Development of a portable qPCR assay for early detection of Plasmopara viticola specie complex

Downy mildew is a devastating grapevine disease worldwide caused by the oomycete Plasmopara viticola. In its native area, five cryptic species have been identified and the aestivalis clade is apparently the unique present in Europe, including southern Italy. A on-field quantitative SybrGreen®-based PCR (qPCR) assay for detection and quantification of P. viticola complex was developed employing an on-site molecular lab station (Generon S.p.A., San Prospero (MO), Italy). A primer set (PLAV19) was designed to target the ITS1 region of the pathogen. Three primer concentrations (200, 300 and 400 nM) and three annealing temperatures (56, 58 and 60°C) were evaluated and 200 nM primer and an annealing temperature of 58°C yielded the most efficient and specific amplification. Under these conditions, the assay showed a limit of detection (LoD) of 1.5 fg µL⁻¹ of P. viticola DNA, corresponding to a quantification cycle (Cq) value of 34. Specificity tests conducted using a panel of more than twenty grapevine pathogens and beneficial microorganisms, and seven table grape cultivars, showed no evidence of unspecific amplification. The assay was further validated using sporangia suspensions of P. viticola at 6 different concentrations (from 10⁵ to 100 ) confirming its high sensitivity (even 1 sporangia). On-field P. viticola quantitative detection was carried out on grape leaves and inflorescences with unspecific symptoms, bunches with larvata-like symptoms, as well as on capture-spores tape, confirming the validity of the protocol for early, rapid and accurate pathogen detection, which can be useful for improving sustainable and effective management of downy mildew