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 [35–37], 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 [41–44].
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 benefit 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 [52–56]. Moreover, exogenous JA has been shown to protect grapevine leaf disks against P. viticola through callose deposition [57]. The dynamics of endogenous phytohormones during compatible interactions with P. viticola have however not been explicitly investigated. Polesani et al. and Li et al. [52, 56] did observe increases in JA, coupled with very strong rises in methyl jasmonate (MeJA), in successful infections. Both endogenous levels increased from 12 to 48 hours post inoculation but perished once the tissue was completely invaded [52], implicating the involvement of JA and MeJA defenses in the early developmental stages of the pathogen in compatible interactions.
Combined stress
Combined abiotic and biotic stress is a different story entirely. When drought and pathogen stress occurred simultaneously, the two stress responses interacted. Remarkably, under continued deficit irrigation, disease symptoms were no longer observed in Chardonnay or Xynisteri. Even more interesting is our finding that the JA and SA contents were low and no longer substantially contributed to the pathogen defense response. Our results reveal that, under concurrent stress, ABA dominated the responses to the pathogen stress occurring under full irrigation, antagonizing JA and SA. Furthermore, a significant, additional rise in ABA was observed in the inoculated compared to the non-inoculated drought-stressed plants, although under full irrigation, the infection did not trigger ABA. Thus, we hypothesize that ABA, rather than JA or SA, is involved in the observed drought-induced resistance to P. viticola. But how can ABA contribute to hindering the infection by P. viticola?
Considered a global regulator of plant stress responses, ABA is crucial in the response of plants to multiple stresses [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, 58–60], 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 pre-invasive defense. Despite their differences in disease susceptibility, both fully and deficit-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 deficit-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 deficit irrigation. Notably, an additional rise in ABA was observed in deficit-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 deficit irrigation period. Because of the striking difference in disease severity during and after exposure to deficit 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 sufficiently 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 deficit irrigation prolonged. This potentially caused lower ABA levels post drought, as a result of the increased ABA catabolism. The previous exposure to deficit 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 field 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 deficit irrigation, Xynisteri was even able to increase its chlorophyll fluorescence (a measure for the maximum Photosystem II quantum efficiency) and chlorophyll content compared to full irrigation. Both cultivars suffered more as the drought stress prolonged. Eventually, the losses in chlorophyll fluorescence, 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 proficiently 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 fluorescence 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 insufficient to affect the chlorophyll fluorescence [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 fluorescence 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 finely tune ROS-dependent 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 final 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 fluorescence 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 H2O2. The drought-tolerant Xynisteri was able to handle the initial drought stress, by activating the antioxidant enzymes at an early stage, keeping H2O2 in balance. Xynisteri even demonstrated slightly higher chlorophyll content and chlorophyll fluorescence 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 H2O2 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 first 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 sufficient oxidative burst can indeed restrain P. viticola [74, 81], the basally higher levels of H2O2 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, H2O2 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 detoxification 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 insufficient to restrain the pathogen.
Combined stress
The infection triggered similar losses of antioxidant enzyme activity in the deficit and fully irrigated plants. Both susceptible, fully irrigated and resistant, deficit-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 deficit-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 field system.