关键词: 青枯菌, 铜抗性, copA, 致病性

Abstract:

【Objective】 Bacterial wilt of plants, caused by Ralstonia solanacearum , is a major soil-borne disease around the world. As an important bactericide to control bacterial diseases such as bacterial wilt, the widespread use of copper-based bactericides has led to the emergence of copper-resistant strains in a variety of plant pathogenic bacterial population. The copper-resistant coding gene copA , homologous with Pseudomonas syringae , was carried on the megaplasmid of R. solanacearum Po82 strain. The objective of this study is to investigate the biological function of copA in copper resistance and pathogenicity of Po82 strain.【Method】The phylogenetic relationship of the copper-resistant gene copA in different strains of R. solanacearum and other phytobacterial strains was analyzed based on neighbor-joining method using MEGA6.0 for constructing the phylogenetic tree of copA . By means of reverse genetics strategy, using the methods of gene homologous recombination and electroporation, copA gene deletion and complementary strains of Po82 were constructed. Copper minimal inhibition concentration (MIC) test, RT-qPCR, Biolog chip analysis, pathogenicity test and other basic biological methods were employed to clarify the relationship between copA and biological characteristics such as response to copper stress, metabolic activity, pathogenicity, and motility of R. solanacearum. 【Result】The results of homology analysis showed that the copA existed widely in the bacterial population, and the copA of R. solanacearum was most closely related to Cupriavidus metallidurans , but far genetic relationship with Xanthomonas oryzae , P. syringae and Escherichia coli . RT-qPCR analysis showed that the expression of copA was induced by copper. The expression of copA increased with the increase of CuSO₄ concentration. The expression level of copA was the highest when the CuSO₄ concentration was 1.0 mmol·L ^-1. By MIC analysis, the result showed that the sensitivity of the copA deletion strain to copper was significantly increased. The MIC value of copA deletion strain was 0.8 mmol·L ^-1, which decreased by 33.3% compared with that of wild-type strain (1.2 mmol·L ^-1). The complementary strain restored copper resistance. The results indicated that copA played an important role in copper resistance of R. solanacearum . Compared with wild-type strain, the logarithmic growth rate of copA gene deletion strain decreased in both NA medium and NA medium containing 0.6 mmol·L ^-1 CuSO₄, indicating that copA was related to the growth rate of R. solanacearum . The absence of copA resulted in a decrease in the pathogenicity of R. solanacearum . On the 10th day of inoculation, the disease index of the copA gene deletion strain decreased by 11.7% compared with that of the wild-type strain Po82. The absence of copA resulted in a reduction of metabolic utilization rate of carbon sources such as α -D-glucose, D-trehalose and nitrogen sources such as L-alanine and glucuronide. Compared with wild-type strain Po82, the expression level of hrpB , hrpG and ripX genes, which are important components of the type Ⅲ secretion system, was also significantly down-regulated in copA gene deletion strain. 【Conclusion】 The copper-resistant gene copA plays an important role in copper stress response and pathogenicity of R. solanacearum . The results provide a theoretical basis for further analysis of copper resistance mechanism and the control of copper-resistant strains.

Key words: Ralstonia solanacearum, copper-resistant, copA, pathogenicity