Thesis (MScAgric (Plant Pathology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: A survey was undertaken on apple and pear trees in the Western Cape Province to determine the aetiology of trunk diseases with reference to trunk diseases occurring on grapevine. Grapevine trunk diseases cause the gradual decline and dieback of vines resulting in a decrease in the vine’s capability to carry and ripen fruit. In recent years, viticulture has been expanding into several of the well established pome fruit growing areas. The presence of trunk pathogens in pome fruit orchards may affect the health of the pome fruit trees as well as cause a threat to young vineyards planted in close proximity to these potential sources of viable inoculum. Several genera containing species known to be involved in trunk disease on pome fruit and grapevine were found, including Diplodia, Neofusicoccum, Eutypa, Phaeoacremonium and Phomopsis. Diplodia seriata and D. pyricolum, were isolated along with N. australe and N. vitifusiforme. Four Phaeoacremonium species, P. aleophilum, P. iranianum, P. mortoniae and P. viticola, two Phomopsis species linked to clades identified in former studies as Phomopsis sp. 1 and Phomopsis sp. 7, and Eutypa lata were found. In addition, Paraconiothyrium brasiliense and Pa. variabile, and an unidentified Pyrenochaetalike species were found. Of these the Phaeoacremonium species have not been found on pear wood and it is a first report of P. aleophilum occurring on apple. This is also a first report of the Phomopsis species and Eutypa lata found occurring on pome trees in South Africa Two new coelomycetous fungi were also found including a Diplodia species, Diplodia pyricolum sp. nov., and a new genus, Pyrenochaetoides gen. nov. with the type species, Pyrenochaetoides mali sp. nov., were described from necrotic pear and apple wood. The combined ITS and EF1-α phylogeny supported the new Diplodia species, which is closely related to D. mutila and D. africana. The new species is characterised by conidia that become pigmented and 1-septate within the pycnidium, and that are intermediate in size between the latter two Diplodia species. Phylogenetic inference of the SSU of the unknown coelomycete provided bootstrap support (100%) for a monophyletic clade unrelated to known genera, and basal to Phoma and its relatives. Morphologically the new genus is characterised by pycnidial with elongated necks that lack setae, cylindrical conidiophores that are seldomly branched at the base, and Phoma-like conidia. The phylogenetic results combined with its dissimilarity from genera allied to Phoma, lead to the conclusion that this species represents a new genus. A pathogenicity trial was undertaken to examine the role of these species on apple, pear and grapevine shoots. N. australe caused the longest lesions on grapevine shoots, while Pyrenochaetoides mali, Pa. variabile, D. seriata and P. mortoniae caused lesions that were significantly longer than the control inoculations. On pears, D. pyricolum and N. australe caused the longest lesions, followed by D. seriata and E. lata. On apples, the longest lesions were caused by N. australe and P. iranianum. D. seriata, D. pyricolum, E. lata, N. vitifusiforme, Pa. brasiliense, P. aleophilum and P. mortoniae also caused lesions on apple that were significantly longer than the control. The study demonstrated that close cultivation of grapevine to apple and pear orchards may have inherent risks in terms of the free availability of viable inoculum of trunk disease pathogens. / No Afrikaans abstract available.

Les maladies fongiques du bois de la vigne que sont le syndrome de l'esca, le Black Dead Arm (BDA) et l'Eutypiose sont particulièrement dommageables à la profession vitivinicole, et sont actuellement en progression. Le temps d'incubation nécessaire à l'expression de ces maladies au champ complique l'évaluation de solutions préventives adaptées en condition contrôlée ainsi qu'en condition de terrain. Ces travaux de thèse ont eu pour objectifs la conception et la validation de tests PCR quantitatifs en temps réel (RtqPCR), permettant la détection et la quantification in planta de cinq champignons associés aux maladies de dépérissement de la vigne, Phaeomoniella chlamydospora et Phaeoacremonium aleophilum (esca), Diplodia seriata et Neofusicoccum parvum (BDA), et Eutypa lata (Eutypiose). Le développement de tests multiplexes a ensuite été entrepris et ces derniers ont été évalués pour la détection de quatre champignons (2 associés à esca et 2 au BDA) dans le bois de jeunes plants issus de pépinière viticole. Enfin, l'étude de l'interaction in planta de deux champignons associés au syndrome de l'esca de la vigne (P.chlamydospora et P.aleophilum) a été réalisée par RT-qPCR, et complétée par la caractérisation histologique de la réponse de la plante à la blessure dans le bois, en inoculation individuelle et en co-inoculation. / Grapevine trunk diseases, among which esca's syndrome, Black Dead Arm (BDA) and Eutypiosis, represent a real threat for grape and wine industry. Incubation time required before symptoms externalization in field complicates the evaluation of the efficacy of preventive solution in control and field conditions. These thesis's works focused on the design and the validation of Real Time quantitative PCR assays (RT-qPCR), in order to detect and quantify in vine plant five fungi associated with grapevine trunk disease, Phaeomoniella chlamydospora et Phaeoacremonium aleophilum (esca), Diplodia seriata et Neofusicoccum parvum (BDA), et Eutypa lata (Eutypiosis). The development of multiplex assays was undertaken and these last were evaluated in order to detect four fungi (esca and BDA) in wood sample from young plants in vine nursery. Finally, a study of the interaction between two fungi associated with esca's syndrome has been determined in planta through RT-qPCR, and completed by a histological analysis of plant response to injury of woody tissues.

