Genetic relationships among strains of Xanthomonas fragariae based on random amplified polymorphic DNA PCR, repetitive extragenic palindromic PCR, and enterobacterial repetitive intergenic consensus PCR data and generation of multiplexed PCR primers useful for the identification of this phytopathogen

Abstract

Genetic relationships among 25 isolates of Xanthomonas fragariae from diverse geographic regions were determined by three PCR methods that rely on different amplification priming strategies: random amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) PCR, and enterobacterial repetitive intergenic consensus (ERIC) PCR. The results of these assays are mutually consistent and indicate that pathogenic strains are very closely related to each other. RAPD, ERIC, and REP PCR assays identified nine, four, and two genotypes, respectively, within X. fragariae isolates. A single nonpathogenic isolate of X. fragariae was not distinguishable by these methods. The results of the PCR assays were also fully confirmed by physiological tests. There was no correlation between DNA amplification product patterns and geographic sites of isolation, suggesting that this bacterium has spread largely through exchange of infected plant germ plasm. Sequences identified through the RAPD assays were used to develop three primer pairs for standard PCR assays to identify X. fragariae. In addition, we developed a stringent multiplexed PCR assay to identify X. fragariae by simultaneously using the three independently derived sets of primers specific for pathogenic strains of the bacteria.

Long PCR-RFLP of 16S-ITS-23S rRNA genes: a high-resolution molecular tool for bacterial genotyping

“…Isolating novel bacterial cultures from this vast majority or tracing a given microbial species in the environment often requires the screening of a great number of strains. A number of DNA-based fingerprinting methodologies have been developed to facilitate such typing work, for instance, amplified fragment length polymorphism (Blears et al 1998), random amplified polymorphic DNA (Cocconcelli et al 1995), amplified ribosomal DNA restriction analysis (ARDRA) (Redecker et al 1997), ribosomal intergenic spacer analysis (Bourque et al 1995), pulsed-field gel electrophoresis (Tanskanen et al 1990) and repetitive extragenic palindromic-PCR (rep-PCR) (Pooler et al 1996).…”

Application of molecular methods for the differentiation of acetic acid bacteria in a red wine fermentation

“…ERIC and REP elements are consensus sequences derived from highly conserved palindromic inverted repeat regions found in enteric bacteria (Pooler et al 1996). However, these sequences seem to be widely distributed in the genomes of various bacterial groups.…”

“…However, these sequences seem to be widely distributed in the genomes of various bacterial groups. The amplification of the sequences between these repetitive elements has generated DNA fingerprints of several Gram-negative and Gram-positive species (Pooler et al 1996;Beyer et al 1998;Sander et al 1998;Wieser and Busse 2000;Guinebretier et al 2001).…”

“…However, these techniques revealed different polymorphism depending on the type of bacteria. Pooler et al (1996) used both techniques to characterize the genetic diversity within Xanthomonas fragariae. In this species both techniques yielded the same number of genotypes among the studied isolates.…”

“…Other PCR techniques have also been proposed for typing strains. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and Repetitive Extragenic Palindromic-PCR (REP-PCR) have, because of their high degree of polymorphism, recently been proposed as suitably accurate techniques for identifying bacterial strains and for determining taxonomic relationships between bacterial species (Versalovic et al 1991;Pooler et al 1996;Matheson et al 1997;Beyer et al 1998;Sander et al 1998;Nanda et al 2001).…”