Responses to drought stress modulate the susceptibility to Plasmopara viticola in grapevine

Climate change will increase the occurrence of plants simultaneously suffering drought and pathogen stress. Although it is well-known that drought can alter the way plants respond to pathogens, knowledge about the effect of concurrent drought and biotic stress in grapevine is scarce. This is especially true for Plasmopara viticola, the causal agent of grapevine downy mildew. This research addresses how vines with different drought tolerance respond to the challenge with P. viticola, drought stress or their combination, and how one stress affects the other.

. Interaction between both stresses is to be expected, since the stomata, the site of entry of P. viticola, are the plants' rst line of defense against drought stress. P. viticola can manipulate stomatal behavior [29], facilitating the infection and thus potentially altering the drought response. The experiment was conducted using an introduced cultivar (Chardonnay) and a drought-tolerant, indigenous cultivar (Xynisteri) in the natural, hot and dry climate of Cyprus.

Experimental set-up
The experimental set-up is given in Fig. 1. More details can be found in Materials and Methods.
Basal differences between cultivars Chardonnay. In contrast, the peroxidase (POD) and superoxide dismutase (SOD) activities and the indole-3-acetic acid (IAA) and proline content were signi cantly higher in Xynisteri than in Chardonnay. The malondialdehyde (MDA) content tended to be slightly higher in Xynisteri. For the catalase (CAT) activity and jasmonic acid (JA) content, no signi cant differences were observed between the cultivars.

Effect of drought stress on disease susceptibility Previous exposure -in vitro inoculation
To assess the effect of a previous exposure to drought stress on the susceptibility to P. viticola, Xynisteri and Chardonnay plants were rst exposed to full or de cit irrigation, either for 7 or 14 days of irrigation treatment (dot), before the in vitro inoculation of leaf disks with the pathogen (previous exposure to full/de cit irrigation -see Fig. 1). The disease evaluation of the leaf disks is shown in Fig. 3a. When watered su ciently (full irrigation), Xynisteri showed a signi cantly higher disease severity than Chardonnay. On disks from non-stressed Chardonnay plants, almost no sporulation was present.
However, Chardonnay leaf disks became more susceptible to P. viticola when exposed to 7 days of de cit irrigation. On Xynisteri leaf disks, P. viticola was able to develop fast, irrespective of this exposure to drought stress. As the duration of the previous drought exposure increased from 7 to 14 days, the disease severity increased in both cultivars. Especially in Chardonnay, the longer exposure to drought stress drastically enhanced susceptibility, reaching a level similar to Xynisteri.

Continuous exposure -in planta inoculation
In parallel, the effect of continuous exposure to drought stress on the susceptibility to P. viticola was examined. Chardonnay and Xynisteri plants were subjected either to 7 or 14 days of full or de cit irrigation, before being sprayed with a P. viticola sporangia suspension or distilled water (continued exposure to full/de cit irrigation -see Fig. 1). The disease evaluation of the plants is shown in Fig. 3b. The irrigation regime was maintained until the moment of disease evaluation, at 7 days post inoculation (dpi). Interestingly, in fully irrigated conditions, the indigenous cultivar Xynisteri showed more severe symptoms than the introduced cultivar Chardonnay. While the fully irrigated plants showed clear disease symptoms, the symptoms on plants challenged with a short drought stress (7 days of de cit irrigation prior to inoculation) remained almost completely absent. Comparing de cit-irrigated plants inoculated at 7 and 14 dot, the resistance to P. viticola did not signi cantly change with longer exposure to de cit irrigation before inoculation.

Field measurements
Single drought stress Fig. 4 shows how the stomatal conductance, chlorophyll uorescence, and soil plant analysis development (SPAD) measurements changed in response to a short (9 dot) or prolonged (16 dot) irrigation treatment, whether or not combined with P. viticola inoculation. The statistical analyses of the single stress treatments are indicated in Additional le 1: Table S1. The temporal changes in the physiological parameters during drought stress are presented in Additional le 2: Fig. S1. The drought stress had a profound effect on the leaf stomatal conductance. After three days of de cit irrigation, the stomatal conductance had already plummeted in both cultivars (Additional le 2: Fig. S1). The drought stress (Fig. 4, FI + Ctrl ¢ vs DI + Ctrl ¢) did not affect the SPAD values, initially (9 dot), though a slight reduction was observed during prolonged drought stress (16 dot) for Chardonnay. The chlorophyll uorescence increased signi cantly in Xynisteri, but not in Chardonnay, after 3 and 7 days of de cit irrigation (Additional le 2: Fig. S1). After 14 days of prolonged stress, the drought caused the chlorophyll uorescence to decrease sharply in both cultivars. The higher loss of chlorophyll uorescence at 16 dot in Chardonnay indicates this cultivar suffered more than Xynisteri (Fig. 4).

Single pathogen stress
No signi cant differences in the physiological parameters were observed between plants inoculated with water and plants inoculated with P. viticola for the same irrigation regime (Additional le 1: Table S1). However, an increasing trend in the stomatal conductance due to the pathogen infection was observed at 16 dot (Fig. 4, ¢ FI + Ctrl vs ¢ FI + Path). The SPAD values were slightly lower in the pathogen-inoculated compared to the water-inoculated plants at 1.5 dpi.

