Sibylle Farine

Esca et Black Dead Arm : deux acteurs majeurs des maladies du bois chez la Vigne

Grapevine trunk diseases are very harmful to wine-growing heritage durability because the fungi responsible, by attacking perennial organs, cause at a more or less long-term the death of the vine stock. Esca and BDA are the two main pathogens inducing such decaying diseases. The infection can be diagnosed by the presence in the wood forming tissues of sectorial and/or central necrosis, which revealed itself by brown stripes or canker, and at the foliar level by discoloration and withering. This review presents an overview of both Esca and BDA, their symptomatology, the characteristics of the associated fungi and host-pathogen interactions. To conclude, a progress report on the control of both diseases is discussed.

Vitis vinifera VvNPR1.1 is the functional ortholog of AtNPR1 and its overexpression in grapevine triggers constitutive activation of PR genes and enhanced resistance to powdery mildew

Studying grapevine (Vitis vinifera) innate defense mechanisms is a prerequisite to the development of new protection strategies, based on the stimulation of plant signaling pathways to trigger pathogen resistance. Two transcriptional coactivators (VvNPR1.1 and VvNPR1.2) with similarity to Arabidopsis thaliana NPR1 (Non-Expressor of PR genes 1), a well-characterized and key signaling element of the salicylic acid (SA) pathway, were recently isolated in Vitis vinifera. In this study, functional characterization of VvNPR1.1 and VvNPR1.2, including complementation of the Arabidopsis npr1 mutant, revealed that VvNPR1.1 is a functional ortholog of AtNPR1, whereas VvNPR1.2 likely has a different function. Ectopic overexpression of VvNPR1.1 in the Arabidopsis npr1-2 mutant restored plant growth at a high SA concentration, Pathogenesis Related 1 (PR1) gene expression after treatment with SA or bacterial inoculation, and resistance to virulent Pseudomonas syringae pv. maculicola bacteria. Moreover, stable overexpression of VvNPR1.1-GFP in V. vinifera resulted in constitutive nuclear localization of the fusion protein and enhanced PR gene expression in uninfected plants. Furthermore, grapevine plants overexpressing VvNPR1.1-GFP exhibited an enhanced resistance to powdery mildew infection. This work highlights the importance of the conserved SA/NPR1 signaling pathway for resistance to biotrophic pathogens in V. vinifera.

Phytotoxic metabolites from Neofusicoccum parvum, a pathogen of Botryosphaeria dieback of grapevine.

Liquid chromatography-diode array screening of the organic extract of the cultures of 13 isolates of the fungus Neofusicoccum parvum, the main causal agent of botryosphaeria dieback of grapevine, showed similar metabolites. One strain was selected for further chemical studies and led to the isolation and characterisation of 13 metabolites. Structures were elucidated through spectroscopic analyses, including one- and two-dimensional NMR and mass spectrometry, and through comparison to literature data. The isolated compounds belong to four different chemical families: five metabolites, namely, (-)-terremutin (1), (+)-terremutin hydrate (2), (+)-epi-sphaeropsidone (3) (-)-4-chloro-terremutin hydrate (4) and(+)-4-hydroxysuccinate-terremutin hydrate (5), belong to the family of dihydrotoluquinones; two metabolites, namely, (6S,7R) asperlin (6) and (6R,7S)-dia-asperlin (7), belong to the family of epoxylactones; four metabolites, namely, (R)-(-)-mellein (8), (3R,4R)-4-hydroxymellein (9), (3R,4S)-4-hydroxymellein (10) (R)(-)-3-hydroxymellein (11), belong to the family of dihydroisocoumarins; and two of the metabolites, namely, 6-methyl-salicylic acid (12) and 2-hydroxypropyl salicylic acid (13), belong to the family of hydroxybenzoic acids. We determined the phytotoxic activity of the isolated metabolites through a leaf disc assay and the expression of defence-related genes in Vitis vinifera cells cv. Chardonnay cultured with (-)-terremutin (1), the most abundant metabolite. Finally, analysis of the brown stripes of grapevine wood from plants showing botryosphaeria dieback symptoms revealed the presence of two of the isolated phytotoxins.

Genetic chimerism of Vitis vinifera cv. Chardonnay 96 is maintained through organogenesis but not somatic embryogenesis

BackgroundGrapevine can be a periclinal chimera plant which is composed at least of two distinct cell layers (L1, L2). When the cell layers of this plant are separated by passage through somatic embryogenesis, regenerated plants could show distinct DNA profiles and a novel phenotype which proved different from that of the parent plant.ResultsGenetically Chardonnay clone 96 is a periclinal chimera plant in which is L1 and L2 cell layers are distinct. Plants obtained via organogenesis through meristematic bulks are shown to be composed of both cell layers. However, plants regenerated through somatic embryogenesis starting from anthers or nodal explants are composed only of L1 cells. These somaclones do not show phenotypic differences to the parental clone up to three years after regeneration. Interestingly, the only somaclone showing an atypical phenotype (asymmetric leave) shows a genotypic modification.ConclusionThese results suggest that the phenotype of Chardonnay 96 does not result from an interaction between the two distinct cell layers L1 and L2. If phenotype conformity is further confirmed, somatic embryogenesis will result in true-to-type somaclones of Chardonnay 96 and would be well suitable for gene transfer.

