Variation in Susceptibility to Downy Mildew Infection in Spanish Minority Vine Varieties

Downy mildew is one of the most destructive diseases affecting grapevines (Vitis vinifera L.). Caused by the oomycete Plasmopara viticola (Berk. and Curt.) Berl. and de Toni, it can appear anywhere where vines are cultivated. It is habitually controlled by the application of phytosanitary agents (copper-based or systemic) at different stages of the vine growth cycle. This, however, is costly, can lead to reduced yields, has a considerable environmental impact, and its overuse close to harvest can cause fermentation problems. All grapevines are susceptible to this disease, although the degree of susceptibility differs between varieties. Market demands and European legislation on viticulture and the use of phytosanitary agents (art. 14 of Directive 128/2009/EC) now make it important to know the sensitivity of all available varieties, including minority varieties. Such knowledge allows for a more appropriate use of phytosanitary agents, fosters the commercial use of these varieties and thus increases the offer of wines associated with different terroirs, and helps identify material for use in crop improvement programmes via crossing or genetic transformation, etc. Over 2020–2021, the susceptibility to P. viticola of 63 minority vine varieties from different regions of Spain was examined in the laboratory using the leaf disc technique. Some 87% of these varieties were highly susceptible and 11% moderately susceptible; just 2% showed low susceptibility. The least susceptible of all was the variety Morate (Madrid, IMIDRA). Those showing intermediate susceptibility included the varieties Sanguina (Castilla la Mancha, IVICAM), Planta Mula (Comunidad Valenciana, ITVE), Rayada Melonera (Madrid, IMIDRA), Zamarrica (Galicia, EVEGA), Cariñena Roja (Cataluña, INCAVI), Mandrègue (Aragón, DGA) and Bastardo Blanco (Extremadura, CICYTEX). The highly susceptible varieties could be differentiated into three subgroups depending on sporulation severity and density.


Introduction
Downy mildew is, on a global level, one of the diseases that most affects grapevines (Vitis vinifera L.) [1]. It is caused by the oomycete Plasmopara viticola (Berk. and Curt.) Berl. and de Toni. Its control largely relies on the use of phytosanitary agents. Copper-based contact-acting products or systemic agents are commonly applied at different times during the vine growth cycle. Indeed, successful modern viticulture is dependent on the repeated application of large quantities of fungicides. Their use in Europe is particularly heavy. Not only does this have a negative environmental impact, it reduces the profitability of viticulture compared to the raising of other crops, calling into question the sustainability of wine production (a problem for both viticulturalists and consumers). The continued (and excessive) use of fungicides could also provoke the development of resistance to them, and the appearance of more aggressive, more virulent 'races' of the causal pathogen.
As far as we know, all grapevine varieties are susceptible to downy mildew, although not all to the same degree [2]; differences in susceptibility may also exist between clones of the same variety [3]. It is not known for sure how many varieties exist, but a figure somewhere between 8000 and 10,000 seems likely [4]. Despite the many planting options this affords, many winemaking regions around the world use only 10-12 varieties. Over the last 20 years, however, market competition has led to increased interest in the recovery of old, 'pre-phylloxera' varieties which, for a variety of reasons, fell into disuse (with some even approaching extinction) [5,6]. Some of these varieties are unknown beyond their local areas, yet they may have great oenological potential, and may even be able to adapt to different climates [5][6][7][8][9][10][11][12][13]. Certainly, our poor knowledge of the characteristics of these varieties extends to their degree of susceptibility to diseases such as downy mildew. Their study might allow some to come into commercial use, diversifying the offer of wines linked to different terroirs. Material that could be used in crop improvement programmes (crossing, genetic transformation, etc.) designed to produce plants resistant to disease, might also be identified. Such programmes, however, have so far only involved wild American (V. riparia, V. rupestris, V. rotundifolia and V. cinerea, etc.) and Far Eastern (V. piasezkii, V. amurensis, V. romanetii; V. vinifera Kishmish vatkana) species. These can confer partial or even total resistance to downy mildew (such is the case of V. rotundifolia and V. piasezkii) [14,15]. To date, 27 genomic regions associated with resistance to the disease (Rpv loci) have been identified [16][17][18][19][20]. Some of the genes that confer this resistance include Rpv1 and Rpv2 in Muscadinia rotundifolia Michaux, Rpv3 and Rpv19 in Vitis rupestris Scheele, Rpv4, Rpv7, Rpv11, Rpv17, Rpv18, Rpv20 and Rpv21 in unspecified American species, and Rpv5, Rpv6, Rpv9 and Rpv13 in V. riparia. Recently, three loci-Rpv29, Rpv30 and Rpv31-have been identified in V. vinifera (Georgian germplasm) that confer resistance.
The aim of the present work was to determine the susceptibility to downy mildew of minority, pre-phylloxera vine varieties from different regions of Spain.

