Oospore germination dynamics and disease forecasting model: an integrated approach for downy mildew management

Downy mildew, caused by Plasmopara viticola (Berk. et Curt.) Berl. e De Toni, is one of the most economically impacting disease of grapevine (Bois et al., 2017). In absence of an adequate control, the disease leads to severe yield losses (Gessler et al., 2011; Toffolatti et al.,2018). Currently, fungicide application to control grapevine downy mildew starts early in spring and is frequently repeated during the vegetative season, resulting in a large number of treatments with plant protection products (PPP), with implications on health, environment and production costs. In the new framework of the Farm to Fork Strategy of the European Green Deal, the European Commission will take additional action to reduce the overall use of chemical pesticides by 50% by 2030. Currently, with 40% share (Eurostat, 2021), fungicides are the most sold group in the EU. Therefore, to achieve the goal of the Farm to Fork Strategy, a drastic decrease in the number of fungicide applications should be accomplished in a relatively short period of time. A key point in improving the effectiveness of grapevine downy mildew control strategies consists in determining the right timing for PPP schedules, which is still difficult for primary infections, to avoid the application of unnecessary treatments. As a consequence, more efforts and researches are needed with the aim of developing novel solutions to achieve a sustainable disease management. P. viticola is a biotrophic and obligate parasite and a polycyclic pathogen, causing both primary and secondary infection cycles (Figure 1), The pathogen survives in absence of the host by differentiating resting structures, the oospores. These survival structures, originated by sexual reproduction, produce the inoculum for primary infections. The oospores are formed into the host tissues and overwinter on the surface litter. Generally, the oospores germinate in spring, differentiating a macrosporangium at the apex of germ tube, where zoospores, the infection spores, are formed (Vercesi et al., 1999). Therefore, primary infections occur in consequence of the oospore germination when the zoospores reach susceptible grapevine tissues and infect the host through stomata (Figure 1). The oospores represent a source of inoculum during the entire host growing season and, frequently, overlap secondary inoculum (Gobbin et al., 2005). Secondary infection cycles are caused by the asexual inoculum, consisting of zoospores differentiated by sporangia. The main ecological factors influencing the oospore germination process are temperature (Ronzon-Tran Manh Sung and Clerjeau; Burruano et al., 1990) water (Burruano et al., 1987; Rossi and Caffi, 2007), soil humidity (Burruano et al., 1992), and location (Galbiati and Longhin, 1984; Burruano et al., 1989) alone or in combination (Ronzon-Tran Manh Sung and Clerjeau, 1987; Rossi et al., 2008; Vercesi et al., 2010). Relating the influence of environmental conditions to the germination dynamics could provide indication on inoculum availability for primary infections and the need for a fungicide treatment. A recent study demonstrated that the number of days required by the oospores to germinate (t) decreases when grapevine reaches susceptibility to P. viticola (Maddalena et al., 2021).


Introduction
Downy mildew, caused by Plasmopara viticola (Berk.et Curt.)Berl.e De Toni, is one of the most economically impacting disease of grapevine (Bois et al., 2017).In absence of an adequate control, the disease leads to severe yield losses (Gessler et al., 2011;Toffolatti et al.,2018).Currently, fungicide application to control grapevine downy mildew starts early in spring and is frequently repeated during the vegetative season, resulting in a large number of treatments with plant protection products (PPP), with implications on health, environment and production costs.In the new framework of the Farm to Fork Strategy of the European Green Deal, the European Commission will take additional action to reduce the overall use of chemical pesticides by 50% by 2030.Currently, with 40% share (Eurostat, 2021), fungicides are the most sold group in the EU.Therefore, to achieve the goal of the Farm to Fork Strategy, a drastic decrease in the number of fungicide applications should be accomplished in a relatively short period of time.A key point in improving the effectiveness of grapevine downy mildew control strategies consists in determining the right timing for PPP schedules, which is still difficult for primary infections, to avoid the application of unnecessary treatments.As a consequence, more efforts and researches are needed with the aim of developing novel solutions to achieve a sustainable disease management.P. viticola is a biotrophic and obligate parasite and a polycyclic pathogen, causing both primary and secondary infection cycles (Figure 1), The pathogen survives in absence of the host by differentiating resting structures, the oospores.These survival structures, originated by sexual reproduction, produce the inoculum for primary infections.The oospores are formed into the host tissues and overwinter on the surface litter.Generally, the oospores germinate in spring, differentiating a macrosporangium at the apex of germ tube, where zoospores, the infection spores, are formed (Vercesi et al., 1999).Therefore, primary infections occur in consequence of the oospore germination when the zoospores reach susceptible grapevine tissues and infect the host through stomata (Figure 1).The oospores represent a source of inoculum during the entire host growing season and, frequently, overlap secondary inoculum (Gobbin et al., 2005).Secondary infection cycles are caused by the asexual inoculum, consisting of zoospores differentiated by sporangia.
The main ecological factors influencing the oospore germination process are temperature (Ronzon-Tran Manh Sung and Clerjeau; Burruano et al., 1990) water (Burruano et al., 1987;Rossi and Caffi, 2007), soil humidity (Burruano et al., 1992), and location (Galbiati and Longhin, 1984;Burruano et al., 1989) alone or in combination (Ronzon-Tran Manh Sung and Clerjeau, 1987; Rossi et al., 2008;Vercesi et al., 2010).Relating the influence of environmental conditions to the germination dynamics could provide indication on inoculum availability for primary infections and the need for a fungicide treatment.A recent study demonstrated that the number of days required by the oospores to germinate (t) decreases when grapevine reaches susceptibility to P. viticola (Maddalena et al., 2021).The promising results obtained in the previous years led the authors to evaluate the effectiveness of EPI model extending its use in other viticultural area.This study aimed at combining biological data on the oospore germination with the use of the EPI model to estimate the risk of infection in Franciacorta, an important Italian viticultural area located in province of Brescia.The analysis on oospore germination data were carried out on three populations of P. viticola oospores overwintered in natural field conditions.The disease forecasting model was used in ten vineyards.The results obtained with the oospores and the model were compared with the real epidemics in field, by estimating disease incidence and severity (Figure 2).

