Sensitivity Analysis of Colletotrichum cereale Isolates from Creeping Bentgrass to Pyribencarb, Metconazole, and Benomyl, and Detection of a TUB2 E198A Mutation

Anthracnose, caused by Colletotrichum cereale, is a destructive disease of turfgrasses, including creeping bentgrass (Agrostis stolonifera) in Japan. Frequent fungicide applications to perennial turfgrasses raise concerns about the development of fungicide resistance in the pathogen, but no reports exist on their fungicide resistance in Japan. We evaluated the fungicidal sensitivity of 43 C. cereale isolates to pyribencarb (quinone outside inhibitor [QoI]), metconazole (demethylation inhibitor [DMI]), and benomyl (methyl benzimidazole carbamate [MBC]). Isolates were categorized into three groups: Group 1 comprised historical isolates deposited before the commercial introduction of major QoI and DMI fungicides; Group 2 comprised isolates from fungicide-free experimental plots; and Group 3 comprised isolates from fungicide-exposed golf courses. EC 50 values were determined using mycelial growth inhibition assays. Group 1 isolates exhibited the lowest EC 50 values for all fungicides. Mean EC 50 values for pyribencarb were 0.29, 1.44, and 1.11 μg/ml for Groups 1, 2, and 3, respectively. For metconazole, the Group 1 mean EC 50 was 1 μg/ml. All Group 1 isolates remained susceptible to benomyl, whereas only 1 of 19 Group 3 isolates exhibited comparable sensitivity. Sequencing of the TUB2 gene from 14 benomyl-resistant isolates identified a single amino acid substitution (E198A). Benomyl-resistant isolates exhibited significantly reduced mycelial growth on potato dextrose agar without benomyl but showed no differences in sporulation or osmotic stress tolerance. These results support continued use of pyribencarb and metconazole for anthracnose management and suggest strategic inclusion of benomyl in nonconsecutive rotational programs.