Combined stress
When both stresses were combined (Fig. 4 Fig. 1). The statistical analysis of the single stress treatments is indicated in Additional le 1: Table S1. Interactions between the cultivar, pathogen stress, and irrigation and its duration were analyzed with a regression model (Additional le 3: Table S2, Additional le 4: Table S3). A short (9 dot) or prolonged (16 dot) de cit irrigation had a profound effect on the phytohormone balance ( Fig. 5, ¢ FI + Ctrl vs ¢ DI + Ctrl). The drought stress caused signi cantly increased ABA and decreased JA levels in both cultivars, resulting in similar levels. Considering the large basal differences in ABA, Xynisteri was producing much more ABA than Chardonnay in response to drought. Indeed, the ABA response to the drought stress was likely to be cultivar-dependent (Additional le 3: Table S2). Moreover, the ABA levels seemed to increase when the drought stress prolonged. Drought stress was also related to a signi cant increase in IAA in both cultivars, which seemed to be higher as the drought stress prolonged. Finally, the SA response to the drought stress was signi cantly dependent on the cultivar (Additional le 3: Table S2).
Chardonnay responded to drought stress by dropping its SA content, especially at 16 dot (Additional le 1: Table S1), while still maintaining levels higher than Xynisteri. No clear SA response to drought was observed for Xynisteri (Fig. 5).

Single pathogen stress
To assess the hormonal changes in the cultivars upon pathogen infection, plants were sprayed with water or P. viticola inoculum. Hormone analysis was performed on samples taken at 1.5 dpi from fully irrigated plants (Fig. 5, ¢ FI + Ctrl vs ¢ FI + Path). Independent repetitions occurred at 9 and 16 dot, with only a slight change in plant age. Although Xynisteri was more susceptible to P. viticola than Chardonnay, the hormonal responses of the cultivars to the pathogen were similar. Compared to water-sprayed plants, the pathogen-inoculated plants accumulated more JA and IAA (Fig. 5). In the linear regression, there are indications that IAA is positively affected by the interaction with the pathogen, at least in Xynisteri (Additional le 4: Table S3). The SA levels seemed slightly elevated and the level of ABA remained unaffected by the pathogen (Fig. 5).

Combined stress
The cultivars were subjected to 7 or 14 days of de cit irrigation, before being sprayed with water or P. viticola inoculum, to determine the combined effect of abiotic and biotic stress. The irrigation regime was maintained and samples for analysis were taken 1.5 dpi at 9 or 16 dot (Fig. 5, ¢ DI + Path). Interestingly, the ABA levels, already strongly increased due to drought, rose even more 1.5 days after inoculation with the pathogen. The interaction between pathogen and drought stress was highly signi cant for ABA (Additional le 3: Table S2). The additional ABA accumulation indicated that drought-stressed plants responded to the pathogen, although no symptoms were observed under drought stress (Fig. 3b).
Similarly, an increasing trend in IAA was observed in drought-stressed Xynisteri, already demonstrating higher levels of IAA due to the drought. In drought-stressed Chardonnay, the presence of the pathogen did not cause an additional rise in IAA. A signi cant interaction between drought and pathogen stress was also observed for JA (Additional le 3: Table S2). The accumulation of JA in response to the pathogen in fully irrigated plants, disappeared when de cit irrigation was applied (Fig. 5). A similar trend was observed for SA.

Chlorophylls and oxidative balance
Single drought stress Fig. 6 demonstrates the impact of drought and pathogen stress on the chlorophyll content and oxidative parameters (continued exposure to full/de cit irrigation -see Fig. 1). The statistical analysis of the single stress treatments is indicated in Supplementary Table S1 (Additional le 1: Table S1). The interactions between cultivar, pathogen stress, and irrigation and its duration were analyzed with a regression model (Additional le 3: Table S2, Additional le 4: Table S3). To assess how short and prolonged drought stress affect the cultivars, plants were subjected to full or de cit irrigation for 9 or 16 days, before samples were collected for analysis of the chlorophyll content and the oxidative parameters ( Fig. 6, ¢ FI + Ctrl vs ¢ DI + Ctrl). Even when drought-stressed, the basally higher activities of SOD and POD in Xynisteri were maintained, just as the chlorophyll levels in Chardonnay remained signi cantly higher than in Xynisteri. At rst, the POD activity increased and the MDA content decreased in Xynisteri in response to drought stress (9 dot), while the chlorophyll, SOD, and CAT activity tended to be elevated. However, when the drought stress persisted (16 dot), the POD and SOD activity seemed to diminish, and the H 2 O 2 levels to rise. Xynisteri's response did no longer include changes in chlorophyll or MDA content or CAT activity.
This response of Xynisteri to 16 days of de cit irrigation, was comparable to the earlier response of Chardonnay to 9 days of irrigation. The only difference in drought response between Chardonnay at 9 dot and Xynisteri at 16 dot, was the trend for the CAT activity. While the CAT activity dropped signi cantly in Chardonnay at 9 dot (Additional le 1: Table S1), it remained unchanged in Xynisteri at 16 dot. Continued drought stress (16 dot) eventually caused a signi cant increase in SOD activity in Chardonnay (Additional le 1: Table S1). Its chlorophyll content seemed to lower and the MDA content and POD and CAT activity seemed to increase.

Single pathogen stress
To determine how the cultivars responded to P. viticola, the plants were sprayed with water or pathogen inoculum. The samples for analysis were taken from fully irrigated plants 1.5 dpi (Fig. 6, ¢ FI + Ctrl vs ¢ FI + Path). In this case, both time points (9 or 16 dot) could be seen as a repetition, with only a small change in plant age. Despite their difference in disease severity (Fig. 3b), the responses of the cultivars to the pathogen were similar concerning the parameters tested. The CAT activities were lowered signi cantly and a decrease was observed for the SOD and POD activities. What stood out most, was the signi cant burst in proline associated with the pathogen-inoculated plants (Additional le 1: Table S1), especially at 16 dot. At 16 dot, the MDA content rose and the chlorophyll level decreased slightly, the SOD activity was signi cantly reduced in Xynisteri and the H 2 O 2 content seemed to go up in Chardonnay.

