Basf Aktiengesellschaft

Margaret Hsien-Fen Kuh Lai

Donald R Kirsch

Sanford Jay Silverman

A method for the identification of agents which inhibit chitin synthesis, thus exhibiting potential fungicidal and insecticidal activity, involves the incubation of test samples in neutral cultures containing calcofluor white, a fluorochrome that causes lethal chitin hyper-polymerization in the yeast. Cultures containing samples that inhibit chitin synthesis exhibit enhanced growth because the growing fungus is rescued from the adverse effects of calcofluor white. In the practice of the invention, the test sample is added to a culture or culture area containing calcofluor white and the culture is incubated with the test sample for such time under such conditions sufficient to observe yeast cell growth inhibition in a corresponding culture or culture area containing calcofluor but no test sample. The extent of growth in the culture or culture area containing test sample is then compared with the extent of growth in the culture or culture area containing no test sample, and the presence of chitin synthesis inhibition is determined by observation of whether culture growth in the presence of test sample exceeds growth in its absence. Preferred embodiments employ test samples on disks or in wells in solidified cultures and a known chitin synthesis inhibitor as a positive control which is compared to the test sample.

Calcafluor screen for chitin biosynthesis inhibitors

A method for screening for potential fungicides identifies biochemical or chemical agents that inhibit the formation of the fungal sterol ergosterol by inhibiting cytochrome P450 reductase or reductase related enzymes or proteins involved in its biosynthesis. Test samples are incubated in two yeast strain cultures, and the extent of growth inhibition is compared between them. One culture is supersensitive to cytochrome P450 reductase inhibitors; the yeast strain typically contains a disrupted or deleted cytochrome P450 reductase gene. The other culture is less sensitive to the compounds, typically exhibiting enhanced permeability such as that observed in erg6 mutants. In preferred embodiments, a known cytochrome P450 reductase related electron-transfer protein inhibitor such as keto-conazole, miconazole, dinaconazole or econazole is employed as a positive control. Preferred embodiments employ solidified media, and test samples are applied to disks or wells. Actives are identified by the production of a zone differential of approximately 8 mm or greater on the yeast culture lawns.

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East Brunswick, NJ, United States of America

Margaret Hsien-Fen Kuh Lai