Plant Materials
Sixty-three minority varieties from 13 regions of Spain ( Figure 1) were studied over 2020 and 2021. In January/February of each year, research groups in each of these regions sent 10-20 cuttings of varieties of interest to the MBG-CSIC for analysis. All cuttings were disinfected, placed in paraffin wax, and held in a cold chamber for four months to promote later root growth. Two rounds of bud break (one in March and one in April) were organised

Plant Materials
Sixty-three minority varieties from 13 regions of Spain ( Figure 1) were studied over 2020 and 2021. In January/February of each year, research groups in each of these regions sent 10-20 cuttings of varieties of interest to the MBG-CSIC for analysis. All cuttings were disinfected, placed in paraffin wax, and held in a cold chamber for four months to promote later root growth. Two rounds of bud break (one in March and one in April) were organised for each variety in each year, thus ensuring sufficient material for testing. For each round, cuttings were placed in water for a few hours in order to hydrate. A hormonebased rooting solution (0.4% indole butyric acid) was then applied to the base of the cuttings, and five of each variety that developed 3-4 buds were planted in a peat-perlite mixture in alveolar trays within a cultivation chamber (hot bed temperature 30

Collection and Preservation of Plasmopara viticola
Plasmopara viticola for inoculations was obtained from natural infections of vines at the MBG-CSIC research vineyard, following the method of Rumbolz et al. [21].

Inoculation of Leaf Discs
The leaf disc technique described by Rumbolz et al. [21] and Staudt et al. [22] was used to determine the susceptibility of the different varieties to downy mildew. Once the plants had produced canes some 15-30 cm in length, five leaves (the 5th or 6th leaves from the apical bud) were collected for each test and control variety. Thirty discs were then punched from the leaves and placed in Petri dishes (one variety per dish). Each disc was

Collection and Preservation of Plasmopara viticola
Plasmopara viticola for inoculations was obtained from natural infections of vines at the MBG-CSIC research vineyard, following the method of Rumbolz et al. [21].

Inoculation of Leaf Discs
The leaf disc technique described by Rumbolz et al. [21] and Staudt et al. [22] was used to determine the susceptibility of the different varieties to downy mildew. Once the plants had produced canes some 15-30 cm in length, five leaves (the 5th or 6th leaves from the apical bud) were collected for each test and control variety. Thirty discs were then punched from the leaves and placed in Petri dishes (one variety per dish). Each disc was inoculated with P. viticola using a 50 µL of suspension of sporangia (50,000 mL −1 ) and left to incubate for six days (25 • C, 95% relative humidity, 8 h dark/16 h light). On the 6th day, each disc was visually inspected for signs of infection, measuring sporulation incidence (percentage of discs showing sporulation), sporulation severity (surface area affected by sporulation), and sporulation density (concentration of sporangia). The latter two variables were quantified using a visual scale (for both variables: 0-25% low; 25-50% moderate; 50-75% high; 75-100%: very high). Inoculations were performed twice, once in June and once in July, using the material produced in the two rounds of bud break (see above).