Combined stress
To examine the combined effect of both abiotic and biotic stress, the plants were subjected to 7 or 14 days of de cit irrigation, before being sprayed with water or P. viticola inoculum. The irrigation regime was maintained and samples for analysis were taken 1.5 dpi at 9 or 16 dot (Fig. 6, ¢ DI + Path). Mostly, the responses of de cit and fully irrigated plants to the pathogen were similar. As the drought prolonged (16 dot) in Chardonnay, however, the chlorophyll content seemed to increase with pathogen inoculation, while it seemed to decrease in full irrigation. Similarly, in Xynisteri, the combination of prolonged drought and pathogen stress seemed to buffer Xynisteri against loss of chlorophyll levels by pathogen stress (Fig.  6). Indeed, for chlorophyll, the interaction between abiotic stress, its duration, and biotic stress is signi cant (Additional le 3: Table S2). Thus, the chlorophyll loss at 1.5 dpi in Xynisteri might be associated with the successful infection by P. viticola. The MDA response to the pathogen depended primarily on the interaction with cultivar, drought stress, and its duration (Additional le 3: Table S2). MDA too seemed rather associated with successful infection: MDA increased in fully irrigated plants at 16 dot when inoculated with the pathogen. De cit-irrigated, inoculated plants at 16 dot exhibited levels of MDA similar to the non-inoculated plants (Fig. 6).
The CAT and POD activities were signi cantly reduced in pathogen-inoculated plants under full irrigation. Although no disease symptoms were seen on the drought-stressed plants (Fig. 3b), a similar reduction was observed in plants subjected to drought and pathogen stress. The clear rise in proline in pathogen-inoculated plants was present in both irrigation regimes, indicating proline was associated with inoculation, rather than disease incidence.
Principal component analysis Fig. 7 shows the principal component analysis (PCA) of the continued exposure to full/de cit irrigation (Fig. 1). Although the cultivars and the irrigation and pathogen treatment were used as supplementary variables, not taking part in the construction of the dimensions, they allow grouping of the data. A strong correlation was found between the rst dimension (Dim1) and the cultivar (Table 1). Indeed, there is a good horizontal separation between both cultivars along the axis of the rst dimension (Fig. 7a). The association with the rst dimension indicates that the differences between the cultivars, both basally and in their response to the stresses, explained most of the variation in our dataset. Chardonnay was mostly associated with higher values for chlorophyll, SA, ABA, and H 2 O 2 , while Xynisteri was associated with higher POD, SOD and CAT activities, and MDA content ( Table 2). The second dimension (Dim2) was mostly correlated to the irrigation regime and, to a lesser extent, the duration of this treatment (Table 1).
In the PCA, a horizontal shift is visible according to drought stress and its duration (Fig. 7b). Droughtstressed plants were mostly associated with ABA, IAA, and CAT activity, but were also positively correlated with the chlorophylls and H 2 O 2 content and SOD and POD activities ( Table 2). Fully irrigated plants were mainly correlated with JA, and SA to a lesser extent (Table 2). Finally, the third dimension was primarily correlated to the pathogen stress (Table 1). In Fig. 7c, the water (mock) and pathogen-inoculated plants separate clearly along the vertical axis (Dim3), irrespective of the disease severity. Proline, IAA, and MDA seem to be strongly correlated and are associated with the pathogen-inoculated plants. Interestingly, this PCA indicates that ABA and IAA play a role in the drought-induced resistance, in which JA is no longer involved.

