Studies on the resistance of different developmental stages in susceptible and tolerant grapevine cultivars against the pathogen Plasmopara viticola

The input of pesticides by agriculture into the environment, leads to increasing pollution and threats to biodiversity. Copper, for example, is deposited in the soil as a heavy metal and cannot be biodegraded. The deposition leads to both a reduction in earthworm population and a decrease in microbial biomass. It also causes stress reactions in soil dwellers (Bünemann et al., 2006; Umweltbundesamt, 2022). Due to the toxic effect, the use of copper is regulated in the European Union to 4 kg/ha/year (Commission Implementing Regulation (EU) 2018/1981). In Germany, the regulations are stricter and are limited to an amount of 3 kg/ha/year. In organic viticulture copper is mainly used to control downy mildew, one of the most important diseases in viticulture (Töpfer et al., 2011). The causative pathogen, Plasmopara viticola (Berk. & M. A. Curtis) Berl. & De Toni, can lead to epidemic infestation in years with suitable warm and humid weather conditions and thus high crop losses (Figueiredo et al., 2017; Unger et al., 2007). In years of high infestation pressure, such as 2012, 2016, and 2021, limited copper levels pose enormous challenges for organic growers to adequately protect their vineyards from downy mildew (Bleyer et al., 2020). A reduction of the copper application amount in such years is not possible at all. For this reason, new strategies must be developed to secure the yield and reduce the use of pesticides to a minimum.


