Physiological and developmental disturbances caused by Botryosphaeria dieback in the annual stems of grapevine.

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.

Comparative RNA sequencing-based transcriptome profiling of ten grapevine rootstocks: shared and specific sets of genes respond to mycorrhizal symbiosis.

Arbuscular mycorrhizal symbiosis improves water and nutrient uptake by plants and provides them other ecosystem services. Grapevine is one of the major crops in the world. Vitis vinifera scions generally are grafted onto a variety of rootstocks that confer different levels of resistance against different pests, tolerance to environmental stress, and influence the physiology of the scions. Arbuscular mycorrhizal fungi are involved in the root architecture and in the immune response to soil-borne pathogens. However, the fine-tuned regulation and the transcriptomic plasticity of rootstocks in response to mycorrhization are still unknown. We compared the responses of 10 different grapevine rootstocks to arbuscular mycorrhizal symbiosis (AMS) formed with Rhizophagus irregularis DAOM197198 using RNA sequencing-based transcriptome profiling. We have highlighted a few shared regulation mechanisms, but also specific rootstock responses to R. irregularis colonization. A set of 353 genes was regulated by AMS in all ten rootstocks. We also compared the expression level of this set of genes to more than 2000 transcriptome profiles from various grapevine varieties and tissues to identify a class of transcripts related to mycorrhizal associations in these 10 rootstocks. Then, we compared the response of the 351 genes upregulated by mycorrhiza in grapevine to their Medicago truncatula homologs in response to mycorrhizal colonization based on available transcriptomic studies. More than 97% of the 351 M. truncatula-homologous grapevine genes were expressed in at least one mycorrhizal transcriptomic study, and 64% in every single RNAseq dataset. At the intra-specific level, we described, for the first time, shared and specific grapevine rootstock genes in response to R. irregularis symbiosis. At the inter-specific level, we defined a shared subset of mycorrhiza-responsive genes.

Sodium arsenite-induced changes in the wood of esca-diseased grapevine at cytological and metabolomic levels.

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.

Biostimulation can prime elicitor induced resistance of grapevine leaves to downy mildew.

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 microbiota of the grapevine holobiont: A key component of plant health.

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 situ Phenotyping of Grapevine Root System Architecture by 2D or 3D Imaging: Advantages and Limits of Three Cultivation Methods.

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-affected grapevine leaf metabolome is clone- and vintage-dependent.

Esca is a complex grapevine trunk disease caused by wood-rotting ascomycetes and basidiomycetes and leading to several foliar and wood symptoms. Given that the esca expression can be influenced by several environmental, physiological, and genetic factors, foliar symptoms are inconsistent in incidence and prevalence and may appear 1 year but not the following. We have previously reported a clone-dependent expression of the disease in cv Chardonnay. Owing to metabolome analysis, we could discriminate the metabolite fingerprint of green leaves collected on diseased vines of clones 76 and 95. These clone-dependent fingerprints were year-dependent in intensity and nature. The present work was conducted to determine if the clone-dependent disease expression observed is specific to Chardonnay or if it also occurs in another cultivar. A plot located in the Jura vineyard (France) and planted with both 1004 and 1026 clones of Trousseau, a cultivar highly susceptible to esca, was thus selected and studied during 2017 and 2018. A year-dependent variation of the symptoms expression was first observed and a possible relationship with rainfall is hypothesized and discussed. Moreover, a higher percentage of the clone 1026 vines expressed disease, compared to the 1004 ones, suggesting the higher susceptibility of this clone. Finally, metabolomic analyses of the remaining green leaves (i.e, without symptom expression) of partial esca-apoplectic vines allowed us to confirm a clone-dependent metabolic response to the disease. The metabolite fingerprints obtained differed in nature and intensity to those previously reported for Chardonnay and also between years.

Cultivar- and Wood Area-Dependent Metabolomic Fingerprints of Grapevine Infected by Botryosphaeria Dieback.

Botryosphaeria dieback is one of the most significant grapevine trunk diseases that affects the sustainability of the vineyards and provokes economic losses. The causal agents, Botryosphaeriaceae species, live in and colonize the wood of the perennial organs causing wood necrosis. Diseased vines show foliar symptoms, chlorosis, or apoplexy, associated to a characteristic brown stripe under the bark. According to the susceptibility of the cultivars, specific proteins such as PR-proteins and other defense-related proteins are accumulated in the brown stripe compared with the healthy woody tissues. In this study, we enhanced the characterization of the brown stripe and the healthy wood by obtaining a metabolite profiling for the three cultivars Chardonnay, Gewurztraminer, and Mourvèdre to deeper understand the interaction between the Botryosphaeria dieback pathogens and grapevine. The study confirmed a specific pattern according to the cultivar and revealed significant differences between the brown stripe and the healthy wood, especially for phytochemical and lipid compounds. This is the first time that such chemical discrimination was made and that lipids were so remarkably highlighted in the interaction of Botryosphaeriaceae species and grapevine. Their role in the disease development is discussed.

