Eutypa dieback of grapevines, caused by Eutypa lata, is a major cause of reduced longevity in vineyards worldwide. The fungus grows in the woody tissue of infected vines, producing translocatable toxins that cause foliar symptoms of the disease. By the time foliar symptoms are evident the pathogen may have become well established in the vine. One aim of this study was to develop DNA markers to allow rapid reliable identification of E. lata and to detect the pathogen in infected wood. The second aim was to analyse secondary metabolite production by E. lata in order to gain information on the compounds responsible for the foliar symptoms of the disease and to identify metabolites which could be used as markers to detect the early stages of the disease prior to the expression of foliar symptoms. In addition, genetic variation of the pathogen was assessed using RFLP and RAPD analysis. Two techniques were used to develop DNA markers; first, SCAR markers derived from RAPD fragments were developed and, second, an E. lata genomic DNA library was constructed, from which DNA fragments specific to E. lata were identified. These markers were used in either PCR- or Southern hybridisation-based assays to detect the pathogen in infected wood. PCR-based detection of the pathogen in infected wood was prone to inhibition by phenolic compounds, however, Southern hybridisation techniques were capable of detecting E. lata in wood. Genetic variation among 38 isolates of E. lata was assessed using six randomly selected clones from the genomic DNA library. A subset of 11 isolates was subjected to RAPD analysis using 10 random primers. Considerable genetic diversity, in terms of RFLP and RAPD profiles, was observed among isolates. There was no apparent correlation between grouping of isolates following neighbour joining analysis and either host species or geographic origin of isolates. The RAPD and RFLP profiles of two isolates differed significantly from the majority of the other isolates. These isolates, which were morphologically similar to all other isolates, were subsequently found not to be E. lata. Secondary metabolite production of 11 isolates was analysed by HPLC following growth on a range of media. A wider range of secondary metabolites was detected in E. lata than has previously been reported. Two of the secondary metabolites, eutypine and an unidentified compound with a retention time of 19.6 min, were produced by eight of nine isolates of E. lata. Neither of the non-E. lata isolates produced these compounds. It was concluded that the remaining isolate of E. lata may have lost the ability to produce these compounds following storage. Whilst a wider range of isolates needs to be screened before a candidate marker can be selected, these results suggest that certain compounds are present in the majority of E. lata isolates and, hence, may prove suitable markers for the detection of the pathogen prior to the expression of foliar symptoms. The molecular probes developed in this study will allow the rapid and reliable identification and detection of E. lata in grapevine cane or wood. These probes also have the potential to be used as a research tool to gather information on the epidemiology of the disease and to assess the efficacy of potential control agents against E. lata. Suitable control measures could then be applied to vines which have been shown by the use of chemical markers to have latent infection. Used in combination, therefore, the DNA and biochemical markers could facilitate improved management of eutypa dieback. / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2003.