Introduction
The input of pesticides by agriculture into the environment, leads to increasing pollution and threats to biodiversity. Copper, for example, is deposited in the soil as a heavy metal and cannot be biodegraded. The deposition leads to both a reduction in earthworm population and a decrease in microbial biomass. It also causes stress reactions in soil dwellers (Bünemann et al., 2006;Umweltbundesamt, 2022). Due to the toxic effect, the use of copper is regulated in the European Union to 4 kg/ha/year (Commission Implementing Regulation (EU) 2018/1981). In Germany, the regulations are stricter and are limited to an amount of 3 kg/ha/year. In organic viticulture copper is mainly used to control downy mildew, one of the most important diseases in viticulture (Töpfer et al., 2011). The causative pathogen, Plasmopara viticola (Berk. & M. A. Curtis) Berl. & De Toni, can lead to epidemic infestation in years with suitable warm and humid weather conditions and thus high crop losses (Figueiredo et al., 2017;Unger et al., 2007). In years of high infestation pressure, such as 2012, 2016, and 2021, limited copper levels pose enormous challenges for organic growers to adequately protect their vineyards from downy mildew (Bleyer et al., 2020). A reduction of the copper application amount in such years is not possible at all. For this reason, new strategies must be developed to secure the yield and reduce the use of pesticides to a minimum.
One possibility for this is the planting of fungus-resistant grape varieties, so-called PIWIs (German: pilzwiderstandsfähige Rebsorten). PIWIs are crosses of wild American or Asian grapevines with European cultivars. Due to coevolution of Vitis species with P. viticola, they have developed resistance to the pathogen. These vines carry resistance-conferring genes (Bove et al., 2019). Since P. viticola was not introduced into Europe until 1878 (Töpfer et al., 2011), the native grape varieties were unable to develop resistance to the pathogen and are thus highly susceptible (Eisenmann et al., 2019). The crosses of American or Asian and European grapevines combine the resistance-bearing properties of the American or Asian vines with the oenological properties of the European vines (Töpfer et al., 2011). PIWIs thus have a certain resistance potential to P. viticola and are therefore an excellent way of saving plant protection products for both organic and conventional viticulture (Eisenmann et al., 2019). Nevertheless, there is a risk that the introduced resistance can be overcome by virulent pathogen strains (Ciubotaru et al., 2021). To avoid this, PIWIs must also be adequately protected. However, in this regard, the potential of the varieties to reduce pesticides should be fully exploited. For this reason, vines should be treated just enough to provide adequate protection and avoid resistance breakthrough. In addition, fungicide use should be just low enough to make the most of the varieties' resistance.
Forecasting platforms represent another helpful tool for reducing plant protection in general. These systems use computer-aided models, real-time weather data and weather forecast models to calculate the possibility of infestation of the most important vine diseases in viticulture. The required weather information is collected via weather stations which have been set up in large numbers throughout the winegrowing regions. Taking into account this data and additional phenology models for vine development, recommendations are made to the winegrowers in order to make plant protection as effective and sustainable as possible (Bleyer et  The aim of this work is the development of a test system that allows characterizing of different PIWIs for their susceptibility to P. viticola. In this work, different PIWIs were evaluated for their susceptibility to P. viticola. For this purpose, six different PIWIs with different resistance loci were selected and two susceptible European cultivars were used as reference. To investigate the inflorescences/grapes, a new test system was developed in which inflorescences/grapes from potted grapevines were inoculated under optimal conditions for P. viticola. In parallel, the investigation was carried out on the leaves of the corresponding cultivars. One focus was the difference between young and old leaves of the vine. Another aspect of this work was to record the phenological development of the grape varieties in a vineyard in Freiburg (Germany), to highlight the differences in development over time. The data collected in this way on the susceptibility of inflorescences/clusters and leaves will be integrated into the VitiMeteo forecasting system in the long term, taking the phenological development into account, in order to expand the forecasts for PIWIs. In the future, this will allow the crop protection strategy to be optimally adapted to the resistance characteristics of the PIWIs. Inflorescences/grapes experiments. A special test system was developed to investigate the susceptibility of the inflorescences and grapes against P. viticola. In this system, potted vines were placed in a climate-controlled chamber. Inoculation was carried out with a sporangia solution (40,000 sporangia / ml) directly onto the inflorescences/grapes (spray inoculated) until they were dripping wet. For the sporangia solution, freshly sporulating leaves were rinsed in desalinated water and adjusted to the desired spore count using a counting chamber. The climate-controlled chamber was then sealed. The vines were spray irrigated at 2 hourly intervals for 30 seconds each. This guaranteed high humidity and wet vine organs for optimal infection conditions. The temperature was between 18 -24 °C. 24 hours after the first inoculation, a second inoculation with the same conditions were performed. After another 24 hours, the vines were placed back in the greenhouse. Evaluation of the disease severity was done 4 weeks after inoculation.
Leaf disc assays. In parallel, leaf discs of the same grape varieties were examined. The vines for this experiment were taken from the greenhouse. Per variant, two leaves from 3 different vines were used. For this purpose, the third leaf at the shoot base and the fifth fully unfolded leaf at the shoot tip were sampled per plant. The leaves were disinfected with 70 % ethanol and washed in distilled water. 12 leaf discs per leaf were punched out with a cork borer and placed upside down on a water agar plate (1 % w/v). Drop Inoculation was performed with 100 µl sporangia suspension (40,000 sporangia / ml) and the plates were put dark for 24 hours. After this time the sporangia droplets were removed, and the plates closed with parafilm. The sporangia solution was produced as described above. Plates were incubated for 7 days in a climate chamber with 14 hours of light and 24 °C degrees Celsius until evaluation.  . With the onset of ripening, the susceptibility of the comparative grape varieties droped rapidly to 0 % ( Figure 1). Surprisingly, the S. Gris variety showed great similarities in the level of resistance on the grapes compared to the two reference varieties (Figure 1). The resistance of the variety started only one developmental stage earlier. The first strong decrease in susceptibility was observed at the stage BBCH 75 with 33 % infestation level. One stage earlier, at BBCH 73, the susceptibility was still almost 90 %. In further development, the grape variety showed almost no susceptibility to P. viticola. While all other investigated grapevine varieties carry two different resistance loci, S. Gris has only one resistance loci. In addition, it should be mentioned at this point that S. Gris vines showed significantly less infestation on the grapes than Pinot Noir in an untreated field trial in 2021. While grape infestation was 94 % for Pinot Noir, S. Gris showed only 25 % infestation (data not shown). Why the PIWI cultivars performed significantly worse in test system could possibly be due to the conditions chosen. A very high inoculum concentration was deliberately chosen to best show the differences between the cultivars. It is unlikely that such a high sporangia concentration would occur in nature. It is important to note that the infestation levels found can therefore not be directly transferred to viticultural practice. Looking at the grape varieties Solaris, Divico and Cabernet Cortis (Figure 1), which all carry the resistance loci Rpv 3.3 and Rpv 10, great parallels can be seen between these cultivar. Until flower set, these cultivars were highly susceptible and recorded a large yield loss. With fruit set, BBCH 73, these cultivars showed a strong level of resistance. As of BBCH 75, all 3 varieties showed no more infestation. Leaves of all PIWI cultivars show high resistance to P. viticola. Leaf disc assays were carried out to investigate the susceptibility of leaves of different PIWIs. The focus was mainly on the differences in the susceptibilities of young (shoot tip) and old (grape zone) leaves. The reference grape variety Mueller-Thurgau showed a clear difference between young and old leaves. The infestation intensity decreased by 26 % from 73 % to 47 % ( Figure 2). Lower infestation levels of older leaves were also observed in PIWIs. The highest infestation of young leaves was observed in the S. Gris variety (7.5 %). This decreases in leaves from the grape zone and droped to 3.8 %. The other PIWIs also showed an infestation, but it is very low throughout. The lowest infestation was found in the Calardis Blanc variety with 0.13 % on young leaves. Looking at the grape zone, the values of older leaves were even smaller with 0.04 % and 0.05 % for Solaris, Divico and Calardis Blanc (Figure 2).
The difference in susceptibility between young and old leaves in Mueller-Thurgau can possibly be attributed to ontogenetic resistance. Studies have shown that the low susceptibility of leaves to other diseases, such as powdery mildew, is due to ontogenetic resistance mechanisms (Ficke et al., 2003;Ficke et al., 2004). Whether the slight difference in infestation levels between young and old leaves of PIWIs is also due to ontogenetic resistance cannot be conclusively determined at this time.

Conclusions
In this work, PIWIs were shown to have higher resistance to P. viticola than standard cultivars, especially on the leaves. This gives a great potential to reduce fungicide use in viticulture in the long term. It is important to determine the right time for plant protection for each variety in order to reduce the use of plant protection products and to protect the vine as efficiently as possible.
Although grapes of PIWIs show under normal field conditions little susceptibility to P. viticola, especially at later stages of development, to avoid resistance overcoming by P. viticola, these grape varieties still need to be subjected to some plant protection at the beginning of the fruit development.
With the help of the data presented and the VitiMeteo "Plasmopara" forecasting model, the aim is to give winegrowers the most optimal plant protection strategy for sustainable and future-oriented organic viticulture.