Phytohormones
Single drought stress Single drought stress severely affected the phytohormone balance of the drought-tolerant cultivar Xynisteri and the drought-sensitive cultivar Chardonnay, respectively native and introduced in the investigated climate. In these conditions, ABA, generally considered the key hormone underpinning mechanisms that regulate drought stress responses in plants, appeared to govern the complex hormone crosstalk by antagonizing JA and SA. For both cultivars, drought stress increasingly triggered ABA as the duration extended, but negatively impacted JA. The SA content was lowered too, but primarily in the drought-sensitive Chardonnay. Although most studies observe that JA and SA are involved in drought stress responses in addition to ABA [30], the negative interaction of ABA with JA and SA has also been reported before [31,32]. Multiple nodes allow interference of ABA with the JA-ethylene pathway [33], but whether their interaction is antagonistic [33] or synergistic [34] strongly depends on the conditions. The suppressive effect of ABA on the SA signaling pathway [35][36][37], has been shown for grapevine in particular by Wang et al. [38], who showed that elicitation with exogenous ABA led to a gradual reduction of SA.
Our data demonstrate that drought stress also caused the levels of IAA to increase in both cultivars. The basally higher IAA in Xynisteri might contribute to its drought tolerance. Although not as thoroughly studied in this context as ABA, endogenous IAA levels have been reported to increase during the grapevine defense response against drought [39]. Through its crosstalk with reactive oxygen species (ROS), IAA can help plants to adjust their growth to unfavorable conditions [40]. Previous studies have associated elevated auxin with the induction of abiotic stress-related genes, the activation of the antioxidant response, and the reduction in ROS accumulation [41][42][43][44].
Single pathogen stress P. viticola was able to infect irrigated vines easily in the extreme weather conditions of Cyprus, with high light intensities and maximum daily temperatures reaching 45°C in the shade, although previous studies have shown both high temperature [45,46] and light intensities [47] inhibit sporulation. The nights, with minimum temperatures between 15 and 25°C and relative humidity reaching 80 to 90%, were optimal for infection. Both in vitro and in planta inoculations demonstrated that Xynisteri was more susceptible to P. viticola than Chardonnay when irrigated. Remarkably, our results for fully irrigated plants indicate the infection by the pathogen was also associated with elevated IAA. IAA appeared to be mainly correlated with proline, which accumulated in both cultivars. The higher basal IAA and proline levels in Xynisteri could be related to its higher disease susceptibility. It is well-known that some pathogens are able to upregulate the plant's auxin signaling, in order to suppress the plant defenses, while others can synthesize IAA themselves through various pathways, to increase their pathogenesis [48]. The IAA levels during P. viticola infection have not been studied before, so the question of the origin and function of the IAA accumulation remains. The accumulation of proline associated with elevated IAA has been observed in IAA-treated plants [49]. Like auxin, proline is also involved in numerous developmental processes [50], which can help maintain sustainable growth under long-term stress. However, because of its positive correlation with pathogen-triggered IAA, the role of proline in the plant-pathogen interaction is ambiguous. As a ROS scavenger, proline might have been produced as part of the host defense mechanism against the oxidative stress caused in response to the pathogen. However, proline might bene t the pathogen in a similar way, by detoxifying ROS, which restrict pathogen development.
Contrary to the abiotic stress, we found pathogen stress acted positively on the JA and SA contents of both cultivars, without an apparent effect on the ABA levels. The upsurge of JA and SA at 1.5 dpi in infected, fully irrigated plants demonstrates the plants were activating their defense mechanism. SA is basally higher in Chardonnay and might be part of its more successful defense against P. viticola. The roles of JA and SA have been extensively studied in resistant cultivars, where both phytohormones strongly accumulate after infection with P. viticola [51]. SA as well as JA-mediated defense responses are implicated in the resistance to P. viticola [52][53][54][55][56]. Moreover, exogenous JA has been shown to protect grapevine leaf disks against P. viticola through callose deposition [57].  [11]. Its role in pathogen defense is poorly understood. Whether ABA acts as a positive or negative regulator of disease resistance, is dependent on the stage of infection and the pathosystem, yet seems to be unrelated to the pathogen lifestyle or mode of attack [16]. Although most studies have established an antagonistic relationship between ABA and disease resistance [33,35,[58][59][60], treatment of detached grapevine leaves with exogenous ABA has been shown to result in a reduction of P. viticola infection, albeit only in high concentrations [57,61].
ABA can be involved in pre-invasive defense, preventing pathogen penetration by controlling rapid stomatal movement [62]. Our data suggest however that the pathogen was not stopped during the preinvasive defense. Despite their differences in disease susceptibility, both fully and de cit-irrigated, pathogen-inoculated plants still showed major changes in IAA and proline content and CAT, POD, and, to a lesser extent, SOD activities. Their independence from the irrigation treatment at this infection stage (1.5 dpi), punctuates the infection in the de cit-irrigated plants ceased post-penetration. It indicates P. viticola was able to penetrate the substomatal cavities, even though the stomatal conductance was markedly reduced in response to de cit irrigation. Notably, an additional rise in ABA was observed in de cit-irrigated plants after inoculation with the pathogen. This additional rise in ABA could be key to the post-invasive resistance to this pathogen. During the post-invasion defense, ABA is implicated in callose [34,63] and stilbene [38] accumulation, thus limiting the pathogen spread. ABA has also been found to accumulate strongly in some genetically resistant Vitis species after P. viticola inoculation [38,64]. In many resistant Vitis species, most infections never advance beyond the assessed developmental stage (24-48 hpi) [52,65].
However, while continued exposure to drought induced resistance, we discovered that leaves detached from drought-stressed plants became more susceptible to this pathogen when inoculated in humid, temperate conditions. This indicates the drought-induced, ABA-mediated resistance depends on a rapid defense response, which can be inverted in a very short time. The fast turnover of the drought-induced resistance could explain why Roatti et al. [25] did not report a reduction in disease severity when P. viticola was inoculated at the end of a de cit irrigation period. Because of the striking difference in disease severity during and after exposure to de cit irrigation, it is unlikely that a physical barrier is the source of the ABA-mediated, post-invasive resistance. Whatever it may be, the response seems strongly dependent on the ABA concentration, which is determined by ABA production, transport, and catabolism.
The rate at which stress ABA is catabolized might be proportional to the amount of stress ABA accumulated [66]. Hence, once the drought stress is lifted and stress ABA is no longer synthesized, the high levels of ABA cannot be sustained. We hypothesize that the recovered disease susceptibility in the detached leaves of drought-stressed plants is linked to their inability to maintain su ciently high ABA levels and to timely restore the adverse effects of drought on its pathogen defense. After all, the drought severely interfered with the pathogen response, including induced IAA and antioxidant enzyme activity and antagonized JA and SA levels. From this point of view, it is not surprising that post drought, Chardonnay partially lost its higher tolerance to the pathogen. Its in vitro susceptibility even increased with the duration of the previous drought stress. Apart from the increased adverse effects, additional ABA accumulated when the de cit irrigation prolonged. This potentially caused lower ABA levels post drought, as a result of the increased ABA catabolism. The previous exposure to de cit irrigation also deteriorated Xynisteri's pathogen defense, but this cultivar was already extremely susceptible under full irrigation.
The changing climate and the practices used to mitigate its effects have a profound impact on plant pathogens. Based on these results, irrigation might render pathogens to become a sudden threat to agroecosystem sustainability. Full irrigation of a drought-tolerant cultivar enhanced its susceptibility to downy mildew infection. The drought-tolerant cultivar can easily be grown with no or ample irrigation, hereby inducing resistance, but for the introduced cultivar, the irrigation is of greater importance. The increasing carbon footprint, coupled with the additional irrigation and disease control measures, underlines the growing importance of the "right plant for the right place". Moreover, the enhanced disease susceptibility found in the in vitro assessment prompts the question as to whether vines, under the studied eld conditions, could become more vulnerable to P. viticola during a rain event following a drought period.

