Quantifying the grapevine xylem embolism resistance spectrum to identify varieties and regions at risk in a future dry climate.

Maintaining wine production under global warming partly relies on optimizing the choice of plant material for a given viticultural region and developing drought-resistant cultivars. However, progress in these directions is hampered by the lack of understanding of differences in drought resistance among Vitis genotypes. We investigated patterns of xylem embolism vulnerability within and among 30 Vitis species and sub-species (varieties) from different locations and climates, and assessed the risk of drought vulnerability in 329 viticultural regions worldwide. Within a variety, vulnerability to embolism decreased during summer. Among varieties, we have found wide variations in drought resistance of the vascular system in grapevines. This is particularly the case within Vitis vinifera, with varieties distributed across four clusters of embolism vulnerability. Ugni blanc and Chardonnay featured among the most vulnerable, while Pinot noir, Merlot and Cabernet Sauvignon ranked among the most resistant. Regions possibly at greater risk of being vulnerable to drought, such as Poitou-Charentes, France and Marlborough, New Zealand, do not necessarily have arid climates, but rather bear a significant proportion of vulnerable varieties. We demonstrate that grapevine varieties may not respond equally to warmer and drier conditions, and highlight that hydraulic traits are key to improve viticulture suitability under climate change.

Biochemical and Histological Insights into the Interaction Between the Canker Pathogen Neofusicoccum parvum and Prunus dulcis.

The number of reports associated with wood dieback caused by fungi in the Botryosphaeriaceae in numerous perennial crops worldwide has significantly increased in the past years. In this study, we investigated the interactions between the canker pathogen Neofusicoccum parvum and the almond tree host (Prunus dulcis), with an emphasis on varietal resistance and host response at the cell wall biochemical and histological levels. Plant bioassays in a shaded house showed that among the four commonly planted commercial almond cultivars ('Butte', 'Carmel', 'Monterey', and 'Nonpareil'), there was no significant varietal difference with respect to resistance to the pathogen. Gummosis was triggered only by fungal infection, not by wounding. A two-dimensional nuclear magnetic resonance and liquid chromatography determination of cell wall polymers showed that infected almond trees differed significantly in their glycosyl and lignin composition compared with healthy, noninfected trees. Response to fungal infection involved a significant increase in lignin, a decrease in glucans, and an overall enrichment in other carbohydrates with a profile similar to those observed in gums. Histological observations revealed the presence of guaiacyl-rich cell wall reinforcements. Confocal microscopy suggested that N. parvum colonized mainly the lumina of xylem vessels and parenchyma cells, and to a lesser extent the gum ducts. We discuss the relevance of these findings in the context of the compartmentalization of decay in trees model in almond and its potential involvement in the vulnerability of the host toward fungal wood canker diseases.

Seasonal and long-term consequences of esca grapevine disease on stem xylem integrity.

Hydraulic failure has been extensively studied during drought-induced plant dieback, but its role in plant-pathogen interactions is under debate. During esca, a grapevine (Vitis vinifera) disease, symptomatic leaves are prone to irreversible hydraulic dysfunctions but little is known about the hydraulic integrity of perennial organs over the short- and long-term. We investigated the effects of esca on stem hydraulic integrity in naturally infected plants within a single season and across season(s). We coupled direct (ks) and indirect (kth) hydraulic conductivity measurements, and tylose and vascular pathogen detection with in vivo X-ray microtomography visualizations. Xylem occlusions (tyloses) and subsequent loss of stem hydraulic conductivity (ks) occurred in all shoots with severe symptoms (apoplexy) and in more than 60% of shoots with moderate symptoms (tiger-stripe), with no tyloses in asymptomatic shoots. In vivo stem observations demonstrated that tyloses occurred only when leaf symptoms appeared, and resulted in more than 50% loss of hydraulic conductance in 40% of symptomatic stems, unrelated to symptom age. The impact of esca on xylem integrity was only seasonal, with no long-term impact of disease history. Our study demonstrated how and to what extent a vascular disease such as esca, affecting xylem integrity, could amplify plant mortality through hydraulic failure.

Behind the curtain of the compartmentalization process: Exploring how xylem vessel diameter impacts vascular pathogen resistance.

A key determinant of plant resistance to vascular infections lies in the ability of the host to successfully compartmentalize invaders at the xylem level. Growing evidence supports that the structural properties of the vascular system impact host vulnerability towards vascular pathogens. The aim of this study was to provide further insight into the impact of xylem vessel diameter on compartmentalization efficiency and thus vascular pathogen movement, using the interaction between Vitis and Phaeomoniella chlamydospora as a model system. We showed experimentally that an increased number of xylem vessels above 100 µm of diameter resulted in a higher mean infection level of host tissue. This benchmark was validated within and across Vitis genotypes. Although the ability of genotypes to restore vascular cambium integrity upon infection was highly variable, this trait did not correlate with their ability to impede pathogen movement at the xylem level. The distribution of infection severity of cuttings across the range of genotype's susceptibility suggests that a risk-based mechanism is involved. We used this experimental data to calibrate a mechanistic stochastic model of the pathogen spread and we provide evidence that the efficiency of the compartmentalization process within a given xylem vessel is a function of its diameter.