Eutypa dieback of grapevines, caused by Eutypa lata, is a major cause of reduced longevity in vineyards worldwide. The fungus grows in the woody tissue of infected vines, producing translocatable toxins that cause foliar symptoms of the disease. By the time foliar symptoms are evident the pathogen may have become well established in the vine. One aim of this study was to develop DNA markers to allow rapid reliable identification of E. lata and to detect the pathogen in infected wood. The second aim was to analyse secondary metabolite production by E. lata in order to gain information on the compounds responsible for the foliar symptoms of the disease and to identify metabolites which could be used as markers to detect the early stages of the disease prior to the expression of foliar symptoms. In addition, genetic variation of the pathogen was assessed using RFLP and RAPD analysis. Two techniques were used to develop DNA markers; first, SCAR markers derived from RAPD fragments were developed and, second, an E. lata genomic DNA library was constructed, from which DNA fragments specific to E. lata were identified. These markers were used in either PCR- or Southern hybridisation-based assays to detect the pathogen in infected wood. PCR-based detection of the pathogen in infected wood was prone to inhibition by phenolic compounds, however, Southern hybridisation techniques were capable of detecting E. lata in wood. Genetic variation among 38 isolates of E. lata was assessed using six randomly selected clones from the genomic DNA library. A subset of 11 isolates was subjected to RAPD analysis using 10 random primers. Considerable genetic diversity, in terms of RFLP and RAPD profiles, was observed among isolates. There was no apparent correlation between grouping of isolates following neighbour joining analysis and either host species or geographic origin of isolates. The RAPD and RFLP profiles of two isolates differed significantly from the majority of the other isolates. These isolates, which were morphologically similar to all other isolates, were subsequently found not to be E. lata. Secondary metabolite production of 11 isolates was analysed by HPLC following growth on a range of media. A wider range of secondary metabolites was detected in E. lata than has previously been reported. Two of the secondary metabolites, eutypine and an unidentified compound with a retention time of 19.6 min, were produced by eight of nine isolates of E. lata. Neither of the non-E. lata isolates produced these compounds. It was concluded that the remaining isolate of E. lata may have lost the ability to produce these compounds following storage. Whilst a wider range of isolates needs to be screened before a candidate marker can be selected, these results suggest that certain compounds are present in the majority of E. lata isolates and, hence, may prove suitable markers for the detection of the pathogen prior to the expression of foliar symptoms. The molecular probes developed in this study will allow the rapid and reliable identification and detection of E. lata in grapevine cane or wood. These probes also have the potential to be used as a research tool to gather information on the epidemiology of the disease and to assess the efficacy of potential control agents against E. lata. Suitable control measures could then be applied to vines which have been shown by the use of chemical markers to have latent infection. Used in combination, therefore, the DNA and biochemical markers could facilitate improved management of eutypa dieback. / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2003.

Les maladies du bois de la vigne, causées par des champignons nécrotrophes, ont un impact considérable sur l'économie viticole au niveau mondial. En effet, tous les cépages Vitis vinifera cultivés actuellement présentent une sensibilité plus ou moins forte à ces champignons.Dans le but d'établir un test rapide d'évaluation de la sensibilité de clones de vigne (nouvellement sélectionnés ou futures obtentions variétales) à Eutypa lata, le champignon responsable de l'Eutypiose, une recherche de marqueurs moléculaires de tolérance a été réalisée. Suite à l'infection in vivo et in vitro d'une douzaine de cépages de sensibilité différente par E. lata, plusieurs gènes candidats ont été identifiés comme marqueurs potentiels de la tolérance à la maladie à partir d'une étude transcriptomique.A l'aide d'un système innovant d'infection in vitro, le dialogue moléculaire sans contact physique entre des disques foliaires de V. vinifera et le mycélium d’E. lata a également été étudié chez les douze cépages. Cette étude a permis de mettre en évidence le rôle potentiel d'éliciteurs dans la mise en place des réponses de défenses. De plus, l'infection par E. lata régule différentiellement l'expression des gènes codant pour un transporteur d'hexoses et des invertases, ainsi que les activités invertasiques associées, chez les cépages de sensibilités variables.Les travaux de recherche présentées dans cette thèse ont ainsi permis l'identification des marqueurs de tolérance à l'Eutypiose et la mise au point d'un test d'infection in vitro efficace et fiable, permettant de diagnostiquer la sensibilité des futures créations variétales. De plus les résultats obtenus démontrent l'importance des éliciteurs dans la mise en place des défenses, de la régulation du transport et du métabolisme des sucres au cours de l'infection par E. lata. / Nowadays, grapevine wood decay diseases cause significant economic losses for the most sensitive varieties and represent a threat to the sustainability of the wine industry.This research focuses on the identification of molecular markers for sensitivity to Eutypa lata, responsible for Eutypiosis that could be used to diagnose the sensitivity of new grapevine clones or cultivars. Using an in vivo and in vitro infection assay, several potential markers genes for tolerance to Eutypiosis have been identified from a gene expression study on twelve different cultivars.Using an innovative in vitro infection system, the molecular dialogue (without physical contact) between Vitis vinfera foliars discs of and Eutypa lata was studied, leading to the identification of elicitors that could potentially play a role in the induction of defense responses in the cultivars. In addition, the expression of several sugar transport and invertase genes and their associated activities were demonstrated to be differentially regulated by the infection in tolerant and susceptible cultivars.Altogether, this research work led to the identification of several tolerance markers genes to Eutypiosis and to the development of a new, efficient and reliable in vitro infection system that could be used to diagnose new cultivar susceptibility. Furthermore, the results obtained demonstrated the importance of sugar transport and defense metabolism regulation during the infection of grapevine by Eutypa lata.

