Emergence of resistance to succinate dehydrogenase inhibitor fungicides inPyrenophora teresf.teresandP. teresf.maculatain Australia

Abstract

The net blotches are among the most economically significant diseases of barley worldwide. There are two forms of the disease: net-form net-blotch (NFNB, causal agent Pyrenophora teres f. sp. teres [Ptt]) and spot-form net blotch (SFNB, causal agent Pyrenophora teres f. sp. maculata [Ptm]). Alongside varietal choice and cultural practices, fungicides form an important part of the regime for net blotch control. The succinate dehydrogenase inhibitors (SDHIs) are a key class of fungicides used in net blotch management. However, resistance to this group of compounds has emerged in the net blotches in recent years. Here, we describe the first cases of resistance to SDHIs in Australian populations of net blotches. This study was prompted by reports of field failures of SDHI fungicides in controlling NFNB in South Australia and SFNB in Western Australia. Target site mutations in the Sdh complex genes, previously associated with reduced sensitivity in European net blotch populations, were found in Australian isolates, and two mutations which have not been previously observed in P. teres, are also described. The mutations found in Ptt included H134R and S135R in SdhC; and H134Y and D145G in SdhD; the SdhC-H134R mutation was the most frequently observed. In Ptm, the mutations found included H277L in SdhB; S73P, N75S, H134R and S135R in SdhC; and D145G in SdhD; the SdhC-N75S mutation was the most common. These mutations were correlated with reduced in vitro SDHI fungicide sensitivity by microtiter assay. The highest resistance factors to fluxapyroxad and bixafen, the most important SDHI fungicides for net blotch control in Australia, were associated with the SdhC-H134R and SdhC-S135R mutations in Ptt, and with the SdhB-H277L, SdhC-H134R, and SdhC-S135R mutations in Ptm. Modelling of the P. teres Sdh complex showed that the two novel mutations, H277L in SdhB and H134Y in SdhD, result in a highly altered binding mode and lower binding affinity of the SDHI compound compared to the wild-type.

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

“…This may be due to decreased sensitivity being present in Australian populations of P. teres f. maculata and P. teres f. teres since at least 2016 and 2013, respectively. 3,6 In contrast, decreased sensitivity to SDHI fungicides was first reported in 2020 for P. teres f. maculata and 2019 for P. teres f. teres in Australia, 5 and was only detected in the South Australian collection in the current study. Alleles associated with DMI and SDHI reduced sensitivity or resistance varied within populations and among states, suggesting population differentiation for fungicide resistance.…”

“…6 Doses were chosen based on reported discriminatory doses, 50% effective concentration (EC 50 ) values and preliminary screening experiments (Supporting Information, Tables S1 and S2). 3,5,6 Growth was assessed at 7 days after plating on each discriminatory dose. Sub-cultures were taken from mycelium growing on the greatest fungicide concentration and placed onto V8-potato dextrose agar (V8-PDA).…”

“…The design of a duplex PCR assay for simultaneous specific identification of P. teres f. maculata and P. teres f. teres was based on formspecific DNA regions described by Mair et al 5 Briefly, a hydrolysis probe sequence was positioned between the forward and reverse primer positions and assessed for oligonucleotide characteristics in Primer3 version 0.4.0 45,46 and sequence similarity by a standard nucleotide Basic Local Alignment Search Tool (BLAST) search of the GenBank Standard databases (nr etc.). 47 PCR optimisation and specificity assessment was then performed using real-time PCR and DNA of standard isolates.…”

“…In Australia, fungicide resistance in the barley (Hordeum vulgare) pathogen Pyrenophora teres has become an important management issue. [3][4][5][6] Pyrenophora teres is recognised as two forms, P. teres f. teres and P. teres f. maculata, causing net form net blotch (NFNB) and spot form net blotch (SFNB), respectively. 7 Hybridisation between the two forms is also possible.…”

“…[28][29][30][31] Genotypes of P. teres associated with sensitivity, reduced sensitivity and resistance have been reported for DMI, SDHI and QoI fungicides. These include single nucleotide polymorphisms (SNPs) in the genes for cytochrome P450 sterol 14⊍-demethylase (CYP51A), 3,6 the succinate dehydrogenase (SDH) B, C and D subunits 5,26,27 and cytochrome b, 29,31 along with a range of 134 bp insertions in the Cyp51A promoter region. 6 Detection of these genotypes may be achieved using methods such as pyrosequencing 32,33 and real-time or digital polymerase chain reaction (PCR).…”