Extracellular compounds produced by fungi associated with Botryosphaeria dieback induce differential defence gene expression patterns and necrosis in Vitis vinifera cv. Chardonnay cells

Three major grapevine trunk diseases, esca, botryosphaeria dieback and eutypa dieback, pose important economic problems for vineyards worldwide, and currently, no efficient treatment is available to control these diseases. The different fungi associated with grapevine trunk diseases can be isolated in the necrotic wood, but not in the symptomatic leaves. Other factors seem to be responsible for the foliar symptoms and may represent the link between wood and foliar symptoms. One hypothesis is that the extracellular compounds produced by the fungi associated with grapevine trunk diseases are responsible for pathogenicity.In the present work, we used Vitis vinifera cv. Chardonnay cells to test the aggressiveness of total extracellular compounds produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with botryosphaeria dieback. Additionally, the toxicity of purified mellein, a characteristic toxin present in the extracellular compounds of Botryosphaeriaceae, was assessed.Our results show that the total extracellular compounds produced by N. parvum induce more necrosis on Chardonnay calli and induce a different defence gene expression pattern than those of D. seriata. Mellein was produced by both fungi in amounts proportional to its aggressiveness. However, when purified mellein was added to the culture medium of calli, only a delayed necrosis and a lower-level expression of defence genes were observed. Extracellular compounds seem to be involved in the pathogenicity of the fungi associated with botryosphaeria dieback. However, the doses of mellein used in this study are 100 times higher than those found in the liquid fungal cultures: therefore, the possible function of this toxin is discussed.

Toxicity of extracellular proteins from Diplodia seriata and Neofusicoccum parvum involved in grapevine Botryosphaeria dieback

Botryosphaeria dieback, esca and Eutypa dieback are three economic major grapevine trunk diseases that cause severe yield reduction in vineyards worldwide. The frequency of disease symptoms has increased considerably over the past decade, and no efficient treatment is currently available to control these diseases. The different fungi associated with grapevine trunk diseases mainly induce necrotic wood and characteristic foliar symptoms. In this context, fungi virulence factors and host invasion are not well understood. We hypothesise that extracellular proteins produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with Botryosphaeria dieback, are virulence factors responsible for the pathogenicity. In our previous work, we demonstrated that the total extracellular compounds produced by N. parvum induced more necrosis on Chardonnay calli and triggered a different defence gene expression pattern than those produced by D. seriata. Furthermore, this aggressiveness was not clearly correlated with the production of mellein, a characteristic phytotoxin of Botryosphaeriaceae, in our in vitro calli model. To characterise other potential virulence factors and to understand the mechanisms of host invasion by the fungus, we evaluated the profile, quantity and the impact of extracellular proteins produced by these fungi on Vitis vinifera calli necrosis and defence gene expression. Our results reveal that, under the same conditions, N. parvum produces more extracellular proteins and in higher concentrations than D. seriata. With Vitis vinifera cv. Chardonnay cells, we showed that equivalent concentrations of proteins secreted by N. parvum were more aggressive than those of D. seriata in producing necrosis and that they clearly induced more grapevine defence genes.

Impact of Quillaja saponaria saponins on grapevine ecosystem organisms

The control of grapevine pathogens is a rising concern in Vitis vinifera culture. The current international trend is toward banning chemicals that are highly toxic to the environment and human workers, and adopting tighter regulations. We evaluated the impact of saponins on three kinds of organisms found in grapevine culture. The ectoparasitic nematode Xiphinema index, the parasitic fungus Botrytis cinerea and various yeast strains representative of the must fermentation population were incubated on synthetic media supplemented with variable concentrations of Quillaja saponaria saponins. Saponins induced reduction in the growth of B. cinerea and showed nematicide effects on X. index. The control of X. index and Botrytis cinerea is discussed in the context of the potential use of these chemicals as environmentally-friendly grapevine treatments. With Saccharomyces cerevisiae and other yeasts, saponins showed higher toxicity against S. cerevisiae strains isolated from wine or palm wine whereas laboratory strains or strains isolated from oak exhibited better resistance. This indicates that Q. saponaria saponins effects against yeast microflora should be assessed in the field before they can be considered an environmentally-safe new molecule against B. cinerea and X. index.

Secreted proteins produced by fungi associated with Botryosphaeria dieback trigger distinct defense responses in Vitis vinifera and Vitis rupestris cells

Grapevine trunk diseases (Eutypa dieback, esca and Botryosphaeria dieback) are caused by a complex of xylem-inhabiting fungi, which severely reduce yields in vineyards. Botryosphaeria dieback is associated with Botryosphaeriaceae. In order to develop effective strategies against Botryosphaeria dieback, we investigated the molecular basis of grapevine interactions with a virulent species, Neofusicoccum parvum, and a weak pathogen, Diplodia seriata. We investigated defenses induced by purified secreted fungal proteins within suspension cells of Vitis (Vitis rupestris and Vitis vinifera cv. Gewurztraminer) with putative different susceptibility to Botryosphaeria dieback. Our results show that Vitis cells are able to detect secreted proteins produced by Botryosphaeriaceae, resulting in a rapid alkalinization of the extracellular medium and the production of reactive oxygen species. Concerning early defense responses, N. parvum proteins induced a more intense response compared to D. seriata. Early and late defense responses, i.e., extracellular medium alkalinization, cell death, and expression of PR defense genes were stronger in V. rupestris compared to V. vinifera, except for stilbene production. Secreted Botryosphaeriaceae proteins triggered a high accumulation of δ-viniferin in V. vinifera suspension cells. Artificial inoculation assays on detached canes with N. parvum and D. seriata showed that the development of necrosis is reduced in V. rupestris compared to V. vinifera cv. Gewurztraminer. This may be related to a more efficient induction of defense responses in V. rupestris, although not sufficient to completely inhibit fungal colonization. Overall, our work shows a specific signature of defense responses depending on the grapevine genotype and the fungal species.