Isolation of an Insertion Sequence from Ralstonia solanacearum Race 1 and Its Potential Use for Strain Characterization and Detection

Abstract

ABSTRACT A new insertion sequence (IS), IS 1405 , was isolated and characterized from a Ralstonia solanacearum race 1 strain by the method of insertional inactivation of the sacB gene. Sequence analysis indicated that the IS is closely related to the members of IS 5 family, but the extent of nucleotide sequence identity in 5′ and 3′ noncoding regions between IS 1405 and other members of IS5 family is only 23 to 31%. Nucleotide sequences of these regions were used to design specific oligonucleotide primers for detection of race 1 strains by PCR. The PCR amplified a specific DNA fragment for all R. solanacearum race 1 strains tested, and no amplification was observed with some other plant-pathogenic bacteria. Analysis of nucleotide sequences flanking IS 1405 and additional five endogenous IS 1405 s that reside in the chromosome of R. solanacearum race 1 strains indicated that IS 1405 prefers a target site of CTAR and has two different insertional orientations with respect to this target site. Restriction fragment length polymorphism (RFLP) pattern analysis using IS 1405 as a probe revealed extensive genetic variation among strains of R. solanacearum race 1 isolated from eight different host plants in Taiwan. The RFLP patterns were then used to subdivide the race 1 strains into two groups and several subgroups, which allowed for tracking different subgroup strains of R. solanacearum through a host plant community. Furthermore, specific insertion sites of IS 1405 in certain subgroups were used as a genetic marker to develop subgroup-specific primers for detection of R. solanacearum , and thus, the subgroup strains can be easily identified through a rapid PCR assay rather than RFLP analysis.