"This thesis is based on the following articles, referred to in the text by the Roman numerals given below. In addition some unpublished results are presented. I. Caiyan Wu ... [et al] Improvement of the secretion of extracellular proteins and isolation and characterization of the amylase I (amyI) gene from Ophiostoma floccosum [pub. in ] Gene 384: 96-103 -- II. Caiyan Wu ... [et al.] Activity-based identification of secreted serine proteases of the filamentous fungus Ophiostoma. Accepted by Biotechnology letters DOI 10.1007/s10529-007-9333-6 -- III. Caiyan Wu ...[et al.] Expression of a thermostable bacterial xylanase in the filamentous fungus Ophiostoma floccosum. Submitted to Letters in applied microbiology in July 2007." - leaf 9. / Thesis (PhD)--Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2007. / Bibliography: leaves 100-123. / Introduction -- Materials and methods -- Results and discussion -- Conclusion and future aspects -- References -- Publications I, II and III. / Ophiostoma spp. belong to the Ophiostomataceae family, a large group of ascomycetes, which are the most frequent blue stain fungi isolated from stained wood. Most Ophiostoma species do not compromise the strength properties of wood, but do reduce the aesthetic quality of timber and therefore decrease the economic value of lumber. Some albino variants of O. floccosum and O. piliferum have been used as biological control agents to prevent blue staining. This successful whole organism approach plus the added capability of extracellular protein secretion makes Ophiostoma spp. attractive for industrial application. In addition, Ophiostoma produces only a small range of abundantly secreted proteins in liquid culture, which can facilitate downstream purification of any recombinant gene product introduced into the system. Genes encoding efficiently secreted proteins provide a potential souce for strong promoters for high-level gene expression. These characteristics provide an excellent starting point for the development of a novel expression system. / In this study, UV-mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein secretion after repeated rounds of mutagenic treatment. Several mutants of O. floccosum derived by UV mutagenesis were isolated and the total amount of secreted protein was increased by 4 to 6 times. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the amount of total secreted protein was about six times greater to that of the parental strain. Proteinase profiles in the culture supernatants of several key mutants were characterized for the future matching of an expression host with a particular gene product. N-terminal sequencing of the five dominant proteins separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an_-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the _-amylase amino acid sequence. The amplified fragment contained the entire gene encoding_-amylase (amyl) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single _-amylase gene locus. / A series of expression vectors containg the _-amylase regulatory sequences and partial amyl gene were constructed. Several selection markers were screened and the hph gene conferring hygromycin resistance under the regulation of the Aspergillus nidulans gpd promoter was chosen and inserted into the amyl expression vectors. The gene encoding a red fluorescent protein DsRed-E5 was used as a reporter gene to test the expression system using mutant MQ.5.1 as host. However, no transformants were obtained by either biolistic transformation or protoplast transformation. Subsequently, an alternative strategy was developed using a thermostable xylanase B as a reporter. Thermostable xylanase activity was detected in the culture supernatants of several transformants. Production of xylanase by transformant SS41 which exhibited high secreted xylanase activity was investigated. Xylanase activity in the culture supernatant of SS41 was visualized by a zymogram gel assay. Two active proteins with molecular masses of around 27 and 30 kDA, which were larger than the predicted Mr of 25 kDA were detected. This is the first report describing successful expression of a recombinant thermostable bacterial enzyme in Ophiostoma. / Mode of access: World Wide Web. / 158 leaves col. ill

Hylurgopinus rufipes, the native elm bark beetle (NEBB), is the major vector of Dutch elm disease (DED) in Manitoba. Dissections of American elms (Ulmus americana), in the same year as DED symptoms appeared in them, showed that NEBB constructed brood galleries in which a generation completed development, and adult NEBB carrying DED spores would probably leave the newly-symptomatic trees. Rapid removal of freshly diseased trees, completed by mid-August, will prevent spore-bearing NEBB emergence, and is recommended. The relationship between presence of NEBB in stained branch sections and the total number of NEEB per tree could be the basis for methods to prioritize trees for rapid removal. Numbers and densities of overwintering NEBB in elm trees decreased with increasing height, with >70% of the population overwintering above ground doing so in the basal 15 cm. Substantial numbers of NEBB overwinter below the soil surface, and could be unaffected by basal spraying. Mark-recapture studies showed that frequency of spore bearing by overwintering beetles averaged 45% for the wild population and 2% for marked NEBB released from disease-free logs. Most NEBB overwintered close to their emergence site, but some traveled ≥4.8 km before wintering. Studies comparing efficacy of insecticides showed that chlorpyrifos gave 100% control of overwintering NEBB for two years as did bifenthrin: however, permethrin and carbaryl provided transient efficacy. NEBB showed a gradual increase in development rate with increasing constant temperature. Lipid content of overwintering NEBB was higher in late fall than in mid-winter, which might show that depletion of fat reserves could jeopardize survival, but could be a result of conversion to cryoprotectants.

