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Botryosphaeria dieback is a grapevine trunk disease caused by fungi of the Botryosphaeriaceae family, which attacks more specifically the woody tissues. The infection leads to different symptoms including a severe form with a leaf drop as well as premature plant death. Botryosphaeria dieback causes major economic losses, since no effective treatment is yet available. A better understanding is necessary to find solutions to fight this disease. In this study, our objective was to characterize the “leaf drop” form by (1) looking for the presence of pathogens in the basal internodes of stems, (2) quantifying blocked vessels by tylosis and/or gummosis, and (3) describing the impact of the disease on vine physiology (gene expression and metabolome) and development (establishment and functioning of the cambium and phellogen) at the level of xylem and phloem of basal stem internodes. Our study has shown that Botryosphaeriaceae were present in both phloem and xylem of the basal internodes of the annual stem, with xylem vessels obturated. We have also clearly demonstrated that gene expression and metabolite profiles were strongly modified in both xylem and phloem of diseased plants. Differences in stems between healthy (control, C) and diseased (D) plants were low at flowering (vines not yet symptomatic), higher at the onset of symptom expression and still present, although less marked, at full disease expression. qRT-PCR analysis showed in both phloem and xylem an overexpression of genes involved in plant defense, and a repression of genes related to meristematic activity (i.e. vascular cambium and phellogen). Metabolomic analysis showed specific fingerprints in stems of healthy and diseased plants from the onset of symptom expression, with an increase of the level of phytoalexins and mannitol, and a decrease of 1-kestose one. At the structural level, many alterations were observed in internodes, even before the onset of symptoms: a classical obstruction of xylem vessels and, for the first time, a disorganization of the secondary phloem with an obstruction of the sieve plates by callose. The disease modifies the development of both secondary phloem (liber) and phellogen. Altogether, this study combining different approaches allowed to highlight deep vine dysfunction in the internodes at the base of stems, that may explain vine decline due to Botryosphaeria dieback.

Background: Grapevine is a woody, perennial plant of high economic importance worldwide. Like other plants, it lives in close association with large numbers of microorganisms. Bacteria, fungi and viruses are structured in communities, and each individual can be beneficial, neutral or harmful to the plant. In this sense, microorganisms can interact with each other and regulate plant functions (including immunity) and even provide new ones. Thus, the grapevine associated with its microbial communities constitutes a supra-organism, also called a holobiont, whose functioning is linked to established plant-microorganism interactions. Aim of review: The overall health of the plant may be conditioned by the diversity and structure of microbial communities. Consequently, an optimal microbial composition will consist of a microbial balance allowing the plant to be healthy. Conversely, an imbalance of microbial populations could lead to (or be generated by) a decline of the plant. The microbiome is an active component of the host also responsive to biotic and abiotic changes; in that respect, a better understanding of the most important drivers of the composition of plant microbiomes is needed. Key scientific concepts of review: This article presents the current state of the art about the grapevine microbiota and its composition according to the plant compartments and the influencing factors. We also focus on situations of imbalance, in particular during plant disease or decline. Finally, we discuss the possible interest of microbial engineering in an agrosystem such as viticulture.

In the past, most grapevine trunk diseases (GTDs) have been controlled by treatments with sodium arsenite. For obvious reasons, sodium arsenite was banned in vineyards, and consequently, the management of GTDs is difficult due to the lack of methods with similar effectiveness. Sodium arsenite is known to have a fungicide effect and to affect the leaf physiology, but its effect on the woody tissues where the GTD pathogens are present is still poorly understood. This study thus focuses on the effect of sodium arsenite in woody tissues, particularly in the interaction area between asymptomatic wood and necrotic wood resulting from the GTD pathogens’ activities. Metabolomics was used to obtain a metabolite fingerprint of sodium arsenite treatment and microscopy to visualize its effects at the histo-cytological level. The main results are that sodium arsenite impacts both metabolome and structural barriers in plant wood. We reported a stimulator effect on plant secondary metabolites in the wood, which add to its fungicide effect. Moreover, the pattern of some phytotoxins is affected, suggesting the possible effect of sodium arsenite in the pathogen metabolism and/or plant detoxification process. This study brings new elements to understanding the mode of action of sodium arsenite, which is useful in developing sustainable and eco-friendly strategies to better manage GTDs.