Woody Plant Declines. What's Wrong with the Microbiome?

Woody plant (WP) declines have multifactorial determinants as well as a biological and economic reality. The vascular system of WPs involved in the transport of carbon, nitrogen, and water from sources to sinks has a seasonal activity, which places it at a central position for mediating plant-environment interactions from nutrient cycling to community assembly and for regulating a variety of processes. To limit effects and to fight against declines, we propose: (i) to consider the WP and its associated microbiota as an holobiont and as a set of functions; (ii) to consider simultaneously, without looking at what comes first, the physiological or pathogenic disorders; and (iii) to define pragmatic strategies, including preventive and curative agronomical practices based on microbiota engineering.

Recognition of Elicitors in Grapevine: From MAMP and DAMP Perception to Induced Resistance.

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.

Hydrophobized laminarans as new biocompatible anti-oomycete compounds for grapevine protection.

Laminaran, a ß-(1→3)-glucan extracted from Laminaria digitata, is a known elicitor of plant defenses, but provides only low level of disease control in vineyard trials. In this context, laminaran was partly hydrophobized by grafting from 1.6 to 7.6 lauryl chains to the native saccharidic chain and the impact of sulfation of the hydrophobized glucans was studied. The activity of the different synthetized laminaran derivatives as antimicrobial agents against Plasmopara viticola, the causal agent of grape downy mildew, and as elicitors of defense reactions in planta, was evaluated. Our results showed that acylation imparts an antimicrobial activity to laminaran which is related to the degree of acylation, AL3, with 7.6 lauryl chains, being the most effective derivative. Sulfation of the acylated laminarans did not further increase the antimicrobial activity. Our results also demonstrated that the efficacy of AL3 against Plasmopara viticola was most likely due to the direct antimicrobial activity of the lauryl chains rather than to an elicitation of plant defenses.

Surfactin and fengycin contribute to the protection of a Bacillus subtilis strain against grape downy mildew by both direct effect and defence stimulation.

Bacillus subtilis GLB191 (hereafter GLB191) is an efficient biological control agent against the biotrophic oomycete Plasmopara viticola, the causal agent of grapevine downy mildew. In this study, we show that GLB191 supernatant is also highly active against downy mildew and that the activity results from both direct effect against the pathogen and stimulation of the plant defences (induction of defence gene expression and callose production). High-performance thin-layer chromatography analysis revealed the presence of the cyclic lipopeptides fengycin and surfactin in the supernatant. Mutants affected in the production of fengycin and/or surfactin were thus obtained and allowed us to show that both surfactin and fengycin contribute to the double activity of GLB191 supernatant against downy mildew. Altogether, this study suggests that GLB191 supernatant could be used as a new biocontrol product against grapevine downy mildew.

Assessment of the impact of PS3-induced resistance to downy mildew on grapevine physiology.

Elicitor-induced resistance against diseases is an attractive strategy that could contribute to reduce the use of fungicides for plant protection. However, activation of defenses has an energetic cost that plants have to fuel by a mobilization of their primary metabolism with possible adverse effect on their physiology. In this context, this study was performed to determine whether elicitor-induced resistance of grapevine leaves against downy mildew impacted its development and metabolism. The elicitor PS3 (sulfated ß-glucan laminarin) was sprayed on grapevine herbaceous cuttings grown in greenhouses once or three times, and its impact was studied on young and older grapevine leaves, prior to, and after Plasmopara viticola inoculation. PS3 did not affect grapevine development during the time course of the experiment. A metabolomic analysis, mainly focused on primary metabolites, highlighted a leaf age dependent effect of PS3 treatment. Nitrogen compounds, and sugars to a lesser extent, were impacted. The results obtained complete the current knowledge of the impact of elicitor-induced resistance on plant physiology. They will be helpful to guide further experiments required to better determine the costs and benefits of elicitor-induced resistance in plants.

A Plant Extract Acts Both as a Resistance Inducer and an Oomycide Against Grapevine Downy Mildew.

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.

Correction: The Sulfated Laminarin Triggers a Stress Transcriptome before Priming the SA- and ROS-Dependent Defenses during Grapevine's Induced Resistance against Plasmopara viticola.

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

Combined enzymatic and metabolic analysis of grapevine cell responses to elicitors.

