Molecular Epidemiology of Xanthomonas euvesicatoria Strains from the Balkan Peninsula Revealed by a New Multiple-Locus Variable-Number Tandem-Repeat Analysis Scheme

Bacterial spot of pepper and tomato is caused by at least three species of Xanthomonas, among them two pathovars of Xanthomonas euvesicatoria, which are responsible for significant yield losses on all continents. In order to trace back the spread of bacterial spot pathogens within and among countries, we developed the first multilocus variable number of tandem repeat analyses (MLVA) scheme for pepper- and tomato-pathogenic strains of X. euvesicatoria. In this work, we assessed the repeat numbers by DNA sequencing of 16 tandem repeat loci and applied this new tool to analyse a representative set of 88 X. euvesicatoria pepper strains from Bulgaria and North Macedonia. The MLVA-16 scheme resulted in a Hunter–Gaston Discriminatory Index (HGDI) score of 0.944 and allowed to resolve 36 MLVA haplotypes (MTs), thus demonstrating its suitability for high-resolution molecular typing. Strains from the different regions of Bulgaria and North Macedonia were found to be widespread in genetically distant clonal complexes or singletons. Sequence types of the variable number of tandem repeats (VNTR) amplicons revealed cases of size homoplasy and suggested the coexistence of different populations and different introduction events. The large geographical distribution of MTs and the existence of epidemiologically closely related strains in different regions and countries suggest long dispersal of strains on pepper in this area.