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

© 2020, Società Italiana di Patologia Vegetale (S.I.Pa.V.). 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.11n

Complete genome assembly of the levan-positive strain PVFi1 of Pseudomonas savastanoi pv. savastanoi isolated from olive knots in Central Italy

Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease, is a fluorescent Gram-negative bacterium classified, according to the specific LOPAT profile, as Ib. However, during the 90s, a number of atypical non-fluorescent levan-positive strains of Pseudomonas savastanoi pv. savastanoi have been unexpectedly isolated from olive knots in Central Italy. Since its first report, several studies were conducted on this species variant, but its genome sequence has never been reported. The complete genome sequence and two additional plasmids of PVFi1, a representative strain, were here obtained using a hybrid sequencing approach with both Oxford Nanopore Technology and Illumina sequencing. A thorough genomic analysis unravelled several genetic features of this peculiar strain, showing a transposase insertion downstream a fragmented copy of the levansucrase gene. The same features were previously reported on levan-negative Pseudomonas savastanoi pv. savastanoi strains. In addition, a second copy of the levansucrase gene fully equipped for a gene expression and comparable to the levan-positive Pseudomonas savastanoi pv. glycinea, may explain the levan-positive test. This result provides a solid genetic demonstration that the bacterial species Pseudomonas savastanoi contains either levan-positive or levan-negative strains, providing insights for an update of the related LOPAT classification.s