Specific oligonucleotide primers for the identification ofPseudomonas syringaepv.pisiyield one of two possible DNA fragments by PCR amplification: evidence for phylogenetic divergence
ABSTRACT Two unique DNA fragments, generated by RAPD-PCR, were used as probes against dot-blots of representative isolates of the seven races ofPseudomonas syringaepv.pisi. DNA from each isolate hybridized only to one of the two probes. Fragments identified from isolates 1691 (race 7) and 203 (race 2), were cloned into pUC18 and sequenced. The resulting sequences were used to design two pairs of oligonucleotide primers which when used in PCR reactions withP. syringaepv.pisicells gave specific amplification products (either a 272 bp or a 132 bp fragment) corresponding to the original cloned fragments. When all four primers were used in combination with specific DNA amplification reactions in 51 isolates ofP. syringaepv.pisithey produced one of the two PCR bands. No bands were detected in a range of closely relatedP. syringaepathovars following PCR amplification. These results suggest thatP. syringaepv.pisican be unambiguously identified using specific oligonucleotide primers and that isolates can be classified into two phylogenetic groups, I and II.
- SourceAvailable from: Jose Antonio Gutiérrez BarranqueroPhytopathology 01/2013; · 2.97 Impact Factor
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ABSTRACT: Genetic diversity among 43 petroleum hydrocarbon-degrading Pseudomonas belonging to four different species and the type strain Pseudomonas aeruginosa MTCC1034 was assessed by using restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified 16S-23S rDNA intergenic spacer regions (ISRs) polymorphism. PCR amplification from all Pseudomonas species yielded almost identical ISR amplicons of "?" 800 bp and in nested PCR of "?" 550 bp. The RFLP analysis with MboI and AluI revealed considerable intraspecific variations within the Pseudomonas species. The dendrogram constructed on the basis of the PCR-RFLP patterns of 16S-23S rDNA intergenic spacer regions differentiated all the species into seven different clusters.Folia Microbiologica 12/2011; 57(1):47-52. · 0.79 Impact Factor
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ABSTRACT: A 1 kb DNA band from strains of Brenneria nigrifluens, as shown by amplification of their genomic DNA by polymerase chain reaction (PCR) using minisatellite primer designed on the minisatellite sequence of the M13 phage, was isolated, cloned and sequenced. Specific oligonucleotides (F1–C3) were selected into this 1 kb DNA sequence and used in a PCR assay to detect and identify strains of B. nigrifluens. Several strains of B. nigrifluens were assessed with F1–C3 primers producing a specific band of approximately 250 bp pairs in length. This target was successfully amplified from purified genomic DNA, from bacterial culture and directly from infected walnut bark tissue. No amplification was obtained when the PCR assay was performed on other plant-pathogenic species from the following genera Brenneria, Erwinia, Agrobacterium, Pseudomonas, Ralstonia, Pectobacterium, Xanthomonas and from walnut-associated bacteria, indicating the specificity of these primers. The PCR assay with the primers described here provides a rapid, specific and sensitive diagnostic method for B. nigrifluens and a useful tool for epidemiological studies.Journal of Phytopathology 04/2008; 156(7‐8):464 - 469. · 1.00 Impact Factor