The fungicides prochloraz and tebuconazole (at concentrations of2 J.lg ml-I ) were shown to reduce the mycelial growth of Fusarium culmorum, F. avenaceum, F. poae, F. gramineanlm and Microdochium nivale in vitro by over 90 % compared to the untreated control. In addition, chlorothalonil inhibited spore germination of all species and pyrimethanil reduced the mycelial growth of M nivale by over 60 % at 2 J.lg ml-I , although it was ineffective against the other species. In the glasshouse, prochloraz and tebuconazole were moderately effective in reducing the severity of fusarium ear blight (FEB) caused by F. culmorum and M nivale. The fungicides gave less effective control of FEB in the field. There was a significant relationship between the incidence and severity of FEB in 1995 but there was no significant relationship between ear blight and yield in either 1995 or 1996. It was proposed that the interactions between saprophytic microflora and ear blight pathogens may account for the poor performance of fungicides against FEB in vivo. Glasshouse and laboratory studies showed that Alternaria alternata, Botrytis cinerea and Cladosporium herbarum reduced the severity of FEB caused by F. culmorum and this antagonism was attributable to both volatile and non-volatile antibiotic production. The saprophytic species showed inherent variability in their sensitivity to the fungicides tested in vitro and in the glasshouse. It was shown that certain fungicides (e.g. pyrimethanil) which reduced mycelial growth of the saprophytic species in vitro allowed the pathogen to grow by reducing the antagonism of the microflora species against the pathogen. This may not be true for all fungicides in practice. It was also proposed that the inefficacy of fungicides to control FEB was due to a failure of the fungicide to reach the site of infection. It was shown, using a fluorescent tracer that retention 11 of spray was influenced by cultivar, time of application and fungicide. The amount of tracer retained was significantly correlated with the number of extruded anthers of wheat. When radio-labelled prochloraz was applied to the ears of wheat, the prochloraz was retained predominantly on the outer glumes, with very small amounts being retained by the florets and rachis. There was no movement of prochloraz between tissues and the half-life of the active ingredient was 48 hours. This work illustrates the efficacy of fungicides against Fusarium spp. and Microdochium nivale in vitro, under glasshouse conditions and in the field, and provides some evidence to explain their poor performance. It is proposed that future work should investigate environmental and biological factors which contribute to ear blight epidemics, in order that a forecasting system for fungicide application can be devised. Also, studies of fungicide activity against antagonistic ear microflora species and studies of fungicide retention and penetration may help to optimise fungicide application to control this disease.

The Botryosphaeriaceae is a family of fungi that includes many species, which are well-known as pathogens, saprophytes and endophytes of plants and especially of trees. As a result of their pathogenic nature and potential threat to plantations and agricultural crops, much research has been devoted to their identification. The main focus of studies that make up this thesis has been on the fungal complex referred to as Diplodia pinea sensu lato. These fungi are members of the Botryosphaeriaceae and studies have specifically concentrated on their taxonomy, host associations and mycovirus infections associated with them. Diplodia pinea sensu lato represents a species complex of highly similar morphological types that mainly infect Pinus spp., world-wide. The species complex includes what have in the past been known as the A, B and C morphological types of D. pinea. Multiple gene genealogies based on sequences of partial protein-coding genes and microsatellite markers were used to resolve the species complex into two genera, D. pinea and D. scrobiculata (= B morphotype). Diplodia-like isolates from Australia, Greece and Cyprus were characterized using both morphological and molecular characteristics. Morphologically, these isolates all have dark, thick-walled conidia (Diplodia-like) but phylogenetically, they could belong to three distinct genera of the Botryosphaeriaceae namely Diplodia, Lasiodiplodia and Dothiorella. Results of this study led to the description of Dothiorella casuarini from Casuarina spp. in