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Titre de l'écran-titre (visionné le 15 décembre 2023) / Les forêts, les agents pathogènes et leurs interactions sont de plus en plus affectés par des facteurs liés au changement climatique et à la mondialisation. Ces changements influencent la répartition géographique des agents pathogènes, la gravité de leurs dommages et les impacts de leurs origines indigènes ou invasives. Dothistroma septosporum est un champignon ascomycète présent sur plusieurs continents (Europe, Afrique, Amériques); cet agent pathogène était auparavant considéré comme étant endémique sur les pins dans l'ouest de l'Amérique du Nord. Des épidémies récentes de cette maladie dans l'hémisphère nord ont été observées et semblent être directement liées au changement climatique, qui favorise la propagation de l'agent pathogène et la gravité de la maladie. Par conséquent, l'expansion des connaissances quantitatives sur la façon dont le changement climatique entraîne le développement de D. septosporum est cruciale. Dans le premier chapitre, nous analysons les gènes différentiellement exprimés de D. septosporum au cours de l'infection et comparons les interactions sensibles et tolérantes avec son hôte, Pinus contorta. On a inoculé D. septosporum à des individus de P. contorta et des échantillons du tissu infecté ont été prélevés au cours des stades précoce, moyen et tardif de l'infection. L'expression de gènes différentiellement exprimés a été comparée entre les hôtes sensibles et tolérants à la maladie. Nous avons identifié de nombreux gènes candidats qui sont différentiellement exprimés, dont ceux qui jouent un rôle dans la virulence, notamment de nombreux gènes encodant des transporteurs, facteurs de transcription et CAZymes. Ces gènes ont également été précédemment observés dans d'autres interactions pathogènes. Pour le chapitre deux, nous avons identifié les gènes et les SNPs liés à la croissance de D. septosporum à différentes températures à l'aide d'une analyse d'association à l'échelle du génome (GWA). La croissance de chaque isolat de D. septosporum provenant de l'ouest de l'Amérique du Nord a été mesurée sur une gélose à l'extrait de malt à 3 % pendant 5 semaines et le taux de croissance calculé. La contribution des SNPs a été évaluée par une analyse de régression avec un modèle linéaire. Nous avons identifié des gènes d'intérêt, associés au taux de croissance; les produits de ces gènes sont dans des classes bien connues, comme des transporteurs et la régulation du métabolisme. Les gènes candidats identifiés par GWA qui jouaient un rôle dans la croissance étaient plus répandus à des températures plus élevées (20ºC et 25ºC) en raison du meilleur ajustement des modèles par rapport à 15ºC. Une population « à risque » recueillie à Vancouver, en Colombie-Britannique, qui a un génotypage européen, a été identifiée à partir des données phénotypiques. La population de Vancouver surpasse les autres populations à toutes les températures de 15ºC, 20ºC et 25ºC. Enfin, le chapitre trois utilise des modèles de distribution des espèces (SDM) pour projeter la probabilité de propagation de D. septosporum selon plusieurs scénarios de changement climatique. La méthode « Ensemble » a été utilisée pour prendre en compte plusieurs modèles uniques, ce qui augmente la puissance de prédiction. Les données d'occurrence de l'agent pathogène ont été divisées en deux populations génétiquement distinctes, représentant les populations côtières et intérieures de l'ouest de l'Amérique du Nord. La population côtière a montré une plus grande probabilité de propagation par rapport à son homologue de l'intérieur, mais les deux semblent augmenter avec le temps et se déplacer vers le sud-est sous les climats futurs. Dans l'ensemble, les cartes de prédiction des distributions futures ont montré une augmentation de la superficie totale pour les deux populations au milieu et à la fin du siècle, 2050 et 2100, respectivement. / Forests, pathogens, and their interactions are increasingly affected by factors related to climate change and globalization. These changes are influencing geographical distribution of pathogens, the severity of their damages and the impacts that pathogens have on their native or invasive ranges. Dothistroma septosporum is an ascomycete fungus that is endemic on pines in Western North America and a pathogen with worldwide distribution. Recent outbreaks of this disease in the Northern hemisphere have been observed and appear to be directly linked to the changing climate, which aids the spread and severity of the pathogen. Therefore, the expansion of quantitative knowledge on how climate change drives the development D. septosporum is crucial. In the first chapter, we analyse differentially expressed genes of D. septosporum during infection and compare both susceptible and tolerant interactions with its host, Pinus contorta. Susceptible and tolerant P. contorta were artificially inoculated with D. septosporum and samples of the infected tissue were collected during its early, middle, and late stages of infection. The expression of differentially expressed genes for susceptible hosts were analysed at each time point and further investigated. We identified many candidate genes that play a role in virulence, including many transporters, transcription factors, and CAZymes. These genes have also been previously observed in other pathogenic interactions. For Chapter Two, we use genome-wide association (GWA) analysis to identify genes and SNPs related to growth of D. septosporum at three different temperatures: 15ºC, 20ºC and 25ºC. We monitored the growth of D. septosporum isolates from across Western North America on 3% malt extract agar for 5 weeks and extracted the growth rate by fitting the observations on a linear model. A « risk » population collected from Vancouver, BC, which has a European genotyping, was identified from the phenotypic data. The Vancouver population outperforms other populations at all three temperatures tested. The GWA showed that candidate genes involved in growth were more prevalent at higher temperatures (20oC and 25ºC) compared to 15ºC. These candidate genes include transporters and genes involved with regulation of metabolism. Finally, Chapter Three discusses our use of species distribution models (SDM) to project the likelihood of D. septosporum spread under several climate change scenarios. Ensemble forecasting techniques were used as opposed to single models to improve prediction power. Occurrence data of the pathogen was divided into two genetically distinct populations, representing the coastal and interior populations of Western North America. The coastal population showed a higher likelihood.

