RESUMO
In order to establish a fast and accurate method for novel DMIs fungicide screening, lanosterol 14alpha-demethylase of Magnaporthe grisea expressed in E. coli was used as target enzyme and the DMI fungicides diniconazole, tebuconazole, triadimenol and triadimefon were used as representative fungicides, the effects of enzyme activity, enzyme purity and concentration on the binding spectra were investigated. The results showed that active enzyme, elimination of interference of other P450s and proper enzyme concentration were necessary for obtaining accurate binding spectra. The Kd values of diniconazole, tebuconazole, triadimenol and triadimefon were 0.143 micromol/L, 0.24 micromol/L, 0.257 micromol/L and 0.307 micromol/L respectively, which significantly correlated to their 120h-EC50 values on the growth of Magnaporthe grisea. The results indicated that the binding spectra of fungicide and lanosterol 14alpha-demethylase can serve as a reliable and fast method for novel fungicide screening.