Distribution of streptomycin-resistant strainsof Erwinia amylovora in Israel and occurrence of blossom blight in the autumn

Following failure in control of fire blight with streptomycin, the distribution of streptomycin-resistant strains ofErwinia amylovora in Israel was surveyed. During 1994–1997 109 pear, apple, loquat and quince orchards were monitored. Streptomycin-resistant strains ofE. amylovora were recovered from flowers and from infected branches collected at 18 locations in the Sharon, Galilee and Golan Heights regions. In the Sharon region all the isolated strains ofE. amylovora were streptomycin-resistant, whereas in the Galilee and Golan Heights, resistant as well as sensitiveE. amylovora strains were recovered at different locations. In the southern coastal plain no resistance could be detected. Streptomycin-resistant strains ofE. amylovora did not hybridize with the DNA probe SMP3, and resistance could not be transferred by mating to a sensitive strain, suggesting that streptomycin resistance in Israel is not plasmid-mediated. Fire blight symptoms were observed, for the first time, on pear blossoms during the autumn of 1994. A high population of 2x 10⁶-6x 10⁷ CFU/flower in the autumn of 1995 and of 1996 was correlated with the appearance of blossom blight symptoms.     

Characterisation of naturally occurring <Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains lacking the common plasmid pEA29 and their detection with real-time PCR

Erwinia amylovora, the causal agent of fire blight, carries the common plasmid pEA29 of 29 kb. To screen for occurrence of natural strains without plasmid pEA29, we applied PCR analysis with primers from the plasmid and the chromosomal ams region. In addition, a described TaqMan probe from pEA29 and newly designed primers from the ams-region were used for identification by real-time PCR. One strain isolated in Iran, one strain from Spain and two strains from Egypt lacked plasmid pEA29. From a recent screening series in southern Germany, in 123 E. amylovora strains from necrotic fire blight host plants, one strain was found without the common plasmid. The strains without pEA29 were virulent in assays with immature pears and on apple seedlings, but showed a reduced growth level in minimal medium without amino acids and thiamine. Transposon-labelled pEA29 was transformed into the plasmid-free strains resulting in restoration of this growth deficiency. The plasmid was stably maintained in these E. amylovora cells. The newly designed chromosomal primers for conventional and for real-time PCR identified E. amylovora strains in field samples lacking pEA29. These variants are apparently rare, but were detected in isolates from different regions in the world with fire blight.     

Erwinia amylovora populations resistant to oxolinic acid in Israel: prevalence, persistence and fitness

Oxolinic acid (OA) has been the only bactericide used against fire blight in pear and quince orchards in Israel since 1998. OA-resistant Erwinia amylovora strains (Ea-OA^R) were detected in several orchards in two restricted areas in the northern Galilee region during 1999–2001. In the following years, resistant strains could not be detected in some of these locations. Documenting the fate of Ea-OA^R strains in commercial orchards at eight sites in northern Israel during 2000/03 revealed that the resistant population appeared irrespective of the number of sprays applied and the severity of the disease. The persistence of the Ea-OA^R populations varied from site to site, ranging from 4 to 20 months; these differences could be attributed to the fire blight management activities of growers. Comparative studies on the fitness of Ea-OA^R and E. amylovora strains sensitive to OA (Ea-OA^S) were conducted in vitro and in planta using two strains of each group. In four of the six comparisons, disease incidence on detached blossoms inoculated with Ea-OA^S was significantly higher than that on blossoms inoculated with Ea-OA^R. In two experiments conducted on 8-year-old pear trees grown under netting, the colonization of Ea-OA^S in blossoms, annual shoots and perennial spurs was significantly higher than that of the Ea-OA^R. In two experiments conducted on 2-year-old trees grown under netting in an experimental station, the incidence of shoots exhibiting fire blight symptoms and the rate of symptom progress within the branches were significantly higher in trees inoculated with Ea-OA^S than in those inoculated with Ea-OA^R. The results of this study suggest that OA-resistant E. amylovora strains have lower fitness than wild-type strains. These findings may have implications for fire blight management.     