Content removed from thesis due to copyright restrictions: Schwelm, A., Barron, N. J., Zhang, S. & Bradshaw, R. E. (in press). Early expression of aflatoxin-like dothistromin genes in the forest pathogen Dothistroma septosporum. / Dothistroma septosporum causes pine needle blight, a foliar disease currently causing epidemics in the Northern hemisphere. D. septosporum synthesizes dothistromin, a mycotoxin similar in structure to the aflatoxin (AF) precursor versicolorin B. Orthologs of AF genes, required for the biosynthesis of dothistromin, have been identified along with others that are speculated to be involved in the same pathway. The dothistromin genes are located on a mini-chromosome in Dothistroma septosporum but, in contrast to AF genes, not in a continuous cluster. The aim of this study was to increase knowledge of the biological role of dothistromin, which was previously a suspected pathogenicity factor. To identify putative roles of dothistromin, the dothistromin gene expression was investigated and green fluorescence protein (GFP) reporter gene strains of D. septosporum were developed. Expression analyses of dothistromin genes revealed co-regulation. More surprisingly, dothistromin is produced at an early stage of growth and gene expression is highest during exponential growth. This is fundamentally different to the late exponential/stationary phase expression usually seen with secondary metabolites such as AF. Strains with a dothistromin gene (dotA) promoter-regulated GFP confirmed early expression of the toxin genes, even in spores and germtubes. Parallel studies with transformants containing a GFP-DotA fusion protein suggest spatial organization of dothistromin biosynthesis in intracellular vesicles. The early expression of dothistromin genes led to the hypotheses that dothistromin is either required in the early stage of the plant/fungi interaction, or for inhibiting the growth of competing fungi. Constitutive GFP strains helped to determine that dothistromin is not a pathogenicity factor. However, a putative role of dothistromin in competition with other fungi, including pine-colonizing species, was detected, supporting the second hypothesis. It was shown that dothistromin-producing strains appear to have a competitive advantage which is lacking in dothistromin-deficient strains. However, some competitors were not affected and have potential as biocontrol agents. In summary, this work has led to the discovery of an unusual pattern of regulation of a secondary metabolite, has made substantial progress in identifying the biological role of dothistromin, and has indicated potential for biocontrol of Dothistroma needle blight.

Content removed from thesis due to copyright restrictions: Schwelm, A., Barron, N. J., Zhang, S. & Bradshaw, R. E. (in press). Early expression of aflatoxin-like dothistromin genes in the forest pathogen Dothistroma septosporum. / Dothistroma septosporum causes pine needle blight, a foliar disease currently causing epidemics in the Northern hemisphere. D. septosporum synthesizes dothistromin, a mycotoxin similar in structure to the aflatoxin (AF) precursor versicolorin B. Orthologs of AF genes, required for the biosynthesis of dothistromin, have been identified along with others that are speculated to be involved in the same pathway. The dothistromin genes are located on a mini-chromosome in Dothistroma septosporum but, in contrast to AF genes, not in a continuous cluster. The aim of this study was to increase knowledge of the biological role of dothistromin, which was previously a suspected pathogenicity factor. To identify putative roles of dothistromin, the dothistromin gene expression was investigated and green fluorescence protein (GFP) reporter gene strains of D. septosporum were developed. Expression analyses of dothistromin genes revealed co-regulation. More surprisingly, dothistromin is produced at an early stage of growth and gene expression is highest during exponential growth. This is fundamentally different to the late exponential/stationary phase expression usually seen with secondary metabolites such as AF. Strains with a dothistromin gene (dotA) promoter-regulated GFP confirmed early expression of the toxin genes, even in spores and germtubes. Parallel studies with transformants containing a GFP-DotA fusion protein suggest spatial organization of dothistromin biosynthesis in intracellular vesicles. The early expression of dothistromin genes led to the hypotheses that dothistromin is either required in the early stage of the plant/fungi interaction, or for inhibiting the growth of competing fungi. Constitutive GFP strains helped to determine that dothistromin is not a pathogenicity factor. However, a putative role of dothistromin in competition with other fungi, including pine-colonizing species, was detected, supporting the second hypothesis. It was shown that dothistromin-producing strains appear to have a competitive advantage which is lacking in dothistromin-deficient strains. However, some competitors were not affected and have potential as biocontrol agents. In summary, this work has led to the discovery of an unusual pattern of regulation of a secondary metabolite, has made substantial progress in identifying the biological role of dothistromin, and has indicated potential for biocontrol of Dothistroma needle blight.