Wood Metabolomic Responses of Wild and Cultivated Grapevine to Infection with Neofusicoccum parvum, a Trunk Disease Pathogen

Grapevine trunk diseases (GTDs), which are associated with complex of xylem-inhabiting fungi, represent one of the major threats to vineyard sustainability currently. Botryosphaeria dieback, one of the major GTDs, is associated with wood colonization by Botryosphaeriaceae fungi, especially Neofusicoccum parvum. We used GC-MS and HPLC-MS to compare the wood metabolomic responses of the susceptible Vitis vinifera subsp. vinifera (V. v. subsp. vinifera) and the tolerant Vitis vinifera subsp. sylvestris (V. v. subsp. sylvestris) after artificial inoculation with Neofusicoccum parvum (N. parvum). N. parvum inoculation triggered major changes in both primary and specialized metabolites in the wood. In both subspecies, infection resulted in a strong decrease in sugars (fructose, glucose, sucrose), whereas sugar alcohol content (mannitol and arabitol) was enhanced. Concerning amino acids, N. parvum early infection triggered a decrease in aspartic acid, serine, and asparagine, and a strong increase in alanine and β-alanine. A trend for more intense primary metabolism alteration was observed in V. v. subsp. sylvestris compared to V. v. subsp. vinifera. N. parvum infection also triggered major changes in stilbene and flavonoid compounds. The content in resveratrol and several resveratrol oligomers increased in the wood of both subspecies after infection. Interestingly, we found a higher induction of resveratrol oligomer (putative E-miyabenol C, vitisin C, hopeaphenol, ampelopsin C) contents after wood inoculation in V. v. subsp. sylvestris

Overexpression of the VvSWEET4 Transporter in Grapevine Hairy Roots Increases Sugar Transport and Contents and Enhances Resistance to Pythium irregulare, a Soilborne Pathogen

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Use of a RT-qPCR Method to Estimate Mycorrhization Intensity and Symbiosis Vitality in Grapevine Plants Inoculated with <i>Rhizophagus irregularis</i>

Assessing the mycorrhization level in plant roots is essential to study the effect of arbuscular mycorrhizal fungi (AMF) on plant physiological responses. Common methods used to quantify the mycorrhization of roots are based on microscopic visualization of stained fungal structures within the cortical cells. While this method is readily accessible, it remains time-consuming and does not allow checking of the symbiosis vitality. The aim of this work is thus to develop an efficient method for assessing the intensity and vitality of mycorrhiza associated with grapevine through gene expression analyses by RT-qPCR. To this end, grapevine plants were inoculated with the AMF Rhizophagus irregularis (Ri). The relationship between mycorrhization level, assessed by microscopy, and expression of several fungus and grapevine genes involved in the symbiosis was investigated. In AMF-inoculated plants, transcript amounts of fungal constitutively-expressed genes Ri18S, RiTEF1α and RiαTub were significantly correlated to mycorrhization intensity, particularly Ri18S. Grapevine (VvPht1.1 and VvPht1.2) and AMF (GintPT, Ri14-3-3 and RiCRN1) genes, known to be specifically expressed during the mycorrhizal process, were significantly correlated to arbuscular level in the whole root system determined by microscopy. The best correlations were obtained with GintPT on the fungal side and VvPht1.2 on the plant side. Despite some minor discrepancies between microscopic and molecular techniques, the monitoring of Ri18S, GintPT and VvPht1.2 gene expression could be a rapid, robust and reliable method to evaluate the level of mycorrhization and to assess the vitality of AMF. It appears particularly useful to identify AMF-inoculated plants with very low colonization level, or with non-active fungal structures. Moreover, it can be implemented simultaneously with the expression analysis of other genes of interest, saving time compared to microscopic analyses

Protoplasma

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