Digital droplet PCR quantificationand field-scalespatial distribution of Plasmopara viticola oospores in vineyard soil

Abstract

Grapevine downy mildew, caused by the oomycete Plasmopara viticola, is one of the most devastating diseases affectinggrapevine worldwide. Primary inoculum (i.e., oospores) plays a decisive role in downy mildew epidemics, but we still know very little about its abundance in vineyard soil. This study presents a novel molecular method for quantifying P. viticola oospore concentration in vineyard soil using digital droplet PCR (ddPCR). The development of this method enabled the characterization of both the abundance and spatial distribution of oospores in a vineyard at the onset of the growing season. Following a regular grid, a total of 198 soil samples (0-15 cm horizon) were collected in March 2022 in grapevine rows in a 0.22 ha vineyard planted with cv. Merlot and conducted according to French organic viticulture specifications.Additional samples were collected from the same fieldwithin fivenested sampling plots with three distance levels, including samples collected in the inter-rows. Using ddPCR, we found P. viticola DNA in all soil samples except one, and we estimated that oospore concentration ranged from 0 to 1,858 oospores per gram of soil (303 ± 308 on average). The distribution of oospores at fieldscale was not random but characterized by 15-m diameter patches of concentrically increasing oospore concentration. Oospores accumulated fivetimes more below the vine stocks than in the inter-row. Using a leaf disc bioassay, we found that soil infectious potential significantlyincreased with oospore concentration assessed by ddPCR. However, the low coefficientof determination of the relationship indicated that DNA-based oospore quantificationlacked clear epidemiological significance.Both ddPCR and bioassay methods are valuable tools that could be used to assess reservoirs of P. viticola primary inoculum across differentagroclimatic contexts, thereby bringing greater genericity. Further methodological improvement will also help refinethe accuracy of DNA-based assessment of primary inoculum reservoir and improve our understanding of the relationship between primary inoculum reservoir and epidemic dynamics. Ultimately, these data will be essential for improving epidemic risk models and evaluating new preventive disease management strategies targeting the primary inoculum.