging to different metabolic pathways. Two aspects, that are usually opposed, were observed: P. oligandrum was perceived by the plant either (i) as a pathogen because certain defence reactions were triggered (e.g. calcium signalling, resistance genes, abscissic acid metabolism) or as (ii) a symbiotic microorganism since several transcriptional changes were similar to those reported in symbiotic interactions (e.g. induction of subtilase genes). An assay aimed at protecting grapevine against a pathogenic fungus involved in esca, and known to be responsible for wood necrosis, i.e. Phaeomoniella chlamydospora, was carried out. The root colonization by P. oligandrum was associated with a reduction in the length of necroses. In line with this result, transcriptomic analyses by microarrays and RT-qPCR showed overexpression of several genes, particularly those of the ethylene pathway. Some of these induced genes could be thus used as resistance markers, but this needs to be validated in further experiments.

Dans le contexte de changement climatique, la compréhension des mécanismes régissant les pertes en eau de la vigne peut permettre d'adapter le matériel végétal pour maintenir la productivité de la vigne et la qualité du vin. L'adaptation à la sécheresse est un caractère complexe faisant intervenir des mécanismes physiologiques liés aux génotypes du greffon et du porte-greffe. Mais les effets du porte-greffe sur la régulation stomatique du greffon sont mal connus. La production par les racines de signaux chimiques tels que l'ABA et/ou hydraulique pourraient y contribuer. La réponse physiologique et moléculaire à la contrainte hydrique a été analysée sur de jeunes boutures pour 7 porte-greffes plus ou moins adaptés à la sécheresse et 2 cépages connus pour leur caractère iso ou anisohydrique. Puis 23 combinaisons greffon/porte-greffe issues de ces génotypes ont été étudiées. Une analyse métabolique sur l'accumulation de l'ABA et ses dérivés a été menée sur feuilles, racines et dans la sève xylémienne. Ces informations ont été couplées à des analyses transcriptomiques sur des gènes du métabolisme et de la signalisation de ABA, et codant des aquaporines de type PIP. L‘analyse conjointe des données physiologiques, métabolomiques et transcriptomiques ont permis d'identifier des composants moléculaires discriminant les porte-greffes selon leur fond génétique et leur adaptation à la sécheresse. Les réponses globales à la contrainte hydrique sont mieux coordonnées au sein d‘un même tissu qu‘entre racines et feuilles. A l‘échelle de la plante greffée, une prépondérance du signal hydraulique est probable. Certains gènes répondent spécifiquement aux interactions greffon/porte-greffe. / In the context of climate change, understanding the mechanisms governing the water loss of the vine is necessary to adapt the plant material to maintain the productivity of the vine and wine quality. The adaptation to drought is a complex trait involving physiological mechanisms related to scion and rootstock genotypes. But the effects of the rootstock on stomatal regulation graft are still unknown. Production by roots of chemical signals such as ABA and / or hydraulic ones be involved. Molecular and physiological responses to water stress were analyzed on young cuttings for 7 rootstocks more or less adapted to drought and 2 varieties known for their iso or anisohydric behaviour. Then 23 combinations scion / rootstock from these genotypes were investigated. Metabolic analyses for ABA and its derivatives was conducted in leaves, roots and in the xylem sap. The information was integrated with transcriptomic analyzes for genes involved in ABA metabolism and signaling, and encoding PIP aquaporins. Joint analyses of physiological data, metabolomic and transcriptomic allow the identification of the molecular components discriminating rootstocks according to their genetic background and their adaptation to drought. Global responses to water stress are better coordinated within the same tissue between roots and leaves. At the scale of the grafted plant, a preponderance of the hydraulic signal is likely. Some genes specifically respond to the scion / rootstock interactions.

