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 25°C 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 to play 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 a melting temperature below 87.5°C that includes phylotypes I, III, and IV strains and the group with a melting temperature above 87.5°C that includes 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 show potential for use as a marker in continued studies of the cool-virulence phenotype. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.