A novel multilocus variable number tandem repeat analysis typing scheme for African phylotype III strains of the Ralstonia solanacearum species complex

Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used. Results and Discussion. Sixteen polymorphic TR loci, which included seven microsatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more discriminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC phylotype III.