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Species diversity and production of antimicrobial compounds by Pacific Northwestern clavicipitalean entomogenous fungi (Cordyceps spp.) King, Brian Christopher

Abstract

Members of the fungal genus Cordyceps and its anamorphic genera are mostly pathogens of arthropods (insects, spiders, mites) with a few species parasitizing hypogeous fungi (Elaphomyces spp.). They produce numerous secondary metabolites with a wide range of biological activities which facilitate their life cycle and ecological roles. Examples include immunosuppressant, antibacterial, antifungal, antiviral, cytotoxic, and insecticidal compounds. This research focused on entomogenous fungi collected from British Columbia, Oregon, and Washington. Twenty-eight isolates were selected for DNA sequencing and phylogenetic analysis. Genomic DNA was extracted from mycelium grown on agar media. PCR was used to amplify the ribosomal ITS and LSU regions which were then sequenced. Examination of morphology and DNA sequences grouped the isolates into twelve distinct taxa within the family Clavicipitaceae (Ascomycota). Most were from the teleomorphic genus Cordyceps and its anamorphic genera Beauveria, Isaria, Tolypocladium, and Metarhizium. One isolate was in Lecanicillium, an anamorph of the spider and scale pathogen Torrubiella. To screen for antibacterial secondary metabolites, seven isolates from four species of entomogenous fungi were grown on different liquid media. Cultures were filtered through a fine mesh screen to separate the mycelium from the liquid broth. They were then frozen, freeze dried, and extracted in 100% methanol. Antibacterial activity was tested using the disc-diffusion bioassay. The crude extracts from five of six fungi showed significant inhibition of the gram-positive bacteria Staphylococcus aureus, S. aureus methicillin resistant, Bacillus subtilis, and Enterococcus faecalis. None showed activity against the gram-negative Escherichia coli, and Salmonella typhimurium. The minimum inhibition concentration for extracts showing bioactivity was determined using serial dilutions in a ninety-six well plate. The phenol-red overlay method allowed for the integration of thin layer chromatography and bioactivity. One fungus was selected for further chemical investigation. Using a combination of liquid-liquid partitioning, preparative column chromatography, and preparative thin layer chromatography, three compounds showing antimicrobial activity were isolated. Structural elucidation of these compounds using MS and NMR is currently underway.

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