Eight phylloplane yeasts were isolated from backyard apple trees in Corvallis, OR. Yeast isolates were classified to genus or species level. All isolates were tested in vitro for antagonistic activity against the postharvest pathogens Botrytis cinerea and Penicillium expansum. Of these isolates, Aureobasidium pullulans, Sporobolomyces roseus Rhodotorula sp., consistently reduced mycelial growth of B. cinerea and P. expansum in nutrient yeast dextrose agar (pH 4.5 or 7.0) incubated for 8 or 30 days at 24 or 1 C, respectively. These three yeasts also were evaluated for their ability to suppress spore germination of B. cinerea and P. expansum in a gradient of apple juice concentrations and to suppress development of gray and blue mold lesions in inoculated fruits of Golden Delicious apple. Germination of B. cinerea and P. expansum was reduced significantly (P���0.05) when incubated with the yeast isolates in 100 or 50% apple juice, but not in 0, 1 or 10% apple juice. S. roseus and A. pullulans reduced significantly (P���0.05) the size of gray mold lesions in wounded fruit stored at 5 C and 24 C by 63 to 72 and 81 to 90%, respectively, when compared to the nontreated control. Size of blue mold lesions in fruit stored at 5 and 24 C also were reduced significantly (P���0.05) by 66 to 38 and 74 to 63%, respectively, when pre-treated with S. roseus and A. pullulans. In general, fruit rot suppression by some yeasts isolated in this study was similar in magnitude to suppression obtained by Cryptococcus laurentii isolate 87-108, a yeast with commercial potential to suppress postharvest rots of pome fruits. Pretreatment of apple wounds with washed cells of A. pullulans, S. roseus, Rhodotorula sp., resulted in disease suppression, but treatment of wounds with cell-free culture supernatant of these isolates did not affect lesion development. Population size of A. pullulans, S. roseus, and C. laurentii increased in apple wounds incubated at 5 or 24 C for up to 25 days, indicating that they colonized the wound site. Data collected in this study support the hypothesis that yeast isolates antagonize fruit pathogens by competing for nutrients in wounds on fruit surfaces. The isolates of A. pullulans and S. roseus show promise for commercial development. / Graduation date: 1997

Hormesis is a dose response phenomenon where low doses of a stress bring about a positive response in the organism undergoing treatment. UV-C hormesis has been known for over three decades and has a broad range of benefits on postharvest produce. Benefits include increased nutritional content, delayed chlorophyll degradation and disease resistance. The beneficial effects have been observed on many varieties of fresh produce including climacteric and non-climacteric fruit, tubers, salads and brassicas. The majority of previous studies have used low-intensity (LIUV) UV-C sources. LIUV sources require lengthy treatment times, which are in the region of 6 minutes for tomato fruit. This has, in part, prevented the commercial application of this technique. High-intensity, pulsed polychromatic light (HIPPL) sources, however, have recently been developed. HIPPL sources may have the potential to drastically reduce treatment times and increase their commercial viability. It was shown, here, that the use of HIPPL can control disease (reduce disease progression) caused by Botrytis cinerea and Penicillium expansum and also delay ripening on tomato fruit. Both disease control and delayed ripening were at similar levels for LIUV and HIPPL treatments on mature green fruit. The HIPPL treatments used in these studies can reduce treatment times for tomato fruit by 97.3%. Both HIPPL and LIUV treatments elicit local responses irrespective of the treatment orientation and tomato fruit, therefore, require full surface irradiation. Furthermore, UV-C in the HIPPL source is not required for disease control or delayed ripening. It does, however, contribute approximately 50% towards the total observed effects. Investigations into the mechanisms underpinning postharvest HIPPL and LIUV hormesis, on tomato fruit, identified that the expression of genes involved in plant hormone biosynthesis, defence, secondary metabolism and ripening were affected. This indicates that disease control is achieved through induced resistance. Changes to expression, following treatment, were highly similar for both HIPPL and LIUV treatments and were mediated by salicylic acid, jasmonic acid and ethylene. This may lead to broad range resistance against necrotrophic and biotrophic pathogens as well as abiotic stresses and herbivorous pests. Recently, the exposure of foliage to UV-C has been shown to induce resistance against B. cinerea on Arabidopsis thaliana. The horticultural applications of such treatments, however, have not been explored. Pre-harvest treatments of lettuce in the glasshouse showed variation in damage threshold and optimal treatment to control disease following LIUV and HIPPL treatment. Further sources of variation included the cultivar, pathogen of interest and the point that treatment was applied during the year. Using a controlled environment allowed seasonal variation to be mitigated and both HIPPL and LIUV treatments controlled disease against B. cinerea. For pre-harvest treatments to be a success in the glasshouse, further studies into how both biotic and abiotic factors influence treatment is required. To circumvent the problems associated with pre-harvest treatments and environmental variation in the glasshouse, LIUV seed treatments were performed on tomato. Control of B. cinerea was established with an approximately 10% reduction in incidence and disease progression with a 4 kJ/m2 treatment. When monitoring the effect of treatment on germination and early seedling development it was also identified that an 8 kJ/m2 treatment led to biostimulation of germination and root and shoot growth.

