Disease risk assessment of poppy downy mildew using qPCR analysis of two Peronospora spp. in naturally-infested soil

Poppy downy mildew, caused by Peronospora meconopsidis and P. somniferi, is one of the most devastating diseases of opium poppy and continues to be a major constraint to commercial production in Australia. A real-time polymerase chain reaction assay was developed for the quantification of the two Peronospora spp. in leaves, soil and in spore traps. Based on the nucleotide differences within the cytochrome oxidase regions, species-specific Taqman and locked nucleotide acid probes were designed to perform a quantitative assay for the specific detection and quantification of P. meconopsidis and P. somniferi, respectively. To normalise between samples, an exogenous internal positive control, the marine bacterium Pseudoalteromonas prydzensis was included with samples prior to DNA extraction, and a primer and probe set targeting the control was also developed. Specificity of the assay to detect P. meconopsidis and P. somniferi was confirmed through testing against nine other closely related Peronospora spp. The assay was able to reliably detect a minimum of 30 pg/μl and 2 ng/μl of DNA for P. meconopsidis and P. somniferi, respectively. The capacity of the P. somniferi assay to quantify pathogen levels in soil was validated by testing 95 naturally-infested field soils collected between 2015 and 2017. Pathogen detection levels were 0.00 - 5,568 ng DNA/g of soil for P. somniferi, with only low levels recorded for P. meconopsidis (<1.0 ng DNA/g of soil). Successful disease transmission was shown in seven of the field soils, through measurement of infection in seedlings grown in tested soils under controlled conditions. A threshold of 20 ng DNA/g of soil was observed for reliable transmission of P. somniferi. No transmission of P. meconopsidis was detected from these samples. Utilization of this assay will enable risk prediction of field sites prior to planting. This assay can also be deployed for monitoring of pathogen dissemination via seed and airborne conidia, through spore trapping.