Application of Real-time PCR for the Detection and Quantification of Oomycetes in Ornamental Nursery Stock

Numerous Phytophthora and Pythium disease outbreaks have oc-curred in Europe following inadvertent introduction of contaminated orna-mental plants. Detection and identification of pathogens are crucial to reduce risks and improve plant biosecurity in Europe and globally. Oomycete diver-sity present in roots and compost was determined in 99 hardy woody plants bought from nurseries, retailers and internet sellers, using both isolations and molecular analyses. Oomycete DNA was quantified using real-time PCR of en-vironmental DNA from the plants using three loci: ITS, trnM-trnP-trnM and atp9-nad9. At least one oomycete species was isolated from 89.9% of plants us-ing classical techniques. In total, 10 Phytophthora spp., 17 Pythium spp. and 5 Phytopythium spp. were isolated. Oomycetes were isolated from 86% of asymp-tomatic plants, but real-time PCR demonstrated that oomycetes were associ-ated with all plants tested. More oomycete DNA occurred in composts in com-parison with roots and filters from baiting water (a mean of 7.91 ng g-1, 6.55 x 10-1 ng g-1 and 5.62 x 10-1 ng g-1 of oomycete DNA detected in compost with ITS, trnM and atp9 probes, respectively); the ITS probe detected the highest quan-tities of oomycete DNA. No significant differences were found in quantities of oomycete DNA detected using real-time PCR in plants purchased online or from traditional retailers.