L'agriculture a besoin de fongicides ayant la propriété d'être transportés par le phloème pour contrôler des pathogènes vasculaires et racinaires. Jusqu'à présent, les tentatives de modulation structurale de fongicides pour les rendre phloème mobiles ont généralement eu comme conséquence une perte d'activité biologique.<br />La première approche de notre travail reprend la stratégie utilisée avec succès pour le développement d'herbicides auxiniques. Nous avons choisi comme molécule modèle le fenpiclonil en raison de la possibilité d'ajouter un groupe acide carboxylique en divers sites de la molécule. L'un de ces dérivés, le N-(1-carboxyéthyl)-3-cyano-4-(2,3-dichlorophényl)pyrrole possède une mobilité phloémienne modérée et montre une activité fongicide contre une souche d'Eutypa lata comparable à celle du fenpiclonil.<br />La deuxième stratégie a consisté à synthétiser des propesticides mobiles dans le phloème en greffant un acide aminé à divers xénobiontes ou à des composés naturels impliqués dans la défense des plantes. Ces conjugués avec une fonction a-aminoacide sont manipulés par des perméases de la membrane plasmique et sont nettement phloème mobiles. Toutefois, les enzymes qui peuvent libérer le composé initial peuvent être plus spécifiques que le système de transport.<br />Enfin, la capacité du phloème de ricin à charger l'acide salicylique (AS) a été étudiée pour deux raisons : 1- l'AS est une molécule signal importante impliquée dans la résistance systémique acquise 2- dans les cellules animales, l'AS est manipulé par un système de transport qui manipule aussi des médicaments de taille importante. L'AS s'accumule fortement dans le phloème et est ambimobile. Quelques résultats conduisent à l'hypothèse de l'intervention d'un système de transporteurs dépendant du pH et contribuant au chargement phloémien de l'AS, outre le phénomène de piégeage d'acide.

L'esca est un syndrome cryptogamique vasculaire de la vigne (Vitis vinifera). Une phase pionnière, sous la dépendance de Phaeoacremonium aleophilum (PA), Phaeomoniella chlamydospora (PC) et éventuellement de Eutypa lata (EL) permet la mise en place d'autres espèces. Depuis l'interdiction de l'arsénite de sodium, cette maladie n'est plus contrôlée.<br /> Nous avons étudié les propriétés de PA, PC et EL, en particulier l'impact sur leur croissance de fongicides commerciaux et de fongicides systémiques synthétisés au laboratoire, ainsi que de molécules naturelles. Ces pathogènes ne présentent pas la même sensibilité à ces molécules et l'un d'eux (PA) est peu affecté par divers traitements. En parallèle, nous avons étudié les propriétés d'ambimobilité de l'acide salicylique (AS) et de quelques-uns de ses dérivés halogénés.<br /> Nous avons montré que F 30, un dérivé acide du fenpiclonil, est mobile dans les boutures de vigne après application foliaire. Il est en partie retenu dans le bois et libère la molécule parent dans les racines. L'acide 5-chlorosalicylique (5-ClAS), connu pour être plus actif que AS pour stimuler les défenses naturelles, présente une mobilité voisine de celle de AS. Sur ces bases, F 30 et 5-ClAS ont été retenus pour des tests préliminaires de traitement par voie foliaire de boutures de vigne infectées.<br /> Cette recherche exploratoire souligne la complexité de la problématique, une lutte chimique (fongicide), génératrice de contraintes, devant s'intégrer dans une stratégie globale de contrôle.