Elicitors trigger plant defense responses, including phytoalexin production and cell-wall reinforcement. Primary metabolism plays an important role in these responses as it fuels the associated energetic costs and provides precursors for the synthesis of the numerous secondary metabolites involved in defenses against pathogens. In this context, we aimed to determine whether oligosaccharidic elicitors differing in their capacity to activate defense-associated secondary metabolism in grapevine would differently impact primary metabolism. To answer this question, cell suspensions were treated with two elicitors: an oligogalacturonide, and the ß-glucan laminarin. Enzymatic activity assays together with targeted (HPLC) and global (GC-MS) analyses of metabolites were next performed to compare their impact on plant primary or secondary metabolism. The results showed that the oligogalacturonide, which induced the highest level of the phytoalexin resveratrol and the highest activity of stilbene synthase, also induced the highest activity of shikimate hydroxycinnamoyltransferase, a key enzyme involved in the synthesis of lignin. The oligogalacturonide-induced defenses had a significant impact on primary metabolism 24 h following elicitor treatment, with a reduced abundance of pyruvate and 2-oxoglutarate, together with an increase of a set of metabolites including carbohydrates and amino acids. Interestingly, an accumulation of galacturonate and gentiobiose was observed in the oligogalacturonide- and laminarin-treated cells, respectively, suggesting that both elicitors are rapidly hydrolyzed in grapevine cell suspension cultures.

Clone-Dependent Expression of Esca Disease Revealed by Leaf Metabolite Analysis.

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.

Alterations in Grapevine Leaf Metabolism Occur Prior to Esca Apoplexy Appearance.

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.

Metabolic Fingerprint of PS3-Induced Resistance of Grapevine Leaves against Plasmopara viticola Revealed Differences in Elicitor-Triggered Defenses.

Induction of plant resistance against pathogens by defense elicitors constitutes an attractive strategy to reduce the use of fungicides in crop protection. However, all elicitors do not systematically confer protection against pathogens. Elicitor-induced resistance (IR) thus merits to be further characterized in order to understand what makes an elicitor efficient. In this study, the oligosaccharidic defense elicitors H13 and PS3, respectively, ineffective and effective to trigger resistance of grapevine leaves against downy mildew, were used to compare their effect on the global leaf metabolism. Ultra high resolution mass spectrometry (FT-ICR-MS) analysis allowed us to obtain and compare the specific metabolic fingerprint induced by each elicitor and to characterize the associated metabolic pathways. Moreover, erythritol phosphate was identified as a putative marker of elicitor-IR.

Proteomics towards the understanding of elicitor induced resistance of grapevine against downy mildew.

Elicitors are known to trigger plant defenses in response to biotic stress, but do not systematically lead to effective resistance to pathogens. The reasons explaining such differences remain misunderstood. Therefore, elicitation and induced resistance (IR) were investigated through the comparison of two modified ß-1,3 glucans applied on grapevine (Vitis vinifera) leaves before and after inoculation with Plasmopara viticola, the causal agent of downy mildew. The sulfated (PS3) and the shortened (H13) forms of laminarin are both known to elicit defense responses whereas only PS3 induces resistance against downy mildew. The analysis of the 2-DE gel electrophoresis revealed that PS3 and H13 induced distinct proteomic profiles after treatment and pathogen inoculation. Our results point out that the PS3-induced resistance is associated with the activation of the primary metabolism especially on amino acids and carbohydrates pathways. In addition, few proteins, such as the 12-oxophytodienoate reductase (OPR-like) related to the OPDA pathway, and an Arsenite-resistance protein (Serrate-like protein) could be considered as useful markers of induced resistance. SIGNIFICANCE: One strategy to reduce the application of fungicides is the use of elicitors which induce plant defense responses. Nonetheless, the elicitors do not systematically lead to resistance against pathogens. The lack of correlation between plant defense activation and induced resistance (IR) requires the investigation of what makes the specificity of elicitor-IR. In this study, the two ß-glucans elicitors, sulfated (PS3) and short (H13) laminarins, were used in the grapevine/Plasmopara viticola interaction since only the first one leads to resistance against downy mildew. To disclose IR specificity, proteomic approach has been employed to compare the two treatments before and after P. viticola inoculation. The analysis of the 2-DE revealed that PS3 and H13 induced distinct proteomic profiles after treatment and pathogen inoculation. Significant increase of the number of proteins regulated by PS3, relative to both H13 and time-points, is correlated with the resistance process establishment. Our results point that the PS3-induced resistance requires the activation of the primary metabolism especially on amino acids and carbohydrates pathways. In addition, few proteins, such as the 12-oxophytodienoate reductase (OPR-like) related to the OPDA pathway, and an Arsenite-resistance protein (Serrate-like protein) could constitute useful markers of PS3 induced resistance.