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 (DfnR ) isolates from vineyards exhibited decreased fitness. Positive cross-resistance was detected between DMI triazoles. Sequence alignment results from the DfnR and DfnS isolates revealed that multiple mutations are distributed at CgCYP51A, concomitant with mutations at CgCYP51B. The half maximal effective concentration (EC50 ) values of single deleted and complemented mutants of CgCYP51A and CgCYP51B showed that ΔCgCYP51A became more sensitive to difenoconazole, but not ΔCgCYP51B. Furthermore, all single complemented mutants had a stronger biological fitness than the progenitor strain. All the defectives of ΔCgCYP51A and ΔCgCYP51B 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 CgCYP51s, 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.
Keywords: Colletotrichum gloeosporioides; DMI resistance; mutations at CYP51A&B; six resistant genotypes; sterol 14α-demethylases; vineyards.
© 2020 Society of Chemical Industry.