Baseline sensitivity and resistance mechanism of Colletotrichum isolates on tea-oil trees of China to tebuconazole

Colletotrichum fungi could cause anthracnose, a destructive disease in tea-oil trees. The sterol demethylation inhibitor (DMI) tebuconazole has been widely used in controlling plant diseases for many years. However, the baseline sensitivity of Colletotrichum isolates on tea-oil trees to tebuconazole has not been determined. In this study, the sensitivity of 117 Colletotrichum isolates from tea-oil tree of 7 provinces in southern China to tebuconazole was tested. The mean effective concentration resulted in 50% mycelial growth inhibition (EC50) was 0.7625 μg/mL. The EC50 of 100 isolates (83%) were lower than 1 μg/mL, 20 isolates (17%) were higher than 1 μg/mL, which implied the resistance has already occurred in Colletotrichum isolates on tea-oil trees. The EC50 values of the most resistant and sensitive isolates (named Ca-R and Cc-S1 respectively) were 1.8848 and 0.1561 μg/mL respectively. The resistance mechanism was also investigated in this study. Gene replacement experiment indicated that CYP51A/B gene of resistant isolates Ca-R and Cf-R1 cannot confer Cc-S1 fully resistance to DMI fungicides, though three point mutants, Cc-S1CYP51A-T306A and Cc-S1CYP51A-R478K exhibited decreased sensitivity to DMI fungicides. This result suggested that resistance of Colletotrichum isolates was partly caused by mutations in CYP51A. Moreover, expression level of CYP51A/B was almost identical among Ca-R, Cf-R1, Cc-S1 and Cc-S1CYP51A point mutants, which indicated that the resistance was irrelevant to the expression level of CYP51A, and other non-target-based resistance mechanism may exist. Our results could help to guide the application of DMI fungicide and be useful for investigating the mechanism of resistance.