聚六亚甲基胍对柑橘溃疡病菌的抑菌活性

doi:10.3864/j.issn.0578-1752.2025.09.008

摘要/Abstract

摘要：

【目的】明确聚六亚甲基胍（PHMG）对重大农业病原细菌柑橘溃疡病菌（柑橘黄单胞菌柑橘致病变种，Xanthomonas citri pv. citri，Xcc）的离体抑菌活性，评估PHMG对柑橘溃疡病的田间防治效果。【方法】在测定离体抑菌活性时，运用浊度法监测菌液浊度的变化，以此量化PHMG对柑橘溃疡病菌生长的抑制程度。通过叶片接种模拟病原菌侵染柑橘叶片，观察PHMG是否能阻挡柑橘溃疡病菌的侵入，同时探究其是否具备防治柑橘溃疡病的作用。田间试验选择秋季新梢即将抽梢时进行，以最接近真实生产问题的环境来检验PHMG对柑橘溃疡病的防治效果。采用扫描电镜和荧光显微镜等微观观测仪器观察PHMG作用后柑橘溃疡病菌表面及形态的变化；碘化丙啶（PI）染色将细胞凋亡过程可视化；结晶紫染色试验揭示柑橘溃疡病菌生物膜形成情况，以探明PHMG对柑橘溃疡病菌菌体的影响。【结果】 PHMG具有广谱抑菌特性，对包括柑橘溃疡病菌在内的5种植物病原细菌均具有较好的离体抑制效果，其EC₅₀值稳定在0.5 μg·mL^-1左右，表明低浓度的PHMG即可对病原菌生长产生抑制。PHMG对51株柑橘溃疡病菌的EC₅₀值介于0.19—0.69 μg·mL^-1，离体抑菌效果较为稳定。叶片接种结果表明PHMG可保护柑橘叶片免受病原菌的侵染，且具有治疗作用。田间试验结果表明以0.5 mg·mL^-1的浓度喷施PHMG对柑橘溃疡病防治效果达到60%左右，略低于对照药剂春雷-噻菌铜和春雷-喹啉铜。此外，PHMG不仅致使柑橘溃疡病菌菌体表面凹陷，菌体大小异常，还促进病原菌凋亡，抑制细胞生物膜形成，并降低病原菌的运动性。【结论】 PHMG对柑橘溃疡病菌具有较强的离体抑菌活性，在实际应用中可有效降低田间柑橘溃疡病的发生。此外，PHMG对柑橘溃疡病菌的菌体形态、细胞凋亡、生物膜形成以及运动性均造成影响。研究结果可为后续PHMG防治柑橘溃疡病以及拓展至其他细菌病害领域具有重要的理论指引作用。

关键词: 柑橘溃疡病, 柑橘溃疡病菌, 聚六亚甲基胍, 抑菌活性, 田间防治效果

Abstract:

【Objective】 The objective of this study is to clarify the in vitro antibacterial activity of polyhexamethylene guanidine (PHMG) against Xanthomonas citri pv. citri (Xcc), a major agricultural pathogenic bacterium that causes citrus canker, and to evaluate the field control effect of PHMG on citrus canker.【Method】 When determining the in vitro antibacterial activity, the turbidity method was used to monitor the changes in the turbidity of the bacterial solution, so as to quantify the inhibitory degree of PHMG on the growth of Xcc. By inoculating the leaves to simulate the infection of citrus leaves by Xcc, whether PHMG could block the invasion of Xcc was observed and whether it had the effect of preventing and treating citrus canker was explored. The field experiment was carried out when the autumn new shoots were about to sprout, in an environment that was closest to the actual production problems, to test the actual effect of PHMG in preventing and treating citrus canker. Microscopic observation instruments such as scanning electron microscopy and fluorescence microscopy were used to show the surface and morphological changes of Xcc after the action of PHMG; PI staining was used to visualize the process of cell apoptosis; the crystal violet staining experiment was used to reveal the formation of biofilms of Xcc. Through these experiments, the effects of PHMG on the cells of Xcc were explored.【Result】 PHMG showed broad-spectrum antibacterial properties and had good in vitro inhibitory effects on five plant pathogenic bacteria, including Xcc. The EC₅₀ values were approximately 0.5 μg·mL^-1, indicating that a low concentration of PHMG could inhibit the growth of pathogenic bacteria. The EC₅₀ values of PHMG against 51 strains of Xcc ranged from 0.19 to 0.69 μg·mL^-1, and the in vitro antibacterial activity was relatively stable and reliable. The results of leaf inoculation showed that PHMG could protect citrus leaves from Xcc and had a therapeutic effect. The results of field trials showed that spraying PHMG (0.5 mg·mL^-1) had a control effect of about 60% on citrus canker, which was slightly lower than that of the control fungicides, kasugamycin-thiodiazole-copper and kasugamycin-oxine-copper. Furthermore, PHMG not only caused depressions on the surface of Xcc and abnormal cell size, but also promoted the apoptosis of Xcc, inhibited the formation of cell biofilm, and reduced the motility of Xcc.【Conclusion】 PHMG has excellent in vitro antibacterial activity against Xcc, and can effectively reduce the occurrence of citrus canker in the field in practical applications. In addition, PHMG affected the cell morphology, cell apoptosis, biofilm formation and motility of Xcc. The results of this study will provide important theoretical guidance for the subsequent application of PHMG for citrus canker and its expansion to other bacterial diseases.

Key words: citrus canker, Xanthomonas citri pv. citri (Xcc), polyhexamethylene guanidine (PHMG), antibacterial activity, field control effect

刘婕, 侯瑞, 周泽华, 易图永. 聚六亚甲基胍对柑橘溃疡病菌的抑菌活性[J]. 中国农业科学, 2025, 58(9): 1779-1790.

LIU Jie, HOU Rui, ZHOU ZeHua, YI TuYong. Antibacterial Activity of Polyhexamethylene Guanidine Against Xanthomonas citri pv. citri[J]. Scientia Agricultura Sinica, 2025, 58(9): 1779-1790.

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病原菌 Pathogen	回归方程 Regression equation (y=)	相关系数 r	EC₅₀ (μg·mL^-1)	MIC (μg·mL^-1)	MBC (μg·mL^-1)
柑橘溃疡病菌Xcc	6.08+5.81x	0.99	0.48	1.00	8.00
野油菜黄单胞菌X. campestris	6.87+6.44x	0.98	0.51	1.00	16.00
水稻黄单胞菌稻致病变种X. oryzae pv. oryzicola	10.94+6.42x	0.95	0.47	1.00	8.00
丁香假单胞菌猕猴桃致病变种P. syringae pv. actinidiae	7.17+7.35x	0.95	0.51	1.00	8.00
青枯雷尔氏菌R. solanacearum	4.15+4.58x	0.98	1.54	3.00	35.00
链格孢A. alternata	3.52+1.77x	0.95	6.89	—	—
玉米小斑病菌B. maydis	3.60+1.30x	0.97	12.13	—	—
稻瘟病菌M. oryzae	4.06+1.33x	0.98	5.08	—	—
柑橘间座壳菌D. citri	3.91+1.22x	0.98	7.86	—	—

处理 Treatment	相对防治效果 Relative control effect (%)
1 mg·mL^-1 PHMG	74.72±1.12a
0.5 mg·mL^-1 PHMG	61.86±2.26b
0.25 mg·mL^-1 PHMG	54.35±1.10c
0.5 mg·mL^-1春雷-噻菌铜 Kasugamycin-thiodiazole-copper	72.94±2.72a
0.5 mg·mL^-1春雷-喹啉铜 Kasugamycin-oxine-copper	74.84±1.89a