Dissertation (Ph.D.)--Auburn University,2009. / Abstract. Vita. Includes bibliographic references (p.222-226).

Granbarkborren, Ips typographus, är den mest destruktiva skadeinsekten för svensk barrskog och orsakar årligen stora ekonomiska förluster för landets skogsägare. Granbarkborren kan endast föröka sig i döda träd. För att döda ett stående friskt träd kan de med feromoner signalera till andra granbarkborrar att tillsammans inleda en massattack. Granbarkborren är även beroende av blånadssvampar för att kunna slå ut friska träd. Med dessa blånadssvampar kombinerat med en massattack kan granbarkborren döda trädet inom några veckor. Blånadssvampen ger en missfärgning i virket vilket innebär en värdeminskning av den erhållna veden. I detta examensarbete har de tillväxthämmande effekterna mellan blånadssvamparterna Ophiostoma picea, Grosmannia penicillata och Ceratocystis polonica undersökts. De tillväxthämmande effekter svamparna utövar på varandra har studerats visuellt med odlig i artificiellt såväl som naturligt medium representerad av bark. Tillväxthastigheterna hos respektive svamp har uppmätts. G. penicillata fanns ha den högsta tillväxthastigheten samt de största tillväxthämmande effekterna mot de andra svamparna. Kemiska analyser har utförts med SPME och GCMS för att se vilka ämnen svamparna och barken producerar. Resultaten tyder på att vissa ämnen som produceras av barken inte finns närvarande efter att svampen vuxit i barken. Många ämnen produceras av alla tre svampar men en del är även unika för en viss svampart. Som exempel var G. penicillata ensam om att producera seskviterpenen β-Guaiene, det aromatiska kolvätet m-Cymene och terpenen Borneol. Barken producerade ämnen, bland dessa seskviterpen-alkoholen δ-Cadinol, som sedan inte gick att finna när svamparna var närvarande. Detta tyder på att svamparna konsumerat dessa ämnen alternativt att barken inte producerar samma ämnen i svamparnas närvaro. / The spruce bark beetle, Ips typographus, is the most destructive insect pest of Swedish pine forests and causes major economic losses annually for the forest owners. The spruce bark beetle can only reproduce in dead trees. To kill a healthy tree they can, with pheromones, signal other spruce bark beetles to launch a mass attack. The spruce bark beetle is also dependent on the blue stain fungi in order to beat out healthy trees. With these blue stain fungus combined with a mass attack, the spruce bark beetles can kill the tree within a few weeks. The fungus causes a discoloration in the wood, resulting in a reduction in economic value of the obtained wood. In this thesis, the growth inhibitory effects of blue stain fungi species Ophiostoma picea, Grosmannia penicillata and Ceratocystis Polonica have been investigated. The growth inhibitory effects that the fungi have on each other have been studied visually by cultivation in an artificial as well as a natural medium represented by bark. The growth rates of the respective fungi have been measured. G. penicillata were found to have the highest growth rate and the largest growth inhibitory effects on other fungi. Chemical analyses were performed with SPME and GCMS to see what chemical substances fungi and bark produces. The results show that some substances produced by the bark are not present when the fungi are growing in the bark. Various substances are produced by all three fungi, but there are also many unique compounds from a particular fungal species. For example, G. penicillata was the only one that produced the sesquiterpene β-Guaiene, the aromatic hydrocarbon m-cymene and the terpene Borneol. The bark also produced unique substances, among these the sesquiterpenoid alcohol δ-Cadinol, which then could not be found when the fungi were present. This suggests that the fungi consume these substances or that the bark does not produce the same substances in fungi presence.