<p>The management of gray mold, caused by the fungus <em>Botrytis cinerea</em>, on ornamental plants relies heavily on fungicide applications. To improve fungicide efficacy, the manipulation of nozzle type, spray volume, and pressure influence droplet size (µm) and density (droplets/cm2) on the leaf’s surface. However, leaf wettability dictates how well the application droplets adhere and spread across the surface. When leaf surfaces are waxy (hydrophobic) or hairy (tomentose), droplets fail to adhere, impacting fungicide sorption.</p> <p>The goal of this research was to evaluate how the interaction of droplet density and leaf wettability impact the efficacy of chemical and biological fungicides against <em>Botrytis cinerea</em>. Leaf surfaces vary between species, within species, leaf age, and leaf sides (abaxial or adaxial). Hydrophobic leaf surfaces influence fungicide efficacy by reducing fungicide droplet spread compared to the wettable and hydrophilic leaf surfaces. The presence of trichomes on the leaf surface can inhibit droplets from reaching the surface.</p> <p>To quantify droplet density, a fine and coarse spray of fungicide treatments was applied with a yellow fluorescent dye. After application, <em>Begonia</em> x <em>hybrida</em> ‘Dragon wing’ leaves were placed on black, blackout curtains below a blacklight. Images were analyzed by ImageJ, using an image processing method. The number of lesions, disease incidence, were counted to observe fungicide efficacy. Results show there was no interaction between the actual droplet density within treatments applied with fine and coarse sprays. However, the interaction between spray type (fine and coarse) and treatments can have a significant effect on disease incidence. Disease incidence was significantly different between the systemic and contact fungicides for fine and coarse sprays. However, the systemic fungicide treatment had the highest disease incidence compared to the contact fungicide.</p> <p>To assess leaf wettability impact on fungicide efficacy, five <em>Begonia </em>species (<em>B. scharffii, B. erythrophylla, B. </em>x<em> hybrida ‘</em>Dragon Wing’<em>, B. epipsila, and B. goldingiana</em>) were used based on their observed leaf surface type. A contact angle goniometer was used to take pictures of a droplet on <em>Begonia</em> leaf surfaces. The quantification of the leaf surface took place by using the ImageJ program ‘Drop-Snake’ within the plugin ‘Drop Analysis’. The number of lesions, an indicator of disease incidence, were counted to observe fungicide efficacy. Results showed the contact angles were different between the <em>Begonia</em> species. There was a significant interaction between the <em>Begonia</em> species and treatments, where <em>Begonia</em> ‘hairy’ and ‘waxy’ leaf surfaces can influence fungicide efficacy. However, there was no significance for the interaction between <em>Begonia</em> species’ contact angles and treatments.</p> <p>These studies advance our understanding of how droplet density and leaf surfaces influence fungicide efficacy, thus improving our ability to manage <em>Botrytis</em> for diverse ornamental plants. </p>

The primary objectives of this research are to determine effective biological and alternative control strategies of insect and disease pests in order to reduce harsh chemical use during greenhouse crop production and transport s. This research includes two separate studies: 1) testing the practical viability of rearing and storing four species of entomopathogenic nematode (EPN), Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora, Heterorhabditis indica; and, 2) the efficacy of UV-C radiation applied, pre-transport, as a preventative disease control strategy against Botrytis cinerea. A study was conducted testing EPN infectious juvenile (IJ) rearing production counts and IJ viability after a six-day storage period. When all four species are compared, S. feltiae had a greater number of infectious juveniles emerge from the wax moth cadavers and S. carpocasae had the least. All four species survived the six day storage period but EPN infectious juvenile counts were significantly different among species. Our second study tested the efficacy of UV-C radiation as an alternative control to traditional fungicides to deactivate B. cinerea in vitro and to determine plant tolerance to UV-C. The crops tested were poinsettia (Euphorbia pulcherrima) and primula (Primula vulgaris). All the UV-C doses, 1.0, 2.8, 3.7 or 4 W/m2, significantly decreased B. cinerea conidial germination in vitro and resulted in zero percent damage on poinsettia bracts. However, all UV-C doses during both replications caused minor damage, 15% or less, to primula flowers. / Master of Science in Life Sciences / Entomopathogenic nematodes (EPN) shows promise in being non-chemical and environmentally friendly solution for greenhouse pest and disease control. These can also be referred to as Biological Controls (Biocontrols). Entomopathogenic nematodes are used widely to control multiple greenhouse plant pests which include both Lycoriella spp., Fungus Gnats, and Frankliniella spp., Western Flower Thrips. However, there are challenges with EPN viability and storage from the manufacture to the greenhouse producer. We studied four EPN species, Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora, Heterorhabditis indica, which were reared and stored to determine differences in production viability between species. Results show that the EPN species do not respond the same to storage and produce different amounts of infectious juveniles during rearing when conditions are the same. Separate from, but just as concerning as greenhouses plant pests are plant diseases. Ultraviolet radiation in the C spectra is known to be germicidal due to its narrow wavelengths. Because of this, UV-C has been shown to deactivate many different plant pathogens on contact and is being considered as a possible Biocontrol alternative to harsh traditional fungicides and bactericides. One disease that is known to contribute to the highest volume of annual crop losses is Botrytis cinerea. Botrytis cinerea is a plant disease that impacts floricultural crops to vegetables during propagation through the production supply chain to shipping and storage. We evaluated UV-C radiation at different doses, to determine if it could be used to replace a traditional fungicide before plants are shipped to reduce B. cinerea infection during transport. We found that UV-C successfully deactivated B. cinerea in vitro, but the viability of the application to plant tissue before transport has yet to be proven successful as a practical method of reducing B. cinerea during transport.

