Esca is a destructive disease that affects vineyards leading to important losses in wine production. Information about the response of Vitis vinifera plants to this disease is scarce, particularly concerning changes in plant metabolism. In order to study the metabolic changes in Vitis plants affected by esca, leaves from both infected and non-affected cordons of V. vinifera cv. Alvarinho (collected in the Vinho Verde region, Portugal) were analysed. The metabolite composition of leaves from infected cordons with visible symptoms [diseased leaves (dl)] and from asymptomatic cordons [healthy leaves (hl)] was evaluated by 1D and 2D (1)H-nuclear magnetic resonance (NMR) spectroscopy. Principal component analysis (PCA) of the NMR spectra showed a clear separation between dl and hl leaves, indicating differential compound production due to the esca disease. NMR/PCA analysis allowed the identification of specific compounds characterizing each group, and the corresponding metabolic pathways are discussed. Altogether, the study revealed a significant increase of phenolic compounds in dl, compared with hl, accompanied by a decrease in carbohydrates, suggesting that dl are rerouting carbon and energy from primary to secondary metabolism. Other metabolic alterations detected comprised increased levels of methanol, alanine, and gamma-aminobutyric acid in dl, which might be the result of the activation of other defence mechanisms.

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Key message Migration ability of the PWN through wood branch tissues of adult Maritime pine trees significantly differed among Iberian provenances and this variation was related to differences in anatomical and chemical defensive traits. Abstract The pinewood nematode or pine wilt nematode (PWN; Bursaphelenchus xylophilus) is one of the most dangerous threats to European coniferous forests, especially for the susceptible Maritime pine (Pinus pinaster), a valuable forest resource in South Western Europe. The PWN is vectored by beetles of the genus Monochamus (Coleoptera, Cerambycidae) and once inoculated in healthy branches, it quickly migrates downward to the main trunk through the resin canal system. Therefore, the anatomy of the resin canal system may modulate its migration and proliferation rates. Using material from nine Maritime pine Iberian provenances established in a common garden trial, we investigated whether these provenances differed in their (1) resin canal anatomy, (2) concentration of chemical defences (non-volatile resin and total polyphenolics) in stems and (3) ability of the PWN to migrate through the pine woody tissues in 'in vitro' bioassays. Whether variation in anatomical and chemical defensive traits relates to differences in PWN migration across populations was also investigated. Significant intraspecific variation in anatomical and chemical defensive traits and in nematode migration rates through pine tissues was observed. Moreover, the variation in nematode migration rate among pine provenances was related to differences in both anatomical and chemical features. Overall, this study highlights the role of plant genetics in the development of defensive traits against this harmful coniferous pest. The observed intraspecific variation should be taken into account when considering breeding as a strategy to provide areas of high risk of PWN with resistant genetic material.

Esca is a complex grapevine trunk disease associated with fungal infection of the xylem. However, the inconstancy of external symptoms and the ability of esca-associated fungi to inhabit grapevines without causing apparent disease suggests that abiotic factors might be involved in the disease. Water stress has been proposed to be one of the factors influencing esca symptom manifestation but the specific role played by water stress on esca development is unknown. We conducted a proton nuclear magnetic resonance spectroscopy-based metabolomic study aiming at unveiling drought-induced modifications in xylem sap composition that could contribute to esca-related infection progression. Vitis vinifera 'Chardonnay' plants were inoculated with Phaeomoniella chlamydospora or Phaeoacremonium minimum and exposed to water stress. Using this approach, 28 metabolites were identified in xylem sap. The results show that water stress induces a concentration increase of most metabolites in xylem sap. An average increase >100% was found for asparagine, isoleucine, leucine, methionine, phenylalanine, proline, tyrosine, valine, sarcosine, and trigonelline. The increase of these compounds seems to be also modulated by fungal infection. This study offers further support to the putative role of drought in esca expression, and opens new avenues of research by extending the current knowledge about metabolites possibly involved in esca disease.

