Specific oligonucleotide primers for the identification of pv. yield one of two possible DNA fragments by PCR amplification: evidence for phylogenetic divergence

Arnold, Douglas L.; Athey-Pollard, AL; Gibbon, Marjorie J.; Taylor, J. Paul; Vivian, Alan

doi:10.1006/pmpp.1996.0051

Cited by 36 publications

(40 citation statements)

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“…This indicated that the two races could not be derived one from another by a simple genomic rearrangement. This conclusion was further confirmed by Arnold et al (1996) , who developed two pairs of specific oligonucleotide primers for the amplification of distinct and unrelated bands in each of these two races. The latter authors showed that strains of Ppi could be separated on the basis of these primer-0002-2762 0 1999 SGM Mansfield (1993) This study specific amplifications into two phylogenetic groups, designated I (containing races 1, 4B, 5, 7 and some nonfluorescent strains of race 3, now regarded as belonging to race 3B) and I1 (containing races 2, 4A, 6 and fluorescent strains of race 3, now regarded as race 3A).…”

Section: Introductionmentioning

confidence: 71%

A dispensable region of the chromosome which is associated with an avirulence gene in Pseudomonas syringae pv. pisi

Arnold

Brown²,

Jackson³

et al. 1999

Microbiology

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Lane, Bristol 8516 IQY, UK Pseudomonas syringae pv. pisi comprises a number of races which fall into two phylogenetically distinct groups (designated I and 11). Races are based on cultivar specificity in the host plant, pea (Pisum sativum), and are specified by the presence of avirulence genes. The avirulence gene avrPpiAl is present on the chromosome of all strains examined in race 2, which belongs to phylogenetic group II. A race 4B strain, from phylogenetic group I , lacks this avirulence gene and a comparative study was made of the chromosome in strains representing these two races. A race 2 cosmid clone (pAV270) carrying avrPpiA1 was used as a basis for collinearity analysis of races 2 and 4B. A region of the chromosome amounting to 8 5 kb and including avrPpiA1 was absent from race 46 compared with race 2. A fragment spanning the junction of the discontinuity in race 4B was isolated, cloned and used to delimit the extent of the additional DNA present in race 2. In both races the borders of the discontinuity contained DNA sequences which showed a high degree of conservation. A 7 bp slightly imperfect direct repeat (CCAGCT/,T) flanked the additional DNA in race 2, with a single copy in race 46. The region flanking the additional DNA was present in all races of P. syringae pv. phi. These results confirm the phylogenetic groupings in P. syringae pv. pisi.

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Section: Introductionmentioning

confidence: 71%

A dispensable region of the chromosome which is associated with an avirulence gene in Pseudomonas syringae pv. pisi

Arnold

Brown²,

Jackson³

et al. 1999

Microbiology

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“…Restricted͞amplified DNA was radiolabeled by using a High Prime radiolabeling kit (Boeringher Mannheim), and pAV511 was radiolabeled (1 h) in agarose by using Ready-to-Go beads (Amersham Pharmacia). Blots were hybridized (65°C, 16 h) with the labeled probes in hybridization solution (33), and then washed to high stringency (34).…”

Section: Methodsmentioning

confidence: 99%

Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogenPseudomonas syringaepathovar phaseolicola

Jackson

Athanassopoulos

Tsiamis

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

302

253

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The 154-kb plasmid was cured from race 7 strain 1449B of the phytopathogen Pseudomonas syringae pv. phaseolicola (Pph). Cured strains lost virulence toward bean, causing the hypersensitive reaction in previously susceptible cultivars. Restoration of virulence was achieved by complementation with cosmid clones spanning a 30-kb region of the plasmid that contained previously identified avirulence (avr) genes avrD, avrPphC, and avrPphF. Single transposon insertions at multiple sites (including one located in avrPphF) abolished restoration of virulence by genomic clones. Sequencing 11 kb of the complementing region identified three potential virulence (vir) genes that were predicted to encode hydrophilic proteins and shared the hrp-box promoter motif indicating regulation by HrpL. One gene achieved partial restoration of virulence when cloned on its own and therefore was designated virPphA as the first (A) gene from Pph to be identified for virulence function. In soybean, virPphA acted as an avr gene controlling expression of a rapid cultivar-specific hypersensitive reaction. Sequencing also revealed the presence of homologs of the insertion sequence IS100 from Yersinia and transposase Tn501 from P. aeruginosa. The proximity of several avr and vir genes together with mobile elements, as well as G؉C content significantly lower than that expected for P. syringae, indicates that we have located a plasmidborne pathogenicity island equivalent to those found in mammalian pathogens.Varietal resistance to halo-blight disease of bean (Phaseolus vulgaris L.) caused by Pseudomonas syringae pv. phaseolicola (Pph) is determined by gene-for-gene interactions involving five resistance (R) genes in the host and five matching avirulence (avr) genes in the pathogen. Depending on the presence or absence of functional avr genes, nine races of Pph have been distinguished (1, 2). The avr genes matching R1, R2, and R3 have been cloned and sequenced. Their full designations are avrPphF.R1, avrPphE.R2, and avrPphB.R3; the terminal R gene designation will not be used here (3-5). Both avrPphE and avrPphB are chromosomal, whereas avrPphF is located on a large plasmid in those races that cause the hypersensitive reaction (HR) in cultivars of bean with the matching R1 gene.

