Six simultaneous point mutations within Cyp51 confer resistance to prochloraz in Alternaria alternata

Alternaria alternata causes leaf brown spot disease on tobacco and threatens the yield and quality of tobacco leaf preharvest and postharvest. Prochloraz, one member of demethylation inhibitors has been registered for controlling various pathogens, including A. alternata . Here, we determine the sensitivities of 88 A. alternata isolates to prochloraz using the mycelial growth rate method. Our results indicate that the mean EC 50 value for the 66 sensitive isolates is 0.552 ± 0.290 μg/ml and that for the resistant 22 isolates is 2.209 ± 0.643 μg/ml. Compared with the prochloraz-sensitive populations, the prochloraz-resistant populations exhibit no significant differences in mycelial growth, sporulation, spore germination, and pathogenicity. A positive cross-resistance is observed between prochloraz and difenoconazole but not between prochloraz and metconazole or tebuconazole. Sequencing of the complete AaCyp 51 gene from A. alternata isolates reveals that the prochloraz-resistant isolates carry a combination of six substitutions at codons 188 (AAC→AAA), 192 (GTC→ATC), 237 (TCG→GCA), 307 (GCT→GGT), 412 (CAC→TAC), and 434 (GAG→GAC), which are not detected in the sensitive isolates. Molecular docking simulations shows that hydrophobic interactions between prochloraz and AaCYP51 are weakened after the six simultaneous substitutions in AaCYP51(N→K, V→I, S→A, A→G, H→Y, and E→D), correlating with a lower binding affinity of prochloraz to the protein. The six-substitution transformants obtained through artificial construction confer prochloraz resistance, with EC 50 values of 1.501 μg/ml and 1.671 μg/ml (0.204 μg/ml for parental isolate), while exhibiting no significant fitness penalty (i.e., sporulation, spore germination, and pathogenicity). To our knowledge, this is the first study to report that six simultaneous point mutations in Cyp 51 confer resistance to prochloraz.