Using plant defense elicitors to protect crops against diseases is an attractive strategy to reduce chemical pesticide use. However, development of elicitors remains limited because of variable effectiveness in the field. In contrast to fungicides that directly target pathogens, elicitors activate plant immunity, which depends on plant physiological status. Other products, the biostimulants, can improve certain functions of plants. In this study, the objective was to determine whether a biostimulant via effects on grapevine physiology could increase effectiveness of a defense elicitor. A new methodology was developed to study biostimulant activity under controlled conditions using in vitro plantlets. Both biostimulant and defense elicitor used in the study were plant extracts. When added to the culture medium, the biostimulant accelerated the beginning of plantlet growth and affected the shoot and root development. It also modified metabolomes and phytohormone contents of leaves, stems, and roots. When applied on shoots, the defense elicitor changed metabolite and phytohormone contents, but effects were different depending on whether plantlets were biostimulated or controls. Defense responses and protection against Plasmopara viticola (downy mildew agent) were induced only for plantlets previously treated with the biostimulant, Therefore, the biostimulant may act by priming the defense elicitor action. In this study, a new method to screen biostimulants active on grapevine vegetative growth was used to demonstrate that a biostimulant can optimize the efficiency of a plant defense elicitor.

The root system plays an essential role in the development and physiology of the plant, as well as in its response to various stresses. However, it is often insufficiently studied, mainly because it is difficult to visualize. For grapevine, a plant of major economic interest, there is a growing need to study the root system, in particular to assess its resistance to biotic and abiotic stresses, understand the decline that may affect it, and identify new ecofriendly production systems. In this context, we have evaluated and compared three distinct growing methods (hydroponics, plane, and cylindric rhizotrons) in order to describe relevant architectural root traits of grapevine cuttings (mode of grapevine propagation), and also two 2D- (hydroponics and rhizotron) and one 3D- (neutron tomography) imaging techniques for visualization and quantification of roots. We observed that hydroponics tubes are a system easy to implement but do not allow the direct quantification of root traits over time, conversely to 2D imaging in rhizotron. We demonstrated that neutron tomography is relevant to quantify the root volume. We have also produced a new automated analysis method of digital photographs, adapted for identifying adventitious roots as a feature of root architecture in rhizotrons. This method integrates image segmentation, skeletonization, detection of adventitious root skeleton, and adventitious root reconstruction. Although this study was targeted to grapevine, most of the results obtained could be extended to other plants propagated by cuttings. Image analysis methods could also be adapted to characterization of the root system from seedlings.

Esca is one of the most widespread grapevine trunk diseases affecting vineyards. This complex disease leads to leaf alterations, wood necrosis and eventually to plant death. Esca symptoms are caused by several fungi inhabiting the xylem of host plants and degrading the wood structure. The main pathogens causing the disease are Phaeomoniella chlamydospora, Phaeoacremonium minimum, Fomitiporia mediterranea and other wood-rotting basidiomycetes. Grapevine susceptibility to esca can be predisposed by several factors, especially climate, vine age, and cultivar. An experiment was carried out (in 2015) to assess if esca expression on leaves could also be clone-dependent. Chardonnay clones 76 and 95 grown in the same plot were compared according to their developmental and physiological traits, metabolome, and foliar symptom expression. Leaves were sampled during summer on visually healthy vines as controls (C), and from asymptomatic (D-) and symptomatic (D+) shoots of esca-affected vines. Analysis of their metabolomes highlighted a clone-dependent metabolite fingerprint associated to esca expression. Opposite variations of specific metabolites were found between C and D+ leaves of both clones. The experiment was repeated (in 2018). Leaf samples could be discriminated, especially the C and D+ samples for each clone, but the differences were less marked than in the first experiment. Discriminant compounds were all different between the two experiments, and showed no opposite variations between C and D+ samples of both clones, which indicated variable metabolite responses from year to year for both clones. These results confirm that the leaf metabolite fingerprint associated to esca expression is clone-dependent, and is year-dependent in intensity and nature.

