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

Anthracnose, caused by <i>Colletotrichum cereale</i>, is a destructive disease of turfgrasses, including creeping bentgrass (<i>Agrostis stolonifera</i>) in Japan. Frequent fungicide applications to perennial turfgrasses raise concerns about the development of fungicidal resistance in the pathogen, but no reports exist on their drug resistance in Japan. We evaluated the fungicidal sensitivity of 43 <i>C. cereale</i> isolates to pyribencarb (QoI), metconazole (DMI), and benomyl (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. EC₅₀ values were determined using mycelial growth inhibition assays. Group 1 isolates exhibited the lowest EC₅₀ values for all fungicides. Mean EC₅₀ values for pyribencarb were 0.29, 1.44, and 1.11 μg/ml for Groups 1, 2, and 3, respectively. For metconazole, Group 1 mean EC₅₀ was <0.01 μg/ml, while Groups 2 and 3 showed 133-fold and 32-fold increases, respectively. Benomyl resistance was widespread, with 89% of isolates showing EC₅₀ values >1 μg/ml. All Group 1 isolates remained susceptible to benomyl, whereas only one of 19 Group 3 isolates exhibited comparable sensitivity. Sequencing of the <i>TUB2</i> gene from 14 benomyl-resistant isolates identified a single amino acid substitution (E198A). Benomyl-resistant isolates exhibited significantly reduced mycelial growth on PDA 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 non-consecutive rotational programs.