Metabolomic Profiling of C. paliurus Cultivars

For further metabolomics analysis of secondary metabolites, 2-year-old C. paliurus seedlings of resistant (Wufeng) and susceptible (Jinggangshan) cultivars were inoculated following the IPI method as described earlier. The sample preparation, extract analysis, metabolite separation, and detection were conducted by MetWare Biological Science and Technology (Wuhan, China) following their standard procedures, which were previously described by Li et al. (2021) (link) and Mei et al. (2020) (link). In brief, 100 mg crushed, freeze-dried sample was extracted overnight at 4°C with 0.6 mL 70% (v/v) aqueous methanol. After centrifuging at 10,000 rpm for 10 min, the extracts were absorbed and filtered, and then analyzed on a UPLC–ESI–MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM30A system; MS, Applied Biosystems 4500 QTRAP). Metabolite quantification was conducted utilizing the multiple reaction monitoring (MRM) method (Chen et al., 2013 (link)). Following data evaluation (quality control and PCA analysis), OPLS-DA, a supervised multivariate method, was used to maximize metabolome differences between sample pairs. Differentially accumulated metabolites (DAMs) were set at fold change (FC) > 2 or FC < 0.5 and OPLS-DA VIP ≥ 1. The identified DAMs were further annotated using the KEGG compound database to reveal the function and content variation of these metabolites.