Fire blight of pears in Israel: infection, prevalence, intensity and efficacy of management actions

The pear production area in Israel is 1500 ha, most of which(ca 1200 ha) is located in the northern part of the country. Fire blight (caused by the bacteriumErwinia amylovora (Burrill) Winslowet al.) was first observed in Israel in that region (in 1985) and the disease has prevailed there since then. In a comprehensive survey conducted in Israel in 1996–1999, data were collected and observations were made yearly in one-third to one-half of the pear production area. The aim was to document the prevalence and intensity of fire blight in commercial orchards and to use the data to evaluate the efficacy of management measures employed for its suppression. Regionwise, a severe fire blight epidemic developed in 1996, moderate epidemics developed in 1998 and 1999, and a mild epidemic developed in 1997. The intensity of fire blight in the preceding season in a specific orchard was more influential on current season severity in a season with a mild epidemic than in a season with a moderate epidemic. Analysis of disease onset records and weather data revealed that only a few (1– 3) infection episodes occurred in individual orchards each year. Comparison of fire blight intensity in orchard-plots treated before green tip with copper hydroxide with nontreated plots revealed that the treatment had no effect on disease intensity during bloom. The efficacy of bactericide sprays applied during bloom was not related to the number of sprays applied but to the timing of spraying. Adequate control was achieved in orchard-plots sprayed soon before or after the occurrence of infection episodes. Contribution no. 508/00 from the Inst. of Plant Protection, ARO, Bet Dagan, Israel.     

Use of a diagnostic medium for<Emphasis Type="Italic">in situ</Emphasis> determination of the response of<Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains to bactericides

Erwinia amylovora, the causal agent of fire blight, is managed by application of bactericides to protect fruit tree blossoms from infection. Monitoring the response ofE. amylovora strains to bactericides is crucial for adequate disease management. The coliform agar medium produced by Merck was recently reported as an effective tool for rapid diagnosis ofE. amylovora (RD-medium). The objective of the present study was to examine the possibility of using the RD-medium forin situ determination of the response ofE. amylovora strains to oxolinic acid and streptomycin. The phenotypic response of 48E. amylovora strains isolated in 2002 to both bactericides was determined with the RD-medium and, for comparison, by a routine laboratory test. The results of 45 samples (93.7%) were in agreement with the findings of the routine laboratory test. Aχ ² test rejected the null hypothesis that the phenotypic characteristics as determined by the two respective methods differed significantly (P=0.389). Thein situ test was implemented on a national scale in 2003 and the results were in agreement with those obtained in laboratory tests, which suggests that this medium can be usedin situ for monitoring the appearance of resistance inE. amylovora populations. http://www.phytoparasitica.org posting Feb. 11, 2004.     

Application of RFLP analysis of recA, gyrA and rpoS gene fragments for rapid differentiation of Erwinia amylovora from Erwinia strains isolated in Korea and Japan

Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified fragments of recA, gyrA and rpoS genes was applied for the characterization of Erwinia amylovora and Erwinia strains, which cause fire blight and Asian pear blight in orchards. Primers, constructed on the basis of the published recA, gyrA and rpoS gene sequences of Erwinia carotovora, allowed us to amplify DNA fragments for RFLP differentiation of E. amylovora and E. pyrifoliae and finally to distinguish strains within these species and relate them to pear pathogens from Japan. Three to seven restriction endonucleases were applied for RFLP analysis of each gene fragment. The electrophoretic patterns generated after PCR–RFLP for each of the tested genes, were characteristic and specific for each species and allowed their differentiation. The data show that PCR–RFLP analysis of the recA, gyrA and rpoS gene fragments may be considered as a useful tool for the identification and differentiation of E. amylovora and E. pyrifoliae. Almost identical restriction patterns of the analyzed gene fragments indicated a high relationship of E. pyrifoliae strains from Korea and pear pathogens from Japan and a divergence to E. amylovora. For quick and effective differentiation of E. amylovora strains from Erwinia strains from Asia without nucleotide sequencing we recommend the amplification of recA and rpoS gene fragments and digestion of each of them with restriction endonuclease Hin6I.     

