BOX-PCR-based identification of bacterial species belonging to Pseudomonas syringae: P. viridiflava group

Abstract

The phenotypic characteristics and genetic fingerprints of a collection of 120 bacterial strains, belonging to Pseudomonas syringae sensu lato group, P. viridiflava and reference bacteria were evaluated, with the aim of species identification. The numerical analysis of 119 nutritional characteristics did not show patterns that would help with identification. Regarding the genetic fingerprinting, the results of the present study supported the observation that BOX-PCR seems to be able to identify bacterial strains at species level. After numerical analyses of the bar-codes, all pathovars belonging to each one of the nine described genomospecies were clustered together at a distance of 0.72, and could be separated at genomic species level. Two P. syringae strains of unknown pathovars (CFBP 3650 and CFBP 3662) and the three P. syringae pv. actinidiae strains were grouped in two extra clusters and might eventually constitute two new species. This genomic species clustering was particularly evident for genomospecies 4, which gathered P. syringae pvs. atropurpurea, coronafaciens, garçae, oryzae, porri, striafaciens, and zizaniae at a noticeably low distance

BOX-PCR is an adequate tool for typing of clinical <i>Pseudomonas aeruginosa</i> isolates

“…In contrast to PFGE, the highly specific rep-PCR technique is an easy procedure, requiring little time for results, and is characterized by low labor costs [6][7][8] [8]. Recently, the BOX-PCR method was used for typing strains of different genera of Pseudomonas (Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas putida and Pseudomonas fluorescens) [6,7,[9][10][11].…”

Molecular characterization of Pseudomonas syringae pvs. from different host plants by repetitive sequence-based PCR and multiplex-PCR

“…rep-PCR analysis was performed using two primer sets for REP (REP IR-I/REP 2-I) and ERIC (ERIC1R/ ERIC2) and two primers for BOX-PCR (BOX A1R and (GTG) 5 ), as recommended by Marques et al (2008) and Gilbert et al (2009 of template DNA. The amplification conditions for REP-PCR consisted of an initial denaturation at 95 for 4 min, followed by 30 cycles (denaturation at 94°C for 55 s, primer annealing at 42°C for 65 s and primer extension at 65°C for 7 min) and a final extension at 65°C for 12 min.…”

“…syringae strains originating from different and the same hosts (Natalini et al, 2006;Gilbert et al, 2009;Kaluzna et al, 2010;Ivanović et al, 2012). Repetitive element sequence-based PCR (rep-PCR) is based on three main sets of repetitive DNA elements: the repetitive extragenic palindromic (REP) elements (REP-PCR), the enterobacterial repetitive intergenic consensus (ERIC) sequences (ERIC-PCR) and BOX elements (BOX-PCR), and this method is successful in typing a variety of bacteria, as well as P. syringae (Vicente, Roberts, 2007;Marques et al, 2008;Kaluzna et al, 2010). The aim of this study was to characterize and compare P. syringae pvs.…”

Genotyping Techniques for Determining the Diversity of Microorganisms

“…In addition to studying diversity, rep-PCR genomic fingerprinting has become a valuable tool for the identification and classification of bacteria, and for molecular epidemiological studies of human and plant pathogens (Louws et al, 1996;Versalovic et al, 1997). It has been applied in the classification and differentiation of strains of many Gram-positive and -negative bacteria including Bartonella (Rodriguez-Barrados et al, 1995), Bacillus subtilis (Pinna et al, 2001), B. sporothermodurans (Herman & Heyndrickx, 2000), E. coli (Leung et al, 2004;Panutdaporn et al, 2004;Silveira et al, 2003), Citrobacter diversus , Enterobacter aerogenes , Salmonella (Chmielewski et al, 2002;Kerouanton et al, 1996;Millemann et al, 1996;Rasschaert et al, 2005), Vibrio cholerae (Colombo et al, 1997;Rivera et al, 1995), Pseudomonas corrugata (Achouak et al, 2000), Vibrio parahaemolyticus (Khan et al, 2002;Son et al, 1998), Pseudomonas syringae-Pseudomonas viridiflava group (Marques et al, 2008), Aeromonas spp. (Taco et al, 2005), Xanthomonas (Rademaker et al, 2000), Rhizobium meliloti (De Bruijn, 1992;Niemann et al, 1997), Pandoraea apista (Atkinson et al, 2006), methicillin-resistant S. aureus (Van Belkum et al, 1992), S. pneumoniae , A. baumanii (Dijkshoorn et al, 1996), Burkholderia cepacia , B. pseudomallei (Currie et al, 2007), L. pneumophilia (Georghiou et al, 1994), Helicobacter pylori (Kwon et al, 1998), N. gonorrhoeae (Poh et al, 1996), N. meningitidis (Woods et al, 1996), Enterococcus spp.…”