Trees and forests are under increasing threat from pathogens which cause huge economic and ecological damage. The unprecedented global movement of pathogens into new areas creates novel pathosystems, while the changing climate affects the dynamics of endemic pathosystems. Co-evolution within endemic pathosystems affects the genetic composition of hosts and pathogens. Spatial heterogeneity in pathogen pressure leads to genetic variation in disease-related traits among host populations. In contrast, novel hosts or populations are expected to be highly susceptible to exotic pathogens as there has been no evolution of defence responses. Host response to disease can therefore be an indicator of a novel or endemic pathosystem. The long term resilience of forests to pathogens depends on the adaptive capacity of both the host and pathogen species. Establishing the extent of genetic and phenotypic variation within both the host and pathogen is therefore fundamental in understanding past, current and future pathosystem dynamics. The most significant current threat to Scots pine (Pinus sylvestris) is Dothistroma needle blight (DNB) caused by the foliar pathogen Dothistroma septosporum which is assumed to be exotic to Great Britain. This study aimed to increase understanding of the genetic and phenotypic variation in this pathosystem. Results from this study show that there are high levels of variation in the Dothistroma – Pinus pathosystem. Genetic variation, elucidated using neutral genetic markers, mating type specific markers and in vitro analysis of phenotypic variation in D. septosporum collected from Scottish pinewoods, was found to be high: there was high allelic diversity, particularly within plantation forests outside the native pinewood range, and high phenotypic plasticity in response to different temperature treatments. Both mating type idiomorphs were found in one forest which demonstrates their potential for sexual as well as asexual reproduction. There is also tentative evidence from this study that the pathogen is either introduced to Great Britain or that endemic pathogen populations have been augmented with introduced pathogens. Artificial and natural inoculations of native Scots pine provenances with D. septosporum indicate that there is considerable variation in susceptibility to DNB across the native range in Scotland and that variation in this trait is both highly heritable and evolvable. Furthermore, provenance mean susceptibility to DNB is negatively and significantly associated with water-related variables at site of origin, a finding that is potentially indicative of a co-evolutionary history between host and pathogen. Genetic differences among individuals which are ‘resistant’ or ‘susceptible’ to DNB were identified in Pinus radiata for which there has been extensive research in this pathosystem, by comparing the transcriptome sequences of the two phenotypic groups. Nearly half of the genetic differences identified among phenotypes were found in genes with a putative defence function. In conclusion, native Scots pine provenances contain the necessary heritable genetic diversity to evolve a decrease in their susceptibility to D. septosporum through natural selection in response to elevated prevalence of this pathogen. However, implementation of key native pinewood management strategies, including encouraging regeneration in particular, are necessary in order to facilitate the adaptive evolution of native forests to increased levels of DNB. The effectiveness of this response will depend on the rapidity of adaptation of the pathogen. Measures to limit adaptation where possible, including the use of pathogen monitoring and control in nurseries and the limitation of pathogen movement into native pinewoods, should be continued.

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.