Over-accumulation of abscisic acid in transgenic tomato plants increases the risk of hydraulic failure.

Climate change threatens food security, and plant science researchers have investigated methods of sustaining crop yield under drought. One approach has been to overproduce abscisic acid (ABA) to enhance water use efficiency. However, the concomitant effects of ABA overproduction on plant vascular system functioning are critical as it influences vulnerability to xylem hydraulic failure. We investigated these effects by comparing physiological and hydraulic responses to water deficit between a tomato (Solanum lycopersicum) wild type control (WT) and a transgenic line overproducing ABA (sp12). Under well-watered conditions, the sp12 line displayed similar growth rate and greater water use efficiency by operating at lower maximum stomatal conductance. X-ray microtomography revealed that sp12 was significantly more vulnerable to xylem embolism, resulting in a reduced hydraulic safety margin. We also observed a significant ontogenic effect on vulnerability to xylem embolism for both WT and sp12. This study demonstrates that the greater water use efficiency in the tomato ABA overproducing line is associated with higher vulnerability of the vascular system to embolism and a higher risk of hydraulic failure. Integrating hydraulic traits into breeding programmes represents a critical step for effectively managing a crop's ability to maintain hydraulic conductivity and productivity under water deficit.

Exploring the Hydraulic Failure Hypothesis of Esca Leaf Symptom Formation.

Vascular pathogens cause disease in a large spectrum of perennial plants, with leaf scorch being one of the most conspicuous symptoms. Esca in grapevine (Vitis vinifera) is a vascular disease with huge negative effects on grape yield and the wine industry. One prominent hypothesis suggests that vascular disease leaf scorch is caused by fungal pathogen-derived elicitors and toxins. Another hypothesis suggests that leaf scorch is caused by hydraulic failure due to air embolism, the pathogen itself, and/or plant-derived tyloses and gels. In this study, we transplanted mature, naturally infected esca symptomatic vines from the field into pots, allowing us to explore xylem integrity in leaves (i.e. leaf midveins and petioles) using synchrotron-based in vivo x-ray microcomputed tomography and light microscopy. Our results demonstrated that symptomatic leaves are not associated with air embolism. In contrast, symptomatic leaves presented significantly more nonfunctional vessels resulting from the presence of nongaseous embolisms (i.e. tyloses and gels) than control leaves, but there was no significant correlation with disease severity. Using quantitative PCR, we determined that two vascular pathogen species associated with esca necrosis in the trunk were not found in leaves where occlusions were observed. Together, these results demonstrate that symptom development is associated with the disruption of vessel integrity and suggest that symptoms are elicited at a distance from the trunk where fungal infections occur. These findings open new perspectives on esca symptom expression where the hydraulic failure and elicitor/toxin hypotheses are not necessarily mutually exclusive.

Modeling of xylem vessel occlusion in grapevine.

Morphological traits of the plant vascular system such as xylem vessel diameter have been implicated in many physiological processes including resistance to drought-induced xylem cavitation and vessel occlusion during infection with vascular wilt diseases. In both events, xylem vessels lose function because they become filled with air or tyloses and gels. Xylem cavitation has been well studied, whereas vessel occlusion remains purely descriptive even though it is a critical response to wounding injuries and compartmentalization of vascular pathogens. The timing of vessel occlusion is a key determinant to a successful compartmentalization of pathogens within the plant vascular system and we hypothesized that xylem vessel diameter is the driving variable. Using a dye injection method coupled with automated image analysis, we parameterized a model to investigate how xylem vessel diameter affects the speed of vessel occlusion in Vitis vinifera L. cv. Cabernet Sauvignon in response to wounding. Our dataset contains observations from 6,646 vessels at five kinetic points following stem pruning, over a time course of 1 week. Using this approach we provide evidence that the diameter of vessels is a key determinant of the timing of their occlusion. We discuss how these findings impact resistance to vascular wilt diseases in perennial woody hosts.

Xylem Vessel Diameter Affects the Compartmentalization of the Vascular Pathogen Phaeomoniella chlamydospora in Grapevine.