Stomatal conductance and photosynthetic parameters
Single drought stress Since Chardonnay originates from French valleys with humid conditions, this cultivar probably lacks adaptive changes to quickly cope with water stress and might have less sensitive stomatal control than Xynisteri [67,68]. The higher basal concentrations of ABA in the leaves of Chardonnay compared to those of Xynisteri might be related to anisohydric behavior [69]. As a native cultivar in Cyprus, Xynisteri likely has developed fast mechanisms to avoid drought stress. Up to 9 days of de cit irrigation, Xynisteri was even able to increase its chlorophyll uorescence (a measure for the maximum Photosystem II quantum e ciency) and chlorophyll content compared to full irrigation. Both cultivars suffered more as the drought stress prolonged. Eventually, the losses in chlorophyll uorescence, chlorophyll content, and SPAD values (a measure for the chlorophyll content per unit leaf area) were higher in Chardonnay, indicating that the drought was a greater burden to Chardonnay than Xynisteri.

Single pathogen stress
In plants without drought stress, P. viticola was able to infect its hosts pro ciently by manipulating them during the infection. Particularly at 16 dot, the pathogen seemed to increase stomatal conductance, potentially as a result of the accumulation of IAA after infection [70], since the ABA levels were not substantially lowered. It is well-known that P. viticola is able to manipulate stomatal movements. Stoll et al. [71] report that stomatal conductance in irrigated plants decreased when infected with P. viticola, while other studies observe the pathogen keeping the stomata open by suppressing ABA [72] or degrading or blocking its transport [29]. The infection also slightly decreased the chlorophyll content and SPAD values, although the chlorophyll uorescence did not appear to be affected. This biotroph has been shown to lower the photosynthetic rate [73,74] through the loss of chlorophyll, the downregulation of chlorophyll a/b binding protein, chlorophyll synthase, and Rubisco, and the upregulation of chlorophyllase [75]. Just 2 days after inoculation, the chlorophyll losses recorded were still small, likely because the chlorophyll content only decreases within the infected lesion [75,76], and might still have been insu cient to affect the chlorophyll uorescence [77].

Combined stress
Our results suggest the pathogen affects the stomatal control of Xynisteri, part of the strategies to tolerate drought. In plants with drought stress, the pathogen-inoculation at 1.5 dpi was associated with an additional rise in ABA. As expected, this resulted in a further decrease of the stomatal conductance in Chardonnay. In contrast, in drought-stressed Xynisteri, the pathogen was associated with a slightly higher stomatal opening despite this pathogen-induced increase in ABA. In this cultivar, the combined stress also caused an additional rise in IAA, which has the ability to counteract ABA-induced closure [70].
Moreover, the drought stress seemed to abolish the loss of chlorophyll by the pathogen stress. This indicates the chlorophyll loss only occurred as part of a successful infection by the pathogen and demonstrates the pathogen development at 1.5 dpi was already hindered compared to the fully irrigated plants. As would be expected when less chlorophyll is lost, the pathogen inoculation also decreased the loss of chlorophyll uorescence due to prolonged drought stress in Chardonnay. In Xynisteri, however, this loss was increased, despite the trends towards higher stomatal conductance and abolished chlorophyll loss. This might be an indication that the response to the pathogen interferes with the adaptive strategies of Xynisteri to cope with drought stress, which are lacking in Chardonnay.

Oxidative stress parameters
Single drought stress Plants generally respond to abiotic and biotic stresses with the production of ROS as signaling molecules. This is typically followed by the activation of the antioxidant system, to nely tune ROSdependent signal transduction and prevent oxidative damage. During drought, the antioxidant system is activated sooner or stronger in a drought-tolerant than in a drought-sensitive cultivar [78]. ROS can severely damage many host cell components, by breaking DNA, destroying the function of proteins, and causing lipid peroxidation [79]. Lipid peroxidation is the most prominent symptom of oxidative stress in animals and plants [80]. It is highly correlated with the concentration of MDA, one of its nal products, which enhances the cell membrane damage, leading to cell death, but also acts as a signaling molecule under stress conditions. Stress can disturb the well-maintained equilibrium between the production and scavenging of ROS.
The drought-sensitive Chardonnay did not respond as fast to the drought stress as Xynisteri. In Chardonnay, the activity of the antioxidant enzymes only increased during prolonged drought stress.
During the initial drought stress, the antioxidant enzymes in Chardonnay even showed lowered activity. Further illustrating the drought-sensitivity of Chardonnay, is the increasing loss of chlorophyll uorescence and the gain in MDA as the drought stress prolonged. The indigenous cultivar Xynisteri, on the other hand, is equipped with a basal toolset to cope with oxidative stress, including higher basal activity of the antioxidant enzymes and lower levels of H 2 O 2 . The drought-tolerant Xynisteri was able to handle the initial drought stress, by activating the antioxidant enzymes at an early stage, keeping H 2 O 2 in balance. Xynisteri even demonstrated slightly higher chlorophyll content and chlorophyll uorescence and reduced MDA levels compared to the fully irrigated control. However, this cultivar also suffered when the drought stress prolonged, with its oxidative responses becoming more similar to the responses of Chardonnay during initial drought stress.

