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

Esca disease, a major threat to global viticulture, involves complex interactions between Vitis vinifera and pathogenic fungi of the trunk microbiome. Despite its economic impact, the biochemical mechanisms underlying plant defense remain poorly elucidated, partly due to limitations in analytical coverage of the plant metabolome. Here, we present a multiplexed metabolomics and lipidomics strategy combining biphasic extraction and complementary LC-MS methods (reverse-phase (RP), HILIC, and lipidomics-oriented RP) to profile grapevine wood in response to fungal infection and biocontrol treatment. This integrative workflow identified 1,425 unique features and unveiled a comprehensive landscape of grapevine chemical responses. Multi-omics data integration using sPLS-DA and DIABLO highlighted lipid metabolism, particularly the production of oxidized fatty acids (oxylipins), as a key component of defense signaling. Hydroxyeicosatetraenoic acids (HETEs) such as 13-HETE, 16(R)-HETE, and 11(R)-HETE emerged as putative biomarkers modulated by fungal pathogens and the biocontrol agent Trichoderma Vintec®. Concurrently, changes in phenylpropanoid, terpenoid, and alkaloid pathways were detected, underscoring the role of diverse metabolite classes in grapevine immunity. Our study establishes a new analytical framework for comprehensive metabolome coverage in plants and reveals the involvement of oxylipin-mediated signaling in grapevine defense against trunk diseases. These findings open avenues for the development of metabolite-based biomarkers and biocontrol strategies in viticulture.