L’orme, un arbre très apprécié pour le paysagisme urbain, est menacé par des champignons ophiostomatoïdes causant la maladie hollandaise de l'orme (MHO). Dans le xylème de l’orme, ces champignons se répandent passivement grâce à leurs cellules levuriformes bourgeonnantes et se servent de la forme mycélienne pour progresser dans les vaisseaux non infectés. Ce dimorphisme pourrait faciliter la colonisation de l’hôte, ce qui indique une possible liaison avec la virulence. Plusieurs stimuli affectent le dimorphisme, y compris des inhibiteurs de lipoxygénases. Les lipoxygénases (Lox), ainsi que les cyclooxygénases (Cox) sont des dioxygénases qui catalysent la formation d’oxylipines à partir d’acide gras. Ces molécules modulent le développement fongique et agissent possiblement dans le dimorphisme et la virulence des Ophiostoma. L’objectif de cette thèse est d’analyser le dimorphisme des agents de la MHO et vérifier si cette transition et la pathogénie sont modulées par des gènes qui encodent des enzymes du groupe des dioxygénases. Nous avons examiné l’effet de différents stimuli sur le dimorphisme des souches d’ophiostomatoïdes qui causent la MHO. Nous avons aussi vérifié si l’acide linoléique induit la transition de levure-mycélium et la formation de structures de reproduction chez ces agents. Également, nous avons identifié et caractérisé des gènes qui encodent des dioxygénases chez les génomes d’O. novo-ulmi H327 et d’O. ulmi W9. De plus, nous avons produit une souche Δppo1 d’O. novo-ulmi pour explorer la fonction de ce gène. Cette transition est probablement modulée par différents mécanismes et voies car la réponse à la manipulation de stimuli variait selon la souche analysée. L’acide salicylique, inhibiteur de cyclooxygénases, réduit la production de mycélium chez les souches testées. Néanmoins, l'acide linoléique a stimulé la formation de mycélium ainsi que la production des structures de reproduction. Aucun gène lox mais deux gènes cox sont présents dans les génomes d’O. novo-ulmi H327 et d’O. ulmi W9. Selon nos résultats, un de ces gènes cox (gène ppo1 ou g7173) ne semble pas lié à la virulence ni aux fonctions essentielles du cycle de vie d’O. novo-ulmi. Toutefois, les mutants Δppo1 produisent moins de mycélium que les souches sauvages en milieu liquide contenant de l’arginine. D’après nos résultats, les cyclooxygénases pourraient être liées au dimorphisme des agents de la MHO. / Elm trees, which are valued for urban landscaping, are threatened by ophiostomatoid fungi causing Dutch elm disease (DED). These fungi are transported passively in the elm xylem as yeast-budding cells and switch to the mycelial form to progress in uninfected vessels. This dimorphism may facilitate colonization of the host, which indicates its possible association with fungal virulence. Several stimuli affect dimorphism, among them lipoxygenase inhibitors. Lipoxygenase (Lox) and cyclooxygenase (Cox) are dioxygenases which catalyze oxylipins from fatty acids. These molecules modulate fungal growth and are possibly involved in dimorphism and virulence in Ophiostoma. The objective of this thesis was to analyze dimorphism of DED agents and verify whether this transition and pathogenesis are modulated by genes that encode dioxygenase enzymes. The effect of different stimuli on dimorphism was examined in ophiostomatoid strains that cause DED. I also verified if linoleic acid induced transition from yeast to mycelium and production of reproductive structures in these agents. In addition, I identified and characterized genes that encode dioxygenases in genomes of O. novo-ulmi H327 and O. ulmi W9. Finally, I produced a Δppo1 strain of O. novo-ulmi to determine the function of this gene. This transition is probably modulated by different mechanisms and pathways since the response to the manipulation of stimuli varied according to the strain analyzed. Salicylic acid, a cyclooxygenase inhibitor, reduced mycelial production in the strains tested. However, linoleic acid stimulated the production of mycelia and reproductive structures. No lox gene but two cox genes are present in the genomes of O. novo-ulmi H327 and O. ulmi W9. According to our results, one of these cox genes (gene ppo1 or g7173) does not seem related to the virulence or essential functions of the O. novo-ulmi life cycle. Nonetheless, the Δppo1 mutants produced less mycelia in liquid medium with arginine compared to the wild-type. As observed with these data, cyclooxygenases could be related to dimorphism of DED agents.