Single pathogen stress
The single pathogen stress caused high lipid peroxidation, more than the drought stress, as indicated by the high correlation between MDA and pathogen-inoculated plants. Apart from ROS, increased lipoxygenase activity can also be implicated in lipid peroxidation. Associated with JA biosynthesis, lipoxygenases are involved in the activation of defense signaling against P. viticola [74]. The course of oxidative stress can be observed particularly at 16 dot, when the H 2 O 2 accumulation due to the pathogen infection at 1.5 dpi led to the highest accumulation of MDA. Interestingly, this was accompanied by strong decreases in antioxidative enzyme activity. The lipid peroxidation and weak oxidative burst during the rst 24 hours of the compatible infection with P. viticola have been associated with slight increases of total antioxidant capacity [74,81]. The increased SA content might have inhibited the activities of the antioxidant enzymes, in order to enhance pathogenesis-related (PR) gene expression [82,83]. Inactivation of the antioxidant capacity to obtain stronger ROS production could be key in boosting the plant defense and limiting the pathogen infection. Since su cient oxidative burst can indeed restrain P. viticola [74,81], the basally higher levels of H 2 O 2 and the potentially SA-mediated, lower activity of antioxidant enzymes could be part of the more successful pathogen defense of Chardonnay.
However, despite the lowered activity of the antioxidant enzymes, H 2 O 2 levels only increased slightly.
Proline, which accumulated with MDA, could have been produced to quench and scavenge ROS, in order to stabilize proteins, DNA, and membranes [26,84]. In the case of drought stress, proline, rather than the antioxidant enzymes, has been associated with the detoxi cation of ROS in vines [85]. Previous studies have shown proline accumulated under stress by P. viticola [86] and by drought [6,26,85,87]. While the net impact of the host-pathogen interaction is clear, it is hard to make a distinction between host response, the pathogens modulation of this response, and the pathogens biosynthesis. Brilli et al. [88] report the P. viticola genome is holding the genes necessary for proline biosynthesis. In that case, P. viticola might have impaired the oxidative burst by producing or triggering the production of proline, restricting ROS to small concentrations which are insu cient to restrain the pathogen.

Combined stress
The infection triggered similar losses of antioxidant enzyme activity in the de cit and fully irrigated plants. Both susceptible, fully irrigated and resistant, de cit-irrigated plants showed a dramatic proline accumulation. This indicates proline levels at 1.5 dpi can be a measure for the pathogen stress, whether the infection was successful or not. The combined stress seemed to revert the MDA and chlorophyll levels, although affected by both single stresses, to levels similar to non-stressed plants. This shows that lipid peroxidation at 1.5 dpi mainly occurred during the successful infection by the pathogen and indicates the pathogen development was already hindered in the de cit-irrigated vines. The inoculation with the pathogen also seemed to mitigate the small changes in MDA and chlorophyll due to drought, an indication of the crosstalk between both responses.

The gap between in vitro and in planta experiments
Interestingly, depending on the inoculation occurring on leaf disks or intact plants, contradicting conclusions were reached about the impact of irrigation on the susceptibility to P. viticola. Because of the perennial nature and size of the grapevine plant, many studies investigating the impact of compounds, microorganisms, resistance genes, or stress, are performed on detached leaves. Understandably, the cutting itself, but also the removal of the leaf from the elicitor of study and the plant system could trigger or inhibit responses in the leaf, resulting in responses different from those occurring in planta. In vitro studies of the plant response could oversimplify the system. This is especially the case when studying the effects of abiotic stress, since placing the leaves in controlled conditions partly relieves the excised leaf disks of the abiotic stresses the plants were experiencing. This study highlights the importance of being careful and critical in generalizing conclusions obtained through in vitro assays. Sometimes, in vitro assays provide an excellent model, like for the comparison of the cultivar susceptibility under full irrigation. In other cases, it proves impossible to extrapolate the results of in vitro studies to the whole plant and eld system.

Conclusions
Because of overlap and crosstalk between the responses to the individual stresses, the response to the concurrent pathogen and drought challenge could not be interpolated from the independent stress response. Single drought stress triggered IAA and ABA, which antagonized JA and SA. Compared to the native cultivar Xynisteri, which boosted chlorophyll uorescence and chlorophyll levels when initially faced with drought, the drought-sensitive Chardonnay activated the antioxidant system later and seemed to suffer more as the drought stress prolonged. Chardonnay, however, proved less susceptible to P. viticola than Xynisteri when irrigated. Under full irrigation, the successful infection by P. viticola at 1.5 dpi was associated with high IAA, SA, and JA levels, strong decreases in antioxidant enzyme activity, and parallel bursts in proline. When both stresses were combined, the response to the pathogen seemed to interfere slightly with the adaptive strategies of Xynisteri to cope with drought stress. Most interesting was the discovery that the de cit irrigation induced resistance to this pathogen in both Chardonnay and Xynisteri. Since drought-induced ABA overruled the SA and JA defense responses, generally implicated in the resistance against P. viticola, ABA is suggested to be involved in this resistance to P. viticola. Supporting this hypothesis is the additional rise in ABA observed in de cit-irrigated plants after inoculation with the pathogen, compared to non-inoculated plants. The nature of this ABA-mediated defense remains to be investigated, but is most likely post-invasive, since the changes in IAA, antioxidant enzyme activity, and proline at 1.5 dpi occurred independently of the irrigation treatment. Other major ndings in this study are the differences between concomitant and consecutive drought and pathogen stress, and as such between in planta and in vitro research. In sharp contrast to the in planta droughtinduced resistance, leaves from drought-stressed plants became more susceptible to the pathogen when inoculated in vitro. This quick turn-over led us to conclude that high ABA concentrations may be most important to the drought-induced resistance. It suggests that, once the ABA concentrations are lowered, the adverse effects of the drought on the pathogen response, such as the lowered JA, can increase the susceptibility to P. viticola. It still stands to question as to whether vines, under the studied eld conditions, could become more vulnerable to P. viticola during a rain event following a drought period.
The irrigation-dependent susceptibility highlights that the practices used to mitigate the effects of climate change may have a profound impact on plant pathogens. For sustainable vineyard management, the effect of de cit and full irrigation on the crops and prevailing plant pathogens should be evaluated. To avoid downy mildew epidemics, the application of de cit rather than full irrigation may be advisable when the drought becomes intolerable. In arid conditions, however, de cit irrigation might not be su cient for introduced cultivars. The increasing carbon footprint, associated with the additional irrigation and disease control measures of the introduced cultivar, underlines the growing importance of the "right plant for the right place". In the context of climate change, the impact on one stress should not be considered without the other. Breeding programs, breeding for the future, should pay special attention to the combination of biotic and abiotic stresses. The expected increase in abiotic stress might also be of importance in selecting resistance-inducing bene cial microorganisms or elicitors since abiotic stress might interfere with the pathways needed to trigger resistance. All of these studies must, however, take into consideration that a simpli ed model, such as the leaf disk model, cannot be used without prior comparison with the whole plant model.

