Detection of Ramularia collo-cygni from barley (Hordeum vulgare) in Australia using triplex quantitative and digital PCR

Ramularia leaf spot (RLS), caused by Ramularia collo-cygni, is an emerging threat to barley (Hordeum vulgare) 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 PCR-based methods for R. collo-cygni-specific identification and detection have been described, however these assays (based upon the internal transcribed spacer [ITS] region) 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. Using the phylogenetically informative RNA polymerase II second largest subunit (rpb2) and translation elongation factor 1- (tef1-) genes, along with the tef1- gene of H. vulgare, a triplex assay was developed for both quantitative and digital PCR. The triplex assay was used to assess DNA of barley leaves from New South Wales, South Australia, Tasmania, Victoria and Western Australia, along with DNA of seeds from Western Australia. Detection of R. collo-cygni DNA was confirmed for leaf samples from New South Wales, South Australia, Tasmania, Victoria and Western Australia, indicating a distribution ranging across the southern barley growing regions of Australia. No R. collo-cygni DNA was detected in seed from Western Australia. The R. collo-cygni-specific assay will be a valuable tool to assist with monitoring the distribution of R. collo-cygni in Australia and other regions.