Submitted to: Acta Horticulture Proceedings
Publication Type: Proceedings
Publication Acceptance Date: December 3, 2002
Publication Date: January 8, 2005
Citation: Wedge, D.E. 2005. Agrochemical discovery: finding new fungicides from natural products. Acta Horticulture Proceedings. 3:109-113. Interpretive Summary: Successful discovery, evaluation, and development of natural product fungicides are totally dependent upon the availability of high quality miniaturized antifungal bioassays. The paper describes two bioassays used for discovery of natural product fungicides for agriculture. The bioautography assays are sensitive to small amounts of compounds, selective enough to determine the best target pathogens, and they work well with plant extracts. The standardized 96-well microtiter plate assay was developed for discovery of natural product fungicidal agents from plants, marine algae, and microorganisms. This 96-well plate assay was designed to evaluate dose-response relationships, commercial fungicide standards, modes of action, and structure activity studies. These methods provide us with a standard operating protocol for natural product discovery that we currently use at the ARS Natural Product Utilization Research Unit.
Technical Abstract: The continuing development of fungicide resistance in plant and human pathogens necessitates the discovery and development of new fungicides. Discovery and evaluation of natural product fungicides is largely dependent upon the availability of miniaturized antifungal bioassays. Essentials for natural product bioassays include; sensitivity to microgram quantities, selectivity to determine optimum target pathogens, and adaptability to complex mixtures. Experimental accuracy and precision must be stable between assays over time. These assays should be relevant to potential pathogen target sites in the natural infection process of the host and applicable to the agrochemical industry. Bioassays should take advantage of current high-throughput technology available to evaluate dose-response relationships, commercial fungicide standards, modes of action, and structure activity studies. As part of a program to discover natural product-based fungicides, two sensitive detection systems were developed for evaluation of antifungal agents as agrochemical plant protectants. Bioautography assays using Colletotrichum acutatum, C. fragariae, and C. gloeosporioides were used to identify antifungal components from plant extracts, eliminate commonly occurring nuisance compounds, and characterize antifungal activity of pure compounds. A microbioassay using conidia from Botrytis cinerea, Colletotrichum spp., Fusarium oxysporum, Phomposis obscurans, and P. viticola in a 96-well microtiter format was also developed to evaluate growth effects of active fractions or pure compounds identified by bioautography. These microbioassays allow for the evaluation of microgram quantities, determine dose-response relationships, compare antifungal activity with fungicide standards of known mode of action. This coupled approach to natural product discovery combines the simple and visual nature of direct bioautography with the rapid, sensitive, and high throughput capabilities of a microtiter system. Ultimately, development of safer plant protectants with selective antifungal activity and low environmental and mammalian toxicity is sought.