Variability in the antifungal activity of wetland plants and implications to natural products research

Plants are prolific sources of secondary metabolites and harbor tremendous potential in the search for novel bioactive compounds. Variability in the distribution of these compounds is a significant challenge in natural products research. The purpose of this study was to investigate variability in the antifungal chemistry of wetland plants and its implications to natural product plant prospecting efforts. The investigation of natural products against many disease targets is hampered a lack of biological assays suitable to the small-scales necessary for evaluation of natural product samples. In particular, members of the genus Phytophthora are pathogens that cause devastating impacts on numerous plant crops. Bioassays available for Phytophthora spp. are unsuitable for natural product discovery; thus, a natural-product-compatible screening protocol was developed using Phytophthora nicotianae. Suspensions of zoospore of P. nicotianae were quantifiable using percent transmittance (620 nm), and subsequent growth of mycelia was monitored by measuring optical density (620 nm). The effects of six media and four zoospore concentrations on growth were evaluated. Both full- and half-strength Roswell Park Memorial Institute mycological broth were identified as suitable synthetic growth media for P. nicotianae, and 1000 zoospores/ml was established as the optimum initial concentration. EC ₅₀ values (i.e., effective concentration for 50% growth reduction) for eight commercial antifungal compounds were predicted by the assay and compared to those obtained by measuring linear growth of mycelia on fungicide-agar amendment. The assay was used to investigate the antifungal activity of 28 herbaceous wetland species against P. nicotianae; antifungal activity against seven medicinally and agriculturally important fungal pathogens was evaluated as well. In vitro antifungal activity was used as an indirect measure of levels of antifungal metabolites. Trends in relative antifungal activity were assessed to determine the importance of environmental parameters on sourcing strategies for the discovery of antifungal metabolites from plants. Antifungal activity was greatest in plants collected from wet-soil areas of high altitude meadows late in the growing season for wetland species, and mesic-soil areas of low altitude meadows early in the season for upland species. Chemical variability was further assessed for two species, Caltha leptosepala and Trollius laxus. Significant temporal and spatial differences were observed in crude extract concentrations, qualitative chemical profiles, and antifungal activity. Reciprocal transplant experiments revealed that this variability was the product of phenotypic plasticity. Changes in extract concentrations, chemical profiles, and antifungal activity occurred within a two-month transplant period and no further adaptation was noted within an additional 12 months. Results from this study reveal that environmental parameters are important considerations in the ecological and economical sourcing of plants for the discovery of novel antifungal compounds.