DEVELOPMENT OF A CELL-FREE ASSAY FROM BOTRYTIS-CINEREA AS A BIOCHEMICAL SCREEN FOR STEROL BIOSYNTHESIS INHIBITORS

An assay for measuring ergosterol synthesis in cell-free extracts of t he filamentous plant pathogen Botrytis cinerea is described. The extra cts capable of synthesizing C4-desmethyl sterols from [2-C-14]mevalona te were derived by mechanical disruption of young conidial germlings i n a Bead-Beater apparatus. The C4-desmethyl sterol fraction consisted of three distinct compounds and totalled 39% of the non-saponifiable l ipids formed. Ergosterol accounted for 63% of the C4-desmethyl sterols . Only small amounts of C4-monomethyl sterols were synthesized, while C4,4-dimethyl sterols made up 29% of the non-saponifiable lipids. The latter fraction mainly consisted of lanosterol (54%) and eburicol (28% ). The cell-free system had a narrow pH optimum for synthesis of C4-de smethyl sterols of pH 7.3-7.4. Cell-free synthesis of C4-desmethyl ste rols was inhibited by the imidazole fungicide imazalil, concomitant wi th an accumulation of eburicol. The IC50 value (concentration of fungi cide which inhibited cell-free synthesis of C4-desmethyl sterols by 50 %) was 9.1 x 10(-9) m. These results are consistent with the hypothesi s that imazalil is a potent inhibitor of the cytochrome P450-dependent sterol 14alpha-demethylase of B. cinerea. The method described may be used to screen compounds biochemically for inhibition of sterol synth esis in an agriculturally important plant pathogen.