胡桃醌对核桃黑斑病病原菌的抑制作用及其机制

doi:10.3864/j.issn.0578-1752.2026.12.007

摘要/Abstract

摘要：

【目的】明确胡桃醌对核桃黑斑病主要病原菌核桃黄单胞杆菌（Xanthomonas arboricola pv. juglandis，Xaj）与成团泛菌（Pantoea agglomerans，Pa）的抑菌活性，并探究其作用机制，为核桃黑斑病绿色防控提供理论依据。【方法】通过体外抑菌活性测定，明确胡桃醌对Xaj和Pa的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）。借助生长曲线与刃天青染色法，明确胡桃醌处理后Xaj与Pa活性的动态变化；使用扫描电子显微镜（SEM）观察胡桃醌处理后的Xaj与Pa菌体表面和细胞形态变化；测定细胞膜相对电导率、细胞表面疏水性等指标，揭示胡桃醌对细胞膜完整性及其表面特性的影响机制；分析病原菌蛋白和核酸电泳变化，阐明胡桃醌对病原菌核酸及蛋白质合成等生物过程的干扰效应；采用紫外吸收光谱分析与分子对接技术，明确胡桃醌与病原菌DNA的互作方式；叶片接种试验评价胡桃醌对核桃黑斑病的叶片防治效果。【结果】胡桃醌对核桃黑斑病主要病原菌均具有明显抑菌活性，且Xaj较Pa更为敏感。胡桃醌对Xaj和Pa的MIC分别为 0.1和0.4 mg·mL^-1，MBC分别为0.2和0.8 mg·mL^-1。生长曲线表明，胡桃醌可浓度依赖性抑制两种病原菌生长；0.5×MIC处理30 min后，Xaj和Pa细胞活性分别降低35.25%和37.43%；SEM观察表明，胡桃醌处理后菌体出现皱缩、凹陷甚至破裂等形态异常；0.5×MIC处理后，两种病原菌细胞膜相对电导率和表面疏水性升高，并伴随胞内核酸和蛋白等物质泄漏增加，表明胡桃醌可破坏细胞膜完整性；紫外吸收光谱和分子对接结果显示，胡桃醌可能通过氢键等非共价相互作用与病原菌DNA发生沟槽结合；叶片试验表明，施用1×MIC胡桃醌能够显著降低核桃叶片侵染后的病斑面积，表现出较好的离体防治效果。【结论】胡桃醌对核桃黑斑病病原菌具有明显的抑菌活性，主要通过破坏细胞膜完整性、提高膜通透性，并进一步影响核酸和蛋白质相关生物过程，从而发挥其抑菌作用；同时在叶片上表现出一定的防治潜力。

关键词: 核桃黑斑病, 胡桃醌, 抑菌机制, 核桃黄单胞杆菌, 成团泛菌

Abstract:

【Objective】This study aimed to determine the antibacterial activity of juglone against the major pathogens causing walnut blight, Xanthomonas arboricola pv. juglandis (Xaj) and Pantoea agglomerans (Pa), and to investigate its mechanism of action, so as to provide a theoretical basis for the green control of walnut blight.【Method】The antibacterial activity of juglone against Xaj and Pa was evaluated in vitro by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Growth curves and resazurin staining were used to characterize the dynamic changes in the viability of Xaj and Pa after juglone treatment. Scanning electron microscopy (SEM) was used to observe alterations in the cell surface and morphology of the two pathogens. Relative electrical conductivity and cell surface hydrophobicity were measured to reveal the effects of juglone on membrane integrity and surface properties. Changes in pathogen protein and nucleic acid electrophoretic profiles were analyzed to assess the interference of juglone with nucleic acids, proteins, and related biological processes. UV absorption spectroscopy and molecular docking were applied to clarify the interaction mode between juglone and pathogen DNA. Leaf inoculation assay was conducted to evaluate the protective effect of juglone against walnut blight on leaves.【Result】Juglone showed obvious antibacterial activity against the major pathogens causing walnut blight, and Xaj was more sensitive than Pa. The MIC values of juglone against Xaj and Pa were 0.1 and 0.4 mg·mL^-1, respectively, while the corresponding MBC values were 0.2 and 0.8 mg·mL^-1. Growth curve analysis showed that juglone inhibited the growth of both pathogens in a concentration-dependent manner. After treatment with 0.5×MIC juglone for 30 min, the cell viability of Xaj and Pa decreased by 35.25% and 37.43%, respectively. SEM observations showed that juglone treatment caused obvious morphological abnormalities in bacterial cells, including shrinkage, depression, and even rupture. After treatment with 0.5×MIC juglone, the relative electrical conductivity and cell surface hydrophobicity of both pathogens increased, accompanied by increased leakage of intracellular nucleic acids, proteins, and other substances, indicating that juglone disrupted cell membrane integrity. Ultraviolet absorption spectroscopy and molecular docking suggested that juglone might interact with bacterial DNA through groove binding mediated by non-covalent interactions such as hydrogen bonding. The detached-leaf assay showed that application of 1×MIC juglone significantly reduced the lesion area on infected walnut leaves, exhibiting good leaf control efficacy.【Conclusion】Juglone showed obvious antibacterial activity against the pathogens causing walnut blight. Its antibacterial effect was mainly achieved by disrupting cell membrane integrity, increasing membrane permeability, and further affecting nucleic acid- and protein-related biological processes. In addition, juglone showed control potential against walnut blight on leaves.

Key words: walnut blight, juglone, antibacterial mechanism, Xanthomonas arboricola pv. juglandis, Pantoea agglomerans

苏一, 孙乐园, 吴承勖, 姬新颖, 聂瑞宁, 夏娟, 韩卓然, 周晔, 张俊佩. 胡桃醌对核桃黑斑病病原菌的抑制作用及其机制[J]. 中国农业科学, 2026, 59(12): 2623-2636.

SU Yi, SUN LeYuan, WU ChengXu, JI XinYing, NIE RuiNing, XIA Juan, HAN ZhuoRan, ZHOU Ye, ZHANG JunPei. Inhibitory Effects and Mechanisms of Juglone Against Walnut Blight Pathogens[J]. Scientia Agricultura Sinica, 2026, 59(12): 2623-2636.

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对接体系 Docking system	结合能Binding energy (kJ·mol^-1)	氢键作用（核苷酸-氢键长度） Hydrogen bonding (nucleotide-hydrogen bond length, Å)	疏水作用（5 Å范围内核苷酸） Hydrophobic interaction (nucleotides<5 Å)
1BNA (Fig. 10-a)	-27.21	DG10 (A) (2.8, 3.0, 3.1); DG16 (B) (2.9, 3.1)	DC11 (A); DA17 (B); DA18 (B)
1CGC (Fig. 10-b)	-26.80	DG3 (A) (2.9, 3.2); DC18 (B) (2.8); DG19 (B) (3.0, 3.1)	DG4 (A); DG5 (A)
1ZEW (Fig. 10-c)	-27.21	DT3 (A) (3.2); DC4 (A) (2.8, 2.9); DG17 (B) (2.8)	DA18 (B); DG19 (B); DG20 (B)
4C64 (Fig. 10-d)	-27.63	DG10 (A) (2.8, 3.0); DC15 (B) (2.7); DG16 (B) (3.0)	DC11 (A); DG12(A); DA17 (B)

病原菌Pathogen	仅接病菌Pathogen inoculation only	1×MIC胡桃醌处理Juglone treatment	对照Control
核桃黄单胞杆菌Xaj	0.030±0.007a	0.005±0.002b	0.001±0.0001c
成团泛菌Pa	0.027±0.013a	0.004±0.003b	0.001±0.0002c