基于超高效液相色谱-串联质谱技术的柑橘黑点病菌(Diaporthe citri)发育关联代谢物分析

Development-associated metabolites of Diaporthe citri: a metabolomics analysis based on UPLC-MS/MS

摘要:

【目的】柑橘黑点病是柑橘间座壳菌(Diaporthe citri)引起的真菌性病害，是危害柑橘的重要病害之一，D. citri在生长发育过程中经历菌丝生长(10 d, T1)、分生孢器形成(20 d, T2)和分生孢器产孢(30 d, T3)三个阶段。通过不同发育阶段代谢组分析，挖掘病原菌发育过程中标记物、关键代谢物，为黑点病菌产孢机制、代谢调控等深入研究提供依据。【方法】利用超高效液相色谱-串联质谱(ultra performance liquid chromatography/tandem mass spectrometry, UPLC-MS/MS)技术分析了D. citri发育过程中的代谢变化，采用主成分分析(principal component analysis, PCA)和正交偏最小二乘判别分析(orthogonal partial least squares discriminant analysis, OPLS-DA)，筛选出了显著差异代谢物并进行了KEGG (Kyoto encyclopedia of genes and genomes)富集分析。【结果】D. citri生长发育的3个阶段共鉴定出1 090种代谢物，基于二重筛选条件(VIP≥1，fold change≥2和fold change≤0.5)，T1 vs. T2、T2 vs. T3、T1 vs. T3比较组分别筛选出265、456和580个显著差异代谢物，差异代谢物主要是有机酸、甘油酯类、脂肪酸和氨基酸等物质，病原菌在生长发育过程中形态发育与代谢物变化密切相关。亚油酸及其代谢产物[inolenic acid、13-过氧化羟基-9Z,11E-十八碳二烯酸[(±)13-HpODE]、(9S,10E,12Z)-9-羟基十八碳-10,12-二烯酸[9(S)-HODE]、9-氧代-10E,12Z-十八碳二烯酸(9-oxoODE)、13-氧代-9Z,11E-十八碳二烯酸(13-oxoODE)、9-过氧化羟基-9Z,11E-十八碳二烯酸[(±)9-HpODE]、9,12,13-三羟基-十八碳单烯酸[9(S),12(S),13(S)-TriHOME]和(±)9,10-二羟基-12Z-十八碳烯酸(9,10-DiHOME)、γ-linolenic acid]和花生四烯酸及其代谢物[脂氧素B4 (LXB4)、15-酮基-前列腺素F2a (15-keto prostaglandin F2α)、20-羧基-白三烯B4 (20-carboxy LTB4)和15-脱氧-δ12,14-前列腺素J2 (15-deoxy-δ-12,14-PGJ2)]可能与D. citri产孢机制有关。【结论】D. citri在生长发育过程中形态发育与代谢物变化密切相关，氧化脂类代谢物(亚油酸及其代谢产物和花生四烯酸及其代谢物)是D. citri产孢的关键代谢物。

Abstract:

[Objective] Melanose caused by Diaporthe citri is one of the major fungal diseases in citrus. D. citri mainly undergoes three developmental stages, including mycelial growth stage (10 d, T1), pycnidium development stage (20 d, T2), and conidial sporulation stage (30 d, T3). The metabolomics analysis of the markers and key metabolites during the development of D. citri can provide clues for clarifying the sporulation mechanism and metabolic regulation of D. citri. [Methods] Ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was employed to analyze the metabolic changes during the development of D. citri. Then, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed to screen the differential metabolites. Finally, KEGG pathway enrichment analysis was conducted for the differential metabolites. [Results] A total of 1 090 metabolites were identified at the three developmental stages of D. citri. According to the thresholds of VIP≥1 and fold change≥2 or ≤0.5, 265, 456, and 580 differential metabolites were screened out for T1 vs. T2, T2 vs. T3, and T1 vs. T3, respectively. The differential metabolites mainly included organic acids, triglycerides, fatty acids, and amino acids, which were closely associated with the morphological development of the pathogen. Linoleic acid and its metabolites (13-HpODE, 9(S)-HODE, 9-oxoODE, 13-oxoODE, 9-HpODE, 9(S),12(S),13(S)-TriHOME, 9,10-DiHOME, and γ-linoleic acid), as well as arachidonic acid and its metabolites (20-COOH-AA, LXB4, 15-keto prostaglandin F2α, 20-carboxy LTB4, 15-deoxy-δ-12,14-PGJ2), might play a role in the sporulation of D. citri. [Conclusion] The morphological development of D. citri was closely associated with the changes of metabolites during different developmental stages, and the oxylipid metabolites (linoleic acid, arachidonic acid and their metabolites) were the key metabolites for sporulation.