Genetic diversity and population structure of Pseudomonas savastanoi, an endemic pathogen of the Mediterranean area, revealed up to strain level by the MLVA assay

Pseudomonas savastanoi is a bacterial species included in the Pseudomonas syringae complex. It is further sub-typed in pathovars which are the causal agents of a group of diseases of woody plants, such as the “knot disease” on olive and oleander and the bacterial canker on ash. Given its long-established presence in the Mediterranean area, the pathogen causing the afore-mentioned diseases can be considered endemic. Here, an MLVA approach was developed to assess the genetic relationships among and within those pathovars, with a specific focus on P. savastanoi pv. savastanoi. By analyzing the genome of the P.savastanoi pv. savastanoi strain NCPPB 3335 (accession n° CP008742), 14 Tandem Repeat (TR) loci were identified and the corresponding primers were designed and used for the amplification of genomic DNAs from 84 strains belonging to Pseudomonas savastanoi pathovars. Data were analyzed using different approaches, such as hierarchical clustering, STRUCTURE, and k-means clustering with DAPC to evaluate the effectiveness of the assay in describing pathovars and population structure of the pathogen. Results reveal a very complex articulation of genetic relationships, as expected from a long-time evolving pathogen, while providing the possibility to discriminate the pathovars between each other. At intra-pathovar level, the MLVA assay clusters isolates mainly according to their hosts and geographic origin. This resulted particularly useful in the identification and tracking of P. savastanoi populations at local level.