Differentiation of cool-virulent strains in Ralstonia solanacearum species complex by melt curve of a DNA fragment from effector gene ripS1

The Ralstonia solanacearum species complex (Rssc) causes bacterial wilt diseases across hundreds of plant species. Phylogenetic analysis of DNA sequences has allowed the grouping of Rssc strains into phylotypes based on genetic relatedness. Although specific phenotypes are associated with corresponding phylotypes, the molecular bases for these differences are not always well understood. The complex trait of cool-virulence that allows several members of phylotype II to cause disease at temperatures below 25oC is one of these poorly understood traits. Here we report that a 96-bp DNA fragment from a gene encoding the type III secretion system effector protein RipS1, which was recently reported as playing a role in the cool-virulence phenotype, has a melting temperature that varies based on the strain of origin. By testing 81 Rssc strains with melt-curve analysis, we found a bimodal distribution with clear separation between the group of strains with Tm below 87.5oC that include phylotypes I, III and IV strains, and the group with Tm above 87.5oC that include all 35 tested select agent race 3 biovar 2 (phylotype IIB sequevars 1 and 2) and seven phylotype IIB sequevar 4 strains, previously known to possess cool-virulence capability, with a few exceptions. This provides further support that sequence differences in this effector differentiate cool-virulent strains from non-cool-virulent strains and shows potential for use as a marker in continued studies of the cool-virulence phenotype.