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.     

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.     

Analysis of Variable Short-sequence DNA Repeats on the 29 kb Plasmid of Erwinia amylovora Strains

The fire blight pathogen Erwinia amylovora has been specifically and sensitively detected by PCR assays with primers derived from plasmid pEa29. The amplified fragment of approximately 1kb can vary in length for individual strains, easily seen in a digest with restriction enzymes Sau3A or HpaII. DNA fragments from this variable region were cloned and DNA sequence analysis revealed short-sequence DNA repeat (SSR) motifs which were reiterated to various extents. The SSR units consisted of eight nucleotides (ATTACAGA), and terminated with ATTA which is part of an SSR. The shortest repetition consisted of four units and the longest one in Austrian E. amylovora strains was 15 units. The number of SSR units was remarkably stable during propagation of strains, but was occasionally changed when a strain was stressed by exposure to antibiotics, copper sulphate or storage at low temperature. Changes in the SSR number could be due to adjustment in bacterial fitness to environmental pressure. We designed oligonucleotide PCR primers from DNA sequences adjacent to the SSR region of pEA29 for rapid analysis of SSR length variations. With this PCR assay, more than 130 strains were classified into at least 11 types based on the number of repeats. E. amylovora strains isolated in Germany carried mostly six repeats in pEA29, which never changed under laboratory conditions. E. amylovora strains from Hungary and the Netherlands were quite divergent for the SSRs and further changes were sometimes observed after plating on agar medium. Homology search of nucleotide sequence data libraries revealed similarities of the SSR motif to partition functions of low copy number plasmids. Amino acid homology searches showed similarity of the deduced amino acid sequence in the ORF adjacent to the SSR motif to replication proteins of plasmids. The SSR may play a role in regulation of plasmid replication and partition as assumed for iterons.     

Genetic analysis of streptomycin-resistant (Sm(R)) strains of Erwinia amylovora suggests that dissemination of two genotypes is responsible for the current distribution of Sm(R) E. amylovora in Michigan

Streptomycin-resistant (Sm(R)) strains of the fire blight pathogen Erwinia amylovora were first isolated in southwest Michigan in 1991. Since that time, resistant strains have progressed northward to other apple-producing regions in the state. A total of 98.7% of Sm(R) strains isolated between 2003 and 2009 in Michigan harbored the strA-strB genes on transposon Tn5393. strA and strB encode phosphotransferase enzymes that modify streptomycin to a nonbactericidal form. Mutational resistance to streptomycin, caused by a point mutation-mediated target-site alteration of the ribosomal S12 protein, occurred in 1.3% of E. amylovora strains from Michigan. Tn5393 was originally introduced to E. amylovora on the plasmid pEa34; thus, the first Sm(R) strains isolated contained both pEa34 and the ubiquitous nonconjugative plasmid pEA29. More recently, we have observed Sm(R) strains in which Tn5393 is present on pEA29, suggesting that the transposon has moved via transposition from pEa34 to pEA29. Almost all of the strains containing Tn5393 on pEA29 had lost pEa34. Of 210 pEA29::Tn5393 plasmids examined, the transposon was inserted at either nucleotide position 1,515 or 17,527. Both of these positions were in noncoding regions of pEA29. Comparative sequencing of the housekeeping genes groEL and potentially variable sequences on pEA29 was done in an attempt to genetically distinguish Sm(R) strains from streptomycin-sensitive (Sm(S)) strains isolated in Michigan. Only 1 nucleotide difference within the total 2,660 bp sequenced from each strain was observed in 2 of 29 strains; multiple sequence differences were observed between the Michigan strains and E. amylovora control strains isolated in the western United States or from Rubus spp. Alterations in virulence observable using an immature pear fruit assay were detected in three of eight Sm(R) strains examined. Our current genetic data indicate that only two Sm(R) strain genotypes (strains containing pEA29::Tn5393 with Tn5393 inserted at either nucleotide position 1,515 or 17,527 on the plasmid) are responsible for the dissemination of Tn5393-encoded streptomycin resistance in Michigan, and that the Sm(R) and Sm(S) strains in Michigan compose a homogenous group.     

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.     

