Methylation sensitive amplification polymorphism of Ralstonia solanacearum strains with different pathogenicity

Abstract:

[Objective] To investigate the changes of pathogenicity and DNA methylation levels and patterns of Ralstonia solanacearum strains with different pathogenicity during consecutive subculture. [Methods] R. solanacearum strains with different pathogenicity were consecutively subcultured for 50 passages. The pathogenicity of different strains was determined by the attenuated index (AI) method and the pot experiments. Methylation sensitive amplification polymorphism (MSAP) analysis was performed to profile the DNA methylation levels of different strains. Moreover, the relative expression levels of genes related to methylases and demethylases were determined by real-time fluorescent quantitative PCR (qRT-PCR). [Results] After 50 passages, both of the virulent strain FJAT15304 and the intermediate strain FJAT445 evolved into avirulent strains, while the avirulent strain FJAT15249 remained to be avirulent. Compared with F₁ strains, FJAT15304.F₅₀ and FJAT445.F₅₀ showed the total methylation rates increasing by 7.82% and 38.22%, respectively. However, both of FJAT15249.F₁ and FJAT15249.F₅₀ had the total methylation rate of 33.33%. Full methylation was the main pattern in the virulent and intermediate strains, while hemi-methylation was the main pattern in all the avirulent strains. Compared with F₁ strains, strains FJAT15304.F₅₀ and FJAT445.F₅₀ showed up-regulated expression of three methylase-related genes dam, dcm, and ftsZ and down-regulated expression of demethylase-related gene alkB, which suggested that the change of DNA methylation might play a key role in the debilitation of pathogenicity. [Conclusion] The pathogenicity of R. solanacearum attenuates during the consecutive subculture, which might be related to the level of DNA methylation. The findings provide a scientific basis for the application of avirulent strains in the biocontrol of bacterial wilt.

Key words:Ralstonia solanacearum;consecutive subculture;DNA methylation;methylation sensitive amplification polymorphism