Development of a molecular method for quantifying oospores in the soil

P. viticola oospores, survival structures produced through sexual reproduction, form in leaves infected with downy mildew in the fall. They persist in the soil and trigger downy mildew outbreaks in the spring. In this study, a method using digital droplet PCR (ddPCR) was developed to quantify oospores in vineyard soils. By fractionating the sample into microdroplets, ddPCR eliminates interference from soil inhibitors and allows for absolute and direct quantification of the target DNA, thereby improving the reliability of analyses in complex matrices such as soils.

For each sample, 2 g of freeze-dried soil was used for DNA extraction (GenoSol protocol¹). After grinding and cell lysis, the DNA was purified by centrifugation, precipitation, and washing, then stored at −20 °C. Amplification of P. viticola DNA was performed using specific primers and a TaqMan probe (Giop kit) targeting the ITS1/5.8S region², and the analyses were conducted on the QX200 system (Bio-Rad).

Mapping the distribution of oospores in an experimental vineyard

This study was conducted in 2022 in an experimental vineyard on sandy-gravelly soil, located at the INRAE Bordeaux research center, within the Pessac-Léognan appellation. This plot, which is managed according to organic farming principles, was planted in 2011 with the Merlot grape variety at a density of 6,600 vines per hectare (1.6 m between rows and 0.95 m between vines within the row). Soil maintenance under the rows was performed mechanically. A total of 318 soil samples weighing 500 g each were collected at a depth of 15 cm on March 23, 2022 (Figure 1a): (i) 198 samples according to a regular grid (3.2 m × 2.85 m) covering the entire plot; (ii) 120 samples with a finer grid (0.8 m and 0.2 m spacing) in 5 plots straddling rows and inter-rows. In addition, the vertical distribution of the inoculum was studied using six soil cores collected from the inter-row area and divided into four horizons: 0–10 cm, 11–20 cm, 21–30 cm, and 31–40 cm, yielding 24 samples. The collected soil samples were stored at 5 °C before being sieved (± 2 mm), homogenized, and freeze-dried.

Downy mildew DNA was present in all soil samples, with a high concentration in the top 10 centimeters

Data analysis revealed the presence of P. viticola DNA in all but one sample, with an average concentration of 6.62 ± 6.68 ITS (Internal Transcribed Spacer) copiers per µL. The ITS copy number corresponds to the quantity of target sequences, specifically identified in order to detect P. viticola DNA, that was present in the analyzed soil sample. It serves as a direct indicator of the amount of primary inoculum present in the soil and is closely correlated with oospore density.

Sample concentrations ranged from 0.05 to 40.4 ITS copies per µL.

The amount of P. viticola DNA, expressed as the number of ITS copies per µL, was converted to the number of oospores per gram of soil based on ddPCR results, taking into account the number of ITS copies in the grapevine downy mildew genome. Estimated concentrations ranged from 0 to 1,858 oospores per gram of soil, with an average of 303 oospores per gram in the study plot.

Five times more oospores in the row than in the inter-row

The distribution of oospores at the plot level is not random and is characterized by clusters approximately 15 m in diameter (Figure 1b). Analysis of the vertical distribution shows that downy mildew oospores are concentrated primarily in the top 10 centimeters of soil (Figure 2a). In soil under the grapevines, oospores are present at levels five times higher than in soil collected from between rows. The results show a higher amount of downy mildew DNA in the row, with a decrease as the distance from the row increases (Figure 2b).

The fact that the highest concentrations of oospores are observed in the row is likely due to the preferential accumulation of leaves in the tilled strip at the base of the grapevines.

A bioassay to evaluate soil infectious potential

To assess the relationship between P. viticola DNA quantification in soil samples and their infectious potential, a leaf disc bioassay was performed. Soil samples (1 g per sample) were placed in water-filled dishes under optimal conditions to promote oospore germination (20 °C, darkness). Leaf discs (n = 10 per dish) were exposed to zoospores during 4 trapping events (on days 0, 2, 4, and 6 after the start of incubation), for a total of 40 discs placed on the water surface per sample. After 48 hours of contact, the discs were transferred to moist filter paper and incubated for 6 days (20 °C, 12-hour light-dark cycle) to assess sporulation development.

This bioassay was conducted on 40 soil samples (representing one-eighth of the 318 samples). The cumulative number of infected leaf discs per sample (out of the 40 discs placed) ranged from 0 to 12. The average infected area per disc ranged from 0 to 10.3 %.

These results show that the soil’s infectious potential, assessed by the cumulative number of infected discs and the average percentage of infected area, increases with the concentration of downy mildew DNA estimated by ddPCR (Figure 3a). However, the correlation coefficients obtained (R² = 0.33 for the infected area and R² = 0.19 for the number of infected discs) indicate a moderate relationship and significant biological variability.

Conclusion

This ddPCR method is a promising tool for quantifying reservoirs of P. viticola oospores in vineyard soils. Both accurate and reproducible, it allows for absolute quantification as well as detection of downy mildew DNA in soil at very low concentrations.

The bioassay results further support the relationship between the quantity of P. viticola DNA in the soil and its infectious potential. In the long term, integrating this new indicator could contribute to improving disease risk forecasting models. It also provides a means to assess the effectiveness of novel agronomic practices designed to reduce the primary inoculum of downy mildew in vineyards.

Acknowledgments: This work was funded by the Alexis Millardet Chair (sponsored by the Châteaux Cheval Blanc, Petrus, Ausone, and Montrose via the University of Bordeaux Foundation https://www.fondation.univ-bordeaux.fr/projet/fonds-isvv/chaire-millardet), as well as by the PROFIL project funded by Ecophyto https://ecophytopic.fr/research-innovation/prevenir/projet-profil and the SAVOIR project under the PARSADA Ecophyto 2030 plan https://ecophytopic.fr/recherche-innovation/proteger/projet-savoir. The authors thank the GenoSol platform https://doi.org/10.15454/L7QN45 of the INRAE Agroecology UMR in Dijon for their assistance with methodological development, as well as the Bordeaux INRAE Vine Experimental Unit https://doi.org/10.17180/75AW-3519 for its participation in the installation and monitoring of the experimental setup in the vineyard.