Alternative methods to describe virulence of Erwinia amylovora and host-plant resistance against fireblight

The evaluation of host-plant susceptibility to Erwinia amylovora and of colonization of host-plant tissue by individual strains was facilitated by labelling the pathogen with green fluorescent protein (GFP). Colonization of apple leaves assayed with a fluorescence microscope was associated with visual disease ratings on plants to describe virulence (= aggressiveness) of the fireblight pathogen. Resistance induced with 2,6-dichloro-isonicotinic acid (INA) and benzo(1,2,3-) thiadiazol-7-carbothioic acid-S-methyl ester (BTH, the active component of BION™) restricted colonization by the pathogen to an area adjacent to the inoculation site. Migration in leaves was associated with symptom formation on pear slices and host plants of mutant strains. Non-virulent E. amylovora mutants did not migrate into the leaf veins and strains with intermediate-to-low virulence moved slowly. To compare the migration efficiency of individual wild-type strains in apple and plum cultivars, a blend of five wild-type E. amylovora strains with specific numbers of short-sequence DNA repeats (SSRs) in the common plasmid pEA29 was applied to distinguish them by PCR. Fast-moving strains identified in the GFP assays were dominant, independent of the apple cultivar. When apple shoots, pear slices or leaves of apple plants were coinoculated with streptomycin (Sm)-resistant strains and the corresponding parent strains, Sm-resistant mutants were able to dominate the wild-type strain for tissue colonization.     

Erwinia pyrifoliae, an Erwinia species different from Erwinia amylovora, causes a necrotic disease of Asian pear trees

Bacteria from necrotic branches of Asian pear trees ( Pyrus pyrifolia ) in Korea were consistently isolated as white colonies on nutrient agar and formed mucoid, slightly yellow colonies on a minimal medium with copper sulphate. Isolates with this colony morphology were studied in a series of microbiological, molecular and pathological tests. Most isolates allowed the verification of Koch's postulate on P. pyrifolia seedlings and on slices from immature pear ( Pyrus communis ) fruits and were also positive in hypersensitivity tests on tobacco leaves. They showed characteristics common to species in the genus Erwinia , but were different from Erwinia amylovora , the agent of fire blight. A relationship between the novel pathogen and E. amylovora was found in microbiological and serological tests. Both organisms had similar but not identical protein patterns in 2-D gel electrophoresis, and in growth morphology the new pathogen produced colonies on MM2 Cu medium that were mucoid and slightly yellow, compared with the clearly yellow colonies of E. amylovora . No similarity was found in the plasmid profiles, and consequently no PCR signal was obtained with primers from the E. amylovora plasmid pEA29. REP-PCR also produced bands differing for the two organisms.     

Complete sequence of Erwinia piriflorinigrans plasmids pEPIR37 and pEPIR5 and role of pEPIR37 in pathogen virulence

Erwinia piriflorinigrans is a newly described pathogen causing necrosis of pear blossoms. Complete sequencing of the 37‐kb plasmid pEPIR37 common to 27 E. piriflorinigrans strains revealed homology to sequences of the ubiquitous plasmids pEA29 of the fire blight pathogen E. amylovora, plasmid pEP36 of E. pyrifoliae, plasmid pEJ30 of Erwinia sp. from Japan, and genomic regions of the related Rosaceae epiphytic Erwinia species E. tasmaniensis and E. billingiae. A second 5·5‐kb cryptic plasmid pEPIR5, found in 12 E. piriflorinigrans strains, was also sequenced revealing mobilization and replication proteins with similarities to many small ColE1‐type plasmids in Erwinia spp. and other enterobacteria. Functional analyses of pEPIR37 introduced into a strain of E. amylovora cured of pEA29 plasmid, which has a reduced virulence, showed a role in increasing symptom development similar to that observed in E. amylovora carrying plasmid pEA29.     

Real-time PCR for detection and quantification of Erwinia amylovora, the causal agent of fireblight

Real-time PCR was used for quantitative detection of Erwinia amylovora , the causative agent of fireblight. Specific primers were created from a DNA fragment of the common plasmid pEA29, successfully used for standard PCR identification of the pathogen. The primers amplified DNA from various E. amylovora strains, but not from other plant-associated bacteria. DNA of E. amylovora was also amplified from field samples and from inoculated apple leaves or flowers. Neither the presence of other bacteria nor low amounts of tissue extracts from bark or leaves changed the signal threshold. Assays with SYBR Green I instead of the Taqman probe showed a similar sensitivity, detecting 50 cells per assay. Real-time PCR could be especially useful for mass screening of commercial products and for resistance studies of transgenic host plants, in breeding experiments and after treatments to control fireblight.     

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.     