Australia and they highlight the fact that similar morphological characteristics and disease etiology does not necessarily provide a true reflection of the evolutionary history of a pathogen. Phylogenetic studies on species of the Botryosphaeriaceae with Diplodia-like anamorphs revealed intriguing host association patterns. The availability of sequence data for many species of the Botryosphaeriaceae made it possible to extend the phylogeny to include six of the ten lineages as previously described for the Botryosphaeriaceae. Angiosperms appeared to be the most common, and possibly ancestral, host group of the Botryosphaeriaceae, with the exception of Macrophomina, Guignardia, Saccharata and “Botryosphaeria” quercuum. Infection of gymnosperms most likely occurred more recently, only in specific groups (Diplodia and Lasiodiplodia) via host shifts. Three distinct viruses have now been characterized from isolates of D. pinea sensu lato. Two of these were previously characterized and are known as Sphaeropsis sapinea RNA virus 1 and 2 (SsRV1 and SsRV2). The third dsRNA element more commonly found in association with D. scrobiculata was characterized in this dissertation and named Diplodia scrobiculata RNA virus 1 (DsRV1). It has a genome of 5018 bp with a unique genome organization characterized by two open reading frames (ORFs). One ORF codes for a putative polypeptide similar to proteins of the vacuolar protein-sorting (VPS) machinery and the other one for a RNA dependent RNA polymerase (RdRp). The hypothetical protein probably has a role in transport or protection of this unencapsulated virus into membranous vesicles. Phylogenetically, DsRV1 groups closest to a dsRNA element from Phlebiopsis gigantea (PgV2) and they both group separately from other families in which fungal viruses have been classified. The frequency and distribution of DsRV1, SsRV1 and SsRV2 were determined in a collection of D. pinea and D. scrobiculata isolates using Real-time PCR. Infections with SsRV1 and SsRV2 occurred in both D. pinea and D. scrobiculata, while DsRV1 was mainly found in D. scrobiculata. DsRV1 was also found to always occur in combination with SsRV1 and/or SsRV2. Therefore, DsRV1 probably selected against a coat protein as the result of a fitness trade-off. Although earlier studies indicated that these viruses have no effect on the phenotype or virulence of D. pinea and D. scrobiculata isolates, the presence of specific viruses in their host populations serve as a useful marker in studying movement of fungal pathogens. The ultimate aim of studies making up this dissertation was to expand the base of knowledge regarding species in the D. pinea species complex. This was justified by the fact that D. pinea is one of the most important tree pathogens in South Africa and that an expanded knowledge might contribute to reducing diseases caused by it. Clearly understanding the identity of the fungus must clearly underpin many elements of a management strategy and this was one of the aims of the suite of studies conducted. Furthermore, I attempted to augment the knowledge base regarding dsRNA elements in D. pinea sensu lato. These studies were of a basic nature and relatively far removed from the practical application level. Nonetheless, it is my hope that they have pushed ahead knowledge barriers and that in some way they will contribute to reducing the impact of Diplodia-associated diseases in the future. / Thesis (PhD)--University of Pretoria, 2011. / Microbiology and Plant Pathology / Unrestricted

Dans le contexte des invasions, l'objectif de ce travail est d'évaluer dans quelle mesure l'origine des espèces végétales conditionne le fonctionnement d'un écosystème en situation de dominance. Cinq paires d'espèces autochtones dominantes (Agrostis stolonifera, Rubus caesius, Populus nigra, Urtica dioica et Salix alba) et introduites envahissantes (Paspalum distichum, Fallopia japonica, Buddleja davidii, Impatiens glandulifera et Acer negundo) ont été comparées en milieu riverain pour les processus de production primaire et de dégradation des litières. Ces études ont été complétées par une analyse détaillée des conséquences de l'invasion des ripisylves par A. negundo. Nos résultats montrent que si les espèces introduites envahissantes peuvent être plus efficaces dans la réalisation de certains processus écologiques, l'absence de coévolution entre les espèces introduites et les organismes des milieux récepteurs n'a pas d'implication systématique pour le fonctionnement des écosystèmes.

