中华蹄盖蕨挥发油对番茄溃疡病菌的抑菌效果及机理研究

doi:10.11924/j.issn.1000-6850.casb2022-0828

中国农学通报 ›› 2023, Vol. 39 ›› Issue (31): 139-146.doi: 10.11924/j.issn.1000-6850.casb2022-0828

中华蹄盖蕨挥发油对番茄溃疡病菌的抑菌效果及机理研究

渠红¹^,²^,³(), 王梦亮³, 闫然¹^,²^,³, 王琪⁴, 蔡瑾³()

¹ 山西大学中医药现代研究中心，太原 030006
² 山西大学化学生物学与分子工程教育部重点实验室，太原 030006
³ 山西大学应用化学研究所，太原 030006
⁴ 山西大学生命科学学院，太原 030006

收稿日期:2022-09-29 修回日期:2023-06-13 出版日期:2023-11-05 发布日期:2023-10-31
通讯作者: 蔡瑾，女，1982年出生，山西太原人，副教授，博士，研究方向：中药植物资源利用。通信地址：030006 山西省太原市小店区坞城路92号山西大学应用化学研究所，E-mail：caijin@sxu.edu.cn。
作者简介:
渠红，女，1995年出生，山西临县人，硕士在读，研究方向：中药植物资源利用。通信地址：030006 山西省太原市小店区坞城路92号山西大学应用化学研究所，E-mail：quhongsxu@163.com。
基金资助:
山西省基础研究计划自然科学研究面上项目“抗番茄溃疡病菌红蓼活性衍生物的设计合成及与DNA的互作关系研究”(20210302123451)

Study on the Inhibitory Activity and Mechanism of Athyrium sinense Essential Oil Against Clavibacter michiganensis subsp. michiganensis

QU Hong¹^,²^,³(), WANG Mengliang³, YAN Ran¹^,²^,³, WANG Qi⁴, CAI Jin³()

¹ Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006
² The Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006
³ Institute of Applied Chemistry, Shanxi University, Taiyuan 030006
⁴ School of Life Science, Shanxi University, Taiyuan 030006

Received:2022-09-29 Revised:2023-06-13 Published-:2023-11-05 Online:2023-10-31

摘要/Abstract

摘要：

为研究中华蹄盖蕨挥发油对番茄溃疡病菌(Cmm)的抑菌效果及机理，以平板打孔法和浊度法测定中华蹄盖蕨挥发油对Cmm的抑菌效果。抑菌机理主要包括细胞壁、细胞膜和细胞周期3个方面，测定胞外碱性磷酸酶(AKP)活性以分析对细胞壁的作用，测定胞内活性氧、胞外可溶性蛋白泄漏量、细胞膜电位、ATP酶活性以分析对细胞膜的作用，最后测定细胞周期。研究发现，中华蹄盖蕨挥发油抑菌圈直径是(19.06±1.83) mm、最小抑菌浓度为0.313 mg/mL，表明中华蹄盖蕨挥发油对Cmm有明显抑制作用。中华蹄盖蕨挥发油作用后，使Cmm胞外AKP活性、活性氧水平和胞外可溶性蛋白泄漏量显著(P<0.05)升高，细胞膜电位、ATP酶活性显著(P<0.05)降低，处于DNA复制期的Cmm占比降低。中华蹄盖蕨挥发油对Cmm具有显著抑菌效果。其抑菌机理为破坏细胞壁导致胞外AKP活性升高，破坏细胞膜导致活性氧水平和可溶性蛋白泄漏量显著(P<0.05)升高，出现细胞膜去极化及ATP酶活性显著(P<0.05)降低现象，最终干扰了正常的细胞周期。

关键词: 中华蹄盖蕨, 挥发油, 番茄溃疡病菌, 抑菌活性, 抑菌机制

Abstract:

