Mutations at sterol 14α‐demethylases ( <scp>CYP51A</scp> &amp;B) confer the <scp>DMI</scp> resistance in <i>Colletotrichum gloeosporioides</i> from grape

Abstract

Abstract BACKGROUND Grape anthracnose caused by the ascomycete fungus Colletotrichum gloeosporioides has been widely controlled by demethylation inhibitors (DMIs) for decades in China. The resistance status and mechanism of C. gloeosporioides against DMIs is not well understood. RESULTS All difenoconazole‐resistant (Dfn R ) isolates from vineyards exhibited decreased fitness. Positive cross‐resistance was detected between DMI triazoles. Sequence alignment results from the Dfn R and Dfn S isolates revealed that multiple mutations are distributed at CgCYP51A , concomitant with mutations at CgCYP51B . The half maximal effective concentration (EC 50 ) values of single deleted and complemented mutants of CgCYP51A and CgCYP51B showed that Δ CgCYP51A became more sensitive to difenoconazole, but not Δ CgCYP51B . Furthermore, all single complemented mutants had a stronger biological fitness than the progenitor strain. All the defectives of Δ CgCYP51A and Δ CgCYP51B could be restored by complementation of the whole corresponding gene from the resistant strains. Relative gene expression of CgCYP51A and CgCYP51B in most of the mutants was greatly upregulated relative to the progenitor isolate when treated with difenoconazole at the same concentration. Moreover, the extension of five amino acids (GNETI) caused by mutation at the stop codon of CgCYP51A , concurrent with other seven amino acid substitutions and the synonymous mutation P10P (CCG → CCT), significantly enhanced DMI resistance. CONCLUSION The DMI resistance of C. gloeosporioides selected in vineyards is conferred by mutations at CgCYP51 s, and validated by a genetics method. The roles of CgCYP51A and CgCYP51B overlap, and are counter‐balanced, but cannot be replaced reciprocally. © 2020 Society of Chemical Industry