INHIBITION OF STEROL BIOSYNTHESIS IN CELL-FREE-EXTRACTS OF BOTRYTIS-CINEREA BY PROCHLORAZ AND PROCHLORAZ ANALOGS

The sensitivity of cytochrome-P450-dependent sterol 14alpha-demethylas e (P450(14DM)) to prochloraz and several prochloraz analogues was stud ied in a cell-free assay of Botrytis cinerea Pers. ex Fr. The EC50 val ues (concentrations which inhibited radial growth of B. cinerea by 50 %) of the compounds tested ranged from 3-3 x 10(-8) to 1.7 x 10(-5) m. The IV50 values (concentrations which inhibited cell-free C4-demethyl sterol synthesis by 50 %) in cell-free assays of B. cinerea ranged fr om 2.6 x 10(-9) to 4.4 x 10(-7) M. Ranking compounds in terms of their relative inhibitory potencies showed quite similar trends to the orde r of fungitoxicity, but the IC50 values did not quantitatively reflect the differences in toxicity. Therefore, the differential inhibition o f cell-free P450(14DM) activity by these compounds cannot fully accoun t for their differences in activity towards B. cinerea. Additional mec hanisms must be involved. The compounds tested were generally more pot ent in the B. cinerea assay than in similar assays developed for Penic illium italicum Wehmer and, in particular, Saccharomyces cerevisiae Me yen. This correlated with the relatively higher activity of most test compounds to B. cinerea. Results suggest that the cell-free assay of B . cinerea is more useful to evaluate candidate fungicides as inhibitor s of sterol 14alpha-demethylase activity than similar assays from mode l organisms. The present study confirms that the affinity of prochlora z analogues for P450(14DM) depends on the nature of the N-1 substituen t of the imidazole and the azole ring. It was also found that addition of an amino group at C-2 of the imidazole moiety of prochloraz gave a compound (6) which inhibited 4,4-demethyl sterol biosynthesis in B. c inerea at a different site from the P450(14DM). This was confirmed by the observation that laboratory-generated triadimenol-resistant isolat es of B. cinerea showed reduced sensitivity to triadimenol and prochlo raz, but not to compound 6.