Vitis Methods to Understand and Develop Strategies for Diagnosis and Sustainable Control of Grapevine Trunk Diseases.

Vitis vinifera is affected by many diseases every year, depending on causal agents, susceptibility of cultivars, and climate region. Some are caused by a single agent, such as gray mold caused by Botrytis cinerea or powdery mildew caused by Erysiphe necator. Others result from the actions of a complex of pathogens such as grapevine trunk diseases (GTDs). GTDs are presently among the most devastating diseases in viticulture worldwide because both the economic losses and the long-term sustainability of vineyards are strongly affected. The complexity of GTDs results from the diversity of associated fungi, the undetermined period of latency within the vine (asymptomatic status), the erratic foliar symptom expression from one year to the next, and, probably correlated with all of these points, the lack of efficient strategies to control them. Distinct methods can be beneficial to improve our knowledge of GTDs. In vitro bioassays with cell suspensions, calli, foliar discs, full leaves, or plantlets, and in vivo natural bioassays with cuttings, grafted plants in the greenhouse, or artificially infected ones in the vineyard, can be applied by using progressive integrative levels of in vitro and in vivo, depending on the information searched. In this review, the methods available to understand GTDs are described in terms of experimental procedures, main obtained results, and deliverable prospects. The advantages and disadvantages of each model are also discussed.

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.

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.

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.

Separation and identification of enzymatic sucrose hydrolysis products by high-performance anion-exchange chromatography with pulsed amperometric detection.

An accurate carbohydrate analysis method, namely high-performance anion-exchange chromatography with pulsed amperometric detection was successfully applied to the study of sucrose hydrolysis under enzymatic (baker's yeast invertase) conditions. The hydrolysis was monitored by determining sucrose degradation and the corresponding formation of D-glucose, D-fructose and five intermediate fructans using a CarboPac PA-100 (Dionex) analytical anion-exchange column. Highly reproducible results were obtained. The unknown fructans were collected from a semi-preparative CarboPac PA-100 (Dionex) column, neutralized and then desalted on a column containing mixed bed resin AG 501-X8 (D) before identification of the chemical structure. This procedure permitted us to obtain about 20 microg of pure product which is not enough for NMR analysis. Detailed GC-MS analytical data of the methylated compounds indicated that these oligosaccharides were beta-D-Fru-(2 --> 1)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (1-kestose), beta-D-Fru-(2 --> 6)-alpha-D-glucopyranoside (6-beta fructofuranosylglucose), beta-D-Fru-(2 --> 1)-beta-D-fructofuranoside (inulobiose), beta-D-Fru-(2 --> 6)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (6-kestose) and beta-D-Fru-(2 --> 6)-alpha-D-Glc-(1 --> 2)-beta-D-fructofuranoside (neokestose) coeluating with a disaccharide.

Application of high performance anion exchange chromatography to study invertase-catalysed hydrolysis of sucrose and formation of intermediate fructan products.

Baker's yeast invertase was found to catalyse transfructosylation reactions in aqueous and anhydrous organic media with sucrose as a substrate, leading to the formation of five intermediate fructans in addition to the release of D-glucose (D-Glc)and D-fructose (D-Fru). All the reaction products were separated and quantitatively estimated using high performance anion exchange-pulsed amperometric detection equipment. The unknown products were subsequently identified by linkage analysis as beta-D-Fru-(2 --> 1)-beta-D-Fru-(2 --> 1)- alpha-D-glucopyranoside (1-kestose), beta-D-Fru- (2 --> 6)-alpha-D-glucopyranoside (6-beta-fructofuranosylglucose), beta-D-Fru-(2 -->1) -beta-D-fructofuranoside (inulobiose), beta-D-Fru-(2 --> 6)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (6-kestose) and beta-D-Fru-(2 --> 6)-alpha-D-Glc-(1 --> 2)-beta-D-fructofuranoside (neokestose); and this last was eluted together with a disaccharide. The time-course of sucrose hydrolysis via fructan production in 2 ml of a 50 mM sodium acetate buffer (pH 4.5) containing 0.2 M sucrose and 25 U of invertase was different from that in 2 ml of anhydrous toluene with 1.46 M sucrose and 1,000 U of invertase as a suspended powder. Under the latter experimental conditions, invertase was found to exhibit cyclic behaviour, where sucrose was degraded and subsequently synthesised. This observation has not yet been reported, as far as we know.

Comparative quantitative analysis of sucrose and related compounds using ion exchange and reverse phase chromatographic methods.

Two analytical methods of sugar determination, namely ion exchange chromatography on an anionic resin coupled with electrochemical detection, and reverse phase chromatography on Nucleosil-NH2 resin equipped with a light scattering detector were tested and compared as regards their rapidity, sensitivity and accuracy with sucrose, fructose, glucose, raffinose, maltose, arabinose, fucose, rhamnose and xylose. Excellent resolution and highly reproducible results were obtained in both cases. Greater sensitivity up to the picomolar range was possible however only with ion exchange chromatography. Reverse phase chromatography was successfully applied to the time course of sucrose hydrolysis under chemical (acid) and enzymatic (invertase) conditions. The hydrolysis was monitored by determining sucrose degradation and the corresponding formation of glucose and fructose.