Fungal wilt diseases are a threat to global food safety. Previous studies in perennial crops showed that xylem vessel diameter affects disease susceptibility. We tested the hypothesis that xylem vessel diameter impacts occlusion processes and pathogen compartmentalization in Vitis vinifera L. We studied the interaction between four grape commercial cultivars with the vascular wilt pathogen Phaeomoniella chlamydospora. We used qPCR and wood necrotic lesion length to measure fungal colonization coupled with histological studies to assess differences in xylem morphology, pathogen compartmentalization, and fungal colonization strategy. We provided evidence that grape cultivar with wide xylem vessel diameter showed increased susceptibility to P. chlamydospora. The host response to pathogen included vessel occlusion with tyloses and gels, deposition of non-structural phenolic compounds and suberin in vessel walls and depletion of starch in parenchyma cells. Pathogen compartmentalization was less efficient in wide xylem vessels than in narrow diameter vessels. Large vessels displayed higher number of tyloses and gel pockets, which provided substrate for P. chlamydospora growth and routes to escape occluded vessels. We discuss in which capacity xylem vessel diameter is a key determinant of the compartmentalization process and in turn grape cultivar resistance to disease caused by P. chlamydospora.

Identification of host factors potentially involved in RTM-mediated resistance during potyvirus long distance movement.

The long distance movement of potyviruses is a poorly understood step of the viral cycle. Only factors inhibiting this process, referred to as "Restricted TEV Movement" (RTM), have been identified in Arabidopsis thaliana. On the virus side, the potyvirus coat protein (CP) displays determinants required for long-distance movement and for RTM-based resistance breaking. However, the potyvirus CP was previously shown not to interact with the RTM proteins. We undertook the identification of Arabidopsis factors which directly interact with either the RTM proteins or the CP of lettuce mosaic virus (LMV). An Arabidopsis cDNA library generated from companion cells was screened with LMV CP and RTM proteins using the yeast two-hybrid system. Fourteen interacting proteins were identified. Two of them were shown to interact with CP and the RTM proteins suggesting that a multiprotein complex could be formed between the RTM proteins and virions or viral ribonucleoprotein complexes. Co-localization experiments in Nicotiana benthamiana showed that most of the viral and cellular protein pairs co-localized at the periphery of chloroplasts which suggests a putative role for plastids in this process.

A Method to Detect and Quantify Eutypa lata and Diplodia seriata-Complex DNA in Grapevine Pruning Wounds.

Trunk diseases are factors that limit sustainability of vineyards worldwide. Botryosphaeria and Eutypa diebacks are caused by several fungi belonging to the Botryosphaeriaceae and Diatrypaceae, respectively, with Diplodia seriata and Eutypa lata being two of the most common species. Previous information indicated that the traditional isolation method used to detect these pathogens from plant samples could underestimate their incidence levels. In the present study, we designed two sets of primers that target the ß-tubulin gene and that are amenable for quantitative real-time PCR (qPCR) Sybr-Green assays for the detection and quantification of D. seriata-complex (DseCQF/R) and E. lata (ElQF/R) DNA. The design of a species-specific assay was achieved for E. lata. For D. seriata, a species-specific assay could not be designed. The low interspecific diversity across ß-tubulin genes resulted in an assay that could not discriminate D. seriata from some closely related species either not yet reported or presenting a low prevalence on grapevine, such as D. intermedia. We validated our technique on grapevine spur samples naturally and artificially infected with D. seriata and E. lata during the dormant season. Experimental grapevines were located in two counties of northern California where the incidence of both pathogens was previously reported. The qPCR assays revealed that a high frequency of pruning wound infections (65%) was achieved naturally by E. lata, while low infection frequency (less than 5%) was observed using the reisolation method. For D. seriata-complex, low (5%) to no natural infection frequencies were observed by the qPCR and the reisolation method, respectively. These results also provided evidence that our qPCR detection methods were more sensitive to assess the incidence of E. lata and D. seriata-complex in plant samples, than traditional isolation techniques. Benefits of molecular methods for the detection of canker pathogens in the field under natural conditions are discussed.

Variations in Early Response of Grapevine Wood Depending on Wound and Inoculation Combinations with Phaeoacremonium aleophilum and Phaeomoniella chlamydospora.