Afin d'assurer un contrôle des dommages causés par la maladie hollandaise de l'orme, une meilleure compréhension de l'interaction hôte-pathogène s'avère nécessaire. Plus spécifiquement, cette étude vise à approfondir les connaissances du pathogène lui-même et des facteurs de virulence utilisés par celui-ci. Deux méthodes permettent d'identifier les gènes impliqués dans la pathogénie du champignon ascomycète Ophiostoma novo-ulmi : 1) la comparaison de souches présentant des niveaux de virulence très différents ou 2) la mutagenèse aléatoire. En milieu naturel il existe très peu de variation de la virulence entre les souches d'O. novo-ulmi; en conséquence, la première alternative n'était pas envisageable. C'est pourquoi nous avons développé une méthode efficace de transformation à partir d'une souche très virulente d'O. novo-ulmi (H327) afin de produire des transformants moins virulents et d'identifier l'un des gènes associés à cette perte de virulence. L'ajout d'enzyme de restriction a permis d'augmenter le taux de transformants mais n'a pas favorisé le taux d'insertions uniques du plasmide tel que mentionné dans d'autres travaux. Des séquences d'ADN génomique proches du site d'insertion du plasmide ont ensuite été isolées à partir des transformants présentant une croissance réduite. Le deuxième volet de ces travaux consiste à développer un test qui permettrait de déterminer rapidement la virulence des transformants et d'éviter l'utilisation de plants d'orme lors du criblage initial. Nous avons démontré l'utilité de mesurer les lésions sur pommes 'Golden Delicious' pour évaluer la virulence des transformants et de souches sauvages d'O. novo-ulmi. Enfin, la dernière partie de la thèse porte spécifiquement sur un transformant moins virulent, KP78, identifié précédemment lors des tests sur pommes et sur ormes. Parmi les six gènes situés dans la région de l'insertion, deux présentent une expression réduite dans le mycélium poussant sur milieu au bois d'orme. L'analyse de ségrégation de l'insertion dans une descendance issue d'un croisement entre KP78 et une souche de type sauvage a montré qu'une mutation non marquée serait responsable des caractères qui distinguent KP78 de la souche de référence H327.

L’analyse de données de transcriptomique par le biais du séquençage d’ARN messagers (RNAseq) offre une perspective globale de la régulation de l’expression génique au cours d’un évènement biologique. Dans cette thèse, nous avons exploité cette technique dans le but de comprendre les mécanismes moléculaires qui régulent la transition morphologique réversible levure-mycélium qui est une caractéristique souvent liée au pouvoir pathogène chez les champignons. Dans un premier temps, par le biais de comparaisons de données de transcriptomique entre sept espèces fongiques dimorphiques, nous avons observé une certaine conservation des processus biologiques associés au changement de morphologie chez des champignons issus de branches très éloignées de l’arbre phylogénétique fongique. Dans un second temps, nous nous sommes concentrée sur notre modèle d’étude principal, Ophiostoma novo-ulmi, le champignon pathogène responsable de la maladie hollandaise de l’orme. Par l’analyse comparée des gènes exprimés en phases levure et mycélienne, nous avons défini les facteurs moléculaires qui sont spécifiques à chacune des phases chez O. novo-ulmi et établi une distinction claire entre les deux phases d’un point de vue du contenu en gènes exprimés. Par la suite, nous avons affiné notre étude en nous focalisant sur l’évènement de transition levure-mycélium afin déterminer les gènes dont l’expression était modulée au cours du temps dans le processus de changement morphologique. Nous avons mis en évidence plusieurs facteurs potentiellement impliqués dans la transition, notamment des gènes liés à la cascade de phosphorylation des MAPKs, connues pour jouer un rôle clé dans le dimorphisme chez plusieurs espèces fongiques. Finalement, dans le but d’évaluer plus précisément le niveau de conservation des processus biologiques liés au dimorphisme chez des espèces non modèles éloignées, nous avons comparé la régulation de l’expression génique au niveau des gènes orthologues entre O. novo-ulmi et l’espèce basidiomycète, Pseudozyma flocculosa. Nous nous sommes concentrée sur les gènes qui étaient différentiellement exprimés entre les phases de germination et de filamentation. Dans l’ensemble, les processus associés aux gènes pour lesquels la régulation de l’expression est conservée chez les deux espèces portent sur des fonctions essentielles du développement fongique. Ainsi, cette comparaison a permis de définir ce qui semble constituer la « base minimale » génique commune nécessaire à la transition asexuée levure-mycélium chez des espèces