纳米氧化锌对成团泛菌 P2生物膜形成的影响

Zinc oxide nanoparticles affect the biofilm formation of Pantoea agglomerans P2

摘要:

背景 核桃黑斑病已成为危害我国核桃生长的主要常见病害，成团泛菌( Pantoea agglomerans)现已成为陕西、云南、山东、河北等地区的核桃黑斑病优势病原菌。 目的 明确抑菌金属离子Zn对核桃黑斑病原成团泛菌P2的防病机制，通过外源添加不同浓度纳米氧化锌，测定其对成团泛菌菌株生长、生物膜形成、胞外多糖和蛋白质含量的影响。 方法 采用纳米氧化锌0.2、0.4、0.6、0.8、1.0 mmol/L 5个处理，测定了不同浓度对成团泛菌P2生长的影响；观察生物膜形成情况并采用称质量法和结晶紫染色法对生物膜形成能力进行定量测定；通过95%冰冻乙醇萃取及考马斯亮蓝加硫酸铵沉淀法对胞外多糖和蛋白质含量进行测定。 结果 不同浓度纳米氧化锌均会抑制菌株生长，0.8 mmol/L和1.0 mmol/L纳米氧化锌处理较对照差异显著，0.2-0.6 mmol/L纳米氧化锌处理与对照无显著差异；各浓度纳米氧化锌处理都显著弱化了生物膜的褶皱和立体程度，尤其表现为1.0 mmol/L时不形成生物膜。同时镜检结果也显示，随着纳米氧化锌浓度的增加，生物膜细胞聚集程度逐渐下降；对生物膜的定量结果表明，各纳米氧化锌处理下的生物膜形成能力逐渐减弱；此外，纳米氧化锌各浓度处理还降低了菌株P2生物膜的胞外多糖和蛋白含量。 结论 本研究揭示了纳米氧化锌在抑制成团泛菌P2防治效果方面的重要作用，以及高效发挥新型纳米制剂的防治潜力，为核桃黑斑病的化学防治提供了一种有效的策略。

Abstract:

Background Walnut black spot disease has become one of the major common diseases affecting walnut growth in China. Pantoea agglomerans has become the dominant pathogen of walnut black spot disease in Shaanxi, Yunnan, Shandong, Hebei, and other regions. Objective To elucidate the antibacterial mechanism of the metal ion zinc (Zn) against Pantoea agglomerans P2, the pathogen causing walnut black spot disease, we assessed the effects of different concentrations of exogenously added nano-zinc oxide (nZnO) on the growth, biofilm formation, and extracellular polysaccharide and protein content of this strain. Methods The experiment was designed with five concentrations of nZnO: 0.2, 0.4, 0.6, 0.8, and 1.0 mmol/L, the effects of which on the growth of P. agglomerans P2 were evaluated. Biofilm formation was observed and quantitatively assessed by weight measurement and crystal violet staining. Extracellular polysaccharide and protein content was determined by 95% cold ethanol extraction and Coomassie brilliant blue with ammonium sulfate precipitation. Results All the concentrations of nZnO inhibited bacterial growth. Notably, 0.8 mmol/L and 1.0 mmol/L nZnO exhibited significant inhibitory effects compared with the control, while the 0.2-0.6 mmol/L nZnO treatment showed no significant difference. All the nZnO treatments significantly reduced the folds and three-dimensional structure of the biofilm, with 1.0 mmol/L completely inhibiting biofilm formation. Microscopy revealed that as the nZnO concentration increased, the degree of cell aggregation within the biofilm decreased. Quantitative analysis indicated a progressive decline in biofilm formation with the increase in nZnO concentration. Furthermore, all the nZnO treatments reduced the extracellular polysaccharide and protein content in the biofilm of P2. Conclusion This study highlights the significant inhibitory effect of nZnO on P. agglomerans P2 and the potential of nZnO as a novel nanomaterial for disease control, offering an effective chemical strategy for managing walnut black spot disease.