A novel MLVA assay to genotype Pseudomonas savastanoi strainsat inter- and intra-pathovar level

Abstract: Pseudomonas savastanoi is a bacterial species subtyped in several pathovars thatare casual agents of olive knot disease on olive and oleander, and bacterial canker on ash. AnMLVA approach was developed to assess genetic diversity between and within thesepathovars. Analysing the genome of P. savastanoi pv. savastanoi strain NCPPB 3335(accession n. CP008742), 20 Tandem Repeat (TR) loci were selected, corresponding primerswere designed and used for the amplification of genomic DNA from 54 strains belonging tothe pathovars savastanoi, nerii, and fraxini. Then, capillary electrophoresis was used toexactly estimate the dimension of the amplicons and, consequently, the TR numbers per eachlocus in each strain. Due to amplification failure, production of multiple amplicons, or lowreproducibility, 5 loci were discarded and the remaining 15 were used for the final MLVAanalysis. Genetic relationship between the strains was assessed in relation to pathovarclassification and to the geographic and time origin of the individuals. The assay represents apromising tool for the identification and tracking of P. savastanoi populations.