Activity of resistance inducers against Plasmopara viticola in vineyard

Plasmopara viticola (Berk. M. A. Curtis) Berl. & De Toni, the causal agent of grapevine downy mildew (DM), is an Oomycete responsible for serious yield and quality losses, and its management still largely depends on the use of fungicides (Gessler et al., 2011). However, numerous alternative products are under investigation to comply with the European restrictions on pesticide use (Directive 2009/128/EC) for a more sustainable and safe agriculture. Among the alternatives to fungicides, the use of plant resistance inducers seems promising. Resistance inducers (RIs) are molecules able to trigger plant defense mechanisms through a cascade of processes and lead to the development of systemic acquired resistance (SAR) or induced systemic resistance (ISR) (Delaunois et al., 2014). These processes are mediated by phytohormones such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and others that activate effective basal plant resistance mechanisms against pathogens (López et al., 2008; Héloir et al., 2019).


Introduction
Plasmopara viticola (Berk. M. A. Curtis) Berl. & De Toni, the causal agent of grapevine downy mildew (DM), is an Oomycete responsible for serious yield and quality losses, and its management still largely depends on the use of fungicides (Gessler et al., 2011). However, numerous alternative products are under investigation to comply with the European restrictions on pesticide use (Directive 2009/128/EC) for a more sustainable and safe agriculture. Among the alternatives to fungicides, the use of plant resistance inducers seems promising. Resistance inducers (RIs) are molecules able to trigger plant defense mechanisms through a cascade of processes and lead to the development of systemic acquired resistance (SAR) or induced systemic resistance (ISR) (Delaunois et al., 2014). These processes are mediated by phytohormones such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and others that activate effective basal plant resistance mechanisms against pathogens (López et al., 2008;Héloir et al., 2019).
In grapevine, a number of compounds were reported to trigger plant resistance. Laminarin (a beta-glucan extracted from the brown alga Laminaria digitata) (Aziz et al., 2003) and its sulfate derivatives, chitosan mixed with oligo-galacturonides (also known as COS-OGA) (van Aubel et al., 2014), cerevisane (extracted from cell walls of Saccharomyces cerevisiae LAS 117) (Pujos et al., 2014), and phosphonates (Lim et al., 2013) have all been reported to induce a plant defense reaction against biotic and abiotic stresses. The metabolic pathways and defense mechanisms following infection by P. viticola in plants treated with RIs have been studied under controlled conditions and their efficacy have long been reported after inoculation experiments (Aziz et al., 2003). In difference, studies devoted to field application tactics and strategies for an effective management in the field are less available (Aziz et al., 2006;Bleyer et al., 2020). Testing RIs under natural conditions is important because plants are already primed by various biotic and abiotic stresses in vineyards, and this may affect the commercial RIs efficacy compared to the laboratory experiments (Delaunois et al., 2014).
In this study, we carried out experiments to evaluate the effect of resistance inducers (RIs) on downy mildew. In a first experiment, we focused on the use of the RIs in a protection strategy based on the combination with copperbased products applied following the indications of the decision support system (DSS) vite.net ® provided by Horta (Rossi et al., 2014). In a second experiment, we studied the efficacy of resistance inducers in controlling DM following artificial inoculation of P.viticola at different timings after treatment.

