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.
"), P. corrugata (Catara et al., 2000), P. syringae pv. pisi (Arnold et al., 1996). This method could be used to verify the efficacy of sanitation of an area and also to monitor the occurrence of the disease. "
[Show abstract][Hide abstract] 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.
"Blots were hybridized ( 65 °C, 16 h) using the labelled probes in hybridization solution (Sambrook et al., 1989), and washed to low (2% SSC, 0.1% SDS) or high (0.1% SSC, 0.1% SDS) stringency, as described by Arnold et al. (1996). "
[Show abstract][Hide abstract] ABSTRACT: SummaryvirPphA is a major determinant of the pathogenicity of Pseudomonas savastanoi pv. phaseolicola to Phaseolus bean. A family of homologues of virPphA was detected in pathovars of P . savastanoi and P . syringae . We examined the structure and activity of alleles designated virPphA , virPphAPgy , and virPphAPsv from P . savastanoi pathovars phaseolicola , glycinea , and savastanoi , respectively, and avrPtoB from P . syringae pv. tomato . Sequencing showed that the virPphAPgy homologue had a 48-bp central deletion in the open reading frame (ORF) compared with virPphA and virPphAPsv , but otherwise all three P . savastanoi alleles had > 98% identity at the DNA level. By contrast, AvrPtoB from P . syringae pv. tomato strain DC3000 was predicted to have only 51% amino acid similarity with VirPphA. All ORFs have an upstream hrp -box promoter indicating potential regulation by HrpL. Each cloned homologue was introduced into the P. savastanoi pv. phaseolicola strain RW60, which lacks a native plasmid carrying virPphA as part of a pathogenicity island (PAI), and which is not pathogenic on bean. The homologues all restored virulence, as measured by the development of water-soaked lesions in bean pods, and increased bacterial populations in leaves compared with RW60 alone. RW60 harbouring virPphA or virPphAPsv elicited a strong hypersensitive reaction (HR) in soybean cv. Osumi; the presence of avrPtoB caused a weak HR, but virPphAPgy did not affect the null reaction observed in soybean with RW60 alone. A second effector gene, avrPphD , was detected on the genomic clones carrying virPphAPgy and virPphAPsv . avrPphD was also present in both P . savastanoi pv. phaseolicola and P . syringae pv. tomato , but elsewhere in their genomes. Comparison of the genomic locations of virPphA and other effectors found in the P. savastanoi pv. phaseolicola PAI revealed the greatest divergence of the sequences surrounding virPphA to be in P . syringae pv. tomato .
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas syringae pv. phaseolicola (Pph) race 4 strain 1302A carries avirulence gene avrPphB. Strain RJ3, a sectoral variant from a 1302A culture, exhibited an extended host range in cultivars of bean and soybean resulting from the absence of avrPphB from the RJ3 chromosome. Complementation of RJ3 with avrPphB restored the race 4 phenotype. Both strains showed similar in planta growth in susceptible bean cultivars. Analysis of RJ3 indicated loss of > 40 kb of DNA surrounding avrPphB. Collinearity of the two genomes was determined for the left and right junctions of the deleted avrPphB region; the left junction is approximately 19 kb and the right junction > 20 kb from avrPphB in 1302A. Sequencing revealed that the region containing avrPphB was inserted into a tRNALYS gene, which was re-formed at the right junction in strain 1302A. A putative lysine tRNA pseudogene (PsitRNALYS) was found at the left junction of the insertion. All tRNA genes were in identical orientation in the chromosome. Genes near the left junction exhibited predicted protein homologies with gene products associated with a virulence locus of the periodontal pathogen Actinobacillus actinomycetemcomitans. Specific oligonucleotide primers that differentiate 1302A from RJ3 were designed and used to demonstrate that avrPphB was located in different regions of the chromosome in other strains of Pph. Deletion of a large region of the chromosome containing an avirulence gene represents a new route to race change in Pph.
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