Site description and plant material
This research was conducted on a sun- This study comprised two cultivars of Vitis vinifera, Xynisteri and Chardonnay. Xynisteri is the main white grape cultivar grown in Cyprus, while Chardonnay, one of the most planted white grape cultivars internationally, has been introduced in Cyprus. In 2014, they respectively covered 30.2% and 1.6% of the ca. 6,142 ha viticultural area of Cyprus [5]. Of each cultivar, 60 self-rooted cuttings were planted in 5-liter polyethylene pots containing soil, originating from the traditional vineyard area in Limassol. The soil properties were previously described by Tzortzakis et al. [87]. Brie y, the soil had a clay-loam texture, an organic matter content of 2.19%, a total CaCO 3 content of 66.9%, a pH of 7.42, and an electrical conductivity (EC) of 0.28 of mS cm −1 . The plants were grown in eld conditions and were automatically irrigated at eld capacity using a drip irrigation system. Three months after planting, the plants were uniformly distributed over 12 treatment groups. The experimental set-up is shown in Fig. 1. For each treatment, ve replicates were used per cultivar. Each group was treated with one of four abiotic stress treatments (7 or 14 days of full or de cit irrigation) to assess the effect of short and prolonged drought stress. Two groups were sampled destructively at 7 and 14 dot. In vitro inoculations were performed on disks of these leaves. In the evening, the remaining intact plants were inoculated with either pathogen or water. For these plants, the irrigation regime was maintained until the disease evaluation 7 days later. Some leaves were sampled at 9 and 16 dot to establish the effect of pathogen attack at 1.5 dpi.

Abiotic stress
Plants were either well-watered, in the full irrigation treatment, or exposed to drought stress by de cit irrigation. The fully irrigated plants received irrigation at eld capacity from an automatic drip system, every 6 hours for 5 min. The de cit irrigation was maintained at 40% of the full irrigation, based on the volumetric water content of the soil (VWC). The de cit-irrigated plants were irrigated manually every two days. To verify and accurately adjust the irrigation, the VWC was measured daily in 8 randomly chosen pots using a portable Time-Domain Re ectometer (TDR) (FieldScout TDR 300 Soil Moisture Probe; Spectrum Technologies) with 4.7-inch rods (Additional le 6: Fig. S3).

Biotic stress
To examine the combined effect of abiotic and biotic stress on the vine, pathogen stress was imposed on the intact plants after 7 or 14 days of drought stress (continued exposure to full irrigation -see Fig. 1). Plasmopara viticola isolate FCHPv1, obtained from Chardonnay in France, was grown for 10 days at 22°C on detached Chardonnay leaves on water agar (0.65%). Sporangia were collected with distilled water and the suspension was adjusted to 2.5x10 4 sporangia mL -1 . The arti cial inoculation was performed in the evening. The abaxial sides of all leaves were sprayed until run-off with 3 mL sporangia suspension of P. viticola. The control plants were sprayed with distilled water. The irrigation regimes were maintained until the disease evaluation. Since P. viticola needs 95-100% relative humidity during the night for an optimal infection and sporulation, each plant was equipped with a container of water and a humid plastic cover in the evening. To prevent extreme temperature development within the cover, the cover was removed in the morning and a light shade was created using a shadow mesh.
The sampling and disease evaluation took place at 1.5 and 7 dpi respectively. Each plant was evaluated according to the following classes: 0, no symptoms; 1, few oil spots with little to no sporulation; 2, moderate symptoms and non-spreading sporulation; 3, clearly diseased with spreading sporulation; 4, severe symptoms with dense sporangiophore carpets.
Field measurements At 3,7,9,14, and 16 dot, the stomatal conductance, chlorophyll uorescence, and chlorophyll content were recorded. The measurements were conducted on the 4 th or 5 th leaf starting from the apical meristem on randomly chosen plants at mid-morning, 4 h after onset of light. The stomatal conductance to water vapor was measured on three to ve plants, using a transient state diffusion porometer (AP4; Delta-T Devices). The chlorophyll uorescence (F v F m -1 ), an indicator of the maximum quantum e ciency of Photosystem II, was monitored on three or four plants after exposure to darkness for 20 minutes with a dark adaptation pin using a chlorophyll uorometer (OS30p; Opti-Sciences). The chlorophyll content per unit leaf area was estimated using a non-destructive SPAD meter (SPAD 502 Plus; Spectrum Technologies). SPAD measurements were conducted twice on the same leaf of ve or six plants.
In vitro assessment of disease susceptibility The 3 rd and 4 th leaf, counted from the apex, sampled at 7 and 14 dot (previous exposure to full irrigation -see Fig. 1), were used to investigate the effect of the previous exposure to drought stress on the susceptibility to P. viticola. Leaf disks (11 mm diameter) were treated with 20 µL distilled water or 20 µL P.

Quanti cation of phytohormones
In leaves sampled at 7, 9, 14,  The proline content was also determined using this frozen ground tissue. The leaf tissue (200 mg) was homogenized in 2 mL of 3% aqueous sulfosalicylic acid (SSA). The extracts were then centrifuged and 1 mL of the supernatant was incubated with 1 mL of acid ninhydrin and 1 mL of glacial acetic acid, for 1 h at 100°C. Then, the formed chromogen was extracted with toluene and the absorbance was measured at 520 nm, using toluene as blank. The proline concentration was determined using serial dilutions (0-100 μg mL -1 ) of D-proline [92].

