New opportunity for early on-site detection of Plasmopara viticola by qPCR assay

Plasmopara viticola, the causal agent of grapevine downy mildew, is a widespread and significant plant pathogen. A quantitative PCR (qPCR) assay using a portable thermocycler was developed to enable rapid and early on-site detection of P. viticola. The internal transcribed spacer 1 (ITS1) region was selected as the target, and the specific primer pairs PLAV19 was designed. The assay was optimized using traditional thermocyclers, testing three different primer concentrations (100, 200, and 300 nM), and two annealing temperatures (58 degrees C and 60 degrees C). Optimal conditions were 200 nM primer concentration and an annealing temperature of 60 degrees C. Under these parameters, the assay yielded a limit of detection (LoD) of 1.5 fg/mu L and a limit of quantification (LoQ) of 15 fg/mu L for P. viticola DNA (strain PLVDisspa1), showing consistent performance across both thermocyclers. Specificity tests confirmed no cross-reactivity with DNA from common grapevine-associated microorganisms, biocontrol agents, other Oomycetes and several grapevine cultivars. The PLAV19 primer set was further validated on DNA extracted from healthy, artificially inoculated, and naturally infected grapevine tissues, including samples exhibiting nonspecific leaf symptoms and latent bunch infections. Three DNA extraction protocols were evaluated to validate the extraction method, and one of these was shown to be suitable for on-field applications. The developed assay was a reliable diagnostic tool for the early detection and monitoring of P. viticola under field conditions, with potential applications in disease forecasting and sustainable management of grapevine downy mildew.