Statistical Analysis
Differences in the recorded variables between the varieties were analysed by two-way ANOVA (significance was recorded at p < 0.05, 0.01 and 0.001). The F test was then used to compare each fixed factor against its error. The means of those variables that returned a significant F value were then subjected to Fisher's least significant difference (LSD) test. Principal components analysis was used to confirm the existence of groups of varieties with different levels of susceptibility. All analyses were made using SAS System v.8.1 software [23].

Results and Discussion
No differences were seen between the results for the plants produced in the first and second rounds of sprouting; the results for both were therefore analysed together. Table 1 shows, for each variety, the mean, standard deviation and coefficient of variation for each of the three measured variables. ANOVA revealed significant differences (p < 0.01) among the varieties and years for all three variables. The interaction variety × year had no significant impact on the variables recorded; thus, the plants of each variety behaved similarly in both years. The control varieties (110-Richter and SO 4 ) showed resistance to the disease, returning sporulation incidence values of <15% and sporulation density values of <20%, confirming what was reported by other authors [2,[24][25][26][27], with SO 4 being the more resistant of the two ( Table 1). The SO 4 leaf discs showed small, dispersed necrotic spots, while those for 110-Richter showed fewer but larger sports. This necrosis is a response to infection seen in resistant vines [28][29][30]; hypersensitivity reactions cause programmed cell death around infection sites, helping to prevent the further spread of the pathogen.  Values followed by the same letter, in each column, and for each variable, are not significantly different.
These results show the great majority of the test varieties to be highly susceptible to downy mildew. The incidence of sporulation, however, was shown to be a poor discriminator for assessing degrees of susceptibility; sporulation severity and density were much better indicators.
Principal components analysis involving all three measured variables showed the first two principle components to explain 91% of the total variance. In the first component (Prin 1), sporulation severity and density had the greatest weight, while in the second component (Prin 2) the incidence of sporulation had the greatest weight ( Figure 3). With respect to Prin 1, Trobat Negre (Cataluña, INCAVI), Hebén (Extremadura, CICYTEX), Zurieles (Castilla Plants 2023, 12, 2638 6 of 12 la Mancha, IVICAM) and Rayada Melonera (Andalucía, IFAPA) group towards the right of the graph given the high severity and density values they returned. Morate (Madrid, IMIDRA), which had the lowest severity and density results, was placed to the extreme left. With respect to Prin 2, Albariño Tinto (Galicia, EVEGA) appears in the upper part of the graph given its high incidence value; Morate (Madrid, IMIDRA), Trobat Negre (Cataluña, INCAVI), and Rayada Melonera (Andalucía, IFAPA) and the rootstock varieties 110-Richter and SO 4 group towards the bottom given the lower incidence values they returned. Thus, three groups of varieties are distinguishable. The first is formed by Morate (Madrid, IMIDRA)-the least susceptible, with sporulation incidence, severity and density values all <40%. The second comprises the varieties showing moderate susceptibility (incidence < 75%, severity < 50%, density < 50%); within this latter group, the varieties Sanguina Note that the last variety clustered with this group despite returning a sporulation density of 59% ( Figure 4). Finally, the third group is formed by highly susceptible varieties. Within this group, three subgroups can be distinguished ( Figure 5). These results show the great majority of the test varieties to be highly susceptible to downy mildew. The incidence of sporulation, however, was shown to be a poor discriminator for assessing degrees of susceptibility; sporulation severity and density were much better indicators.
Principal components analysis involving all three measured variables showed the first two principle components to explain 91% of the total variance. In the first component (Prin 1), sporulation severity and density had the greatest weight, while in the second component (Prin 2) the incidence of sporulation had the greatest weight ( Figure 3). With respect to Prin 1, Trobat Negre (Cataluña, INCAVI), Hebén (Extremadura, CICYTEX), Zurieles (Castilla la Mancha, IVICAM) and Rayada Melonera (Andalucía, IFAPA) group towards the right of the graph given the high severity and density values they returned. Morate (Madrid, IMIDRA), which had the lowest severity and density results, was placed to the extreme left. With respect to Prin 2, Albariño Tinto (Galicia, EVEGA) appears in the         (B): incidence > 75%, severity < 50%, density > 50%; (C): incidence > 75%, severity and density < 50%.
