Genetic stability of virulent, intermediate, and avirulent strains of Ralstonia solanacearum after extensive, consecutive subculturing

Abstract

An avirulent strain, FJAT1458, was demonstrated to possess a high level of biocontrol efficiency against bacterial wilt (BW). Assessing the genetic stability of this avirulent strain is essential for ensuring its safety and efficacy for use in field applications. In the present study, the genetic stability of three R. solanacearum strains with different levels of virulence was evaluated over the course of 50 generations of subculturing. Pathogenicity tests revealed that the virulent strain, FJAT91, and the intermediate strain, FJAT445, transformed into avirulent strains, but FJAT1458 remained avirulent after the same number of subcultures. Colonization tests revealed that the colonization pattern of the F1 generation of the avirulent FJAT1458 strain was similar to that of the F50 generation, but the virulent strain was rarely detected in later stages of infection. High performance liquid chromatography revealed that the number and type of chromatographic peaks produced by the avirulent strain did not exhibit any changes over the course of subculturing, however, the pattern of peaks produced by the virulent and intermediate strains changed noticeably over the course of 50 generations of subculturing. Moreover, exopolysaccharide I (EPS I) content, β-1,4-endoglucanase (Egl) activity, and swimming and swarming motility significantly decreased in the virulent and intermediate strains but were stable in the avirulent strain over the course of subculturing. The relative expression level of hrpB, phcA, eps, and egl, genes associated with virulence, significantly decreased in the virulent and intermediate strains over the course of 10 to 20 generations of subculturing but remained relatively stable in the avirulent strain across 50 generations. In conclusion, the avirulent strain remained genetically stable, while the virulent and intermediate strains exhibited genetic instability, over the course of 50 generations of subculturing.