Cette étude s’inscrit dans le cadre d’un programme de recherche destiné à évaluer le potentiel d’utilisation en horticulture des bactéries Bacillus pumilus souche PTB180 et Bacillus subtilis souche PTB185. Elle avait pour objectifs (1) d’évaluer in vitro l’activité antagoniste contre Botrytis cinerea des souches PTB180 et PTB185, (2) d’estimer leur capacité à survivre sur la phyllosphère de la tomate et du concombre et 3) d’évaluer leur effet sur le développement de la pourriture grise (B. cinerea) sur des plants de tomate et de concombre cultivés en serre. L'activité antagoniste de PTB180 et PTB185 a été évaluée en boîtes de Pétri sur géloses, sur tissus foliaires de tomate et de concombre et sur fruits de tomate. Les deux souches ont inhibé très fortement la croissance mycélienne et la germination des spores de B. cinerea sur géloses. Sur feuilles de tomate et sur disques foliaires de concombre, PTB185 et le mélange (1:1) des deux souches ont réduit significativement (p ≤ 0,01) la croissance mycélienne de B. cinerea comparativement aux témoins. PTB180 a réprimé significativement la croissance mycélienne de B. cinerea sur les fruits de tomate. Afin d'estimer la survie de PTB180 et PTB185 sur la phyllosphère, des plants de tomate et de concombre ont été pulvérisés jusqu'à ruissellement avec une suspension (1×107 unités formatrices de colonies [UFC]/mL) de PTB180, PTB185 ou d'un mélange (1:1) des deux souches. Les populations de chaque souche ont ensuite été suivies au cours du temps sur les feuilles. Les résultats obtenus montrent que les souches survivent au moins 21 jours sur les plants de tomate et de concombre avec un taux de survie variant de 43% à 100%. De plus, pratiquement aucune variation dans les proportions de chaque souche n'a été observée au fil du temps lorsque PTB180 et PTB185 étaient appliquées en mélange. Enfin, l’application foliaire de PTB180, PTB185 et du mélange (1:1) des deux souches a permis une réduction significative de l’incidence et de la sévérité de la pourriture grise chez des plants de tomate et de concombre inoculés avec B. cinerea et cultivés en serre. Les souches PTB180 et PTB185 ont montré au cours de cette étude une forte activité antagoniste envers B. cinerea, la capacité de survivre sur la phyllosphère de plants de tomate et de concombre et de réprimer le développement de la pourriture grise chez ces derniers. Ces souches pourraient éventuellement être utilisées comme agents de lutte biologique contre la pourriture grise du concombre et de la tomate de serre.

La lutte chimique est la principale méthode utilisée pour contrôler les maladies causées par les champignons phytopathogènes. Dans certains cas, desphénomènes de résistance envers les fongicides se développent au sein despopulations, altérant parfois l’efficacité des molécules. La compréhension du moded’action des fongicides et des mécanismes de résistance sous-jacents participe à élaboreret à adapter des stratégies de management anti résistance ; et ainsi permettre depérenniser la durée de vie des molécules. Le fenhexamid est un fongicide récent (BayerCropScience, 2000), avec un mode d’action unique. Il est le seul fongicide commercialisébloquant l’étape de C4-déméthylation de la biosynthèse de l’ergostérol. Plusieurs typesde résistance (naturelle et acquises) ont été détectées dans les vignobles européens chez lecomplexe d’espèces Botrytis spp. responsable de la pourriture grise de la vigne. Lestravaux développés durant la thèse s’inscrivent dans l’objectif de la caractérisation dumode d’action et de l’élucidation des mécanismes de résistance. Le premier axe s’estattaché à la caractérisation fonctionnelle de deux gènes impliqués dans la C-4déméthylation de la biosynthèse de l’ergostérol : le gène erg27 codant la 3-céto réductase,cible du fenhexamid, et le gène erg28 codant une protéine qui interagirait en partie avecla 3-céto réductase. Concernant la résistance au fenhexamid, il a été démontré que, pargénétique inverse, les mutations détectées dans le gène erg27 de différents types d'isolatsrésistants issus du vignoble (phénotypes de résistance HydR3- et HydR3+) conféraient larésistance. Par ailleurs, une analyse de fitness du phénotype le plus préoccupant(phénotype HydR3+) a été réalisée en conditions contrôlées sur des souches isogéniquesartificielles afin d’apporter une réponse sur la persistance possible de ces souches auvignoble. Une méthode fine de quantification moléculaire de ces mêmes isolats aégalement été mise au point pour faciliter le suivi de leur évolution et de la persistancedes populations naturelles à l’échelle des vignobles. Cette nouvelle méthode, nomméeASPPAA PCR, exploite le polymorphisme nucléotidique du gène erg27, à l’origine de larésistance. Enfin, la résistance naturelle au fenhexamid de l’espèce apparentée à Botrytiscinerea, appelée Botrytis pseudocinerea a été élucidée. La résistance au fongicide de cetteespèce a été expliquée par la combinaison de modifications de cible (mécanismeminoritaire) et d’une dégradation du fongicide par un cytochrome P450 nomméCyp68.4 (mécanisme majeur). Il s’agit de la première identification et caractérisationgénétique d’un mécanisme de résistance à un fongicide conférée par un processus dedétoxification chez un champignon phytopathogène. / Chemical control is the main method used to control diseases caused byphytopathogenic fungi. In some cases, the resistance phenomena towardfungicides occur within fungal populations, which