| Summary: | Pepper leaf spot is a common disease of <i>Zanthoxylum schinifolium</i>. When it is serious, it directly affects the growth of <i>Z. schinifolium</i>, making the plant unable to blossom and bear fruit, which seriously restricts the development of the <i>Z. schinifolium</i> industry. Therefore, the pathogenic mechanism of leaf spots should be explored to provide a basis for a comprehensive understanding of the disease. Using liquid chromatography–mass spectrometry (LC–MS) technology combined with the data-dependent acquisition, the full spectrum analysis of pathogen mycelium samples was carried out. Partial least squares discriminant analysis (PLS-DA) was used to reveal the differences in metabolic patterns among different groups. Hierarchical clustering analysis (HCA) and PLS-DA were used to reveal the relationship between samples and metabolites, which reflected the metabolomics changes of <i>Pestalotiopsis kenyana</i> in the logarithmic growth phase of mycelia, the stable growth phase of mycelia, the massive spore stage, the induction culture conditions of PDA and <i>Z. schinifolium</i> leaves, and the possible pathogenic substances were selected for pathogenicity detection. PLS-DA had a strong predictive ability, indicating a clear analysis trend between different groups. The results of the metabolomics analysis showed that the differential metabolites of pathogenic bacteria were abundant at different stages and under different medium conditions, and the content of metabolites changed significantly. There were 3922 differential metabolites in nine groups under positive and negative ion modes, including lipids and lipid molecules, organic acids and their derivatives, organic heterocyclic compounds, organic oxygen compounds, carbohydrate polyketides, nucleosides, nucleotides, and analogs. The results of the pathogenicity test showed that the leaves treated with 3,5-dimethoxy benzoic acid, <i>S</i>-(5-adenosy)-<span style="font-variant: small-caps;">l</span>-homocysteine, 2-(1<i>H</i>-indol-3-yl) acetic acid, <span style="font-variant: small-caps;">l</span>-glutamic acid, and 2-(2-acetyl-3,5-dihydroxy phenyl) acetic acid showed different degrees of yellowish-brown lesions. This indicated that these substances may be related to the pathogenicity of <i>P. kenyana</i>, and the incidence was more serious when treated with 3,5-dimethoxybenzoic acid and <i>S</i>-(5-adenosy)-<span style="font-variant: small-caps;"> l</span> -homocysteine. This study provides a basis for further analysis of differential metabolites and provides a theoretical reference for the prevention and treatment of <i>Z. schinifolium</i> leaf spot.
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