Increasing the tolerance of Trichoderma harzianum T-22 to DMI fungicides enables the combined utilization of biological and chemical control strategies against plant diseases

[Display omitted] •MgCYP51B-v, a variant of CYP51B, exhibits a decreased binding affinity with DMIs.•Tebuconazole-resistant Trichoderma harzianum mutants were successfully obtained.•Fungicide resistance was stably inherited in the mutants with no fitness penalty.•The ability to use biological and chemical control agents together was enhanced. Demethylase inhibitors (DMIs) targeting CYP51 proteins in fungi are often used for plant disease management. Trichoderma harzianum, a widely used biocontrol agent, however, is sensitive to DMIs. This negates their combined use as a strategy in practices. Increasing the tolerance of T. harzianum to DMIs represents a feasible solution to this problem. In this regard, the overexpression of a CYP51B variant in Mycosphaerella graminicola (MgCYP51B-v) conveys increased tolerance to DMIs. Molecular docking indicated a distinct decrease in the affinity of MgCYP51B-v with DMIs. Since CYP51B proteins are conserved in M. graminicola and T. harzianum, we attempted to increase DMI tolerance in T. harzianum by heterologously expressing MgCYP51B-v. A series of T. harzianum mutants were obtained that were able to develop colonies on tebuconazole-amended medium. Two representative mutants were selected for further studies that confirmed that they were resistant to tebuconazole with resistance factors (RFs) of approximately 10.0. The mutants maintained a similar level of resistance after ten rounds of subculture without fungicide, indicating that the fungicide resistance was stably inherited. The mutants also exhibited increased tolerance to other DMIs, including metconazole, ipconazole, hexaconazole, and prochloraz. Further assessment indicated that the DMI resistance in the mutants did not have any impact on their fitness. The improved compatibility between T. harzianum and DMIs enables the utilization of a combined strategy in which these disease control agents are used together. Such a strategy has the potential to provide synergistic disease control and prevent or delay the development of fungicide resistance.