The aim of this study was to investigate the antibacterial effect and mechanism of Athyrium sinense essential oil against Clavibacter michiganensis subsp. michiganensis (Cmm). The antibacterial effect of A. sinense essential oil on Cmm was determined by the plate punching method and the turbidimetric method. The mechanism of bacterial inhibition was mainly studied from three aspects including cell wall, cell membrane and cell cycle. The activity of extracellular alkaline phosphatase (AKP) was measured to analyze the effect on cell wall. The level of intracellular reactive oxygen species, the content of extracellular soluble protein, the potential of cell membrane, and the ATPase activity were measured to analyze the effect on cell membrane. Finally, the cell cycle was measured. The diameter of Cmm inhibition circle was (19.06±1.83) mm and the minimum inhibitory concentration (MIC) was 0.313 mg/mL, indicating that A. sinense essential oil had a strong inhibitory effect on Cmm. Analysis of the inhibition mechanism showed that under the treatment of A. sinense essential oil, extracellular AKP activity, ROS level, and extracellular soluble protein leakage increased significantly (P<0.05), cell membrane potential and intracellular ATPase activity were significantly (P<0.05) decreased, and the proportion of Cmm in DNA replication was decreased. The results showed that A. sinense essential oil had significant antibacterial effect on Cmm. The antibacterial mechanism was as follows. The destruction of Cmm cell wall led to the increase of extracellular AKP activity. The destruction of cell membrane led to a significant (P<0.05) increase in the level of reactive oxygen species and the leakage of soluble protein, the occurrence of cell membrane depolarization and the significant (P<0.05) decrease in ATPase activity. Finally, A. sinense essential oil also interfered with the normal cell cycle of Cmm.

Key words: Athyrium sinense, essential oil, Clavibacter michiganensis subsp. michiganensis, antibacterial activity, antibacterial mechanism

渠红, 王梦亮, 闫然, 王琪, 蔡瑾. 中华蹄盖蕨挥发油对番茄溃疡病菌的抑菌效果及机理研究[J]. 中国农学通报, 2023, 39(31): 139-146.

QU Hong, WANG Mengliang, YAN Ran, WANG Qi, CAI Jin. Study on the Inhibitory Activity and Mechanism of Athyrium sinense Essential Oil Against Clavibacter michiganensis subsp. michiganensis[J]. Chinese Agricultural Science Bulletin, 2023, 39(31): 139-146.

图/表 8

图1. 中华蹄盖蕨挥发油对番茄溃疡病菌的抑菌效果通过T-检验进行0.05水平的显著性分析，用*表示其结果；对照组为40% DMSO的抑菌效果；抑菌圈直径为10 mm时，代表无抑菌效果

挥发油浓度/(mg/mL)	番茄溃疡病菌	挥发油浓度/(mg/mL)	番茄溃疡病菌
5	－	0.08	++
2.5	－	0.04	+++
1.25	－	0.02	+++
0.625	－	0.01	+++
0.313	－	对照	+++
0.156	+

表1. 中华蹄盖蕨挥发油对Cmm的抑菌效果

图2. 中华蹄盖蕨挥发油对Cmm胞外AKP活性的影响 1/4MIC、1/2MIC、MIC、2MIC分别代表不同浓度的中华蹄盖蕨挥发油；不加入中华蹄盖蕨挥发油仅含3% DMSO培养基为对照组；每组试验重复开展3次，通过方差分析，进行0.05水平的显著性分析，用不同小写字母表示其结果；下同

图3. 中华蹄盖蕨挥发油对Cmm胞内活性氧水平的影响

图4. 中华蹄盖蕨挥发油对Cmm胞外可溶性蛋白泄漏量的影响

图5. 中华蹄盖蕨挥发油对Cmm细胞膜电位的影响

图6. 中华蹄盖蕨挥发油对Cmm ATP酶活性的影响

图7. 中华蹄盖蕨挥发油对Cmm细胞周期的影响

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中华蹄盖蕨挥发油对番茄溃疡病菌的抑菌效果及机理研究

Study on the Inhibitory Activity and Mechanism of Athyrium sinense Essential Oil Against Clavibacter michiganensis subsp. michiganensis

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