Contre la pourriture grise et les maladies du bois (MdBs), qui sont des maladies cryptogamiques majeures de la vigne, la lutte biologique a un potentiel de développement considérable dans le contexte actuel de réduction des intrants chimiques en viticulture.L’objectif de cette thèse est de sélectionner et d'étudier des souches bactériennes antagonistes de Botrytis cinerea (Pourriture grise) et de deux champignons pathogènes clefs liés aux MdBs: Phaeomoniella chlamydospora et Neofusicoccum parvum. Les expériences de screening principales sont réalisées in vivo et in planta sur 46 souches bactériennes isolées dans le vignoble bordelais. Le niveau de protection par les souches antagonistes dépend significativement de la souche bactérienne, de l’espèce de champignon pathogène ciblée, du tissu ou organe végétal hôte, mais aussi pour N. parvum, du mode d’application de la souche bactérienne et, pour B. cinerea, du génotype lié aux transposons : transposa ou vacuma.Une réduction significative de 40 à 64% de la taille des nécroses dues à P. chlamydospora et/ou N. parvum est induite par trois souches bactériennes Pantoea agglomerans (S1), Paenibacillus sp. (S19) et Bacillus pumilus (S32) sur des boutures de vigne non greffées. Ces souches ont fait l'objet d'investigations approfondies pour déterminer leurs principaux modes d’action : Antibiose, production de composés volatils qui ont été identifiés et/ou induction de différents gènes de défense de la vigne.Concernant B. cinerea, les souches Enterobacter cowanii (S22), Enterobacter sp. (S23), Bacillus ginsengihumi (S38) et Bacillus sp. (S43, S46) présentent un pouvoir antagoniste important par production de composés volatils et diffusibles anti-Botrytis, ainsi que par compétition pour les nutriments par E. cowanii (S22). / Biological control of gray mold and grapevine trunk diseases (GTDs), which are major fungal diseases of grapevine, has a considerable potential development in the current context of reduction of chemical input in viticulture.The aim of this study was to select and study bacterial strains for antagonism against Botrytis cinerea, the causal agent of gray mold, and two key pathogens involved in GTDs: Phaeomoniella chlamydospora and Neofusicoccum parvum. The main screening experiments for antagonistic activity of 46 bacterial strains, isolated from Bordeaux vineyards, have been carried out under different in vivo and in planta conditions. The efficacy of protection by the antagonistic strains significantly depended on the bacterial strain, the targeted pathogen species, the host plant tissue or organ and, for N. parvum, also on the application mode of the bacterial strain and, for B. cinerea, on the transposon genotype: transposa or vacuma.A significant reduction in length of necrosis due to P. chlamydospora and/or N. parvum, ranging between 40 and 64% in non-grafted vine cuttings, resulted from three bacterial strains: Pantoea agglomerans (S1), Paenibacillus sp. (S19) and Bacillus pumilus (S32). These strains were thoroughly further investigated to determine their major modes of action by i) Antibiosis ii) production of antifungal volatile organic compounds, which have been identified, and/or iii) induction of different grapevine defense genes. Concerning B. cinerea, Enterobacter cowanii (S22), Enterobacter sp. (S23) Bacillus ginsengihumi (S38), Bacillus sp. (S43, S46) were of prime importance in the biocontrol by producing anti-Botrytis volatile and diffusible compounds or by competing for nutrients (case of E. cowanii S22).

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.

The overall goal of this research was to determine what, if any, role grapevine rhizosphere bacteria play in the differing susceptibilities of New Zealand grown rootstocks to Cylindrocarpon black foot disease. The size and diversity of bacterial populations associated with the rhizospheres of grapevine rootstocks: 101-14, 5C, Schwarzmann and Riparia Gloire were evaluated. Dilution plating showed that total bacterial (P=0.012, P=0.005 for NA and KB, respectively) and fluorescent Pseudomonad (P=0.035) rhizosphere counts differed between rhizosphere and bulk soils but did not correlate with the differing susceptibilities of the rootstock varieties to black foot. No varietal differences were found for spore forming bacteria (P=0.201). SSCP banding patterns showed that species diversity was similar for most rootstocks, but that there were some differences in the composition of bacterial populations, probably attributable to vigour. Some functional characteristics of the bacteria isolated from the rhizospheres of the most and least susceptible rootstock varieties were assessed to investigate their potential to suppress the pathogen. In dual culture, bacteria from Riparia Gloire, 101-14 and the control soil all had little ability to antagonise Cylindrocarpon destructans. However, they differed in their degrees of activity for glucanase (P=0.000), protease (P=0.001) and siderophores (P=0.000). In all tests, bacterial