phylogénétiquement éloignées. / Large-scale transcriptomic analyses via messenger RNA sequencing (RNAseq) give access to the information on expression regulation of all the genes present in a sample at a given time and in a given experimental condition. In this thesis, we took advantage of this technology in order to investigate the molecular mechanisms that regulate the reversible yeast-to-hypha morphological switch which is a characteristic often linked to virulence in fungal pathogens. To begin with, we compared transcriptomic data among seven dimorphic fungi and found conserved biological processes associated with the morphological switch among species from very distant branches of the fungal phylogenetic tree. Later, we focused on our model species, Ophiostoma novo-ulmi, the causal agent of Dutch elm disease. We first compared the gene expression levels in yeast and mycelium growth phases. We defined the molecular factors that are specific to each growth phase and highlighted a clear molecular distinction between the two phases in terms of expressed gene contents. We further narrowed down our analysis by focusing on the yeast-to-hypha transition in a time course experiment. We determined the set of genes for which the expression was regulated during the morphological switch, thus potentially involved in the yeast-to-hypha transition. In particular, we identified genes that could be related to the MAPK cascade, known to play a crucial role in the dimorphic switch in many fungal species. Finally, in order to address the level of conservation in the biological processes linked to dimorphism in highly divergent non-model species, we compared the gene expression regulation of the orthologous genes between O. novo-ulmi and the basidiomycete Pseudozyma flocculosa. We focused on the genes that were differentially expressed between the germination and the filamentation phases. We identified several factors for which the regulation of expression seems conserved during the switch from germinating spore to filamentous growth. Overall, these genes are associated with biological processes that play essential roles in fungal development. Hence, our comparison here highlighted core components necessary for the yeast-to-hypha transition in phylogenetically distant species.

Thesis (PhD (Botany and Zology))--University of Stellenbosch, 2006. / The well-known, and often phytopathogenic, ophiostomatoid fungi are represented in South Africa by the two phylogenetically distantly related genera Ophiostoma (Ophiostomatales) and Gondwanamyces (Microascales). They are commonly associated with the fruiting structures (infructescences) of serotinous members of the African endemic plant genus Protea. The species O. splendens, O. africanum, O. protearum, G. proteae and G. capensis have been collected from various Protea spp. in South Africa where, like other ophiostomatoid fungi, they are thought to be transported by arthropod vectors. The present study set out to identify the vector organisms of Protea-associated members of mainly Ophiostoma species, using both molecular and direct isolation methods. A polymerase chain reaction (PCR) and taxon specific primers for the two Protea-associated ophiostomatoid genera were developed. Implementation of these newly developed methods revealed the presence of Ophiostoma and Gondwanamyces DNA on three insect species. They included a beetle (Genuchus hottentottus), a bug (Oxycarenus maculates) and a psocopteran species. It was, however, curious that the frequency of these insects that tested positive for ophiostomatoid DNA was very low, despite the fact that ophiostomatoid fungi are known to colonise more than 50% of Protea infructescences. Subsequent direct isolation methods revealed the presence of reproductive propagules of Ophiostoma spp. on four Protea-associated mite species (Oodinychus sp., two Tarsonemus spp. and Proctolaelaps vandenbergi). These mites are numerous within Protea infructescences and Ophiostoma spp. were isolated from a high frequency of these individuals. The Oodinychus sp. mite was found to vector most of the Protea-associated Ophiostoma species. It was thus postulated that the mites (in particular the Oodinychus sp.) act as primary vectors of the Protea-associated Ophiostoma species. The association between Oodinychus mites collected from P. repens and O. splendens proved to be mutualistic. Mites feeding on this fungus showed significantly higher population growth than mites feeding on any of the other fungal species tested. The short- and long-distance dispersal methods of these mites were also investigated. Firstly the ability of mites to move from drying infructescences to moist and sheltered areas such as provided by intact infructescences on the same plant was investigated