The present results thus show that the majority of the tested varieties were either highly or moderately susceptible to downy mildew, although it should be noted that the severity of sporulation (i.e., the surface area occupied by sporulating oomycete) was often <50% ( Figure 6). Thus, once the pathogen has entered the plant, response mechanisms would seem to come into play that are able to defend the host to a degree depending on 1 (CF Navarra, EVENA + UPNA), Cagarrizo (Extremadura, CICYTEX), Albana (Aragón, DGA), Terriza, Hebén and Castellana Blanca (Madrid, IMIDRA), and Riera 43 (Cataluña, INCAVI).
The present results thus show that the majority of the tested varieties were either highly or moderately susceptible to downy mildew, although it should be noted that the severity of sporulation (i.e., the surface area occupied by sporulating oomycete) was often <50% ( Figure 6). Thus, once the pathogen has entered the plant, response mechanisms would seem to come into play that are able to defend the host to a degree depending on the variety. Similar results were reported by Hernández et al. [31] for some of the same varieties when challenged with the same pathogen. Plants of the variety Rayada Melonera from the IFAPA were more susceptible (in terms of sporulation incidence, severity and density) compared to those from the IMIDRA. Similarly, Hebén material from Extremadura was more susceptible than the same from the IMIDRA, and significant differences were seen in terms of incidence between Castellana Blanca material from the IVICAM and both the IMIDRA and EVENA. In a further difference between these latter materials, the plants from the EVENA returned significantly higher sporulation density values. Other authors [32] have reported similar behaviour within the varieties Chasselas Doré and Doña Blanca, with resistances to downy mildew and powdery mildew (caused by Erysiphe necator) varying between low and moderate depending on the material's origin. This phenomenon might be explained by clonal differences, agronomicor climate-linked factors, etc. Some authors attribute host-pathogen interactions to the adaptation of plants to the damage caused to the former by the latter [33]. Thus, plants of the same variety but of different origin, and perhaps therefore living in different environments or exposed to pathogen strains of different aggressiveness, may develop different levels of resistance to further attack.
The clustering of the present varieties in terms of their susceptibility was independent of berry colour. It might be thought that red varieties, with their higher contents in phenolic compounds such as resveratrol [34,35], might be less susceptible to downy mildew [28,36]. Certainly this has been reported with respect to other pathogens such as Botrytis cinerea [37,38]. However, no such correlation was seen in the present work, nor in previous work was any detected in different vine varieties growing in the field [2].
Colleagues working in tandem within the same major project (RTI 2018-101085-RC32see Acknowledgements) detected no relationship between susceptibility to P. viticola and agronomic or ampelographic factors such as earliness or the density of reclining or erect hairs (which provide a barrier to zoospores trying to enter the plant via the stomata) (at press). Other reports [39,40] also indicate a lack of relationship between susceptibility to downy mildew and leaf morphology variables such as hair density. However, these authors indicated associations between factors such as stomatal density and the production of stilbenes; these factors would appear to influence the progress of disease.

Conclusions
Importantly, 2% of the tested varieties-in particular Morate-showed low susceptibility to the pathogen. If cultivated, they might require fewer fungicide treatments than other varieties, reducing costs as well as the environmental impact associated with the use of these agents. They might also be good material for the production of resistant varieties. Their cultivation would not infringe current viticultural legislation, and might improve the selection of wines available to consumers.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.