isolates from the rhizosphere of 101-14 had the largest number of active isolates (P≤0.002); however, those from Riparia Gloire had the greatest degree of positive responses for the glucanase and siderophore assays. Bacterial isolates from the control soil produced few glucanases and no siderophores, but had the highest degree of protease activity. Bands excised and sequenced from SSCP gels frequently matched to other ‘uncultured bacteria’ in GenBank, as well as to other bacterial phyla, classes and genera commonly isolated from soil and sediment samples. These included members of the Firmicutes, Proteobacteria (α, δ, γ), Verrucomicrobia, Acidobacteria and Chromatiales. The pathogenicity of C. destructans and Fusarium oxysporum was investigated by inoculating soil containing wounded ungrafted rootstocks of 101-14, 5C, Schwarzmann and Riparia Gloire. Results indicated that F. oxysporum might be a more aggressive pathogen than C. destructans. Inoculation with F. oxysporum or C. destructans increased disease severity, P=0.018 and P=0.056, respectively at 0 cm. Rootstock variety influenced disease severity caused by C. destructans (P<0.001) and F. oxysporum (P=0.090), with rootstocks 101-14 and 5C being most susceptible to C. destructans, and Riparia Gloire and Schwarzmann most susceptible to F. oxysporum. There was also an indication that inoculation with one pathogen increased plant susceptibility to the other, with increased F. oxysporum infection in the C. destructans inoculated treatments of Riparia Gloire and Schwarzmann (P<0.05). The effect of carbohydrate stress (leaf trimming) and inoculation on C. destructans disease severity, incidence, and rootstock rhizosphere bacterial populations was evaluated by inoculating the soil containing one year old plants of Sauvignon Blanc scion wood grafted to rootstocks 101-14 and Schwarzmann. Disease severity and incidence was similar for both Schwarzmann (8.4% and 29.3%, respectively) and 101-14 (14.9% and 31.0%, respectively). When data for the moderate and no stress treatments were combined, because their effects were similar, the disease severity was significantly higher for the highly stressed plants(P=0.043). Stress did not influence disease incidence (P=0.551). Infection occurred in the non-inoculated plants, but disease severity was higher in the plants inoculated with C. destructans than those that were not. Root dry weight of highly stressed plants was lower than in both the moderately stressed (P=0.000) and unstressed plants (P=0.003). An interaction between inoculation and stress (P=0.031) showed that inoculated and highly stressed plants had the lowest root dry weight but there was no effect of rootstocks (P=0.062). There was no significant effect of carbohydrate stress (P=0.259) or inoculation (P=0.885) on shoot dry weight. SSCP banding patterns showed that bacterial diversity was generally similar between rootstocks, but stress and inoculation altered rhizosphere bacterial communities. This study has demonstrated that functionality of grapevine rhizosphere bacteria do differ between grapevine rootstock varieties that have different susceptibilities to black foot disease, but that this role needs to be further investigated if more accurate and practically relevant conclusions are to be drawn.

La sequera és una de les majors limitacions per a l’agricultura en general, factor que pot incrementar d’acord amb les prediccions del canvi climàtic. Per això, reduir l’ús de l’aigua en el reg i augmentar l’eficiència en l’ús de l’aigua (EUA) constitueix una de les majors prioritats per aconseguir una agricultura sostenible. L’EUA és un balanç entre guanys de biomassa i les despeses d’aigua. Aquesta Tesi s’ha centrat en l’estudi de 3 dels processos fisiològics que afecten a l’ús de l’aigua i a la producció de la planta en el cas de la vinya, i que per tant poden ser considerats factors potencials per millorar l’EUA: (1) conductància del mesòfil, (2) transpiració nocturna (3) respiració. Els resultats d’aquesta Tesi revelen que la conductància del mesòfil i la respiració són els principals components per millorar l’EUA permetent millorar l’assimilació de carboni o minimitzant les pèrdues del carboni fixat per la fotosíntesis

Thesis (MSc (Genetics))--Stellenbosch University, 2008. / South Africa is one of the top ten wine producing countries in the world. The South African wine industry contributes approximately R16.3 billion to South Africa’s annual gross domestic product with 42.8% of wine being exported. To compete with the top wine producing countries and to ensure a viable export market, South Africa needs to ensure that healthy, virus free propagation material is produced and sold. One of the viruses that need to be tested for is Grapevine fanleaf virus (GFLV). Grapevine fanleaf virus causes degeneration and malformation of berries, leaves and canes and is responsible for significant economic losses by reducing crop yields by as much as 80%, reducing the longevity of the vines and