QUINONE OUTSIDE INHIBITOR (QOI) FUNGICIDE RESISTANCE AND MATING-TYPE DISTRIBUTION OF CERCOSPORA SOJINA POPULATIONS ON SOYBEAN FROM INDIANA

Frogeye leaf spot (FLS) is a foliar disease in soybean (Glycine max (L.) Merr.) caused by the fungal pathogen Cercospora sojina Hara. FLS is commonly found in hot and humid regions of the southern United States but has become more common in the North Central states. Foliar application of quinone outside inhibitor (QoI) fungicides has been one of the major tools used in the management of this disease, but QoI-resistant C. sojina isolates have been already confirmed in 21 states, including Indiana. We hypothesized that resistant populations of C. sojina to QoIs fungicides are widespread in Indiana and that sexual reproduction is occurring within C. sojina populations, likely contributing to the dissemination of fungicide resistance. The main objectives of this research were to determine the distribution of QoI-resistant C. sojina isolates on soybean from Indiana and to evaluate C. sojina populations for potential sexual reproduction. In the summer of 2019 and 2020, 406 isolates of C. sojina were collected from 32 counties across Indiana and screened for QoI-fungicide resistance using a PCR-RFLP method. An in vitro fungicide sensitivity test was performed on a subset of isolates to evaluate the sensitivity of C. sojina isolates to azoxystrobin, pyraclostrobin, picoxystrobin, and prothioconazole. A discriminatory dose of picoxystrobin (QoI) and prothioconazole (demethylation inhibitor- DMI) were established at 1 μg/ml and 10 μg/ml, respectively, to distinguish between QoI-resistant and sensitive isolates and to identify a reduction in sensitivity to DMI fungicides, respectively. Discriminatory doses were estimated by testing five concentrations (0.001, 0.01, 0.1, 1, and 10 µg/ml) of each fungicide. QoI-resistant isolates were found in 29 out of the 32 counties. Two hundred and fifty-one (251) out of the 406 isolates (61.8%) were confirmed as QoI-resistant. Partial nucleotide sequences of the cyt b gene from four resistant and four sensitive C. sojina isolates corroborated the presence and absence of the G143A mutation, respectively. Results from the sensitivity assays with azoxystrobin and pyraclostrobin discriminatory doses supported the findings from the PCR-RFLP assay as all QoI-resistant mutants were inhibited less than 50% when exposed to these doses. Results from this study indicated that QoI-resistant C. sojina isolates are spread throughout Indiana and that prothioconazole (DMI) could be a potential supplemental or alternative fungicide to control FLS. Additionally, mating type distribution was determined in 43 C. sojina populations for assessment of potential sexual reproduction. Fifteen (15) populations did not deviate significantly from the expected 1:1 ratio, suggesting potential for cryptic sexual reproduction in these populations, but further research on genetic diversity is required to verify these results.