Metabolomic fingerprint of Reynoutria japonica (Polygonaceae) using suspect screening with LC-HRMS and untargeted approach with GC-HRMS

Knotweed (Reynoutria japonica) is an invasive plant whose ecological dominance in its invasive range may partly rely on allelopathic interactions mediated by root exudates and especially secondary metabolites of wide polarity range. To elucidate the chemical diversity of these exudates, a metabolomic strategy combining liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) and gas chromatography coupled to high-resolution mass spectrometry (GC-HRMS) was employed. After optimization of EcoRoot methodology with design of experiment (DoE), root exudates were sampled with 3 different sorption materials and analyzed using LC-ESI-MS/MS and GC-HRMS. Molecular networking was applied to MS/MS data to visualize chemical structural similarities and facilitate the identification of metabolites with a confidence annotation scale. Diverse families of compounds, including flavonoids, alkaloids, organic acids, terpenoids, and phenolics, were annotated and many of which could be implicated in allelopathic interactions or rhizospheric communication. Notably, molecular networks highlighted the presence of lineage-specific metabolites (Polygonaceae). Also, suspect screening was performed and led to confirmation of allelochemicals as root exudates: chlorogenic acid, catechin, and polydatin. GC-HRMS further revealed volatile organic compounds such as terpenoids or carbon disulfide, which may contribute to belowground chemical interference. These VOCs were trapped using another sorbent called Sorb-Star® and then thermodesorbed. This study underscores the value of integrated HRMS-based metabolomics for exploring the chemical diversity of R. japonica and provides candidate metabolites (isomers of selinene, carbon disulfide) for future studies in both fundamental and applied ecology and agronomy. Finally, this study presents the first metabolomic fingerprint of R. japonica grown in soil.