might alter practicalefficiency of molecules. Understanding modes of action of fungicides andunderlying resistance mechanisms participate to the development and adaptationof management strategies against resistance, and thus help to sustain the life ofmolecules. Fenhexamid is a recent fungicide (Bayer CropScience, 2000), with aparticular mode of action. It is the only fungicide marketed blocking the C4-demethylation step of ergosterol biosynthesis. Several types of resistance (naturaland acquired) were detected in European vineyards in the Botrytis spp speciescomplex, causing grey mold disease. This work focused on the characterization ofthe mode of action and the elucidation of resistance mechanisms. The first aspectinvestigated the functional characterization of two genes involved in the C4-demethylation of ergosterol biosynthesis. The erg27 gene potentially encoding the3-keto reductase which is the fenhexamid target and the erg28 gene encoding aprotein that interact in part with the 3-ketoreductase. Concerning fenhexamidresistance, we shown by reverse genetics that mutations detected in the erg27 genefrom different resistant isolates from the vineyards (phenotypes HydR3- andHydR3+) confer resistance. Furthermore, a fitness analysis under controlledconditions on the most worrying resistant phenotype (HydR3+) was performed onisogenic artificial strains in order to predict the possible persistence of these strainsin vineyards. A fine molecular method to quantify these isolates was developed tofacilitate the follow-up of evolution and persistence of resistant populations in thevineyard. This new method, named ASPPAA PCR is based on the nucleotidepolymorphism of the erg27 gene, responsible for fenhexamid resistance. Finally,the natural resistance to fenhexamid of the related species to Botrytis cinerea, B.pseudocinerea, was elucidated. Fungicide resistance of this species is explained bythe combination of target site modifications (minor mechanism) and fungicidedegradation mediated by a cytochrome P450 named Cyp68.4 (major mechanism).This is the first characterization of a genetic resistance mechanism to a fungicideconferred by detoxification in a phytopathogenic fungus.

La sélection naturelle constitue un processus clé de l’adaptation des populations à leur environnement, favorisant les variants présentant les meilleures valeurs sélectives. Les champignons présentent généralement des traits biologiques (diversité des modes de reproduction, grandes tailles de populations, fortes capacités de dispersion, entre autres) qui favorisent leur adaptation à des environnements variés. La compréhension des mécanismes qui sous-tendent l’évolution de leurs populations sous les contraintes, naturelles et anthropiques, qu’elles subissent constituent donc un enjeu majeur pour la protection des plantes, en particulier dans le contexte actuel de durabilité des méthodes de lutte. Dans cette thèse, nous avons décrit la structure et la diversité des populations Botrytis cinerea à l’aide de marqueurs neutres et sélectionnés et d’un échantillonnage emboîté, et avons proposé des mécanismes pouvant expliquer les résultats observés. Puis nous avons analysé la réponse adaptative des populations de B. cinerea en Champagne, aux applications de fongicides. Premièrement, nous avons montré que la pourriture grise était causée par un complexe de deux espèces cryptiques, vivant en sympatrie sur des hôtes communs. De plus, les populations françaises de B. cinerea sont structurées en cinq dèmes, caractérisés par le système de culture (sélection directionnelle), la plante-hôte (adaptation écologique), et dans une moindre mesure, par la géographie. Sur vigne, nous avons mis en évidence une entité dont l’isolement génétique semble lié à un isolement temporel. Par ailleurs, nous avons montré que l’application de fongicides conduit à la sélection de phénotypes résistants spécifiquement à quasiment tous les modes d’action homologués, selon des proportions variant suivant les vignobles et les usages. Plus particulièrement, la résistance aux fongicides inhibiteurs de la succinate déshydrogénase (SDHI) est causée par au moins sept mutations affectant les gènes encodant la protéine cible de ces fongicides, déterminant ainsi une grande variété de phénotypes. Enfin, nous avons montré que les fongicides ne modifiaient pas la structure neutre des populations mais qu’ils pouvaient conduire à une perte de richesse allélique dans les populations traitées ainsi qu’à un équilibre sélection-migration détectable dans certaines situations sous forme de clines au loci sous pression de sélection contemporaine tels que ceux déterminant la résistance multidrogues. La modélisation de l’évolution des fréquences de résistance hivernale a permis d’estimer le coût de la résistance pour quatre loci déterminant la résistance aux fongicides. Cette thèse a permis d’appréhender le fonctionnement des populations de B. cinerea et de comprendre et quantifier partiellement les mécanismes sélectifs opérant in natura. Ces informations seront utilisées pour raisonner des stratégies anti-résistance adaptées localement et durables. / Natural selection is the most powerful force driving population adaptation to their environment, favoring the variants with the best fitness. Fungi generally exhibit biological traits (diversity of reproduction modes, large population sizes, and intense dispersion) that favor their adaptation to changing