| A collection of more than 2000 endophytes were isolated from natural white (Picea glauca) and red (Picea rubens) spruce trees located in New Brunswick, Nova Scotia and Maine, USA. A random selection of both white and red spruce endophytes were grown in one litre fermentations. Strains of interest due to their ability to grow and produce substantial amounts of secondary metabolites under laboratory conditions, were further screened by LC-MS. The extracts that showed interesting peaks in the chromatograms were tested for toxicity to spruce budworm ( Choristoneura fumiferana) using diet incorporated bioassays and for antifungal properties to Saccharomyces cerevisae using disc diffusion testing. The taxonomy of these strains that displayed toxicity were then examined using DNA sequencing to try and determine the identity of the endophytes present in the needles.;This study had two primary foci; the first was the characterization and structure elucidation of secondary metabolites from foliar fungal endophytes from both white and red spruce trees using MS and NMR. The work was highlighted by the discovery of two novel secondary metabolites, compounds 1 and 2a; two new fungal metabolites, compounds 3 and 4; and the known metabolites 5-10 from the three strains of white spruce endophytes studied in detail. Additionally, compounds 11 and 15 are reported as new structures, 12 and 13 as natural products for the first time, 14 is reported here for the first time as a fungal metabolite and 16-22 were identified as known fungal metabolites in new genera, from the six strains of red spruce endophytes examined. The second focus was on the analysis of the toxins and the presence of the endophyte in inoculated seedlings. Antibody based ELISA methods were developed for the detection of five white spruce endophytes in needles, to provide a reliable method for determining inoculation success. Analytical methods were also developed using HPLC and LC-MS/MS in multiple reaction monitoring (MRM) mode to determine the presence and concentration of the characterized toxins in infected tree needles for three of the white spruce endophytes. The results indicated that needles of trees colonized with these endophytes contain the toxins. These toxins in turn are responsible for reducing the growth rate of spruce budworm larvae. The presence of the endophyte and its toxin is thought to increase the development time of larvae, put the populations out of reproductive synchrony and expose them to a greater risk of death from parasitoides, pathogens and predation. |
