Multiplexed LC-MS analysis reveals novel insights into grapevine defense mechanisms by expanding metabolome coverage

Abstract Introduction Grapevine trunk diseases (GTDs), such as esca, pose a major threat to viticulture worldwide and are associated with complex biochemical responses in woody tissues. Comprehensive metabolome coverage remains a challenge, as conventional methods often overlook non-polar metabolites critical to plant defense mechanisms. Objectives This study aimed to expand metabolome and lipidome coverage of grapevine wood by integrating complementary LC-MS approaches, in order to identify metabolic signatures linked to pathogenic fungi and to a biocontrol agent. Methods Woody tissues of Vitis vinifera cv. Cabernet-Sauvignon were inoculated with Phaeomoniella chlamydospora , Phaeoacremonium minimum , and/or the biocontrol fungus Trichoderma atroviride (Vintec ® ). A biphasic extraction was coupled with three orthogonal LC-MS methods—reverse-phase (RP), hydrophilic interaction chromatography (HILIC), and lipidomics-focused RP. Data were processed through the MSCleanR workflow and integrated using the DIABLO multi-block statistical framework. Compound classification was performed with NPClassifier. Results The multiplexed strategy enabled the annotation of 1,425 unique features, representing an 83% increase compared to previous studies. Distinct metabolomic and lipidomic signatures were associated with fungal infection and biocontrol treatments. Lipidomic analysis highlighted oxidized fatty acids (oxylipins) —specifically hydroxy-eicosatetraenoic acids (13-HETE, 16(R)-HETE, and 11(R)-HETE)—as potential signaling molecules in defense responses. NPClassifier revealed diverse biosynthetic classes, including phenylpropanoids, terpenoids, and sphingolipids, underscoring the chemical heterogeneity of grapevine responses. Conclusion This multiplexed LC-MS workflow provides a versatile analytical pipeline for untargeted metabolomics and lipidomics in plants. By integrating complementary methods, the study uncovered novel biomarkers of grapevine defense, particularly oxylipins, emphasizing the critical role of lipidomics in deciphering plant–pathogen interactions.