environments. Therefore, disentangling the mechanisms that explain their evolution under natural and anthropic constraints constitute a major challenge for plant protection, especially in the actual context of agriculture sustainability. In this thesis, we described Botrytis cinerea population structure and diversity, using neutral and selected markers and a hierarchical sampling, and proposed mechanisms that may explain these observations. We then analyzed the adaptive answer of this species towards fungicide applications. First, we showed that grey mold populations were caused by a complex of two cryptic species, living sympatrically on the same hosts. Second, B. cinerea populations are divided into five demes, according to the cropping system (directional selection), the host-plant (ecological adaptation), and to a lesser extent, by geography. On grapevine, we identified a specific populations exhibiting temporal isolation, as an evidence of extreme exploration of the viticultural conditions. Moreover, fungicide applications select resistance towards all unisite modes of action, with few exceptions, but at varying proportions according to vineyards and fungicide use. More specifically, resistance to succinate dehydrogenase inhibitors (SDHIs) is caused by at least seven mutations altering the target genes of these fungicides, and determines a large variety of phenotypes in the field. At last, we showed that fungicides did not shape population structure but that they could decrease allele richness in treated areas and lead to migration-selection equilibrium, detectable in some situation and for loci under contemporary selective pressures as clines. Modeling the evolution of resistance during winter allowed estimating fitness cost of four loci involved in contemporary fungicide resistance, such as multidrug resistance. As a conclusion, this thesis helped to understand how B. cinerea populations evolve and to detect and quantify selective mechanisms at work in natura. This information will be useful to deign sustainable and locally-adapted anti-resistance strategies.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Botrytis cinerea Pers. Fr. [teleomorph Botryotinia fuckeliana (de Bary) Whetzel] causes serious losses of over 200 crops worldwide, including rooibos seedlings and pears. This pathogen is characterized by morphological, physiological and genetic diversity. The genetic diversity and population structure have not been investigated for B. cinerea populations in South Africa. Botrytis cinerea collected from rooibos seedlings and in pear orchards in the Western Cape of South Africa were investigated in the present study. The study was done with the aid of microsatellite markers, the amplification of mating type alleles MAT1-1 and MAT1-2 and determination of resistance towards various fungicides. Population dynamics was inferred and a similar picture emerged in both production systems. Botrytis cinerea annually causes severe losses of rooibos seedlings (Aspalathus linearis) in nurseries situated in the Clanwilliam region. Sampling was done in five nurseries and the cryptic species status of the isolates obtained was determined through restriction enzyme digestion of the Bc-hch gene. All but one (206 out of 207) of the isolates belonged to Group II or B. cinerea ‘sensu stricto’. Analysis of the B. cinerea Group II population, using seven microsatellite loci, was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.67. Two of the nurseries populations’ sample sizes were severely limited after clone correction, yet 100 genotypes were discerned among the 206 isolates genotyped. The percentage of maximal genotypic diversity (G) ranged between 16 and 68 for the five populations, with a total value of 17 for the 100 genotypes. One genotype, represented by 27 clones, was isolated from four nurseries. Relatively low but significant population differentiation was observed in total between nurseries (mean FST = 0.030, P = 0.001). The distribution of mating types MAT1-1 and MAT1-2 differed significantly from the ratio of 1:1 for the total population plus two of the nurseries’ populations. Three nursery populations had an equal mating type distribution. The index of association (IA) analyses suggests that the populations are asexually reproducing. Analysis of molecular variance (AMOVA) indicated that 97% of the total genetic variation is distributed within subpopulations. Fungicide resistance frequency against iprodione for 198 of the genotyped isolates displayed highly varying levels of resistance amongst the five nurseries. The mean total incidence of resistance towards iprodione was 43%, ranging from 0% to 81% for the five nurseries. Baseline sensitivity towards pyrimethanil yielded an average EC50 value of 0.096 mg/L. Botrytis cinerea isolates were collected from pear blossoms (Pyrus communis) in four orchards. Two orchards in the Ceres area and two in the Grabouw area were sampled from. A total of 181 isolates were collected from the four orchards. Incidence of blossom infection in the orchards ranged from 3% to 17%. Overall, there was a high incidence of isolates that had only the Boty transposable element (74%) compared to those harbouring both (Boty and Flipper), simultaneously (transposa, 24%). One isolate examined had the Flipper element only. Cryptic species status according to restriction enzyme digestion of the Bc-hch gene indicated that all the isolates belonged to Group II