Levels of fire blight (<Emphasis Type="Italic">Erwinia amylovora</Emphasis>) susceptibility of native apple,pear and quince germplasm from Lake Van Basin,Turkey

Fire blight resistance of apple, pear and quince genetic resources from Lake Van Basin (eastern Turkey) was tested using Erwinia amylovora strain Ea Van. Shoot tips of 92 native accessions (48 accessions for apple, 38 accessions for pear and 6 accessions for quince) were wounded for inoculation, and artificially inoculated with pathogenic bacteria under greenhouse conditions. The levels of resistance of accessions were classified in comparison with control varieties according to the genotype susceptibility index (GSI%) scores based on the lesion length on shoots of each genotype. Fire blight resistance of accessions consisted of five classes: resistant (R), moderately resistant (MR), moderately susceptible (MS), susceptible (S) and highly susceptible (HS). GSI% scores differed significantly among accessions from each fruit species (p < 0.01). GSI values ranged from 12.4% to 64.1% for apple genotypes, from 17.2% to 55.1% for pear genotypes, and from 17.8% to 43.4% for quince genotypes. No resistant genotypes of apple, pear and quince were observed. Seven accessions of apple, two accessions of pear and one accession of quince were MR. 25 accessions of apple, 14 accessions of pear and one accession of quince were MS. These findings indicate a considerable variation in fire blight resistance and could contribute to breeding efforts regarding fire blight resistance in apple, pear and quince.     

Enhanced colonization and pathogenicity of Erwinia amylovora strains transformed with the near‐ubiquitous pEA29 plasmid on pear and apple

Three plasmid‐free strains of Erwinia amylovora, the causal agent of fire blight disease of pome trees, one from Iran, one from Egypt and one from Spain, were transformed with the near‐ubiquitous nonconjugative pEA29 plasmid from a wild‐type strain and characterized. The plasmid‐deficient strains were levan‐ and slime‐positive, motile, chemotaxis‐positive, induced HR on Nicotiana tabacum var. xanthi but produced several‐fold less amylovoran and were weakly pathogenic on pear slices and apple seedlings compared to plasmid‐bearing wild‐type strains. When inoculated onto wounded young apple (cv. Royal Gala) leaves, the plasmid‐free strains labelled with green fluorescent protein gene (gfp) were mainly restricted to the inoculation site at the leaf tips, in contrast to the plasmid‐carrying wild‐type strains that moved into the midrib xylem vessel and colonized the adjacent parenchyma cells. Upon introduction of the transposon‐labelled pEA29 plasmid, amylovoran production, degree of oozing and tissue necrosis on pear slices were significantly elevated in all three strains, whilst the levels of levan and levansucrase declined. Only the strains from Iran and Egypt gained the ability to invade and colonize the young apple leaves following the introduction of pEA29. It is concluded that acquisition of the nonconjugative near‐ubiquitous plasmid may not necessarily confer increasing pathogenicity in all bacterial strains.     

Erwinia amylovora hrpN mutants,blocked in harpin synthesis,express a reduced virulence on host plants and elicit variable hypersensitive reactions on tobacco

Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting many rosaceous plants and especially pear tree and apple tree. A protein named harpin, secreted through the Hrp secretion pathway and able to elicit an hypersensitive reaction (HR) on tobacco has recently been isolated. Mutants inhrpN, the gene encoding harpin were described as non pathogenic on immature pear fruit and unable to elicit an HR on tobacco [Weiet al., 1992; Wei and Beer, 1993]. In this paper, the phenotype on plant ofhrpN mutants was carefully determined.hrpN mutants expressed a weak but significant virulence on host plants. Furthermore, when infiltrated into tobacco leaf mesophyll, thehrpN mutants elicited varied responses that fluctuated from null reaction to full necrosis of the infiltrated area. These results show that harpin is not absolutely required neither for pathogenicity on host plant nor for elicitation of an hypersensitive reaction on tobacco. Furthermore, in all the tests performed, mutant blocked in harpin secretion remained non pathogenic and unable to elicit an HR on tobacco. This suggests that factor(s), different from harpin, involved both in pathogenicity and HR eliciting ability is (are) secreted through the Hrp secretion pathway.Abbreviations HR hypersensitive reaction - NSI necrosis severity index - CFU colonie forming units     

Microsatellite primers indicate the presence of asexual populations of<Emphasis Type="Italic">Venturia inaequalis</Emphasis> in coastal Israeli apple orchards