affecting fruit quality. It is widespread in the Breede River Valley of the Western Cape where the nematode vector, Xiphinema index, is prevalent. The Breede River Valley contributes approximately 30% of the total production of the local wine industry, and severe losses in this region could threaten the viticulture. The Plant Improvement Act states that all propagation material sold must be tested for GFLV by a reputable scientific technique. The technique commonly used in South Africa is the Double Antibody Sandwich - Enzyme-linked Immunosorbent Assay (DAS-ELISA) and the kits are imported from Europe at a significant cost to the South African viticulture industry. The objective of this study was to produce a reliable and sensitive diagnostic assay specific for the South African strains of GFLV. This project aimed to develop and optimize a DAS-ELISA, by using recombinant DNA technology to produce antibodies against bacterially expressed viral coat protein. Total RNA was extracted from GFLV infected grapevine material and the viral coat protein (CP) amplified. The CP was cloned into the pGex-6P-2 expression vector, fusing a Glutathione STransferase (GST) partner to the viral coat protein enhancing solubility and protein purification. Insufficient amounts of the soluble protein were expressed and purified, preventing the production of antibodies and thus the development of the DAS-ELISA. An alternative diagnostic rapid-direct-one-tube-RT-PCR assay was developed. This rapid-directone- tube-RT-PCR assay was compared to commercially available DAS-ELISA and ImmunoStrip tests (Agdia) to assess the reliability, sensitivity and specificity of the rapid-direct-one-tube-RTPCR assay. Twelve GFLV isolates from South Africa were sequenced to investigate the variability between the isolates as well as the variability between the South African isolates and GFLV sequences available in Genbank. Sequence identities between clones from different GFLV isolates from South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the coat protein gene sequences showed that the South African isolates form two distinct clades or sub-populations. No significant correlation was found between geographical origin and symptoms, nor between geographical origin and sequence variability or between grapevine cultivar and symptom expression. Of the 23 samples tested with all three tests, 21 tested positive with rapid-direct-one-tube-RT-PCR, 19 with the ImmunoStrips and 17 with an imported DAS-ELISA kit (Agdia). Rapid-direct-one-tube-RT-PCR was found to be the most reliable technique for GFLV detection. Although the establishment of a DAS-ELISA directed to the South African strain(s) of GFLV was not successful, an alternative PCR based diagnostic system was developed, and proved to be sensitive and reliable. RT-PCR based diagnostic assays are generally accepted to be more sensitive than DAS-ELISA, but the latter is still used as the diagnostic assay of choice for routine testing due to ease of use. This rapid-direct-one-tube-RT-PCR assay is a rapid, sensitive and reliable diagnostic test, reducing the prevalence of false negatives, contributing to a virus free viticulture industry. The rapid-direct-one-tube-RT-PCR assay is as easy to use as DAS-ELISA, faster and can be performed by semi skilled workers, thus providing all the advantages associated with DAS-ELISA.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The first aim of this study was to reliably and rapidly detect Grapevine rupestris stem pittingassociated virus (GRSPaV) in grapevine. This was achieved by screening 94 grapevines using crude plant extracts in both quantitative and conventional reverse transcription polymerase chain reaction (RT-PCR). The second aim was to establish a technique capable of differentiating GRSPaV sequence variants. The application of this technique is for the largescale screening of diseased vines to associate sequence variants of GRSPaV with disease symptoms. Nested quantitative polymerase chain reaction and high resolution melting assays (qPCR-HRM) were developed for three regions of the GRSPaV genome (coat protein, RNAdependant RNA-polymerase and triple gene block movement protein). The qPCR-HRM technique using the high saturation dye, EvaGreen™, and the Rotor-Gene™ 6000 analyzer was validated with a panel of sixteen sequence-characterized viral isolates. Diluted RT-PCR products and cloned cDNA gave the most consistent amplification plots and dissociation profiles. RT-PCR products generated from total RNA extracts were used as template for qPCR-HRM assays and for direct sequencing of sixteen samples in the three aforementioned regions. The average amplification efficiency for qPCR was 1.52±0.04. Auto-calling of userdefine genotypes was performed at a confidence interval of 70%. Phylogenetic analysis of the three regions of the GRSPaV genome was performed with published GenBank sequences to confirm the HRM data. The dominant sequence variants found in the South African sample set radiated