Variation in extragenic repetitive DNA sequences in Pseudomonas syringae and potential use of modified REP primers in the identification of closely related isolates

Abstract

In this study, Pseudomonas syringe pathovars isolated from olive, tomato and bean were identified by species-specific PCR and their genetic diversity was assessed by repetitive extragenic palindromic (REP)-PCR. Reverse universal primers for REP-PCR were designed by using the bases of A, T, G or C at the positions of 1, 4 and 11 to identify additional polymorphism in the banding patterns. Binding of the primers to different annealing sites in the genome revealed additional fingerprint patterns in eight isolates of P. savastanoi pv. savastanoi and two isolates of P. syringae pv. tomato . The use of four different bases in the primer sequences did not affect the PCR reproducibility and was very efficient in revealing intra-pathovar diversity, particularly in P. savastanoi pv. savastanoi . At the pathovar level, the primer BOX1AR yielded shared fragments, in addition to five bands that discriminated among the pathovars P. syringae pv. phaseolicola , P. savastanoi pv. savastanoi and P. syringae pv. tomato . REP-PCR with a modified primer containing C produced identical bands among the isolates in a pathovar but separated three pathovars more distinctly than four other primers. Although REP- and BOX-PCRs have been successfully used in the molecular identification of Pseudomonas isolates from Turkish flora, a PCR based on inter-enterobacterial repetitive intergenic concensus (ERIC) sequences failed to produce clear banding patterns in this study.

Bacterial halo blight of coffee crop: aggressiveness and genetic diversity of strains

“…Isolates from the same pathovar may have identical REP, BoX and ERIC fingerprints or they may differ in some banding patterns (Louws et al 1994). Çepni and Gürel (2012) found that ERIC-PCR could not differentiate strains of P. savastanoi pv. savastanoi from strains of P. syringae.…”

“…juglandis (Scortichini et al 2001), P. syringae pv. syringae (natalini et al 2006) and studies of inter-and intraspecificity in strains of P. syringae (Vicente and Roberts 2007;Çepni and Gürel 2012;Gašić et al 2012).…”

Isolation and characterization of Pseudomonas syringae pv. syringae which induce leaf spot on walnut

“…ERIC and BOX profiles have been used successfully to characterize a large number of bacteria and they differentiate among closely related strains (Versalovic et al 1991(Versalovic et al , 1994. These fingerprinting techniques generated characteristic profiles that can be used to differentiate pathogen isolates of Pseudomonas from different species and pathovars and even from the same pathovar (Weingart and Volksch 1997;Little et al 1998;Joana et al 2007;Marques et al 2008;Gašić et al 2012;Kaluzna et al 2010;Çepni and Gürel 2012;Abbasi et al 2013). In this work we confirmed that strains from the same pathovar are differentiated by ERIC and BOX fingerprinting technique since different patterns were obtained for strains causing walnut seedling leaf spot isolated in Hamedan province and strains isolated from Tuyserkan, while the strains of the same region showed identical pattern.…”

Identification and Characterization of Pseudomonas syringae pv. syringae, a Causative Bacterium of Apple Canker in Korea

“…Based on the ERIC- and REP-PCR amplifications, strains belonging to Pss appeared as the most heterogeneous to the host plant and their origin ( Kaluzna et al, 2010 ; Scortichini et al, 2003 ). The discriminatory potential of REP-PCR for different P. syringae pathovars have been shown in previous reports ( Kaluzna et al, 2010 ; Vicente and Roberts, 2007 ), and another study by Çepni and Gürel (2012) examined a limited number of strains that use modified REP-PCR primers to detect intra-pathovar diversity. Tobes and Pareja (2006) reported that the P. syringae genome contains high copy numbers of REP elements that are related to insertion sequence elements.…”