Development of an extraction protocol and untargeted UHPLC-HRMS Orbitrap method for uncovering the polar metabolome of olive xylem tissue

Xylella fastidiosa subsp. pauca (Xfp) is a xylem-limited phytopathogenic bacterium implicated in Olive Quick Decline Syndrome, which has severely affected olive orchards in Apulia (southern Italy) over the past decade. The significant economic and ecological consequences of this disease, along with the absence of effective treatments, highlight the need for in-depth investigation of the molecular and biochemical bases of host-pathogen interaction mechanisms, to develop containment strategies. While most metabolomic studies on the Xfp-olive pathosystem have focused on leaves, metabolic changes within the xylem vessels, the primary site of bacterial colonization, remain largely unexplored during disease progression. In this study, we tried different extraction protocols suitable for olive woody tissue, coupled with an untargeted UHPLC-HRMS Orbitrap metabolomics approach to directly characterize the metabolome within xylem vessels. For method development, different experimental parameters were assessed, including sample matrix selection, pretreatment and storage conditions, and extraction solvent choice. The resulting data were initially assessed qualitatively for extraction efficiency, repeatability, and chromatographic quality. Subsequently, univariate and multivariate statistical analyses were performed using Compound Discoverer™ (Thermo Scientific™) and MetaboAnalyst software to compare experimental conditions, enabling the identification of optimal parameters for the extraction protocol. Overall, this study provides a method for metabolomic analysis suitable for investigating molecular interactions occurring directly within the xylem vessels, offering new insights into the pathogenesis of Xfp in olive trees and supporting future efforts to control this devastating disease.