Exploring the diversity of Ralstonia solanacearum strains from mainland France using a novel multiple loci VNTR analysis scheme

The Ralstonia solanacearum species complex (RSSC), one of the most damaging pests worldwide, includes species Ralstonia solanacearum , Ralstonia pseudosolanacearum , and Ralstonia syzygii . Since 1990, R. solanacearum has been reported in several European countries, including France. However, the genetic diversity of R. solanacearum in mainland France remains poorly characterized. In this study, a novel 14-variable number of tandem repeats multilocus variable-number tandem-repeat analysis (MLVA) scheme specifically designed for IIB-1 strains of R. solanacearum is presented. This scheme effectively differentiated 384 global strains, isolated between 1963 and 2023, into 66 distinct haplotypes. The near clonality of the IIB-1 genetic group was highlighted by the presence of a predominant clonal complex encompassing 82% of the haplotypes. The 346 strains from mainland France used in this study, isolated between 1994 and 2023, were differentiated into 46 haplotypes, which suggests that, while French R. solanacearum IIB-1 strains have been isolated from a relatively confined geographical area over time, they still exhibit some level of genetic diversity. Moreover, this MLVA scheme enabled clear discrimination between global and French/European strains which did not cluster together, allowing the inference of potential invasion routes for certain haplotypes in Europe. This MLVA scheme is thus a valuable tool for epidemiological studies, providing deeper insights into the genetic diversity of global IIB-1 strains. IMPORTANCE The RSSC causes bacterial wilt on over 250 plant species, including major ones like potato, and includes species R. solanacearum , R. pseudosolanacearum , and R. syzygii , all characterized as quarantine organisms in the European Union. R. solanacearum has been reported in several European countries over the last century with major outbreaks confirmed to be IIB-1 strains in the 1990s, and recent outbreaks of R. pseudosolanacearum raised concerns about the emergence of additional RSSC species in Europe. Understanding the genetic diversity of RSSC strains present in mainland France, along with the structure and the dynamics of their population, is critical for maintaining effective epidemiological surveillance. In particular, MLVA is known to be a suitable molecular typing tool to trace bacterial introduction pathways, monitor pathogen spread at multiple geographical scales, and assess evolutionary potential. The insights provided by our study, with the development of a new MLVA scheme, are key to maintaining robust plant health surveillance and implementing targeted, evidence-based management strategies.