Identification of a DNA region associated with the cool virulence of Ralstonia solancearum strain UW551 and its utilization for specific detection of the bacterium’s race 3 biovar 2 strains

Authors: Stulberg, Michael; CAI, XUEQING - Fujian Agricultural & Forestry University; EBRAHIM, ABDELMONIM ALI - Minia University; Huang, Qi

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2018
Publication Date: 11/4/2018
Citation: Stulberg, M.J., Cai, X., Ebrahim, A., Huang, Q. 2018. Identification of a DNA region associated with the cool virulence of Ralstonia solancearum strain UW551 and its utilization for specific detection of the bacterium’s race 3 biovar 2 strains. PLoS One. 13(11). https://doi.org/10.1371/journal.pone.0207280.
DOI: https://doi.org/10.1371/journal.pone.0207280

Interpretive Summary: Ralstonia solanacearum is a bacterial pathogen that causes millions of dollars of crop losses in a wide range of plant species worldwide. One strain in particular, the r3b2 subgroup, is such a threat to U.S. agriculture that is has been designated a select agent in the U.S. and is a quarantine pathogen in Europe and Canada. This strain is adapted to cool temperatures and is responsible for brown rot of potato, among other diseases. ARS scientists in Beltsville, MD identified a region in the genome of the bacterium that is associated with the bacterium’s ability to cause disease at low temperature. They also developed an accurate and efficient detection method by targeting this particular region for specific detection and identification of this group of bacterium. Accurate detection methods are critical in preventing the r3b2 strain from entering the U.S., and also to avoid unnecessary exclusion of non-r3b2 strains in order to effectively safeguard U.S. Agriculture.

Technical Abstract: The cool temperature adapted Ralstonia solanacearum race 3 biovar 2 (r3b2) strains cause destructive brown rot of potato, and are quarantined pathogens in Europe and Canada and select agent pathogens in the United States. We previously identified 115 500-bp r3b2-unique fragments that clustered into 32 regions in the genome of R. solanacearum. In this study, we targeted six of those regions for mutagenesis in order to determine whether they are involved in cool temperature-related biological functions. Knockout mutants of four regions produced no changes to the biology of the r3b2 strain UW551. Region 20, a relatively large 12.5 kb knockout that contains six to nine predicted genes, may contain genes involved in cell replication, particularly under cool conditions. Region 13, which is 3,407 bp in size, seems to be involved in cool virulence of the r3b2 strain UW551, since the mutant displayed significantly reduced twitching motility, attachment to the roots of tomato seedlings, and virulence under cool temperature conditions (18 – 24oC), although no significant difference was found under warm temperature conditions (24 – 30oC) as compared to the wild type strain. As a result, we designed primer pair Rs-CV-F and Rs-CV-R to target the cool virulence-related region 13 for specific detection of r3b2 strains of R. solanacearum. Our assay specifically detected all the 34 r3b2 strains and none of the 56 non-r3b2 strains of R. solanacearum, nor any other plant- or soil-associated bacteria including Enterobacter cloacae, Pseudomonas syringae pv. syringae, Xanthomonas campestris pv. campestris, X. citri, and R. pickettii. Our PCR assay is the first to specifically detect r3b2 strains of R. solanacearum by targeting a DNA region involved in cool-virulence that makes r3b2 strains highly regulated pathogens.