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

Abstract

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.

Exploiting long read sequencing to detect azole fungicide resistance mutations in Pyrenophora teres using unique molecular identifiers

“…As expected, we recovered two high depth clusters for each field sample (Cyp51A1 and Cyp51A2), one of which had two non-synonymous SNPs, I133V and F489L, the latter correlates with decreased fungicide sensitivity, while the former does not 10 . The codon which gave rise to the F489L mutation was c1465t which was originally reported in Ptm 11 but only recently in Ptt 25 . If this codon change had not been discovered prior, the result of an allele-specific assay would have been a false negative, highlighting the utility of our assay for comprehensive resistance profiling.…”

“…This is especially important when profiling biologically complex field samples. We applied our pipeline to infected barley leaf samples and used a culturing method on fungicide media developed by Knight, et al 25 . This method has three main advantages: (i) bulking of fungal mycelia for PCR, (ii) removal of plant DNA, and (iii) selection of only fungicide resistant isolates.…”

“…This suggests that quantitative platforms such as qPCR or digital PCR are still complementary for the detection process, especially as they are sensitive and able to detect fungicide resistance mutations as low as 0.2% 22 . Also, our current field sample pipeline relies on a lengthy culturing step for selective growth of resistant isolates, similar to detection pipelines using qPCR or digital PCR 25 . Ideally improvements will be devised which would allow DNA to be extracted directly from diseased leaves for UMI sequencing and amplicon diversity assessments.…”

“…To validate the mutations in the Cyp51A mock community, we analysed the input DNA used for ONT sequencing with qPCR assays specifically designed to detect the genetic changes in each haplotype (Knight et al 25 ; Fig. 1, Table 2) with the pure genomic DNA from each isolate as positive controls (Supplemental Table S1).…”

“…Five net-blotch symptomatic leaf samples collected in 2021 from barley crops in Minlaton, South Australia, were assessed for fungicide resistance following a culturing method described by Knight et al 25 . Briefly, infected barley leaves were surface sterilised for 30 s in 70% (v/v) ethanol, 60 s in 0.125% (w/v) NaOCl and 2 × 60 s in autoclaved filtered water, lesions excised, and placed on 2 mL PDA supplemented with streptomycin (10 µg mL -1 ), ampicillin (100 µg mL -1 ) and neomycin (50 µg mL -1 ) dispensed into a 12 well plate.…”