Esca disease is one of the major grapevine trunk diseases in Europe and the etiology is complex, since several inhabiting fungi are identified to be associated with this disease. Among the foliar symptom expressions, the apoplectic form may be distinguished and characterized by sudden dieback of shoots, leaf drop, and shriveling of grape clusters in a few days that can ultimately induce the plant death. To further understand this drastic event, we conducted transcriptomic and metabolomic analyses to characterize responses of leaves during the period preceding symptom appearance (20 and 7 days before foliar symptom expression) and at the day of apoplexy expression. Transcriptomic and metabolomic analyses provide signatures for the apoplectic leaves and most changes concerning the metabolism of carbohydrates, amino acids, and phenylpropanoids. In deciphering glutathione-S-transferase (GST), its preferential location in phloem, correlated with the upregulation of GST genes and a decrease of the glutathione level, offers further support to the putative role of glutathione during apoplexy expression.

In a context of a sustainable viticulture, the implementation of innovative eco-friendly strategies, such as elicitor-triggered immunity, requires a deep knowledge of the molecular mechanisms underlying grapevine defense activation, from pathogen perception to resistance induction. During plant-pathogen interaction, the first step of plant defense activation is ensured by the recognition of microbe-associated molecular patterns, which are elicitors directly derived from pathogenic or beneficial microbes. Vitis vinifera, like other plants, can perceive elicitors of different nature, including proteins, amphiphilic glycolipid, and lipopeptide molecules as well as polysaccharides, thanks to their cognate pattern recognition receptors, the discovery of which recently began in this plant species. Furthermore, damage-associated molecular patterns are another class of elicitors perceived by V. vinifera as an invader’s hallmark. They are mainly polysaccharides derived from the plant cell wall and are generally released through the activity of cell wall–degrading enzymes secreted by microbes. Elicitor perception and subsequent activation of grapevine immunity end in some cases in efficient grapevine resistance against pathogens. Using complementary approaches, several molecular markers have been identified as hallmarks of this induced resistance stage. This review thus focuses on the recognition of elicitors by Vitis vinifera describing the molecular mechanisms triggered from the elicitor perception to the activation of immune responses. Finally, we discuss the fact that the link between elicitation and induced resistance is not so obvious and that the formulation of resistance inducers remains a key step before their application in vineyards.

Grapevine trutk diseases, especially Esca, are of major concern since they gradually alter vineyards worldwide and cause heavy economic losses. The expression of Esca disease symptoms depends on several factors, including the grapevine cultivar. In this context, a possible clone-dependent expression of the Esca disease was studied. Two clones of ‘Chardonnay’ grown in the same plot were compared according to their developmental and physiological traits, metabolome, and foliar symptom expression. Analysis of their leaf metabolome highlighted differences related to symptom expression. Interestingly, the content of a few specific metabolites exhibited opposite variations in leaves of symptomatic shoots of clones 76 and 95. Altogether this study showed a clone-dependent expression of Esca disease in ‘Chardonnay’ and the relevance of GC-MS and 3D fluorescence methods to analyze the impact of the disease on the leaf metabolome.

Protecting vineyards from cryptogamic diseases such as downy mildew, caused by Plasmopara viticola, generally requires a massive use of phytochemicals. However, the issues on unintentional secondary effects on environment and human health, and the occurrence of P. viticola resistant strains, are leading to the development of alternative strategies, such as the use of biocontrol products. In this paper, we evidenced the ability of a plant extract to protect grapevine from P. viticola. Further experiments carried out both on cell suspensions and on plants revealed that plant extract activates typical defense-related responses such as the production of H2O2, the up-regulation of genes encoding pathogenesis-related proteins and stilbene synthase, as well as the accumulation of resveratrol or its derivative piceid. We also brought to light a strong direct effect of PE on the release and motility of P. viticola zoospores. Furthermore, we found out that PE application left dried residues on leaf surface, impairing zoospores to reach stomata. Altogether, our results highlight the different modes of action of a new biocontrol product able to protect grapevine against downy mildew.