Pine wilt disease (PWD), recently introduced into Europe, is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus and is a devastating illness that affects mainly pine trees. It is known that the PWN is capable of infecting other conifers; however, there is currently no information on which other plant species may be susceptible to PWD. In this study, the potential susceptibility of two common species of European forests, Picea abies and Cupressus lusitanica, to PWN was assessed through the monitoring of visual external symptoms, dimension and localization of the nematode population in stems, quantification of total chlorophyll, total soluble phenolics and lignin, at 7, 14, 21 and 28 days after inoculation. The degree of susceptibility was established through the comparison of symptoms with Pinus pinaster, a well-known PWN host. Furthermore, the stem ultrastructure of P. abies, C. lusitanica and Pn. pinaster was analysed by scanning electron microscopy. The results suggest that P. abies and C. lusitanica are resistant to PWN, and that lignin biosynthesis in these species is affected at an early stage of the infestation. Nevertheless, P. abies seems to be a compatible host that could act as a repository for PWN.

Iron (Fe) deficiency is an important agricultural concern that leads to lower yields and crop quality. A better understanding of the condition at the metabolome level could contribute to the design of strategies to ameliorate Fe-deficiency problems. Fe-sufficient and Fe-deficient soybean leaf extracts and whole leaves were analyzed by liquid (1)H nuclear magnetic resonance (NMR) and high-resolution magic-angle spinning NMR spectroscopy, respectively. Overall, 30 compounds were measurable and identifiable (comprising amino and organic acids, fatty acids, carbohydrates, alcohols, polyphenols, and others), along with 22 additional spin systems (still unassigned). Thus, metabolite differences between treatment conditions could be evaluated for different compound families simultaneously. Statistically relevant metabolite changes upon Fe deficiency included higher levels of alanine, asparagine/aspartate, threonine, valine, GABA, acetate, choline, ethanolamine, hypoxanthine, trigonelline, and polyphenols and lower levels of citrate, malate, ethanol, methanol, chlorogenate, and 3-methyl-2-oxovalerate. The data indicate that the main metabolic impacts of Fe deficiency in soybean include enhanced tricarboxylic acid cycle activity, enhanced activation of oxidative stress protection mechanisms and enhanced amino acid accumulation. Metabolites showing accumulation differences in Fe-starved but visually asymptomatic leaves could serve as biomarkers for early detection of Fe-deficiency stress.

Cell suspension cultures of Vitis vinifera cv. Vinhão (Vv) were used to study the putative response of V. vinifera to Phaeomoniella chlamydospora (Pc), a fungus frequently associated with esca and grapevine decline. Cells were elicited with a Pc autoclaved biomass extract and methyl jasmonate (MeJ). Phenolic production was evaluated by HPLC-DAD and HPLC-MS/MS. Phenolic production of Vv cells significantly changes after elicitation. Compared to control, Vv cells elicited by Pc extract increase their stilbene production 20-fold and those elicited by MeJ increase stilbenic production 9-fold. In both cases, there is de novo production of viniferin type compounds. We also analyzed the oxidative burst of Vv cells after elicitation with Pc extract and MeJ, using the probe 2′,7′-dichlorodihydrofluorescein diacetate. Adding Pc extract induces an oxidative burst that shows a biphasic pattern in Vv cells. Moreover, the induction of 7 defence-related genes expression in Vv cell cultures upon Pc extract elicitation was investigated employing semi-quantitative RT-PCR. Elicitation increases the expression of class 6 and class 10 pathogenesis-related proteins, β-1,3-glucanase, class III chitinase, lipoxygenase, phenylalanine ammonia lyase and stilbene synthase. Therefore, Vv in vitro cell cultures could be an important tool to study esca disease, since they offer a simple, rapid and selective way to evaluate plant/fungus interactions.

Pine wilt disease, caused by the pinewood nematode Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, is originating severe infections in pine trees. The disease is detected when external symptoms appear (e.g. needle chlorosis), but trees could remain asymptomatic for long periods and serve as a long-term host. The primary goal of this study was to assess the effect of inoculation with an avirulent isolate of B. xylophilus (C14-5) on different Pinus spp. seedlings (P. sylvestris, P. nigra, P. pinea and P. pinaster). At the same time, seedlings were also inoculated with a virulent strain, HF, in order to compare the phenotypic and genomic results of the two types of inoculations. The effect of inoculation was determined in terms of expression of various Pinus genes potentially involved in the response to the disease.The results suggest that P. pinea and P. nigra are more resistant to infection by the nematode than P. sylvestris and P. pinaster. The phenotypic and genetic differences were more marked among P. pinea and P. pinaster.