Polymerase chain reaction fingerprinting of Erwinia amylovora has a limited phylogenetic value but allows the design of highly specific molecular markers.

Erwinia amylovora, the causal agent of fire blight, is genetically very homogeneous, and current methodologies provide insufficient or contradictory information about the probable dispersal routes of the pathogen. With the final aim to obtain specific and reliable molecular markers for different lineages of the pathogen, we studied the molecular basis of rep-polymerase chain reaction (PCR) polymorphism using seven different arbitrary primers to fingerprint 93 E. amylovora strains from different countries, including Spain. Polymorphism was very low, and was displayed by only 11 E. amylovora strains, which produced 22 polymorphic bands. Five of 11 polymorphic bands cloned contained DNA that was present in more than 85% of the strains, whereas six bands were due to DNA present exclusively in the strains producing the rep-PCR polymorphism. Also, five of the polymorphic bands were due to the possession of either the ubiquitous plasmid pEA29, of plasmid pEU30, which was exclusively found in strains from North America, or of a 35-kb cryptic plasmid, present only in 28 strains from Northern Spain. We designed primer pairs from several cloned polymorphic bands that allowed the specific identification of the strains producing the polymorphism. Our results indicate that rep-PCR is not adequate for constructing genealogies of E. amylovora, although the strategy illustrated here, as well as the designed primers, can be used effectively in epidemiological studies with this pathogen.

Location and activity of members of a family of virPphA homologues in pathovars of Pseudomonas syringae and P. savastanoi.

Summary virPphA 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, virPphA(Pgy), and virPphA(Psv) from P. savastanoi pathovars phaseolicola, glycinea, and savastanoi, respectively, and avrPtoB from P. syringae pv. tomato. Sequencing showed that the virPphA(Pgy) homologue had a 48-bp central deletion in the open reading frame (ORF) compared with virPphA and virPphA(Psv), 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 virPphA(Psv) elicited a strong hypersensitive reaction (HR) in soybean cv. Osumi; the presence of avrPtoB caused a weak HR, but virPphA(Pgy) did not affect the null reaction observed in soybean with RW60 alone. A second effector gene, avrPphD, was detected on the genomic clones carrying virPphA(Pgy) and virPphA(Psv). 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.