Vineyards
Experimental activities were carried out in 2021 in ten vineyards located in Franciacorta, in province of Brescia (Northern Italy, Lombardy region).Meteorological data (hourly temperatures, rain and relative humidity) were collected throughout in situ weather stations (Figure 3, Table 1).In each vineyard, a plot consisting in 3-4 rows (75 plants on average) was not treated against P. viticola, to assess the epidemic development weekly.

Oospore germination dynamics
The oospore germinations dynamics was evaluated for three  Moreover, the length of incubation period was calculated (Goidanich et al., 1957), to evaluate ascertain the most probable date of disease infection occurrence.
Weekly bulletins reporting information about infection risk, the oospores germination assays and the real epidemic development observed in field, were provided to the farmers.Overall, the EPI model predicted a medium-high infection risk since the end of April to the first ten days of June and, in some cases, at the beginning of July.The first symptoms in field were observed between 6 th and 21 st of May as a result of infection that occurred between 28 th of April and 11 th of May, as predicted by the model.Interestingly, in correspondence with the infection risk indicated by the EPI model, the oospores showed a reduction in t (with minimum values ranging from two to five days).This confirms that t significantly decreases when the plant reaches susceptibility to P. viticola (Maddalena et al., 2021).It must be point out that, since the oospores are the only overwintering structures of the pathogen and the source of the vital inoculum for primary infection, obtaining real biological data on oospore germination is particularly important also from an epidemiological point of view since it provides information on the availability of inoculum for primary infections.Indeed, the germination of the oospores monitored in laboratory, was observed until 21 st of June, indicating the possibility of primary and secondary infections overlaps during the season.The results obtained by field evaluation carried out weekly on untreated plots showed a slow but progressive increase of the disease until the middle of June.Therefore, the conditions in 2021 resulted favourable for disease development, as demonstrated by the medium-high average values of disease severity and incidence observed in the untreated plots, reported in Table 2.The a posteriori evaluation of the model, highlighted a high accuracy of FTA index, calculated by the EPI model, in identifying periods when conditions were favourable for grapevine downy mildew development.Indeed, the FTA index correctly described the epidemic trend of grapevine downy mildew over the vegetative season that was investigated by field observations.

Conclusions
To conclude, the oospore germination assay, integrated with the forecasting model, allowed to identify the time window for the occurrence of infection during the whole vegetative season.Overall, the adoption of the EPI forecasting model combined with the analysis of oospore germination dynamics could contribute to definition of a rational treatment strategy, by identifying the right moment for the fungicide applications.However, the high variability of grapevine downy mildew incidence across years, highlights the necessity of considering several seasons to validate the model.To this purpose, the present study will be repeated in the next vegetative season in the same vineyards.

Figure 1 :
Figure 1: Life cycle of Plasmopara viticola, the causal agent of downy mildew of grapevine.Many epidemiological models have been developed to identify which conditions favour the disease development (Rossi et al., 2008; Caffi et al., 2011; Legler et al., 2011; González-Domínguez et al., 2011; Orlandini et al., 1993; Rodríguez-Rajo et al., 2010; Hill et al., 2019).EPI (Etat Potentiel d'infection), the heuristic model designed for the assessment of P. viticola infection, was formulated by Strizyk in 1981 and thanks to numerous experiments carried out subsequently on grapevine downy mildew in France and in Northern Italy(Fremiot et al., 2008;Parisi et al., 2012), the EPI algorithm has undergone a series of modifications, also enriching with the results of the research carried out on the oospore germination(Vercesi et al., 1999).The model, developed by "Sesma", is currently included in the Epicure system adopted by the Institut Français de la Vigne et du Vin

Figure 2 :
Figure 2: Experimental procedure pattern 2 Materials and methods

Figure 3 :
Figure 3: Map of the province of Brescia representing locations of weather stations and vineyards vineyards located in Adro, Provaglio d'Iseo and Erbusco.Grapevine leaves, showing downy mildew mosaic symptoms, were collected at the middle of October 2020.Twenty nylon bags containing leaf fragments rich in oospores were prepared(Maddalena et al., 2021).Germination assays were carried out once or twice a week starting from grapevine sprouting (second half of March 2021) until bunch closure (end of June 2021) as described by(Maddalena et al., 2021).For each nylon bag, three agar plates (technical replicates) were inoculated with four 10 l droplets of 100 oospores and incubated at the optimal temperature of 20°C.Macrosporangium formation was checked daily at the stereomicroscope (Leica Wild M10) from 1 to 16 days after incubation (dai).These data were used to calculate, for each germination assay, the minimum number of days required by the oospores to germinate (t)(Maddalena et al., 2021).

Table 1 :
List of the vineyards with relative agronomic characteristics

Table 2 :
I%I and I%D average values for leaves and bunches at bunch closure.