Screening of fungicide resistance in Cercospora beticola populations in Michigan using PCR-RFLP and spiral plate gradient methods.
There are multiple fungicide groups that are commonly used and registered for Cercospora leaf spot management in sugar beet including methyl benzimidazole carbamates (MBC or benzimidazole, FRAC group 1), quinone outside inhibitors (QoI or strobilurin, FRAC group 11), demethylation inhibitors (DMI or triazole, FRAC group 3), and multi-site contact activity (FRAC group M03) fungicide classes. In Michigan, reduced sensitivity to QoI, MBC, and DMI fungicides has been detected and extensively monitored in C. beticola populations in Michigan. In 2021 and 2022, testing was conducted using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays to detect point mutations in the C. beticola genome associated with fungicide resistance and compared to the effective concentrations to inhibit 50% of mycelial growth (EC50) determined in spiral plate gradient assays. In 2021, 29 field locations were sampled across nine counties in east-central Michigan (n = 418 lesions tested); in 2022, 16 field locations were sampled with submissions continuing at present (n = 223 lesions tested). Resistances were determined by detection of the point mutations present in i) the fungal mitochondrial cytochrome b gene (G143A) for QoI fungicides, ii) in the beta-tubulin gene (E198A) for MBC fungicides, and iii) in the C-14 alpha-demethylase gene (Glu169 (GAA to GAG)) for DMI fungicides. In 2021, high frequencies of QoI resistance were observed across all locations and dates; of the 386 isolates tested, 385 were found to contain the G143A mutation. By the final sampling date of the season, mutations for resistance to QoIs and MBCs were present in 100% of the fields tested, while the mutation conferring high levels of resistance to DMIs was found in 83% of fields. In 2021, in vitro screening of corresponding C. beticola isolate sensitivities was conducted for eight fungicide active ingredients including pyraclostrobin (PYR), thiophanate-methyl (TPM), difenoconazole (DIF), fenbuconazole (FEN), mefentrifluconazole (MEF), prothioconazole (PRO), tetraconazole (TET), and triphenyltin hydroxide (TPH) (n = 134 isolates). Based on resulting EC50 values, the majority of isolates (≥90%) were sensitive to DIF, FEN, MEF, and TPH at concentrations of ≤10 µl ml-1. Elevated frequencies of isolates with EC50 values >10 µl ml–1 were identified for PYR (43%), TPM (62%), PRO (69%), and TET (40%). Current year testing is in progress, however, correlations between spiral plate and PCR-RFLP methods will be evaluated for each active ingredient to optimize future screening efforts.