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This thesis describes aspect of the epidemiology and pathogenicity of the soil-borne phase of <i>Rhizoctonia solani </i>AG-3 (Kuhn) fungus on potatoes and its competitive saprophytic colonisation ability in the soil. Under controlled environmental conditions, stem canker incidence and severity increased with increasing levels of soil-borne inoculum but plateaued after ¼ inoculum level (i.e 1 Petri dish of <i>R. solani  </i>AG-3 per kg soil).  Up to the 1/20 inoculum stem canker occurred at a low level.  A significant increase occurred at 1/10 and 1/8 inoculum levels.  A similar pattern was observed for the competitive saprophytic colonisation ability of the fungus.  The fungus was attracted more by seed tubers than by stems and the incidence of black scurf was higher than stem canker at all inoculum levels tested.  Sclerotia developed on seed tubers even at low inoculum levels.  Favourable soil conditions for infection on stems and seed tubers were 10^oC and soil moisture content of 40% water holding capacity.  Optimum moisture content for saprophytism was between 20-50% water holding capacity, although optimum levels of the individual soils tested varied.  Pot and laboratory experiments indicated that conditions for development of <i>R. solani</i> were more conducive in heavy than in light textured soils.  When fine sand was added in increasing quantities to a sandy clay loam soil, the disease initiated by the fungus was steadily reduced with an increase in sand content.  Potato cultivars differed in their susceptibility to <i>R. solani</i> at early stages of growth but  none of the cultivars tested showed complete resistance to the disease.  Depth of planting had no significant effect of <i>Rhizoctonia </i>stem and seed tuber infection.

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

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-10T14:20:09Z No. of bitstreams: 1 Eliane Mayumi Inokuti.pdf: 262975 bytes, checksum: 46e8a0576767e569d0817560c99aae40 (MD5) / Made available in DSpace on 2017-03-10T14:20:09Z (GMT). No. of bitstreams: 1 Eliane Mayumi Inokuti.pdf: 262975 bytes, checksum: 46e8a0576767e569d0817560c99aae40 (MD5) Previous issue date: 2012-07-30 / The fungi Rhizoctonia spp. is an important soilborne plant pathogen. The objective of this study was to evaluate the effectiveness of baits to quantify the saprophytic activity of Rhizoctonia in soil and determine the relationship between saprophytic and pathogenic activities in order to fit an equation of pathogenic activity risk in soils for cowpea and common bean planting. In the evaluation of baits, soils from three locations were packed in trays and infested with an isolate of R. solani (50 mg colonized substrate kg-1 soil). After seven days, soil samples were transferred to gerboxes and sown six baits: beet, cowpea, maize and sorghum seeds, cowpea segment stalks and toothpick segments. After 48 h at 25 ° C, the baits were transferred to the Ko & Hora modified medium. The wood toothpick bait led to the detection of higher levels of saprophytic activity in all soils. The bait toothpick was evaluated against eight isolates and six inoculum densities of R. solani, demonstrating highly effective in all situations. In the analysis of the relationship between saprophytic and pathogenic activities, were used 12 soils collected in areas for cowpea and common bean planting. The saprophytic activity was evaluated using toothpick baits and the pathogenic activity was assessed by the distribution of soils in trays, planting of cowpea seeds and assessment of Rhizoctonia canker severity. There was a significant (P≤0.05) positive correlation (r = 0.7698) between the saprophytic (ATS) and pathogenic (ATP) activities. The regression equation ATP = 1 / (0.5822 to 0.0056 ATS) was estimated with high precision (R2 = 0.9930, P≤0.05), indicating that the risk of pathogenic activity of Rhizoctonia in soils for cowpea and common bean planting can be estimated from the analysis of saprophytic activity. / O fungo Rhizoctonia spp. é um importante fitopatógeno habitante do solo. O objetivo deste trabalho foi avaliar a eficácia de iscas para quantificação da atividade saprofítica de Rhizoctonia no solo e determinar a relação entre atividade saprofítica e atividade patogênica, visando ajustar uma equação de risco de atividade patogênica em áreas destinadas ao plantio de feijão-caupi e feijão-comum. Na avaliação das iscas, solos de três localidades foram acondicionados em bandejas e infestados com um isolado de R. solani (50 mg de substrato colonizado kg-1 solo). Após sete dias, amostras dos solos foram transferidas para caixas gerbox e semeadas seis iscas: sementes de beterraba, feijão-caupi, milho e sorgo, segmentos de talos de feijão-caupi e segmentos de palito de dente. Após 48 h a 25 ºC, as iscas foram transferidas para o meio de Ko & Hora modificado. A isca de palito de dente de madeira propiciou a detecção dos maiores níveis de atividade saprofítica em todos os solos. A isca de palito de dente foi avaliada em relação a oito isolados e seis densidades de inóculo de R. solani, demonstrando elevada eficácia em todas as situações. Na análise da relação entre as atividades saprofítica e patogênica, foram utilizados 12 solos coletados em áreas destinadas ao cultivo de feijão-caupi e feijão-comum. A atividade saprofítica foi avaliada com iscas de palito de dente e a atividade patogênica foi avaliada pela distribuição dos solos em bandejas, plantio de sementes de feijão-caupi e avaliação da severidade da rizoctoniose. Houve correlação positiva (r = 0,7698) significativa (P≤0,05) entre as atividades saprofítica (ATS) e patogênica (ATP). A equação de regressão ATP=1/(0,5822-0,0056 ATS) foi estimada com elevada precisão (R2 = 0,9930; P≤0,05), indicando que o risco de atividade patogênica de Rhizoctonia nos solos destinados ao cultivo de feijão-caupi e feijão-comum pode ser estimado a partir da análise da atividade saprofítica.

The work developed in this thesis aimed to increase understanding about the variability and stability in eleven biotypes of Pochonia chlamydosporia, a facultative parasitic fungus with potential as a biological control agent against root-knot (Meloidogyne spp.), false root-knot (Naccobus spp.) and cyst nematodes (Heterodera and Globodera, spp.). Differences in performance were assessed by measuring saprophytic and parasitic growth using in vitro bioassays. Information on virulence (in vitro) was collected for a range of biotypes with the objective to relate in vitro parasitic growth with rhizosphere colonisation ability and secretion of extracellular enzymes. Results showed differences between biotypes in their ability to colonise the rhizosphere of plants, parasitise nematode eggs and to produce a range of extracellular enzymes but no significant relationships were found between saprophytic or parasitic growth and enzyme production. For the first time, the specific activity of protease, chitinase, esterase and lipase enzyme production by eleven biotypes of the fungus was examined. Enzymatic activity was shown to vary with the biotype and type of enzyme assayed and biotypes could be ranked according to their similarities in enzyme production A novel bioassay to estimate egg parasitism using liquid media highlighted the importance of nutrition in infection processes and suggested that all biotypes are able to infect large numbers of eggs rapidly if the conditions are favourable. The assay reliably detected fungal infection in nematode eggs within 48 hours and provided a simple, rapid assay to test the effect of specific nutrients at controlled concentrations on the infection process. Differences in infection rates between biotypes observed in previous tests on agar were not detected in the new assay in which nematode eggs and fungal conidia were added in suspension. Internal colonisation of individual whole Meloidogyne spp. eggs by P. chlamydosporia was observed using microscopy studies. The destruction of nematode eggs infected with the fungus within seven days, was confirmed. The in vitro formation of appressoria was studied in a range of P. chlamydosporia biotypes. for the first time. Biotypes were found to differ in their ability to produce appressoria but this ability was not related to differences in virulence (in vitro) against nematode eggs. Cont/d.

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