进境苹果果实中梨火疫病菌的套式PCR检测

 针对进境商用苹果果实携带梨火疫病菌Erwinia amylovora数量有限的特点,选取源于病菌pEA29质粒的2对引物P29A/P29B和PEANT1/PEANT2配对组合成套式PCR,其检测灵敏度可达0.15 pg菌体DNA,检测灵敏度高于EPPO推荐的单管套式PCR方法和常规PCR方法。分别利用这3种PCR检测方法对美国、新西兰、日本和智利等国进境的166批苹果样品进行检测,3种检测方法的样品阳性率分别为53.6%、38.0%和8.4%,试验结果表明此套式PCR检测方法可用于进境商用苹果的梨火疫病菌快速检测。进境样品的检测结果证实了进境商用苹果果实中存在梨火疫病菌的可能性。     

Diversity and detection of Korean Erwinia pyrifoliae strains as determined by plasmid profiling, phylogenetic analysis and PCR

Twenty-five strains of Erwinia pyrifoliae were investigated for their plasmid profiles and genetic relatedness. Four types of plasmid profile were observed for the first time, suggesting intraspecific plasmid profile diversity in E. pyrifoliae . Moreover, BOX-PCR and phylogenetic analysis based on the 16S-23S intergenic transcribed spacer (ITS) region showed genetic variations among E. pyrifoliae strains, although all strains were clustered in one group and separated from E. amylovora . On the other hand, ERIC-PCR and phylogenetic analysis based on partial groEL gene sequences revealed close genetic relatedness among the strains. Amplification with EpSPF and EpSPR primers of a fragment of approximately 0·65 kb from the genomic DNA of all E. pyrifoliae strains, but not from E. amylovora strains, suggested that this primer set is useful for identification of this pathogen.     

Transfer from Erwinia herbicola to Escherichia coli of a plasmid associated with biocontrol of fire blight

Strains of Erwinia herbicola effective in the biocontrol of fire blight of hawthorn were used to investigate the possibility that the antagonistic activity is coded by plasmid-born genes. Agarose gel electrophoresis of isolated plasmids from four antagonistic Erw. herbicola strains showed a band of a supercoiled 12 kb plasmid in each strain, with a second band greater than 16.2 kb consistently seen in two strains. Erw. herbicola strains showed resistance to penicillin-G, which could be conferred on penicillin-G sensitive Escherichia coli TG1 by transformation with a pure Erw. herbicola plasmid preparation. Transformed strains of Esc. coli appeared to contain the Erw. herbicola 12 kb plasmid, but not the > 16.2 kb plasmid. In an agar plate assay, Esc. coli transformants produced an inhibition zone against Erw. amylovora similar to those produced by the original Erw. herbicola strains. In two biocontrol assays, the transformed Esc. coli strains had a suppressive effect on disease development on infected pear fruit slices and hawthorn blossoms.     

Eop1 from a Rubus strain of Erwinia amylovora functions as a host-range limiting factor

Strains of Erwinia amylovora, the bacterium causing the disease fire blight of rosaceous plants, are separated into two groups based on host range: Spiraeoideae and Rubus strains. Spiraeoideae strains have wide host ranges, infecting plants in many rosaceous genera, including apple and pear. In the field, Rubus strains infect the genus Rubus exclusively, which includes raspberry and blackberry. Based on comparisons of limited sequence data from a Rubus and a Spiraeoideae strain, the gene eop1 was identified as unusually divergent, and it was selected as a possible host specificity factor. To test this, eop1 genes from a Rubus strain and a Spiraeoideae strain were cloned and mutated. Expression of the Rubus-strain eop1 reduced the virulence of E. amylovora in immature pear fruit and in apple shoots. Sequencing the orfA-eop1 regions of several strains of E. amylovora confirmed that forms of eop1 are conserved among strains with similar host ranges. This work provides evidence that eop1 from a Rubus-specific strain can function as a determinant of host specificity in E. amylovora.     

Improved fireblight diagnostics using quantitative real-time PCR detection of Erwinia amylovora chromosomal DNA

Specific and sensitive TaqMan real-time PCR assays were developed targeting chromosomal DNA of Erwinia amylovora ( ams C gene and ITS region). These assays increased the reliability of detection of E. amylovora strains, regardless of their plasmid profile, and have the ability to differentiate between Erwinia spp. strains from Hokkaido, Erwinia pyrifoliae and Erwinia spp. isolated from necrotic pear blossoms in Spain. The assays were used for testing the efficiency of three different extraction methods to remove plant-based PCR inhibitors. Combined with an automated DNA-extraction method based on magnetic beads (QuickPick™), the real-time PCR assays reliably detected at least 10³ cells mL^−l ( c. four cells per reaction) of the pathogen from blighted woody plant material. In testing of symptomless samples, absolute quantification of E. amylovora before and after enrichment in liquid media provided proof of E. amylovora viability and its ability to multiply, including in cases when subsequent isolation in pure culture was unsuccessful.     