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

Cette thèse aborde le potentiel de la télédétection en tant qu'outil pour la détection automatique de la Flavescence dorée (FD) de la vigne. L'approche repose sur l'analyse des variables (bandes spectrales, indices de végétation et paramètres biophysiques) calculables à partir des images multispectrales à très haute résolution (10 cm) acquises par drone pendant la période d'expression maximal des symptômes. L'analyse de la performance de discrimination des variables est réalisée à partir d'une méthode supervisée basée sur la courbe ROC. Les zones d'entrainement et de validation utilisées dans cette étude ont été acquises sur 14 parcelles situés dans le sud de la France. La performance des variables a été testée sur trois échelles d'analyse (par parcelle, par cépage et par couleur) et pour deux niveaux d'analyse. Le premier niveau d'analyse repose sur le potentiel des variables utilisées dans la détection des zones symptomatiques de la Flavescence dorée des zones asymptomatiques. Le deuxième niveau d'analyse consiste à tester la performance des variables dans la discrimination spécifique la Flavescence dorée (cépages noirs) en faisant une distinction avec les maladies du bois. À l'issue de ces expériences, dans un point de vue méthodologique les résultats ont permis de mettre en évidence (1) une plus faible performance de discrimination pour la discrimination des zones symptomatiques de FD des zones symptomatiques des maladies du bois plus particulièrement à l'échelle par couleur ; (2) la présence des pixels mixtes mal classés plus particulièrement dans les bords des rangs de vigne et (3) une faible discrimination des zones symptomatiques (FD ou MB) avec une proportion du feuillage symptomatique faible (niveau d'infection). Dans un point de vue thématique les résultats obtenus ont mis en évidence les différences dans l'intensité de la coloration anormale des feuilles atteintes de Flavescence dorée en fonction de l'année et leur lien avec la teneur en chlorophylles et anthocyanes des feuilles. Les perspectives ouvertes par ces travaux concernent la création d'un indice spécifique à la Flavescence dorée en fonction de la couleur du cépage (noir ou blanc) ou l'intensité dans la coloration des feuilles (faible ou forte) identifiés à partir des données hyperspectrales et l'amélioration du masquage des pixels mixtes à partir des algorithmes complexes qui prennent en compte la répartition spatiale des pixels dans le feuillage de vigne. / This work investigates the potential of remote sensing as a tool for the automatic detection of Flavescence dorée (FD) grapevine disease. The approach is based on the analysis of variables (spectral bands, vegetation indices, and biophysical parameters) computed from high resolution (10 cm) multispectral images and acquired by Unmanned Aerial Vehicle (UAV) during the period of maximum expression of symptom. The analysis of the variables discrimination performance is evaluated by a supervised method based on the Receiver Operating Characteristic curve (ROC curve). The training and validation areas used in this study were acquired from 14 vineyards located in southern France. The performance of the variables was tested on three different scales of analysis (one by plot, by cultivar and by berry color). Two levels of analysis have been implemented. The first level involves the potential of variables to discriminate Flavescence dorée symptomatic vines areas from asymptomatic ones. The second level of analysis is related to test the performance of the variables for the specific discrimination of Flavescence dorée vines (for the red cultivars) and the discrimination from Grapevine Trunk Diseases (GTD). The results obtained showed (1) a lower discrimination performance for discrimination of FD symptomatic vines areas from GTD symptomatic ones, more pronounced on the color level; (2) the presence of misclassified mixed pixels especially in the edges of the rows of vines and (3) a low discrimination of symptomatic vines areas (FD or MB) with a low proportion of symptomatic foliage (level of infection). From a thematic point of view, the results obtained showed the differences in the intensity of leaf discoloration affected by Flavescence dorée by year and their link with the chlorophylls and anthocyanins content of the leaves. Future prospects for this work concern the creation of a specific Flavescence dorée index depending on the color of the cultivars (red or white) and the intensity of leaf discoloration (attenuated or marked), identified from the hyperspectral data and improving the masking of mixed pixels from complex algorithms that consider the spatial distribution of pixels in the vine foliage.

Eutypa dieback, one of several grapevine trunk diseases (GTDs), is of serious concern to the grape industry globally. This disease is caused by the fungus Eutypa lata but it is often seen in consortia growth with Phaeoacremonium minimum and Phaeomoniella chlamydospora. It is vital to understand the mechanisms for how this disease functions to develop control measures to combat it. Brown rot fungi are able to use a complex of low molecular weight (LMW) metabolites to induce a Fenton reaction to deconstruct woody tissue. These metabolites are part of a chelator mediated Fenton (CMF) chemistry that produces reactive oxygen species that are capable of depolymerizing wood polymers. We propose that a mechanism similar to CMF chemistry may be occurring in grapevine trunk disease pathogens. This thesis investigates how LMW metabolites produced by the fungi contribute to the disease and decay progression in GTDs. Research on Mite control in the laboratory with abamectin was also investigated, as research in this area was required when mites infested our fungal cultures and suitable laboratory controls were not available. Research on the GTD fungi was initiated by first examining whether metabolites produced by the three fungi can function in a manner to promote reactions like the CMF system. We separated and identified specific metabolites that potentially could contribute to CMF chemistry. We found that all three GTD fungi were able to produced LMW metabolites that promoted CMF chemistry, and we hypothesized that this mechanism contributes to processes leading to tissue necrosis in grapevine trunk wood. To explore the development of effective control measures based on this newly discovered mechanism for pathogenesis, we also explored the use of antioxidant/chelator compounds, BHA and BHT, in the control of the consortia fungi. Biocontrol organisms, Bacillus subtilis and Trichoderma atroviride, that produce antioxidants were also tested as biocontrols against the fungi involved in Eutypa Dieback disease. We found that BHA was highly effective in inhibiting fungal growth for all three fungi at concentrations higher than 0.5mM, and both B. subtilis and T. atroviride proved to be effective biocontrol agents in inhibiting E. lata, P. minimum, and P. chlamydospora.