or B. cinerea ‘sensu stricto’. Analysis of the Group II population, through the use of seven microsatellite loci, was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.69 across all populations. Although two of the subpopulations displayed a high clonal proportion, overall 91 genotypes were discerned among the 181 isolates. The percentage of maximal genotypic diversity (G) ranged between 18 and 33 for the four populations, with a total value of 14 for the 91 genotypes. One genotype, represented by 27 clones, was isolated from all orchards. Moderate, but significant population differentiation was present in total among orchards (mean FST = 0.118, P = 0.001). The distribution of the mating types, MAT1-1 and MAT1-2, did not differ significantly from a 1:1 ratio for the total population as well as the subpopulations. Index of association (IA) analyses, on the other hand, suggests that the populations reproduce asexually. Analysis of molecular variance (AMOVA) indicated that 88% of the total genetic variation is distributed within subpopulations, 9% between subpopulations and only 3% between production areas. Fungicide resistance frequency against fenhexamid, iprodione and benomyl varied, with the highest levels of resistance present against benomyl and low levels of resistance seen towards iprodione and fenhexamid. In conclusion, this study has shown that there exist within the studied populations of B. cinerea, obtained from rooibos nurseries and pear orchards, an adaptive capacity to overcome current means of control. The use of population genetics to further our understanding of how plant pathogens interact and spread throughout a given environment is of cardinal importance in aiding the development of sustainable and integrated management strategies. Knowledge of the dispersal of B. cinerea in the two studied cropping systems has shed light on the inherent risk that it poses, and this together with knowledge of the levels of resistance that occurs should serve as an early warning to help divert possible loss of control in future. / AFRIKAANSE OPSOMMING: Botrytis cinerea Pers. Fr. [teleomorf Botryotinia fuckeliana (de Bary) Whetzel] veroorsaak ernstige verliese van meer as 200 gewasse wêreldwyd, insluitende rooibossaailinge en pere. Hierdie patogeen word deur morfologiese, fisiologiese, asook genetiese diversiteit gekenmerk. Die genetiese diversiteit en populasie-struktuur van B. cinerea populasies wat in Suid-Afrika voorkom, is nog nie ondersoek nie. Botrytis cinerea verkryg vanaf rooibossaailinge en in peerboorde in die Wes-Kaap van Suid-Afrika is ondersoek. Hierdie studie is met behulp van mikrosatellietmerkers, amplifikasie van die twee paringstipe gene (MAT1-1 en MAT1-2), asook die bepaling van weerstandsvlakke teenoor verskeie swamdoders, uitgevoer. Populasie-dinamika is afgelei en ‘n soortgelyke tendens is in beide produksie-sisteme waargeneem. Botrytis cinerea veroorsaak jaarliks ernstige verliese van rooibossaailinge (Aspalathus linearis) in kwekerye in die Clanwilliam-area. Monsters is in vyf kwekerye versamel en die kriptiese spesiestatus van die verkrygde isolate is deur restriksie-ensiemvertering van die Bc-hch geen bepaal. Almal behalwe een (206 uit 207) isolaat het aan Groep II of B. cinerea ‘sensu stricto’ behoort. Analise van die B. cinerea Groep II populasie, deur middel van sewe mikrosatellietmerkers, is uitgevoer om die genetiese populasiestruktuur te bepaal. Totale geendiversiteit (H) was hoog, met ‘n gemiddelde van 0.67. Alhoewel twee van die kwekerye se monstergrootte erg ingeperk is ná kloonverwydering, is daar nogtans 100 genotipes onder die 206 isolate wat geïsoleer is, waargeneem. Die persentasie van maksimale genotipiese diversiteit (G) het tussen 16 en 68, vir die vyf populasies, gewissel, met ‘n totaal van 17 vir die 100 genotipes. Een genotipe, verteenwoordig deur 27 klone, is uit vier kwekerye geïsoleer. Relatief lae dog noemenswaardige populasie-differensiasie is in totaal tussen kwekerye waargeneem (gem. FST = 0.030, P = 0.001). Die verspreiding van die twee paringstipes (MAT1-1 en MAT1-2) het beduidend verskil van ‘n 1:1 verhouding vir die totale populasie, asook twee van die kwekerye se populasies. Die drie oorblywende kwekerye se populasies het egter ‘n gelyke verdeling van die twee paringstipes getoon. Die indeks van assosiasie (IA) analises toon dat die populasies ongeslagtelik voortplant. Analise van molekulêre variasie (AMOVA) het aangedui dat 97% van die totale genetiese variasie binne die subpopulasies versprei is. Hoogs variërende vlakke van weerstand tussen die vyf kwekerye teenoor die swamdoder iprodioon, is vir die 198 isolate wat getoets is, gevind. Die totale gemiddelde frekwensie van weerstand teenoor iprodioon was 43%, wat tussen 0% en 81% vir die vyf kwekerye gevarieer het. Fondasie-vlak-sensitiwiteit vir pyrimethanil het ‘n gemiddelde EC50 waarde van 0.096 mg/L opgelewer. Botrytis cinerea isolate is ook vanuit peerbloeisels (Pyrus communis L.) vanuit vier boorde versamel, twee uit elk van die Ceres- en Grabouw-areas. In totaal is 181 isolate vanuit die vier boorde versamel. Die frekwensie van bloeiselinfeksie het tussen 3% en 17% gewissel. Oor die algemeen was daar ‘n hoë frekwensie van isolate wat slegs die Boty transponeerbare element teenwoordig gehad het (74%) in vergelyking met dié wat tegelykertyd beide (Boty en Flipper) teenwoordig gehad het. Een isolaat het slegs die Flipper element gehad. Bepaling van die kriptiese