Workflows for detecting fungicide resistance in net form and spot form net blotch pathogens

Fungicide resistance in Pyrenophora teres f. maculata and P. teres f. teres has become an important disease management issue. Control of the associated barley foliar diseases, spot form and net form net blotch, respectively, relies on three major groups of fungicides, demethylation inhibitors (DMI), succinate dehydrogenase inhibitors (SDHI) and quinone outside inhibitors (QoI). However, resistance has been reported for the DMI and SDHI fungicides in Australia. To enhance detection of different resistance levels, phenotyping and genotyping workflows were designed. The phenotyping workflow generated cultures directly from lesions and compared growth on discriminatory doses of tebuconazole (DMI) and fluxapyroxad (SDHI). Genotyping real-time PCR assays were based on alleles associated with sensitivity or resistance to the DMI and SDHI fungicides. These workflows were applied to a net blotch collection from 2019 consisting predominantly of P. teres f. teres from South Australia and P. teres f. maculata from Western Australia. For South Australia the Cyp51A L489-3 and SdhC-R134 alleles, associated with resistance to tebuconazole and fluxapyroxad, respectively, were the most prevalent. These alleles were frequently found in single isolates with dual resistance. This study also reports the first detection of a 134 base pair insertion located at position -66 (PtTi-6) in the Cyp51A promoter of P. teres f. maculata from South Australia. For Western Australia, the PtTi-1 insertion was the most common allele associated with resistance to tebuconazole. These workflows will be valuable for screening P. teres populations for fungicide resistance, and informing appropriate management strategies.