This study was initiated to determine whether differences in genotypic diversity among populations ofVenturia inaequalis (Cke.) Wint., as detected using neutral genetic markers, were related to the ecological conditions in which apples are grown in Israel. Since sexual reproduction in this fungal pathogen has an obligate requirement for sustained low winter temperatures, and since these requirements in Israel are met only on the Golan Heights, we were interested in whether lower elevation populations of this pathogen might be comprised of asexual clonal lineages. Unlike temperate apple growing regions, where the primary spring inoculum is ascosporic derived from overwintered pseudothecia, Israeli apple orchards at lower elevations in the Hula Valley and along the coastal plain rarely if ever experience low winter temperatures and pseudothecia have never been recovered. Two orchards were sampled from the Golan Heights (El Rom and Ortal, n=38) and three orchards from the Hula Valley and coastal plain (Sede Eliezer, Ginaton and Be’er Tuvia, n=40). Microsatellite primers were used to analyze population structure and the resulting binary data analyzed by both cluster and parsimony analysis. Populations from the coastal plain were genetically uniform within each of the orchards sampled, whereas populations from the Golan Heights showed levels of genotypic diversity ten times as high. The data support field observations that this pathogen does not reproduce sexually in regions characterized by the absence of low winter temperatures and is instead composed of clonal lineages. This may have bearing on control strategies for the disease in Israel. http://www.phytoparasitica.org posting April 21, 2003.     

Taxonomic Position of the Causal Pathogen of Bacterial Shoot Blight of Pear

Bacteriological properties and DNA-DNA homology values were compared among the pathogen causing bacterial shoot blight of pear (BSBP) isolated in 1994–1996, Erwinia amylovora isolated outside of Japan, and other Amylovora group bacteria. Bacteriological properties of BSBP strains were identical to those of E. amylovora in the majority of tests, but differed distinctively in several tests, including hydrolysis of esculin and acid production from salicin, etc. BSBP strains differed from the others in the Amylovora group in many other tests. DNA homology among the strains of BSBP ranged from 85 to 103% and from 83 to 110% among strains of E. amylovora. In contrast, the values between BSBP strains and E. amylovora strains were 55 to 81%, while those between BSBP strains and other Amylovora group strains were 42% or less. We consider, therefore, that the BSBP pathogen may well be included in E. amylovora at the species level. E. amylovora, including BSBP strains, however, can be classified into four biovars based on differences in nine tests such as growth factor requirements and crater formation on high sucrose medium. Namely, there are two biovars from Maloideae sources, one from Rubus idaeus, and one from the source of BSBP in Hokkaido. The presence of these biovars suggests a correlation with geographical, serological, and pathogenic differentiations in the species of E. amylovora. The BSBP pathogen in Hokkaido was identified as E. amylovora bv. 4 which is distinct from E. amylovora bv. 1, 2 and 3 isolated in countries outside of Japan. Received 29 July 1999/ Accepted in revised form 12 October 1999     

The undiscovered potential of dehydroproline as a fire blight control option

The non‐protein amino acid 3,4‐dehydro‐l ‐proline (DHP) significantly reduced the incidence of fire blight infection on immature pear fruits infected with wildtype Erwinia amylovora. DHP also inhibited biofilm formation in both streptomycin‐sensitive and ‐resistant strains of E. amylovora and induced dispersal of preformed biofilms in the streptomycin‐sensitive strain. The investigations shed light on the hitherto undiscovered ability of DHP to inhibit bacterial biofilms and its potential as a chemical control option for fire blight.     

Biological control of fire blight of apple and pear with antagonistic Lactobacillus plantarum

Lactic acid bacteria (LAB) can be a source of biological control agents (BCA) of fire blight disease. Several species of LAB are inhabitants of plants and are currently used as biopreservatives of food because of their antagonistic properties against bacteria, and are considered as generally safe. Candidates to BCA were selected from a large collection of LAB strains obtained from plant environments. Strains were first chosen based on the consistency of the suppressive effect against E. amylovora infections in detached plant organs (flowers, fruits and leaves). Lactobacillus plantarum strains PC40, PM411, TC54 and TC92 were effective against E. amylovora in most of the experiments performed. Besides, strains PM411, TC54 and TC92 had strong antagonistic activity against E. amylovora and also other target bacteria, and presented genes involved in plantaricin biosynthesis (plnJ, plnK, plnL, plnR and plnEF). The strains efficiently colonized pear and apple flowers; they maintained stable populations for at least 1 week under high RH conditions, and survived at low RH conditions. They were effective in preventing fire blight on pear flowers, fruits and leaves, as well as in whole plants and in a semi-field blossom assay. The present study confirms the potential of certain strains of L. plantarum to be used as active ingredient of microbial biopesticides for fire blight control that could be eventually extended to other plant bacterial diseases.     

