Fungi are biologically diverse organisms known to live in competitive environments. To survive in these conditions, fungi produce an array of biologically active secondary metabolites including antimicrobial, antiinsectan, phytotoxic and cytotoxic compounds. As such, fungi are a viable source for new antibiotics. Conversely, crops colonized by fungi are at risk of mycotoxin contamination. It is important to understand the chemistry of these toxins, and what will result from their entry into the food chain. The following studies describe the isolation and identification of secondary metabolites from an isolate of Alternaria metachromatica in the search for new antimicrobial agents and characterization of fungal mycotoxins in food crops. Studies of alfalfa, a valued cash crop in Minnesota, have led to the identification of several fungal pathogens and endophytes. One isolate from St. Paul, MN was identified as A. metachromatica (S3-9 DNA12) based on DNA analysis. This isolate was cultured on jasmine rice for 30 days and then extracted with ethyl acetate. The resulting crude extract (0.9539 g) was then tested in disk diffusion assays and found to be active against S. epidermidis, S. saprophyticus, B. subtilis, E. coli, and V. trifolii N29, warranting chemical investigations. The crude extract was first partitioned using acetonitrile, hexanes, and methanol to remove fat. The acetonitrile-soluble fraction was then separated using vacuum liquid chromatography (VLC) resulting in 9 fractions. Five fractions from this chromatography displayed interesting 1H NMR profiles and were next tested in antimicrobial assays. One of these fractions (Bsi; 338mg) was the largest in mass and active against S. pyogenes. For these reasons it was prioritized for further purification in an effort to identify the metabolites responsible for the displayed antibacterial effects. Fraction Bsi was separated using gravity silica column chromatography resulting in 41 fractions based on TLC analysis. Fraction 14 (Bsi.14; 9.9mg) from this chromatography appeared to contain mostly one metabolite with minor components from the same family after analysis of its 1H NMR data. Due to its small sample size and relative purity, it was decided to collect more spectroscopic data (13C NMR, DEPT, HMQC, HMBC, 1H-1H COSY, and LCMS data) prior to testing in antibiotic assays. Spectroscopic analysis of fraction Bsi.14 as well as bioassay-guided fractionation of other VLC fractions will be discussed.

Spectroscopic analysis of secondary metabolites from extracts of Alternaria metachromatica

251st National Meeting (2016)

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