Les champignons saprophytes sont les acteurs principaux du recyclage de la matière organique morte au sein des écosystèmes forestiers. Ces microorganismes possèdent la capacité unique de dégrader la totalité des polymères constitutifs du bois. L'analyse de la structuration des communautés durant les stades initiaux de la colonisation du bois par séquençage à haut débit a révélé que celui-ci influence la distribution et la dynamique des communautés qui lui sont associées. A l'échelle de l'organisme, les différents groupes écologiques de champignons décomposeurs du bois possèdent des systèmes de dégradation extracellulaires reflétant cette complexité chimique. Le séquençage du génome d'un grand nombre de ces organismes a permis l'identification de superfamilles d'enzymes impliquées dans les mécanismes de résistance et de détoxication des composés toxiques exogènes. Parmi elles, la superfamille des glutathion transférases présente une extension de classes spécifiques au sein des champignons décomposeurs du bois. La détermination des propriétés biochimiques et structurales d'une isoforme, issue d'une de ces classes spécifique (les Etherase-like), présente chez Phanerochaete chrysosporium a révélé des caractéristiques particulières. Cette enzyme possède un mode de dimérisation atypique ainsi que la capacité à séquestrer des composés phénoliques toxiques via une propriété ligandine unique. La comparaison des propriétés de plusieurs isoformes de cette classe d'enzymes appartenant aux champignons C. cinereus et P. chrysosporium a démontré que celle-ci exhibe une grande versatilité intra- et interspécifique, de leurs activités enzymatiques et de leur propriété ligandine / Saprophytic fungi are key players of dead organic matter recycling in forest ecosystems. These microorganisms possess the unique ability to degrade the integrality of wood constitutive polymers by secretion of complex oxydative and hydrolytic enzymatic systems. Communities structuration analysis during the initial stages of wood colonisation by high throughput sequencing revealed that the latter beyond being a source of nutrients, influences the distribution and dynamic of communities by its broad chemical variability. At the organism level, the different ecological groups of wood decomposing fungi possess extracellular degradation systems reflecting this chemical complexity. Genome sequencing of these organisms allowed the identification of enzymes superfamilies involved in resistance and detoxification mechanisms towards exogenous toxic compounds. Among them, the glutathione transferases superfamily exhibit extension of specific classes in wood decaying basidiomycetes. Biochemical and structural properties determination of one isoform belonging to one of these specific classes (the Etherase-like), found in Phanerochaete chrysosporium revealed unusual characteristics. This enzyme possesses an atypical dimerization mode as well as the ability to sequestrate toxic phenolic compounds resulting from wood degradation through a unique ligandin property. Properties comparison of several isoforms from this class belonging to C. cinereus and P.chrysosporium demonstrated a huge intra- and interspecific versatility of their enzymatics activities and ligandin property in response to environmental constraints arising from the great chemical heterogeneity of wood composition

Phanerochaete chrysosporium est un champignon forestier faisant partie des organismes saprophytes capables de recycler la matière organique morte. Grâce à l'excrétion de nombreuses enzymes de dégradation, en particulier des lignine peroxydases, il est capable de décomposer la matière végétale dont la lignine, un polymère complexe de composés phénoliques très résistant. L'élimination de la lignine permet la libération des autres composants du bois tels que la cellulose et l'hémicellulose qui peuvent être utilisés dans l'industrie papetière ou pour la production de bioéthanol de deuxième génération. La structure des intermédiaires et produits de dégradation de la lignine est souvent proche de celle denombreux polluants, d'où l'intérêt biotechnologique de P. chrysosporium dans les processus de bioremédiation. Cependant, les systèmes de dégradation engendrent des composés plus ou moins toxiques pour le champignon et contre lesquels il doit faire face. C'est pourquoi il possède un système de détoxication impliquant des enzymes telles que les cytochrome P450 monooxygénases ou encore les glutathion transférases (GST). Les Ure2p forment une classe de GST étendue chez Phanerochaete et d'autres basidiomycètes saprophytes. Leur étude par des approches phylogénétiques, biochimiques, structurales et transcriptomiques a permis de mieux comprendre les mécanismes d'évolution que peut subir une classe d'enzymes potentiellement soumises à une forte pression de sélection / Phanerochaete chrysosporium is a forest fungus being part of saprophytic organisms able to recycle dead organic matter. Thanks to the excretion of numerous wood decaying enzymes, and especially lignin peroxidases, this fungus is able to break down plant material including lignin, a