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.

Analysis of mutations in West Australian populations of Blumeria graminis f. sp. hordei CYP51 conferring resistance to DMI fungicides.

BACKGROUND: Powdery mildew caused by Blumeria graminis f. sp. hordei (Bgh) is a constant threat to barley production but is generally well controlled through combinations of host genetics and fungicides. An epidemic of barley powdery mildew was observed from 2007 to 2013 in the West Australian grain belt. RESULTS: We collected isolates across Australia, examined their sensitivity to demethylation inhibitor (DMI) fungicides and sequenced the Cyp51B target gene. Five amino acid substitutions were found, of which four were novel. The most resistant haplotypes increased in prevalence from 0% in 2009 to 16% in 2010 and 90% in 2011. Yeast strains expressing the Bgh Cyp51 haplotypes replicated the altered sensitivity to various DMIs and these results were complemented by in silico protein docking studies. CONCLUSIONS: The planting of very susceptible cultivars and the use of a single fungicide mode of action was followed by the emergence of a major epidemic of barley powdery mildew. Widespread use of DMI fungicides led to the selection of Bgh isolates carrying both the Y137F and S524T mutations, which, as in Zymoseptoria tritici, account for resistance factors varying from 3.4 for propiconazole to 18 for tebuconazole, the major azoles used at that time in WA. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Mycotoxins produced by Fusarium spp. associated with Fusarium head blight of wheat in Western Australia.

An isolated occurrence of Fusarium head blight (FHB) of wheat was detected in the south-west region of Western Australia during the 2003 harvest season. The molecular identity of 23 isolates of Fusarium spp. collected from this region during the FHB outbreak confirmed the associated pathogens to be F. graminearum, F. acuminatum or F. tricinctum. Moreover, the toxicity of their crude extracts from Czapek-Dox liquid broth and millet seed cultures to brine shrimp (Artemia franciscana) was associated with high mortality levels. The main mycotoxins detected were type B trichothecenes (deoxynivalenol and 3-acetyldeoxynivalenol), enniatins, chlamydosporol and zearalenone. This study is the first report on the mycotoxin profiles of Fusarium spp. associated with FHB of wheat in Western Australia. This study highlights the need for monitoring not just for the presence of the specific Fusarium spp. present in any affected grain but also for their potential mycotoxin and other toxic secondary metabolites.

A short insert in the leader sequence of RNA 3L, a long variant of Alfalfa mosaic virus RNA3, introduces two unidentified reading frames.

N20-RNA 3L, a large form of RNA 3 associated with Alfalfa mosaic virus (AMV) strain N20 comprises 2281 nt and has approximately 97% overall sequence similarity to the longest previously described RNA 3 of AMV strain YSMV (YSMV-RNA 3; 2188 nt). Compared with YSMV-RNA 3, N20-RNA 3L contains an additional 97 nt in the 5' leader upstream of the open reading frames for movement protein (MP) and coat protein (CP). Two overlapping unidentified reading frames (URF1 and URF2) result from this modification, each of which code for putative translation products of 21 amino acids. The URF1 putative peptide has a hydrophilic N-terminus and a hydrophobic C-terminus, indicating a possible association with both host cell membrane and cytosol whereas the putative URF2 product is predominantly hydrophobic. A further structural modification found in N20-RNA 3L is a new tandem repeat of 243 nts which overlaps with the MP open reading frame.