Mature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea, while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infection process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus spreading. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes upregulated during infection of MB are enriched in functional categories related to necrotrophy, such as degradation of the plant cell wall, proteolysis, membrane transport, reactive oxygen species (ROS) generation, and detoxification. Quantitative-polymerase chain reaction on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but is stopped at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathway switch during berry ripening. In response to B. cinerea inoculation, VB activated a burst of ROS, the salicylate-dependent defense pathway, the synthesis of the resveratrol phytoalexin, and cell-wall strengthening. On the contrary, in infected MB, the jasmonate-dependent pathway was activated, which did not stop the fungal necrotrophic process.

β-(1→3)-Glucans can be found as structural polysaccharides in cereals, in algae or as exo-polysaccharides secreted on the surfaces of mushrooms or fungi. Research has now established that β-(1→3)-glucans can trigger different immune responses and act as efficient immunostimulating agents. They constitute prevalent sources of carbons for microorganisms after subsequent recognition by digesting enzymes. Nevertheless, mechanisms associated with both roles are not yet clearly understood. This review focuses on the variety of elucidated molecular interactions that involve these natural or synthetic polysaccharides and their receptors, i.e., Dectin-1, CR3, glycolipids, langerin and carbohydrate-binding modules.

[This corrects the article DOI: 10.1371/journal.pone.0088145.].

The oomycete Plasmopara viticola is responsible for downy mildew, a severe grapevine disease. In infected grapevine leaves, we have observed an abnormal starch accumulation at the end of the dark period, suggesting modifications in starch metabolism. Therefore, several complementary approaches, including transcriptomic analyses, measurements of enzyme activities, and sugar quantification, were performed in order to investigate and to understand the effects of P. viticola infection on leaf starch and—to a larger extent—carbohydrate metabolism. Our results indicate that starch accumulation is associated with an increase in ADP-glucose pyrophosphorylase (AGPase) activity and modifications in the starch degradation pathway, especially an increased α-amylase activity. Together with these alterations in starch metabolism, we have observed an accumulation of hexoses, an increase in invertase activity, and a reduction of photosynthesis, indicating a source-to-sink transition in infected leaf tissue. Additionally, we have measured an accumulation of the disaccharide trehalose correlated to an increased trehalase gene expression and enzyme activity. Altogether, these results highlight a dramatic alteration of carbohydrate metabolism correlated with later stages of P. viticola development in leaves.

The molecular mechanisms underlying the process of priming are poorly understood. In the present study, we investigated the early signaling events triggered by β-aminobutyric acid (BABA), a well-known priming-mediated plant resistance inducer. Our results indicate that, in contrast to oligogalacturonides (OG), BABA does not elicit typical defense-related early signaling events nor defense-gene expression in grapevine. However, in OG-elicited cells pretreated with BABA, production of reactive oxygen species (ROS) and expression of the respiratory-burst oxidase homolog RbohD gene were primed. In response to the causal agent of downy mildew Plasmopara viticola, a stronger ROS production was specifically observed in BABA-treated leaves. This process was correlated with an increased resistance. The NADPH oxidase inhibitor diphenylene iodonium (DPI) abolished this primed ROS production and reduced the BABA-induced resistance (BABA-IR). These results suggest that priming of an NADPH oxidase–dependent ROS production contributes to BABA-IR in the Vitis-Plasmopara pathosystem.

Stomata, natural pores bordered by guard cells, regulate transpiration and gas exchanges between plant leaves and the atmosphere. These natural openings also constitute a way of penetration for microorganisms. In plants, the perception of potentially pathogenic microorganisms or elicitors of defense reactions induces a cascade of events, including H2O2 production, that allows the activation of defense genes, leading to defense reactions. Similar signaling events occur in guard cells in response to the perception of abscisic acid (ABA), leading to stomatal closure. Moreover, few elicitors were reported to induce stomatal closure in Arabidopsis and Vicia faba leaves. Because responses to ABA and elicitors share common signaling events, it led us to question whether stomatal movements and H2O2 production in guard cells could play a key role in elicitor-induced protection against pathogens that use stomata for infection. This study was performed using the grapevine–Plasmopara viticola pathosystem. Using epidermal peels, we showed that, as for ABA, the elicitor-induced stomatal closure is mediated by reactive oxygen species (ROS) production in guard cells. In plants, we observed that the protection against downy mildew induced by some elicitors is probably not due only to effects on stomatal movements or to a guard-cell-specific activation of ROS production.

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.