Defense mechanisms in woody tissue are poorly understood, especially in vine colonized by trunk pathogens. However, several investigations suggest that molecular mechanisms in the central tissue of Vitis vinifera L. may be involved in trunk-defense reactions. In this work, the perception of Phaeoacremonium aleophilum and Phaeomoniella chlamydospora alone or together were investigated in cuttings of Cabernet Sauvignon trunks. Plant responses were analyzed at the tissue level via optical microscopy and at the cellular level via plant-gene expression. The microscopy results revealed that, 6 weeks after pathogen inoculation, newly formed vascular tissue is less developed in plants inoculated with P. chlamydospora than in plants inoculated with P. aleophilum. Co-inoculation with both pathogens resulted in an intermediate phenotype. Further analysis showed the relative expression of the following grapevine genes: PAL, PR10.3, TL, TLb, Vv17.3, STS, STS8, CWinv, PIN, CAM, LOX at 10, 24, 48, and 120 h post-inoculation (hpi). The gene set was induced by wounding before inoculation with the different pathogens, except for the genes CAM and LOX. This response generated significant noise, but the expression of the grapevine genes (PAL, PR10.3, TL, TLb, Vv17.3, STS, STS8, CWinv, and PIN) still differed due to perception of mycelium by the plant. Furthermore, at 48 hpi, the induction of PAL and STS8 differs depending on the pathogen, and a specific pattern emerges from the different inductions associated with the different treatments. Based on these results, we conclude that V. vinifera L. trunk perceives the presence of pathogens differently depending on the inoculated pathogen or even on the combination of co-inoculated pathogens, suggesting a defense orchestration in the perennial organs of woody plants.

Can vessel dimension explain tolerance toward fungal vascular wilt diseases in woody plants? Lessons from Dutch elm disease and esca disease in grapevine.

This review illuminates key findings in our understanding of grapevine xylem resistance to fungal vascular wilt diseases. Grapevine (Vitis spp.) vascular diseases such as esca, botryosphaeria dieback, and eutypa dieback, are caused by a set of taxonomically unrelated ascomycete fungi. Fungal colonization of the vascular system leads to a decline of the plant host because of a loss of the xylem function and subsequent decrease in hydraulic conductivity. Fungal vascular pathogens use different colonization strategies to invade and kill their host. Vitis vinifera cultivars display different levels of tolerance toward vascular diseases caused by fungi, but the plant defense mechanisms underlying those observations have not been completely elucidated. In this review, we establish a parallel between two vascular diseases, grapevine esca disease and Dutch elm disease, and argue that the former should be viewed as a vascular wilt disease. Plant genotypes exhibit differences in xylem morphology and resistance to fungal pathogens causing vascular wilt diseases. We provide evidence that the susceptibility of three commercial V. vinifera cultivars to esca disease is correlated to large vessel diameter. Additionally, we explore how xylem morphological traits related to water transport are influenced by abiotic factors, and how these might impact host tolerance of vascular wilt fungi. Finally, we explore the utility of this concept for predicting which V. vinifera cultivars are most vulnerable of fungal vascular wilt diseases and propose new strategies for disease management.

Identification of Eutypa spp. Causing Eutypa Dieback of Grapevine in Eastern North America.

Eutypa dieback of grapevine is caused by Eutypa lata in production areas with Mediterranean climates in California, Australasia, Europe, and South Africa. Eutypa dieback has also been described in the colder, eastern North American vineyards where cultivars adapted from native Vitis spp. (e.g., Vitis × labruscana 'Concord') are primarily grown. However, the causal agents associated with the diseases in this region have not been conclusively identified. Examination of 48 vineyards showing symptoms of dieback in the northeastern United States (Connecticut, Massachusetts, Michigan, New York, Ohio, and Rhode Island) and Ontario, Canada revealed that vineyards were mainly infected by Eutypa spp. other than E. lata. Multigene phylogenies (internal transcribed spacer ribosomal DNA, ß-tubulin, and RNA polymerase II) of isolates recovered from these vineyards indicated that Eutypa dieback is caused primarily by an undescribed Eutypa sp. and E. laevata. Eutypa sp. was recovered from 56% of the vineyards examined, whereas E. laevata and E. lata were less far common (17 and 6%, respectively). Fruiting body morphology and spore dimensions supported phylogenetic separation of the three taxa. Pathogenicity tests conducted on Vitis vinifera 'Chardonnay' in the greenhouse and in the field verified that all three species were able to cause wood canker and to infect pruning wounds, respectively.

A method to detect and quantify Phaeomoniella chlamydospora and Phaeoacremonium aleophilum DNA in grapevine-wood samples.

Grapevines are sensitive to a wide range of fungal pathogens, including agents such as Phaeomoniella chlamydospora and Phaeoacremonium aleophilum that cause tracheomycosis. In the present study, a procedure for DNA extraction from grapevine woody tissue is first evaluated and shown to be suitable for quantitative analysis. Next, a multiplex real-time PCR method targeting the ß-tubulin gene of the pathogens and the actin gene of plant material is developed and its quantitative capability is verified. This protocol was evaluated in inoculated grapevine-wood samples and in young vines from a nursery and was found to be reliable and highly specific. Results obtained from inoculated cuttings show that the fungal colonization process must be considered regardless of the wood phenotype. An analysis of samples of young vines from the nursery shows that a high rate of contamination occurs at the basis of plants and that this contamination is associated with low quantitative values. This finding provides evidence that in vine nurseries, these fungi may be efficient soil-borne pathogens.