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

Abstract

BACKGROUNDFungicide 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.RESULTSThe 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 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.CONCLUSIONThe 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 programs. The knowledge of fungicide resistance in regional P. teres collections will be important for informing appropriate management strategies.This article is protected by copyright. All rights reserved.

Fungicide Sensitivity Profile of Pyrenophora teres f. teres in Field Population

“…In Rehfus et al's [21] study, the field dose of fluxapyroxad showed stable control for P. teres isolates with mutations leading to B-H277Y, C-N75S, C-S135R, D-D124N, and D-D145G. Higher resistance factors have been associated with mutations C-G79R, D-H134R, D-D124E, and C-H134R, with reduced fluxapyroxad efficacy [21,35]. In Argentina, the detection of Ptt isolates carrying double mutation C-N75S + D-D145G raises concerns in fluxapyroxad field efficacy [36].…”

“…For further isolation, a QIAcube ® HT DNA extractor (Qiagen, Düsseldorf, Germany) and a DNeasy mericon 96 QIAcube HT Kit were used according to the manufacturer's instructions [37]. After DNA isolation, the Ptt isolates were confirmed with PCR and Sanger sequencing using ITS1 and ITS4 primers according to White et al [38] and a Ptt form-specific assay with primers according to Knight et al [35] (Table 1). PCR amplification was performed with a total volume of 20 µL mix consisting of 2 µL of 10× DreamTaq Green PCR buffer (Thermo Fisher Scientific, Waltham, MA, USA), 2 µL 10× GC-rich Enhancer (Solis BioDyne, Tartu, Estonia), 100 µM of each dNTP, 0.4 µM of specific forward and reverse primers (Table 1), 1 unit DreamTaq Hot Start DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, United States), 10 ng of genomic DNA, and the remaining amount of MilliQ water (Merck KGaA, Darmstadt, Germany).…”

“…The sequences obtained were analysed, and the target-site mutations were identified using blastn and blastx search tools (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed 21 December 2023)) available at NCBI. We focused on previously published relevant SNPs in the target protein-coding gene sequences associated with fungicide insensitivity [19,21,[34][35][36]. The representative sequences of the fungicide target protein genes of the studied Ptt isolates are deposited in NCBI GenBank with accession numbers OR530176, OR761967 to OR761973, and OR777246 to OR777248 (Table S1).…”

“…In this first comprehensive study of the Estonian Ptt population, we focused on the current status of fungicide sensitivity and its association with relevant SNPs in target protein-coding gene sequences linked to fungicide insensitivity [19,21,[34][35][36]. Therefore, the objectives of this research were to assess the Estonian Ptt population for (i) fungicide sensitivity level in vitro microtiter plate assays and subsequently associate insensitivity with (ii) Cyt b mutations, (iii) SDH subunit mutations, (iv) molecular changes in the Cyp51A gene and promoter region; and (v) potential sexual recombination by the analysing mating type prevalence rate.…”