spesiestatus met behulp van restriksie-ensiemvertering van die Bc-hch geen het aangedui dat alle versamelde isolate tot Groep II of B. cinerea ‘sensu stricto’ behoort het. Analise van die Groep II populasie, deur middel van sewe mikrosatellietmerkers, is uitgevoer om genetiese populasie-struktuur te bepaal. Totale geendiversiteit (H) was hoog, met ‘n gemiddelde van 0.69 oor alle populasies. Alhoewel twee subpopulasies ‘n hoë klonale fraksie getoon het, is 91 genotipes tussen die 181 isolate wat verkry is, onderskei. Die persentasie van maksimale genotipiese diversiteit (G) het tussen 18 en 33 vir die vier populasies gewissel, met ‘n totale waarde van 14 vir die 91 genotipes. Een genotipe, verteenwoordig deur 27 klone, was in al vier boorde teenwoordig. Gematigde dog beduidende populasie differensiasie was in totaal tussen boorde teenwoordig (gem. FST = 0.118, P = 0.001). Die verspreiding van die paringstipes (MAT1-1 en MAT1-2) het nie betekenisvol van ‘n 1:1 verhouding vir die totale populasie, insluitende die subpopulasies, verskil nie. Indeks van assosiasie (IA) analises het egter aangedui dat die populasies ongeslagtelik voortplant. Analise van molekulêre variasie (AMOVA) het aangedui dat 88% van die totale genetiese variasie in subpopulasies te vinde was, 9% tussen subpopulasies en slegs 3% tussen produksie-areas. Frekwensie van swamdoder weerstandbiedendheid vir fenhexamid, iprodioon en benomyl het gewissel, met die hoogste vlakke teenoor benomyl waargeneem, maar baie lae vlakke teenoor fenhexamid en iprodioon. Samevattend het hierdie studie getoon dat die populasies van B. cinerea wat in hierdie twee produksie-sisteme, op rooibossaailinge en in peer boorde, ondersoek is, ‘n aanpasbaarheid toon om huidige metodes van beheer te oorkom. Die gebruik van populasiegenetika as ‘n hulpmiddel om ons kennis van patogeen-interaksies en -verspreiding te verbreed, is van kardinale belang in die ontwikkeling van geïntegreerde en volhoubare beheermaatreëls. Kennis van die verspreiding van B. cinerea in die bestudeerde gewasproduksiestelsels, werp lig op die inherente risiko wat dié patogeen inhou. Dít, tesame met kennis van die weerstandsvlakke wat voorkom, kan as ‘n vroegtydige waarskuwing dien ten einde moontlike verlies van beheer in die toekoms te help teenwerk.

La succinate déshydrogénase (SDH) est à la fois une enzyme clé du cycle de Krebs oxydant le succinate en fumarate et le complexe II de la chaîne respiratoire mitochondriale impliqué dans le transfert des électrons et la réduction de l’ubiquinone. Des inhibiteurs de cette enzyme (SDHI) ont été développés ou sont en cours de développement comme antifongiques. Cette famille de fongicides est notamment utilisée pour lutter contre Botrytis cinerea, champignon phytopathogène responsable de la pourriture grise sur de nombreuses cultures dont la vigne. Des souches résistantes aux SDHI ont été isolées chez B. cinerea et d’autres champignons phytopathogènes. Chez ces isolats résistants, des mutations ont été identifiées dans les gènes codant la SDH. Au cours de cette thèse, nous avons étudié l’impact de mutations affectant la sous-unité B (SdhB) de la succinate déshydrogénase sur l’activité de l’enzyme, la biologie du champignon B. cinerea et la résistance aux inhibiteurs ciblant cette enzyme. Par mutagénèse dirigée du gène sdhB, nous avons obtenu des mutants dits « isogéniques » qui ont permis de confirmer l’implication de ces mutations dans la résistance aux différentes molécules SDHI. Par ailleurs, nos résultats montrent que les modifications de la sous-unité SdhB affectent l’affinité des SDHI pour la SDH et les niveaux d’inhibition de l’activité SDH par les molécules inhibitrices ; ce qui explique - in fine - les spectres de résistance des mutants aux SDHI. Actuellement, tous les mutants sont résistants au boscalid et les mutants les plus fréquemment retrouvés au vignoble, sdhBH272R/Y, sont sensibles au fluopyram. Les travaux réalisés sur les mutants sdhB montrent que les mutations étudiées ont également un impact sur l’activité de l’enzyme et sur le développement du champignon, conséquences dépendantes du résidu substitué et de la substitution. En particulier, les mutations sdhBH272L/R affectent fortement l’activité de l’enzyme et la fitness du champignon alors que le mutant sdhBH272Y est peu affecté. Enfin, l’analyse de populations de pourriture grise de différentes origines (région, plantes hôtes) par rapport à la résistance aux SDHI réalisée sur les années 2009/2010 montre que les mutants sdhBH272R/Y sont toujours les plus fréquents mais leurs fréquences varient en fonction des situations agronomiques. Notamment la fréquence du mutant sdhBH272R augmente avec la pression de sélection exercée par les fongicides. Ce mutant attire particulièrement notre attention du fait de sa relation non linéaire entre fitness et fréquence au champ. / Succinate dehydrogenase is both a key enzyme of the TCA cycle, oxidizing succinate into fumarate and complex II of the mitochondrial respiratory chain involved in electron transfer and ubiquinone reduction. Inhibitors of this enzyme (SDHIs) have been developed or are in the developmental process as fungicides. Actually, SDHIs are registered to deal with Botrytis cinerea, a phytopathogenic fungus responsible for grey mold on many crops including grapevine. Strains of B. cinerea and other pathogenic fungi have been isolated for their resistance to SDHI. They mainly harbor mutations in genes