Detection of Ramularia collo-cygni from barley in Australia using triplex quantitative and droplet digital PCR.

BACKGROUND: Ramularia leaf spot (RLS), caused by Ramularia collo-cygni, is an emerging threat to barley (Hordeum vulgare L.) production. RLS has been reported in Australia, however only minimal information is available regarding its detection and distribution. Due to initial asymptomatic growth in planta, slow growth in vitro and symptomatic similarities to net blotch and physiological leaf spots, detection of this pathogen can be challenging. Quantitative polymerase chain reaction (PCR)-based methods for R. collo-cygni-specific identification and detection have been described, however these assays have been demonstrated to lack specificity. False-positive detections may have serious implications, thus we aimed to design a robust R. collo-cygni-specific PCR method. RESULTS: Using the phylogenetically informative RNA polymerase II second largest subunit (rpb2) and translation elongation factor 1-alpha (tef1-α) genes, along with the tef1-α gene of H. vulgare, a triplex assay was developed for both quantitative and droplet digital PCR. The triplex assay detected R. collo-cygni DNA in barley leaves from New South Wales, South Australia, Tasmania, Victoria and Western Australia. No R. collo-cygni DNA was detected in barley seed grown in Western Australia. CONCLUSION: The presence of R. collo-cygni DNA has been confirmed in Australian barley crops, suggesting a distribution ranging across the southern barley growing regions of Australia. The R. collo-cygni-specific assay will be a valuable tool to assist with monitoring the distribution and impact of R. collo-cygni in Australia and other regions. © 2021 Society of Chemical Industry.

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.