Ergosterol biosynthesis in a cell-free preparation of Penicillium italicum and its sensitivity to DMI fungicides

Abstract

A method has been developed to study ergosterol biosynthesis in cell-free extracts of the filamentous plant pathogen Penicillium italicum. The method is based on a mild mechanical disruption of conidial germlings in a Bead-Beater apparatus. The cell-free extract was effective in synthesizing C4-desmethyl sterols from [2- 14C]mevalonate. Ergosterol was the only C4-desmethyl sterol synthesized and amounted to 25.6% of total nonsaponifiable lipids. Other sterols identified in the nonsaponifiable lipid fraction were lanosterol and a trace amount of 24-methylenedihydrolanosterol. Inhibition of ergosterol synthesis by fungicides which inhibit sterol 14α-demethylation (DMIs) led to accumulation of 24-methylenedihydrolanosterol indicating inhibition of cytochrome P450-dependent sterol 14α-demethylase activity. IC 50 values (concentrations which inhibit incorporation of [2- 14C]mevalonate into ergosterol for 50%) of the highly toxic DMI fungicides imazalil, itraconazole, ketoconazole, penconazole and propiconazole ranged from 6.5 ± 0.5 × 10 −9 to 1.7 ± 0.7 × 10 −8 M. This indicates that DMI fungicides are very potent inhibitors of sterol 14α demethylase activity in cell-free extracts of the fungus. Less toxic DMI analogues had much higher IC 50 values, suggesting that these compounds have a significantly lower potency to inhibit sterol 14α-demethylase activity.