encoding SDH subunits. During this thesis, we studied the impact of mutations modifying subunit B of succinate dehydrogenase on enzyme activity, fungal biology and resistance to SDHIs. “Isogenic” mutants obtained through site-directed mutagenesis and homologous recombination allowed us to confirm the role of sdhB mutations in SDHIs resistance. Our results also show that the substitutions in the SdhB subunit impact respectively the affinity of SDHIs to SDH and the inhibition levels of SDH activity by inhibitors, which explain – in fine – the resistance spectra observed for the mutants. Up to now, all sdhB mutants are resistant to boscalid and the most frequent mutants observed in grapevines, sdhBH272R/Y, are susceptible to fluopyram. Studies on sdhB mutants reveal that the mutations also impact the enzymatic activity and the fungal development depending on the substitution. In particular, sdhBH272L/R mutations have the strongest impact on enzyme activity and the fitness of the fungus, whereas these parameters are almost not altered in the sdhBH272Y mutant. Finally, grey mold populations from different origins (country, plant host) were analyzed for their SDHI resistance pheno- and genotypes. Yet, the sdhBH272R/Y mutants were the most frequent, but these frequencies varied according to the agronomical situation. Interestingly, the frequencies of the sdhBH272R mutant seem to increase with the selective pressure exerted by fungicides. This mutant is of particular interest because of the absence of correlation between the fitness we measured and the frequencies we observed in natura.

Organic mulches and cover crops mulched in situ were assessed for their effects on B. cinerea primary inoculum and disease levels in inflorescences at flowering and/or bunches at harvest. Organic mulches were used to enhance biological degradation of vine debris to reduce levels of B. cinerea primary inoculum the following season. Four mulch types (anaerobically and aerobically fermented marc (grape pressings), inter-row grass clippings and shredded office paper) were applied under ten-year-old Riesling vines in a ten-replicate randomized block design in New Zealand over two consecutive years. Plastic mesh bags, each containing naturally infected vine debris, were placed under vines on bare ground (control) and at the soil-mulch interface, in winter (July) 2003 and 2004. In each year, half the bags were recovered at flowering (December) and the remainder at leaf plucking (February), for assessment of B. cinerea sporulation from the vine debris and debris degradation rate. Bait lamina probes, which measure soil biological activity, were placed in the soil-mulch interface three weeks before each of the two bag-recovery dates in both years and were then removed and assessed at the same times as were the bags. All mulches led to a reduction in B. cinerea sporulation. This reduction was significantly correlated with elevated rates of vine debris decomposition and increased soil biological activity. Over both years, compared with the controls, all treatments gave a 3-20-fold reduction in B. cinerea sporulation, a 1.6-2.6-fold increase in vine debris degradation and in the two marc and the paper treatments, a 1.8-4-fold increase in activity of soil organisms. The mulches also altered vine characteristics and elevated their resistance to B. cinerea through changes to the soil environment. Functional soil biological activity, as measured by Biolog Ecoplates and bait lamina probes, was increased 2-4 times in the two marc and paper treatments, compared with the control, an effect relating to the elevated soil moisture and reduced temperature fluctuations under these mulches. Soil nutrient levels and the C:N ratios were also affected in these treatments. The mulched paper lowered vine canopy density by up to 1.4 times that of the other treatments, an effect which probably led to elevated light penetration into the canopy and consequent increased canopy temperature, photosynthesis and lowered canopy humidity. These changes to soil and vine characteristics increased grape skin strength by up to 10% in the paper treatment and sugar concentrations by 1.2-1.4 °Brix in the two marc and paper treatments. The severity of B. cinerea infections in the anaerobic marc, aerobic marc and paper treatments were reduced to 12%, 3% and 2.2% of the control, respectively, in field assessments averaged over two consecutive harvests. Cover crops mulched in situ had similar effects to those of the organic mulches, increasing soil biological activity and reducing B. cinerea primary inoculum and the severity of B. cinerea infection in grapes at harvest (2006). Inter-row phacelia and ryegrass were mulched in winter 2005 and compared with a bare ground control, under 10-year-old Chardonnay vines in a ten-replicate randomized block design. Functional soil biological activity increased by 1.5-4.5 times in the two cover crop treatments compared with the control, an effect possibly related to elevated soil moisture in these treatments. This increase in soil moisture and soil biological activity increased vine debris degradation, reduced B. cinerea primary inoculum on the debris and decreased B. cinerea severity at flowering (December 2005) and harvest (April 2006). These results show the potential of organic mulches and cover crops mulched in situ to enhance soil ecosystem services and improve the sustainability of viticultural practices.