Quanti cation of antioxidant enzymes
The ground leaf samples were also used for the determination of the activity of the antioxidant enzymes. The tissue (200 mg) was homogenized with 3 mL ice-cold 50 mM PPB (pH 7.0), including 1 mM ethylenediaminetetraacetic acid (EDTA), 1% w/v polyvinylpolypyrrolidone (PVPP), 1 mM phenylmethylsulfonyl uoride (PMSF) and 0.05% polyethylene glycol tert-octylphenyl ether (Triton X-100). The homogenate was centrifuged at 16,000 g for 20 min, at 4°C. The supernatant was collected and an aliquot was rst used to determine the protein content via the Bradford method [93], with bovine serum albumin (BSA) as the protein standard.
The CAT (EC 1.11.1.6) activity was determined by following the consumption of H 2 O 2 (extinction coe cient 39.4 mM cm -1 ) at 240 nm for 3 min, as assayed by Jiang and Zhang [94]. The reaction mixture The SOD (EC 1.15.1.1) activity was assayed using the photochemical method. The reaction mixture (1.5 mL) contained 50 mM PPB (pH 7.5), 13 mM methionine, 75 μM nitro blue tetrazolium (NBT), 0.1 mM EDTA, 2 μM ribo avin and an enzyme aliquot. The reaction started after the addition of ribo avin. Tubes containing the reaction were then placed under a light source of two 15-watt uorescent lamps for 15 min. The reaction was stopped by placing the tubes in the dark. The reaction without the extract developed maximal color (control) and a non-irradiated mixture was used as a blank. The absorbance was determined at 560 nm and activity was expressed as SOD units per mg of protein. One unit of SOD activity was de ned as the amount of enzyme required to cause 50% inhibition of the NBT photoreduction rate [95].
The POD activity was assayed using pyrogallol, following the increase in absorbance at 430 nm, after the oxidation to purpurgallin. The reaction mixture of 2 mL contained 1,665 μL of 100 mM PPB (pH 6. The twelve treatments used in this experimental set-up. Each treatment group, represented by one timeline, consisted of ve plants of both cultivars, drought-resistant Xynisteri and drought-sensitive Chardonnay. The plants were rst exposed to 7 days of full/de cit irrigation (short full irrigation/short de cit irrigation), to establish the effect of short drought stress, or to 14 days of full/de cit irrigation (prolonged full irrigation/prolonged de cit irrigation), to examine the effect of prolonged drought stress.
At day 7 or 14, the plants were either sampled destructively or were maintained under the current irrigation regime. Some of the sampled leaves of the rst group were used for in vitro inoculation with water (Ctrl) or P. viticola (Path) to assess the effect of previous exposure to drought stress on the disease development. The other groups were inoculated in planta, either with water (Ctrl) or with P. viticola (Path), to determine the effect of continued exposure to drought stress on the disease development. These plants were sampled at 9 or 16 days of irrigation treatment (dot), corresponding to 1.5 days post inoculation (dpi), and were maintained under the full/de cit irrigation until the disease evaluation at 7 dpi.  In uence of drought stress on the susceptibility of Xynisteri (XYN) and Chardonnay (CHAR) to P. viticola.
The control plants were fully irrigated (FI), while the drought-stressed plants were exposed to de cit irrigation (DI). Different letters indicate signi cant differences (Mann-Whitney U-test; p<0.05). a Previous exposure to drought stress: Plants were exposed to 7 or 14 dot before the leaves were detached (previous exposure to full/de cit irrigation -see Fig. 1). Leaf disks were inoculated with a P. viticola sporangia suspension and each disk was evaluated 5 days later by counting the sporangiophores. Five plants of each cultivar were used per treatment, resulting in an average of 60 disks per treatment; b Continued exposure to drought stress: Plants were exposed to 7 or 14 dot prior to being sprayed with a P. viticola sporangia suspension (continued exposure to full/de cit irrigation -see Fig. 1). The irrigation regime was maintained until the disease severity was evaluated, 7 days later. Five plants of each cultivar were used per treatment.

Figure 4
The impact of pathogen and drought stress on the plants' physiological parameters. Xynisteri (XYN) and Chardonnay (CHAR) plants had been subjected to 7 or 14 days of full (FI) or de cit (DI) irrigation before in planta inoculation with water (Ctrl) or P. viticola (Path) (continued exposure to full/de cit irrigation -see Fig. 1). The stomatal conductance, chlorophyll uorescence, and SPAD were measured at 1.  The impact of drought and pathogen stress on the leaf phytohormone balance. Xynisteri (XYN) and Chardonnay (CHAR) plants had been subjected to 7 or 14 days of full (FI) or de cit (DI) irrigation before in planta inoculation with water (Ctrl) or P. viticola (Path) (continued exposure to full/de cit irrigation -see  Impact of drought and pathogen on the photosynthetic pigments and oxidative parameters. Xynisteri (XYN) and Chardonnay (CHAR) plants had been subjected to 7 or 14 days of full (FI) or de cit (DI) irrigation before in planta inoculation with water (Ctrl) or P. viticola (Path) (continued exposure to full/de cit irrigation -see Fig. 1). Samples for analysis were taken at 1.5 days post inoculation (dpi), corresponding to 9 or 16 days of irrigation treatment (dot). Each treatment consisted of ve repetitions. For the effect of drought stress, the fully irrigated and de cit-irrigated control plants are to be compared.
For the effect of the pathogen stress, the water-inoculated and pathogen-inoculated, fully irrigated plants