Parallel evolution of multiple mechanisms for demethylase inhibitor fungicide resistance in the barley pathogen Pyrenophora teres f. sp. maculata.

The fungal pathogen Pyrenophora teres f. sp. maculata (Ptm), responsible for spot-form of net blotch (SFNB), is currently the most significant disease of barley in Australia and a major disease worldwide. Management of SFNB relies heavily on fungicides and in Australia the demethylase inhibitors (DMIs) predominate. There have been sporadic reports of resistance to DMIs in Ptm but the mechanisms remain obscure. Ptm isolates collected from 1996 to 2019 in Western Australia were tested for fungicide sensitivity levels. Decreased sensitivity to DMIs was observed in isolates collected after 2015. Resistance factors to tebuconazole fell into two classes; moderate resistance (MR; RF 6-11) and high resistance (HR; RFs 30-65). Mutations linked to resistance were detected in the promoter region and coding sequence of the DMI target gene Cyp51A. Solo-LTR insertion elements were found at 5 different locations in the promoter region. Three different non-synonymous mutations encoded an altered protein with a phenylalanine to leucine substitution at position 489, F489L (F495I in the archetype CYP51A of Aspergillus fumigatus). F489L mutations have also been found in DMI-resistant strains of P. teres f. sp. teres. Ptm isolates carrying either a LTR insertion element or a F489L allele displayed the MR1 or MR2 phenotypes, respectively. Isolates carrying both an insertion element and a F489L mutation displayed the HR phenotype. Multiple mechanisms acting both alone and in concert were found to contribute to DMI resistance in Ptm. Moreover, these mutations have emerged repeatedly in Western Australian Ptm populations by a process of parallel evolution.

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

BACKGROUND: 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 (DMIs), succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs). 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. RESULTS: The phenotyping workflow generated cultures directly from lesions and compared growth on discriminatory doses of tebuconazole (DMI) and fluxapyroxad (SDHI). Genotyping real-time polymerase chain reaction (PCR) assays were based on alleles associated with sensitivity or resistance to the DMI and SDHI fungicides. These workflows were applied to spot form and net form net blotch collections 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. CONCLUSION: The workflow and PCR assays designed in this study have been demonstrated to efficiently screen P. teres collections for both phenotypic and genetic resistance to DMI and SDHI fungicides. The distribution of reduced sensitivity and resistance to DMI and SDHI fungicides varied between regions in south-western Australia, suggesting the emergence of resistance was impacted by both local pathogen populations and disease management programmes. The knowledge of fungicide resistance in regional P. teres collections will be important for informing appropriate management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51.

Pyrenophora teres f. sp. teres is the cause of net form of net blotch (NFNB), an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of NFNB in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modeling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localized constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2, and Cyp51A1 was shown to be 1.6-, 3,- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of fungicides in use.