Specific oligonucleotide primers for the identification of Pseudomonas syringae pv pisi yield one of two possible DNA fragments by PCR amplification: Evidence for phylogenetic divergence

Physiological and Molecular Plant Pathology (Impact Factor: 1.99). 10/1996; 49(4):233-245. DOI: 10.1006/pmpp.1996.0051

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.

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    ABSTRACT: The diploid annual legume species, Lathyrus sativus L., also known as grass pea, and L. cicera L., also known as chickling pea, are neglected species although they are widely grown as pulse crops for human food and also as grain feed or as forage in many of the harshest agro-environments of the world. The surface devoted to these two crops is increasing in Spain, where they were traditionally grown. The presence of bacterial blight among these crops was detected in fields located in the North part of the Central Spanish Meseta during the 2008–9 and 2009–10 seasons. Metabolic-biochemical tests, inoculation experiments in different hosts and molecular markers identified the causal agent as Pseudomonas syringae pv. syringae. This work represents the first description of the occurrence of P. syringae pv. syringae in Lathyrus spp. Bacteria isolated from these two plant species were also pathogenic and very aggressive in other cool season legume species. Rep-PCR patterns showed a close similarity among the P. syringae pv. syringae samples collected within each Lathyrus species, and that the bacterial isolates from Lathyrus spp. were very similar to strains of this pathogen isolated from common and hairy vetch, and from pea. Multilocus sequencing typing confirmed the similarity of one of these isolates with database P. syringae pv. syringae sequences. Most of the L. cicera landraces evaluated for resistance to P. syringae pv. syringae were susceptible or highly susceptible, but some promising resistance sources to this pathogen were found under controlled conditions.
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    ABSTRACT: Pseudomonas syringae pv. pisi is a seedborne pathogen distributed worldwide that causes pea bacterial blight. Previous characterization of this pathogen has been carried out with relatively small and/or geographically limited samples. Here, a collection of 91 strains are examined that include strains from recent outbreaks in Spain (53 strains) and from 14 other countries, and that represent all races and the new race 8, including the type race strains. This collection was characterized on the basis of 55 nutritional tests, genetic analysis (rep‐PCR, amplification of AN3 and AN7 specific markers, and multilocus sequence typing (MLST)) and pathogenicity on the differential pea cultivars to identify races. Principal component analysis and distance dendrograms confirm the existence of two genetic lineages within this pathovar, which are clearly discriminated by the AN3/AN7 markers, rep‐PCR and MLST. Strains from races 1 and 7 amplified the AN3 marker; those from races 2, 6 and 8 amplified AN7, while strains of races 3, 4 and 5 amplified either AN3 or AN7. Nevertheless, strains were not grouped by race type by any of the genetic or biochemical tests. Likewise, there was no significant association between metabolic and/or genetic profiling and the geographical origin of the strains. The Spanish collection diversity reflects the variability found in the worldwide collection, suggesting multiple introductions of the bacteria into Spain by contaminated seed lots.
    Plant Pathology 12/2012; 61(6). DOI:10.1111/j.1365-3059.2012.02604.x · 2.97 Impact Factor