茶树精油对番茄青枯雷尔氏菌的抑菌机制

Antimicrobial mechanism of tea tree essential oil against Ralstonia solanacearum in tomato

摘要:

背景 青枯雷尔氏菌(Ralstonia solanacearum)是引起番茄青枯病的土传性致病细菌，具有极大的危害性。目的 研究茶树精油对番茄青枯雷尔氏菌的抑菌作用及其机制，为植物源生物抑菌剂开发提供参考。方法 采用水蒸气蒸馏法提取茶树精油，并通过气相色谱-质谱(gas chromatography-mass spectrometry, GC-MS)联用仪检测，结合NIST库比对分析其成分。测定茶树精油对番茄青枯雷尔氏菌最小抑菌浓度(minimum inhibitory concentration, MIC)、最小杀菌浓度(minimum bactericidal concentration, MBC)，考察其对菌株生长的影响以及熏蒸杀菌活性，探究茶树精油作用下青枯雷尔氏菌DNA、蛋白质、生存活力、膜电位、活性氧(reactive oxygen species, ROS)和生物膜的变化，阐明茶树精油对番茄青枯雷尔氏菌抑菌作用及其机制。结果 茶树精油的主要成分为4-萜烯醇(39.83%)，对番茄青枯雷尔氏菌的MIC和MBC分别为8 mg/mL和16 mg/mL。熏蒸试验表明，208.4 mg/L茶树精油能够完全杀灭番茄青枯雷尔氏菌。细胞外DNA浓度从0.10 μg/mL增加到0.16 μg/mL，蛋白质浓度从3.5 mg/mL增加到6.4 mg/mL，膜电位荧光强度从1 335.6 a.u.降低到177.2 a.u.，细菌生存活力下降，表明茶树精油能够破坏细胞膜，增加细胞膜通透性。荧光光谱检测和共聚焦显微镜观察说明茶树精油能作用于DNA，降低DNA的含量。ROS荧光强度从638.5 a.u.增加到1 595.4 a.u.，ROS累积增加，损伤细菌。荧光光谱和分子对接分析表明茶树精油能够与蛋白质结合，4-萜烯醇能作用于潜在靶蛋白DNA促旋酶亚基B、二氢叶酸还原酶和肽脱甲酰酶，产生抗菌作用。结论 茶树精油通过多种作用机制对番茄青枯雷尔氏菌产生抑菌作用，为开发植物源生物抑菌剂防治番茄青枯病提供理论依据。

Abstract:

Background Ralstonia solanacearum is a soil-borne pathogenic bacterium that causes tomato bacterial wilt and is extremely destructive.Objective To investigate the antimicrobial activity and mechanism of tea tree essential oil against R. solanacearum, thereby providing a reference for the development of plant-derived biological antimicrobial agents.Methods Tea tree essential oil was extracted by steam distillation, and its composition was analyzed by gas chromatography-mass spectrometry (GC-MS) and identified through NIST library comparison. The antimicrobial effects and underlying mechanism of tea tree essential oil against R. solanacearum were investigated by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), effects on the bacterial growth, and fumigation bactericidal activity, as well as measuring its effects on bacterial DNA, proteins, viability, membrane potential, reactive oxygen species (ROS), and biofilm formation.Results The predominant component of tea tree essential oil was terpinen-4-ol (39.83%), and its MIC and MBC against R. solanacearum were 8 mg/mL and 16 mg/mL, respectively. The fumigation test showed that 208.4 mg/L of tea tree essential oil could completely eliminate R. solanacearum. The extracellular DNA concentration increased from 0.10 μg/mL to 0.16 μg/mL, the protein concentration rose from 3.5 mg/mL to 6.4 mg/mL, and the fluorescence intensity of membrane potential decreased sharply from 1 335.6 a.u. to 177.2 a.u., accompanied by a decline in bacterial viability. These results indicated that tea tree essential oil could damage the cell membrane and increase its permeability. Fluorescence spectroscopy analysis and confocal laser scanning microscopy observations demonstrated that tea tree essential oil could target bacterial DNA and reduce its content. Additionally, the fluorescence intensity of ROS increased from 638.5 a.u. to 1 595.4 a.u., suggesting that excessive ROS accumulation induced bacterial damage. Fluorescence spectroscopy and molecular docking analysis showed that tea tree essential oil could bind to proteins, and that terpinen-4-ol could act on the target protein DNA gyrase subunit B, dihydrofolate reductase and peptide deformylase, thereby exerting antimicrobial effects.Conclusion Tea tree essential oil exerts an antimicrobial effect on R. solanacearum through multiple mechanisms, providing a theoretical basis for the development of plant-derived biological antimicrobial agents to prevent and control tomato bacterial wilt.