complex polymer of phenolic compounds. Lignin removal allows the release of other wood components such as cellulose and hemicellulose, which can be further used in paper industry or to produce second generation bioethanol. The structure of intermediates and products from lignin decomposition is close to that of numerous pollutants making P. chrysosporium biotechnologically interesting for bioremediation purposes. Moreover, the fungus has to deal with more or less toxic compounds created by degradation mechanisms. It thus presents a detoxification pathway involving enzymes including cytochrome P450 monooxygenases and glutathione transferases (GST). Ure2p enzymes belong to an extended GST class in Phanerochaete genus as well as in other saprophytic basidiomycetes. Their study based on phylogenetic, biochemical, structural and transcriptomic approaches provides a better understanding of evolution mechanisms of a class of enzymes potentially subject to strong selection selection pressure

Durant ces dernières décennies, les systèmes enzymatiques de dégradation du bois sécrétés par les champignons ont fait l’objet de nombreuses études aboutissant à la caractérisation fonctionnelle et biochimique des enzymes extracellulaires majeures agissant directement sur le polymère. Cependant, les systèmes annexes associés au processus de dégradation n’ont à l’heure actuelle été que peu étudiés. En particulier, les systèmes de détoxication et de réponses des champignons au stress généré par le processus de dégradation ainsi que les mécanismes lui permettant de croître dans cet environnement hostile sont encore peu connus. Ce stress est majoritairement dû à la présence de radicaux et d’extractibles. Les extractibles sont des molécules issues du métabolisme secondaire de l’arbre qui sont synthétisés pour augmenter la durabilité du bois face aux attaques biotiques et abiotiques. Une analyse transcriptomique réalisée au laboratoire a mis en évidence la surexpression de gènes codant des petites protéines sécrétées (SSP) chez Phanerochaete chrysosporium lors d’une culture en présence d’extractibles de chêne. La fonction de ce type de protéines chez les champignons lignolytiques est inconnue. Mon projet de thèse a porté sur la caractérisation d’une de ces SSP (SSP1). Les résultats obtenus ont révélé des propriétés biochimiques atypiques pour cette protéine qui est capable de former une structure fibrillaire, notamment grâce à la présence d’un domaine C-terminal riche en alanine et glycine. De plus, nous avons pu montrer que cette protéine présentait une activité β-glucuronidase in vitro, qui est dépendante de son état d’oligomérisation. Une approche physiologique a également été abordée grâce à l’obtention de mutants knock-out de SSP de Podospora anserina. La caractérisation de ces mutants a montré un défaut de croissance en condition de stress oxydant et en présence de molécules perturbant l’intégrité de la paroi cellulaire. Enfin, une analyse in silico des orthologues de SSP1 a montré la présence de ce gène dans les génomes d’organismes saprophytes, ectomycorhiziens ou pathogènes suggérant un rôle indirect de cette protéine dans les processus de dégradation du bois, probablement en lien avec la gestion du stress associé / During the last decades, the enzymatic systems involved in wood degradation have been intensively studied in fungi. This has led to functional and biochemical characterization of the main extracellular enzymes that are involved in the process. However, other systems associated to the degradation mechanisms have been poorly studied. In particular, the detoxification and stress response pathways allowing the fungus to grow in and resist the toxic conditions that are associated to the degradative process are still unknown. This stress is mostly due to the presence of radicals and extractives. Extractives are putative toxic compounds produced as secondary metabolites in tree to enhance wood durability against biotic and abiotic attacks. A transcriptomic analysis performed in the laboratory highlighted the up-regulation of genes coding for small secreted proteins (SSP) in Phanerochaete chrysosporium in presence of oak extractives. The functions of these SSP are unknown in lignolytic fungi. My PhD project was focused on the characterization of one of these SSP (namely SSP1) of P. chrysosporium. The biochemical data revealed atypical features for SSP1. Indeed, it is able to form fibrilar structure, thanks to an alanine-rich and glycine-rich C-terminal domain. Moreover, we have shown that this protein exhibits β-glucuronidase activity in vitro which is dependent on its oligomerization state. Physiological data were obtained thanks to the obtention of SSP knock-out mutants in Podospora anserina. These mutants have growth defect in oxidizing stress condition and in presence of cell wall-disruptive compounds. Finally, the in silico analysis of SSP1 orthologues revealed the presence of this gene in genomes of saprophytic, ectomycorrhizal or pathogenic fungi, suggesting an indirect role of this protein in wood degradation processes, probably linked to the associated stress