Genetic Analysis of a Pathogenic Erwinia sp. Isolated from Pear in Japan.

ABSTRACT Four Erwinia strains, originally isolated in Japan from pear trees with bacterial shoot blight symptoms, were analyzed to determine their genetic relationship with Erwinia amylovora and E. pyrifoliae. When genomes were characterized with amplified fragment length polymorphism markers and by comparative groEL sequence analysis, the Japanese Erwinia sp. and South Korean E. pyrifoliae strains were placed in the same group, which was phylogenetically distinct from a group of 15 strains of E. amylovora. Sequencing of the 29,593-bp plasmid pEJ30 from Erwinia strain Ejp556 revealed that this plasmid was nearly identical to plasmid pEP36 from E. pyrifoliae and was closely related to the nontransferable ubiquitous plasmid pEA29 from E. amylovora. Twenty-one presumptive genes and their order in pEP36 were highly conserved in pEJ30; however, transposon Tn5394, which was present in pEP36, was not found in pEJ30. Short-sequence DNA repeats were conserved between pEJ30 and pEP36, and were different from short-sequence repeats in pEA29. Despite base-pair mismatches, primer pairs used in pEA29 polymerase chain reaction assays for E. amylovora amplified plasmid DNA from the Japanese Erwinia Ejp556 and Ejp562. Like E. pyrifoliae and a few strains of E. amylovora, Japanese Erwinia Ejp617 contained plasmids related to E. pyrifoliae ColE1-related plasmid pEP2.6. Based on these genetic analyses, we conclude that the Erwinia pathogen of pear in Japan is closely related to E. pyrifoliae and that both of these pathogens are demonstrably distinct from E. amylovora.

Relatedness of chromosomal and plasmid DNAs of Erwinia pyrifoliae and Erwinia amylovora.

The plant pathogen Erwinia pyrifoliae has been classified as a separate species from Erwinia amylovora based in part on differences in molecular properties. In this study, these and other molecular properties were examined for E. pyrifoliae and for additional strains of E. amylovora, including strains from brambles (Rubus spp.). The nucleotide composition of the internal transcribed spacer (ITS) region was determined for six of the seven 16S-23S rRNA operons detected in these species with a 16S rRNA gene probe. Each species contained four operons with a tRNA(Glu) gene and two with tRNA(Ile) and tRNA(Ala) genes, and analysis of the operons from five strains of E. amylovora indicated a high degree of ITS variability among them. One tRNA(Glu)-containing operon from E. pyrifoliae Ep1/96 was identical to one in E. amylovora Ea110, but three tRNA(Glu) operons and two tRNA(Ile) and tRNA(Ala) operons from E. pyrifoliae contained unique nucleotide changes. When groEL sequences were used for species-specific identification, E. pyrifoliae and E. amylovora were the closest phylogenetic relatives among a set of 12 bacterial species. The placement of E. pyrifoliae distinct from E. amylovora corroborated molecular hybridization data indicating low DNA-DNA similarity between them. Determination of the nucleotide sequence of plasmid pEP36 from E. pyrifoliae Ep1/96 revealed a number of presumptive genes that matched genes previously found in pEA29 from E. amylovora and similar organization for the genes and origins of replication. Also, pEP36 and pEA29 were incompatible with clones containing the reciprocal origin regions. Finally, the ColE1-like plasmid pEP2.6 from strain Ep1/96 contained sequences found in small plasmids in E. amylovora strains IL-5 and IH3-1.

Effect of Treating Apple Trees with Acibenzolar-S-Methyl on Fire Blight and Expression of Pathogenesis-Related Protein Genes.

Acibenzolar-S-methyl (ASM, Actigard 50 WG), a synthetic inducer of systemic acquired resistance (SAR) and pathogenesis-related (PR) proteins, was evaluated for the control of fire blight on apple trees in the field and for PR protein gene expression in apple seedlings in the greenhouse. Expression of putative genes related to SAR induction was elevated in ASM-treated apple seedlings 2 to 7 days after treatment; levels of PR-1 and PR-8 RNA were increased 10-fold and PR-2 RNA was increased 100-fold in ASM-treated seedlings over levels in untreated seedlings. Spurs and shoots on Jonathan trees sprayed with ASM at 75 mg a.i./liter at pink and at weekly intervals thereafter had significantly less fire blight than untreated trees, both from natural infection and following artificial inoculation with the pathogen. Less fire blight was observed on trees sprayed weekly with ASM than on trees sprayed biweekly. The severity of fire blight on inoculated shoots of Fuji apple decreased with increasing rates of ASM (0 to 300 mg a.i./liter), and ASM combined with streptomycin showed enhanced activity over ASM at 75 mg a.i./liter alone. In general, ASM was not superior to streptomycin for fire blight control, but integrating a weekly schedule of ASM, preferably at 150 mg a.i./liter, with a schedule of streptomycin designed for blossom blight control appears promising for overall improvement in fire blight control.