experimentally. Significantly more mites were found to actively disperse from drying infructescences to artificially manufactured infructescences containing moistened filter paper shreds than to artificially manufactured infructescences containing dry filter paper shreds. The frequent fires associated with the habitat of these mites would, however, require movement over larger areas than what would be possible through self-dispersal. Dispersal of mites via air currents was thus investigated using sticky traps, but no Ophiostoma-vectoring mites were captured in this way. Self-dispersal aided by air currents could thus be ruled out, and our investigations shifted to vectored dispersal. Numerous insects emerging from Ophiostoma-containing P. repens and P. neriifolia infructescences were collected using specially designed emergence cages. Scanning electron microscopy and stereo-microscopy revealed that all three Ophiostoma-vectoring mite genera were phoretic on the beetle G. hottentottus. Tarsonemus spp. and P. vandenbergi were also phoretic on the beetles Trichostetha fascicularis and T. capensis associated with P. repens and P. neriifolia flowers. Mites collected from the surface of these beetles were found to vector reproductive propagules of various Ophiostoma spp. This thus seems to be the only method of long-distance dispersal of these mites and subsequently also the Protea-associated Ophiostoma species. Molecular phylogenetic reconstruction based on large subunit, ITS and beta-tubulin DNA sequence data suggests a polyphyletic origin for the Protea-associated members of Ophiostoma, which proposes multiple invasions of this unusual niche by these fungi. These studies also revealed the presence of four new species of Ophiostoma associated with Protea spp. The new species O. palmiculminatum, O. phasma, O. gemellus and Sporothrix variecibatus were thus described. Ophiostoma palmiculminatum is associated with P. repens infructescences and the Oodinychus mites collected from them. Ophiostoma phasma was collected from various Protea and mite species. Ophiostoma gemellus and Sporothrix variecibatus were initially only isolated from mites, but have subsequently also been isolated from Protea spp. The present study clarifies many aspects pertaining to the phylogeny and ecology of the interesting members of Ophiostoma associated with Protea hosts. As such this study will form the platform for further studies on the co-evolution of these insect / mite / fungi / plant associations.

Ophiostoma novo-ulmi is the causal agent of Dutch elm disease (DED) which has had a severe impact on the urban landscape in Canada. This research program focused on developing molecular genetic strategies to control this pathogenic fungus. The first strategy involved the development of RNA interference (RNAi) for the down-regulation of genes involved in pathogenicity. An efficient RNAi cassette was developed to suppress the expression of the endopolygalacturonase (epg1) locus which encodes a cell-wall degrading enzyme. This epg1-RNAi cassette significantly reduced the amount of polygalacturonase activity in the fungus and resulted in almost complete degradation of epg1 mRNA. The need for a native promoter to selectively down-regulate specific gene loci was addressed by developing a carbon-catabolite regulated promoter (alcA) to drive the expression of the epg1-RNAi cassette. The expression of an alcA-driven epg1-RNAi cassette resulted in the down-regulation of epg expression under glucose starvation but normal levels of expression in high glucose. The expression could therefore be controlled by culture conditions. The second strategy explored the potential of using dsRNA viruses to vector disruptive RNAi cassettes. An isolate of O. novo-ulmi strain 93-1224 collected in the city of Winnipeg, was infected by two dsRNA mitoviruses which upon sequence characterization were named OnuMV1c and OnuMV7. To assess the transmissibility of this dsRNA virus the infected isolate 93-1224 was paired with three naive isolates of the related fungi O. ulmi and O. himal-ulmi. Through the use of nuclear and mitochondrial markers it was determined that the virus OnuMV1c may not rely on mitochondrial fusion for transmission but may have a cytoplasmic transmission route. This investigation of gene expression and manipulation has provided tools to help understand gene regulation in O. novo-ulmi. It has also added to our knowledge of mitoviruses, their transmission and potential use as a biological control. By enhancing our understanding of transmissible hypovirulence this work contributes to efforts to develop a new approach to target DED as well as a potential model for the control of other fungal diseases. / Graduate / 0307 / 0306 / 0369 / jscarneiro@hotmail.com