with Group II, reference full-length variant GRSPaV-SG1. GRSPaV-infected samples can in future be subjected to qPCR-HRM assays developed during this study. This can be performed to establish similarity to known genotypes and therefore phylogenetic groups. Mixed infection of sequence variants and quasi-species were a common occurrence. The assay will be useful in establishing correlation of specific genotypes to different phenotypical expression of viral disease. This could provide insight into the etiology of diseases associated with GRSPaV. / AFRIKAANSE OPSOMMING: Die eerste doel van hierdie studie was om die virus wat met Rupestris-stamverpitting (Grapevine rupestris stem pitting-associated virus of “GRSPaV”) in wingerd verbind is, vinnig en betroubaar op te spoor. Dit is bereik deur 94 wingerdstokke vir die teenwoordigheid van die virus te toets met beide kwantitatiewe en konvensionele trutranskripsie polimerase kettingreaksies (RT - PCR) vanaf ongesuiwerde plant-ekstraksies. Die tweede doel was die daarstelling van ’n tegniek om onderskeid te tref tussen variante van GRSPaV met verskillende nukleotiedvolgordes. Hierdie tegniek kan op groot skaal gebruik word om ge-affekteerde wingerdstokke te toets om sodoende siektesimptome met spesifieke variante van GRSPaV te verbind. Ge-neste kwantitatiewe polimerase-kettingreaksies (qPCR) en hoë-resolusie smelt-analises (HRM) is ontwikkel vir drie streke van die GRSPaV-genoom (mantelproteïen, RNS-afhanklike RNS-polimerase en trippelgeenblok bewegingsproteïen). Die tegniek van qPCR-HRM met die hoë-versadingingskleurstof EvaGreen™ en die Rotor- Gene™ 6000 ontleder se geldigheid is bevestig deur vergelyking met ’n paneel van sestien virus-isolate waarvan die volgorde reeds bepaal is. Verdunde RT-PCR-produkte en gekloneerde DNS het die mees konsekwente amplifikasie-uitstipping en dissosiasieprofiele opgelewer. RT-PCR-produkte wat vanuit totale RNS-ekstrakte verkry is, is as templaat vir qPCR-HRM-analises gebruik. Dieselfde produkte is ook gebruik, om die volgorde van sestien monsters in drie streke direk te bepaal. Die gemiddelde amplifikasiedoeltreffendheid van die qPCR was 1.52±0.04. Gebruiker-gedefinieerde genotipes is deur middel van outooproeping teen ’n vertroue-interval van 70% uitgevoer. Filogenetiese analises vir drie streke van die GRSPaV-genoom is uitgevoer met gepubliseerde GenBank-volgordes om die HRMdata te bevestig. Die dominante volgorde-variante in die stel Suid-Afrikaanse monsters het ooreengestem met Groep II, vollengte-verwysingsvariant GRSPaV-SG1. Monsters wat met GRSPaV besmet is kan in die toekoms onderwerp word aan die qPCR-HRM-analises wat in hierdie studie ontwikkel is. Dit kan uitgevoer word om ooreenkomste met bekende genotipes te bepaal, en dus ook met filogenetiese groepe. Die besmetting van plante met meer as een volgorde-variant het algemeen voorgekom. Die kwasi-spesies populasie-struktuur van die virus het ook gedurig na vore gekom. Die toets sal nuttig wees in die bepaling van korrelasies tussen spesifieke genotipes en verskillende fenotipiese voorkomste van virussiektes. Dit kan insig verleen in die etiologie van siektes wat met GRSPaV verbind word.

Les effets du changement climatique ont d’ores et déjà été observés sur la vigne : avancement des stades de développement, augmentation des teneurs en alcool des vins, baisse excessive de leur acidité. Une des voies d’adaptation possible de la viticulture est la création de nouvelles variétés. J’ai caractérisé la variabilité phénotypique de 120 descendants de croisements entre Riesling (RI) et Gewurztraminer (GW) pour (1) les stades de développement, décrits à l’aide de sommes de températures (2) la capacité à accumuler des sucres dans les raisins (3) les paramètres de l’acidité des raisins. L’utilisation de marqueurs moléculaires sur l’ADN a permis de mettre en évidence des relations entre données génétiques et phénotypiques pour tous les caractères étudiés (QTLs ou Quantitative trait loci). La principale conclusion est que la variabilité génétique pour les paramètres de l’acidité des raisins est la voie à valoriser en priorité pour l’adaptation de la vigne au changement climatique. / The effects of climate change have already been observed on the grapevine : advance of phenological stages, increase in the alcohol content of the wines, excessive decrease of their acidity. Breeding new varieties is one of the possible means of adaptation. I have characterized the phenotypic variability of 120 genotypes, offspring from crossings between Riesling (RI) and Gewurztraminer (GW)for (1) the developmental stages, described with heat sums (2) the ability to accumulate sugars in the berries (3) the parameters for acidity. The use of DNA molecular markers allowed the detection of quantitative trait loci (QTLs) for all the traits studied. The main conclusion is that the genetic variability for the parameters determining the acidity of the berries is the most promising for the adaptation of grapevine cultivation to climate change.