Histological observation of cucumber infected with <Emphasis Type="Italic">Corynespora cassiicola</Emphasis>

Erwinia amylovora is the causative agent of fire blight, which is a destructive bacterial disease of rosaceous plants. In Hungary Erwinia amylovora (Burrill) Winslow et al. was first detected in 1996. Since the appearance of fire blight, E. amylovora samples have been collected from different host plats from various geographic locations. A motif of eight nucleotides (ATTACAGA) is repeated 3–15 times in the PstI fragment of the pEa29 plasmid in Erwinia amylovora strains, and represents a valuable tool for strain classification. The number of short-sequence DNA repeats in plasmid pEa29 of 30 Hungarian isolates were determined by PCR assays and they ranged from five to ten. The SSR test is suitable for distinguishing the individual strains between the E. amylovora isolates. The examined isolates showed high pathogenicity on immature pear fruits. Several biochemical techniques, such as miniaturized API 20E, were applied on the samples. Differences were also revealed in microbiological assays like levan formation and colony morphology on semi-selective media. Examining the Hungarian Erwinia amylovora population by molecular analysis we can draw the conclusion that the population consists of different strains, which shows great diversity. E. amylovora is a widespread pathogen in Hungary, which is supported by the 30 strains isolated from various host plants from many parts of the country. The phenotypic diversity-evaluation of the E. amylovora strains showed, that they differ metabolically, like other plant pathogenic bacteria as reported by several authors. This is the first report on the diversity of E. amylovora strains isolated from Hungary.     

Differentiation of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains from Bulgaria by PCR-RFLP analysis

Fifty strains of Erwinia amylovora isolated in Bulgaria from different host plants and locations as well as in different years were analysed by RFLP analysis of the pEA29 PstI amplified fragment with HpaII. All the strains formed three well-resolved fragments (large—from 365 to 440 bp, medium—about 341 bp and small—about 180 bp).The strains were classified into three RFLP groups based on the polymorphism in the length of the largest fragment. This fragment was of intermediate size for 63% of the strains, and it was the longest (from 410 to 440 bp) for 29% of the strains. The variable region was sequenced for five strains. The DNA sequence analysis confirmed the different size of the largest fragment. Ten or more than ten SSRs were found for the strains in the group with the largest size of the largest fragment. Some correlation between the RFLP profiles and the origin of the strains was revealed. The RFLP profiles displayed stability in certain strains isolated from the same trees and orchards, but in different years. The number of SSRs was different in strains isolated from one and the same host plant, orchard and year, and also in strains isolated from the same host plant and orchard, but in different years. This could indicate that under natural conditions the fire blight symptoms might be caused by a mixture of E. amylovora strains with different SSR numbers, and so coexistence of distinguishable strains or a change in the population could be assumed.     

Detection of streptomycin resistance in Erwinia amylovora strains isolated from apple orchards in Chihuahua, Mexico

Fire blight, one of the most severe diseases of apple and pear, is caused by the bacterium Erwinia amylovora. One control method is the use of antibiotics like streptomycin; however, streptomycin is the only antibiotic registered to control fire blight. A total of 107 E. amylovora strains were isolated from apple orchards located in Cuauhtémoc and Guerrero, Chihuahua, two major apple-producing areas in Mexico, showing 40 and 24 % streptomycin-resistant strains, respectively. The identification of E. amylovora strains was performed by polymerase chain reaction (PCR) amplification of a 900-bp region located within the non-transferable pEA29 plasmid and by amplification of a specific 1,269-bp region located on the E. amylovora chromosome. The 107 isolates tested carried the pEA29 plasmid, and 36 % of the isolates from both locations showed high resistance to streptomycin at levels that ranged from 200 to ≥1,000 μg ml^?1 streptomycin. The strA-strB and aadA genes, which encode enzymes that inactivate streptomycin, and a mutation in codon 43 of the rpsL gene that confers high resistance to the antibiotic were examined to determine the mechanism of streptomycin resistance. In total, 95 % of the resistant strains showed a single base pair mutation in codon 43 of the rpsL gene, causing an amino acid substitution in ribosomal protein S12. The presence of strA-strB and aadA genes or the rpsL mutation was not identified in the other 5 % of resistant strains, suggesting the existence of a new streptomycin resistance mechanism in E. amylovora.     

