Submitted to: Phytopathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/5/2002
Publication Date: 1/5/2003
Citation: KUHAJEK, J.M., JEFFERS, S.N., SLATTERY, M., WEDGE, D.E. A RAPID MICROBIASSAY FOR DISCOVERY OF NOVEL FUNGICIDES FOR PHYTOPHTHORA SPP.. PHYTOPATHOLOGY. 2003. V. 93(1). P. 46-53. Interpretive Summary: A new microbioassay for discovery of natural compounds that inhibit the growth of Phytophthora species was developed. This assay uses a standardized format in a 96-well plate to evaluate antifungal agents. The assay was used to evaluate eight commercially available fungicides and proved to be a rapid, reproducible, and efficient method to antifungal compounds against Phytophthora species. The assay uses small amounts of compound compared to conventional petri dish assays and is therefore suitable for discovery research.
Technical Abstract: A novel microbioassay for the discovery of compounds that inhibit Phytophthora spp. was developed. This assay uses a 96-well format for high-throughput capability and a standardized method for quantitation of initial zoospore concentrations for maximum reproducibility. Zoospore suspensions were quantifiable between 0.7 and 1.5 (105 zoospores/ml) using percent transmittance (620 nm). Subsequent growth of mycelia was monitore by measuring optical density (620 nm) at 24-h intervals for 96 h. Full- and half-strength preparations of each of three media (V8 broth, Roswell Park Memorial Institute mycological broth, and mineral salts medium), and four zoospore concentrations (10, 100, 1000, and 10,000 zoospores/ml) were evaluated. Both full- and half-strength Roswell Park Memorial Institute mycological broth were identified as suitable synthetic media for growing P. nicotianae, and 1000 zoospores/ml was established as the optimum initial lconcentration. The assay was used to determine EC50 values (i.e., effectiv concentration for 50% growth reduction) for eight commercial antifungal compounds (azoxystrobin, fosetyl-aluminum, etridiazole, metalxyl, pentachloronitrobenzene, pimaricin, and propamocarb). These EC50 values were compared to those obtained by measuring linear growth of mycelia on fungicide-amended medium. The microbioassay proved to be a rapid, reproducible, and efficient method for testing the efficacy of compounds against P. nicotianae and should be effective for other species of Phytophthora as well. The assay requires relatively small amounts of a test compound and is suitable for the evaluation of natural product samples.