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“…Molecular markers have been used to identify P. syringae pathovars and races pathogenic on legume and other plant species (Arnold et al 1996;Sorensen et al 1998;González et al 2003;Scortichini et al 2003) and the advantages and limitations of molecular characterization in identification of plant pathogenic bacteria have been reviewed (Alvarez 2004). The identification of Psy and Ppi was first carried out on the basis of biochemical characteristics.…”

Section: Discussionmentioning

confidence: 99%

“…In order to ascertain whether the high frequency of homoserine positive Psy isolates and the high frequency of Psy as a causal agent of pea disease in North-Central Spain are associated, further tests will be necessary. Arnold et al (1996) described the AN3 and AN7 markers obtained from RAPD markers as specific for Ppi. Among our isolates, the AN3 marker (132 pb, see Table 1) was amplified from races 3, 4, and 5, while AN7 (272 pb) was amplified from races 2, 4, 6, and the putative new race 8, but from none of the Psy isolates or from any reference strain of other Pseudomonas species.…”

Section: Discussionmentioning

confidence: 99%

“…Polymerase chain reactions (PCR) were carried out using a primer set specific for the AN3 and AN7 markers of Pseudomonas syringae pv. pisi (Arnold et al 1996) (Table 1) in a thermal cycler (Perkin Elmer 9600) using the following procedure: initial strand separation at 95°C (15 min), 40 cycles of 72°C (1 min), 94°C (30 s), 60°C (1 min), final extension at 72°C (5 min). The amplifications were carried out in a volume of 25 μl containing 10 ρM of each primer, 1X Buffer II AmpliTaq Gold (Applied Biosystems), 0.2 mM of each dNTP, 1.5 mM of MgCl 2 and 1U AmpliTaq Gold polymerase.…”

Section: Molecular Identificationmentioning

confidence: 99%

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Identification of pathovars and races of Pseudomonas syringae, the main causal agent of bacterial disease in pea in North-Central Spain, and the search for disease resistance

et al. 2010

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A total of 298 bacterial isolates were collected from pea cultivars, landraces and breeding lines in North-Central Spain over several years. On the basis of biochemical-physiological characteristics and molecular markers, 225 of the isolates were identified as Pseudomonas syringae, either pv. pisi (110 isolates) or pv. syringae (112), indicating that pv. syringae is as frequent as pv. pisi as causal agent of bacterial diseases in pea. Most strains (222) were pathogenic on pea. Further race analyses of P. syringae pv. pisi strains identified race 4 (59.1% of the isolates of this pathovar), race 2 (20.0%), race 6 (11.8%), race 5 (3.6%) and race 3 (0.9%). Five isolates (4.6%) showed a not-previously described response pattern on tester pea genotypes, which suggests that an additional race 8 could be present in P. syringae pv. pisi. All the isolates of P. syringae pv. syringae were highly pathogenic when inoculated in the tester pea genotypes, and no significant pathogenic differences were observed. Simultaneous infections with P. syringae pv. pisi and pv. syringae in the same fields were observed, suggesting the importance of resistance to both pathovars in future commercial cultivars. The search for resistance among pea genotypes suitable for production in this part of Spain or as breeding material identified the presence of resistance genes for all P. syringae pv. pisi races except for race 6. The pea cultivars Kelvendon Wonder, Cherokee, Isard, Iceberg, Messire and Attika were found suitable sources of resistance to P. syringae pv. syringae.

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Specific oligonucleotide primers for the identification of pv. yield one of two possible DNA fragments by PCR amplification: evidence for phylogenetic divergence

Cited by 36 publications

References 0 publications

A dispensable region of the chromosome which is associated with an avirulence gene in Pseudomonas syringae pv. pisi

A dispensable region of the chromosome which is associated with an avirulence gene in Pseudomonas syringae pv. pisi

Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogenPseudomonas syringaepathovar phaseolicola

Identification of pathovars and races of Pseudomonas syringae, the main causal agent of bacterial disease in pea in North-Central Spain, and the search for disease resistance

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