Efficacy of resistance inducers in combination with copper-based fungicides
In the years 2020 and 2021, we tested different protection strategies in a cv. Barbera vineyard located in the experimental farm Res Uvae in Castell'Arquato (Piacenza, Italy). The following RIs and copper-based products were applied: Fosetyl-Al (Aliette, Bayer Cropscience, 80%) at 2.5 kg/ha; Potassium phosphonates (Century, Basf, 51.7%) at 2 L/ha; COS-OGA (Ibisco, Gowan, 12.5 g/l) at 2 -3 L/ha; Pythium oligandrum (Polyversum, Gowan, 1x10 6 Table 1: Timing of application of resistance inducers in combination with copper-based fungicides May 2020 was dry and the average temperature was around 19°C; in June, there were 85.8 mm rain, and the average temperature raised to 21°C. In July, there were five isolate rainfall events, and a temperatures peak at 36°C. The flowering started in the second decade of May, while veraison at the end of July. The year 2021 was characterized by lower rainfall than 2020, with a total of 268 mm, mainly distributed in April, May and September. In June there was only one rain of 15.8 mm, and the average temperature was 25.7°C; in July, there were 11 rainfall events with a total of 49.3 mm rain, with maximum temperatures below 35°C. In August there were 3 rainfall events for a total of 11.2 mm rain. The beginning of flowering was observed in the second decade of June, while veraison started between the end of July and the beginning of August. Disease severity was assessed at weekly intervals from the flowering at 65 BBCH (Growth stage) to berries ripe at 85 BBCH in the two years. The area under the disease progress curve (AUDPC) (Madden et al., 2007) was calculated for the different treatments based on disease severity assessments. These data were used in an analysis of variance, and means were separated using the Student-Newman-Keul (SNK) test with α=0.05.
Overall, the untreated control (TNT) showed higher disease severity on leaves in 2021 than in 2020, while the disease severity on clusters was similar for the two years ( Figure 1).
The mean severity of downy mildew on the TNT leaves in 2020 approximated 35% for the trial at BBCH 71, and 5% at BBCH of 79; in 2021, it was 15% at BBCH of 73. Our evaluations then covered a wide range of disease severities.
The analysis of variance (AOV) showed a highly significant (P≤0.01) effects of the treatments, timings (DPT), and their interaction. The interaction accounted for 28% of the total variance, demonstrating that the different RIs had varying effects on the disease when applied at different timings with respect to P. viticola infection. The area of inoculated leaves was used as a covariate in the AOV, and had a highly significant (P≤0.01) effect, indicating that although the leaves selected for inoculation had a similar position on the shoot (the 4 th position from the apex), the leaf area influence the response to the infection; in particular, smaller leaves were more affected than larger leaves. Overall, leaves treated with Potassium phosphonate and Laminarin showed the highest efficacy and some efficacy even when P. viticola was inoculated at 1 DPT ( figure 3). Potassium phosphonate had about 40% efficacy as early as 1 DPT, and 80% efficacy from 3 to 12 DPT; efficacy then decreased to 60% at 19 DPT. The laminarin-based product showed 73% efficacy at 1 DPT and > 80% efficacy at 3 and 6 DPT; efficacy then decreased to 57% at 12 DPT and 30% at 19 DPT. Fosetyl Al and Cerevisane had no efficacy at 1 DP1, and 50% to 70% efficacy for inoculations at 3 to 19 DPT ( figure 3). Finally, COS-OGA and P. oligandrum overall showed a lower efficacy compared to other RIs (figure 3).  Abbott, 1925) of RIs on downy mildew severity on grapevine leaves (cv. Barbera) following artificial inoculations of Plasmopara viticola at 1, 3, 6, 12 and 19 days post-treatment (DPT). Whiskers indicate standard errors. Letters indicate significant differences between treatments based on the SNK test with α=0.05.

Conclusions
Our experiments based on vineyard applications of RIs in combination with copper-based fungicides applied at low dosages confirmed that alternatives to the chemical fungicides may contribute to downy mildew control (Bleyer et al., 2020) when used as preventative, i.e., prior P. viticola infection periods predicted by mathematical models. This is a new finding, because in previous research and in technical advices, these products have been and still are recommended as calendar-based, repeated applications (Romanazzi et al., 2016;Gutiérrez-Gamboa et al., 2019).
The artificial inoculation experiments confirmed the preventive efficacy of RIs when applied 1 to 19 before infection. In general, the efficacy increased towards the infections occurring from 3 to 6 DPT and then decreased in subsequent days even if, in some cases, high efficacy was registered at 19 DPT too. Laminarin and Potassium phosphonate were characterized by a high, fast, and longlasting efficacy. Unlike all the other products, they provided effective downy mildew control as early as 24 hours post-treatment. Studies on Laminarin had already documented a rapid reaction (several hours) in the plant after treatment (Aziz et al., 2003). Potassium phosphonate is known to have also a direct action on the pathogen, and this may explain a prompt efficacy. Fosetyl-Al and Cerevisane also showed good efficacy, less prompt but long-lasting, which is still > 50% at 19 DPT (similar to the Potassium phosphonate). These observations are, overall, consistent with what has been previously documented about the ability of these active ingredients to induce resistance against downy mildew (Lim et al., 2013;Pujos et al., 2014). The COS-OGA and P. oligandrum showed a lower and later efficacy. It may be considered that these products are usually recommended for the control of Erysiphe necator and Botrytis cinerea (Mohamed et al., 2007;van Aubel et al., 2014); therefore, this activity against P. viticola is noteworthy. Cerevisane (commercial product Romeo) is registered in Italy for downy mildew, powdery mildew, and grey mold, and Laminarin (commercial product Vacciplant) for downy mildew and powdery mildew; their broad spectrum makes them interesting from an integrated pest management perspective.
The integration of RIs in vineyard protection strategies along with the use of DSSs for precise and preventive interventions against P.viticola may help overcome current strategies based on conventional (calendar-based) plant protection rather than on actual disease risk. Finally, moving to a pre-infectional use of these substances allows also compensate the metabolic cost related to the plant immunity activation, energy allocation and metabolites associated with priming (Nogueira Júnior et al., 2020), which is otherwise unjustified in the absence of infection risk.