Note: Comparison of three laboratory methods to evaluate the pathogenicity and virulence of tenPseudomonas syringae pv.syringae strains on apple, pear, cherry and peach trees

The pathogenicity and virulence of ten GreekPseudomonas syringae pv.syringae strains from different hosts (citrus, pear, apple, peach and cherry) were evaluated using three different laboratory methods, which produced results in good agreement. All ten strains were virulent on apple, pear, cherry and peach trees. The extent of tissue colonized varied considerably among strains and cultivars. On excised shoots and twigs of apple and pear, strains BPI 176, BPI 203, PI 2 and PI 14 were the most virulent and strains BPI 689, BPI 992, BPI 4, BPI 20, PI 18 and PI 19 were the least virulent. On excised shoots and twigs of peach and cherry, strains BPI 176, BPI 203, PI 2, PI 14, PI 18 and PI 19 were the most virulent and strains BPI 4 and BPI 20 were the least virulent. Moderate virulence was evinced by strains BPI 689 and BPI 992. These pathogenicity assays are proposed as rapid and reproducible screening systems to evaluate the susceptibility of apple, pear, cherry and peach cultivars to this bacterial pathogen.     

Infection frequency of mature apple fruit with Erwinia amylovora deposited on pedicels and its survival in the fruit stored at low temperature

The infection frequency of mature apple fruit by Erwinia amylovora and the survival of E. amylovora in the fruit stored at low temperature were investigated. The fruit stems (pedicels) of 460 mature apple fruit were inoculated with 10⁵ or 10⁴ cfu of bioluminescent E. amylovora, tagged with lux genes. Nine days after inoculation, 43% and 27% of the fruit inoculated with 10⁵ and 10⁴ cfu, respectively, were infected. All infected fruit looked healthy. After 6 months of storage at 5°C, almost all of the 142 infected fruit had viable E. amylovora. Of the fruit containing E. amylovora internally, 19.5% had latent infections and the rest had blight symptoms. E. amylovora was not uniformly distributed in the fruit flesh, and internal brown lesions were observed where E. amylovora was densely distributed. These findings showed that mature apple fruit may be infected with E. amylovora, especially as latent infections, and act as a source for long-range dissemination.     

Tracking Agrobacterium strains by a RAPD system to identify single colonies from plant tumours

A molecular typing system for Agrobacterium strains based on the polymerase chain reaction–random amplified polymorphic DNA (PCR–RAPD) procedure was developed. It employs one to four different 10-mer primers and the results are highly reproducible. The band patterns obtained with the four primers for each of the 39 Agrobacterium strains analysed were different enough to differentiate the strains from each other. Strains with similar chromosomal background but different plasmid content, e.g. strains C58 and A281, gave the same band pattern with all the primers. Ten host plants were inoculated with eight Agrobacterium strains and the isolates obtained from the resulting tumours were analysed using the RAPD system developed here. The procedure allowed rapid identification of isolates recovered from tumours by comparison of their band patterns with band patterns of strains used as inoculum. The procedure also discriminated the various strains analysed. Purified bacterial cell suspensions, used for RAPD analyses, produced the same results as purified DNA, and greatly simplified the procedure. This system can be applied for rapid screening of Agrobacterium-like colonies isolated from plant tumours for epidemiological and genetic diversity studies.     

Erwinia isolates from the bacterial shoot blight of pear in Japan are closely related to Erwinia pyrifoliae based on phylogenetic analyses of gyrB and rpoD genes

The phylogenetic relationships among Erwinia amylovora biovar 4 (the pathogen of bacterial shoot blight of pear in Japan), other biovars of E. amylovora, and Erwinia pyrifoliae were investigated using the sequences of 16S rRNA, gyrB, and rpoD genes. The tested isolates formed two distinct monophyletic groups in the phylogenetic trees constructed based on the gyrB gene, rpoD gene, or a combination of the three genes: group 1 contained E. amylovora biovars 1, 2, and 3; group 2 contained E. amylovora bv. 4 and E. pyrifoliae. This phylogenetic analysis showed that E. amylovora bv. 4 was more closely related to E. pyrifoliae than to other biovars of E. amylovora. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB242876 to AB242925.