Effect of nectar on microbial antagonists evaluated for use in control of fire blight of pome fruits

ABSTRACT Under warm, dry conditions, Erwinia amylovora can become established in relatively high populations on apple (Malus domestica) or pear (Pyrus communis) flower stigmas, and subsequent wet conditions facilitate its movement to the flower hypanthium where infection generally is initiated through the nectarthodes. Research on biological control of fire blight has focused mainly on the flower stigma, and knowledge is lacking regarding the effect of nectar on microbial antagonists in the flower hypanthium. The biocontrol agents Pseudomonas fluorescens strain A506 and Pantoea agglomerans strain C9-1 were cultured in a basal liquid medium with various concentrations (0 to 50% total sugar) of sucrose or synthetic nectar (sucrose/glucose/fructose, 2:1:1). Strain A506 showed less growth and lower survival than strain C9-1 at high sugar levels, and A506 was less effective than C9-1 as a preemptive antagonist of E. amylovora in high-sugar media. Both antagonist strains were less tolerant to high sugar levels than E. amylovora (strain Ea153). The same bacteria were cultured in a medium with 25% total sugar consisting of various proportions of sucrose, glucose, and fructose, and growth response correlated strongly with solute potential. When 28 microbial strains were cultured in synthetic nectar (25% total sugar) and ranked based on growth, strains clustered according to taxonomic group. Yeasts were most osmotolerant, followed by strains of E. amylovora, Pantoea agglomerans, Bacillus spp., and Pseudomonas spp. Further studies done in planta are necessary to determine whether osmotolerance of antagonists is advantageous in the biological control of fire blight.     

Duplex real-time polymerase chain reaction reveals competition between Erwinia amylovora and E. pyrifoliae on pear blossoms

Erwinia amylovora and E. pyrifoliae are the causative agents of fire blight and Asian pear blight, respectively. The pathogens are closely related, with overlapping host ranges. Data are unavailable on the current distribution of E. pyrifoliae and on the interaction between the two species when they are present together on the same host. In this study, a duplex real-time polymerase chain reaction (PCR) protocol was developed to monitor the population dynamics of E. amylovora and E. pyrifoliae on the surface of Bartlett pear blossoms. Bacterial cells washed from blossoms were used directly as the PCR template without DNA extraction. Primers and a probe based on the E. amylovora levansucrase gene detected all E. amylovora strains. All E. pyrifoliae strains, including the Japanese Erwinia strains previously described as E. amylovora, were detected with a primer and probe combination based on the E. pyrifoliae hrpW gene. Disease development and severity were not significantly different in blossoms inoculated with individual Erwinia species or with a mixture of the two species. However, E. amylovora grew to greater population sizes than did E. pyrifoliae in both single species inoculations and in mixtures, suggesting that E. amylovora has a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae.     

梨园气溶胶中梨火疫病菌的定量检测

为系统研究梨园气溶胶中梨火疫病菌的含量, 本研究于2019年－2021年在新疆库尔勒市人和农场梨园, 利用病原菌孢子捕捉器在每年春季(4月下旬)、夏季(6月中旬)、秋季(9月中旬)收集梨园气溶胶, 检测梨火疫病菌。结果显示, 健康梨园气溶胶中未检测到梨火疫病菌, 不同发病程度的梨园气溶胶中均能检测到梨火疫病菌, 携菌量均值在102 cfu/(24cm2·h)以上, 其中, 气溶胶中梨火疫病菌含量最高值为2.81×104 cfu/(24cm2·h), 最低值为8.50×102 cfu/(24cm2·h); 重度、中度、轻度发病果园收集的气溶胶中含梨火疫病菌总菌落数均值分别为8.74×103、4.55×103、2.36×103 cfu/(24cm2·h)。此外, 在同一高度收集的气溶胶中, 梨火疫病菌菌落数随收集时间的延长而增加。不同季节气溶胶携菌量检测结果表明, 秋季发病梨园中气溶胶携菌量明显高于夏季和春季, 与梨园梨火疫病发病规律相符。致病性测定结果表明, 气溶胶中分离的梨火疫病菌具有致病性。     

Identification by PCR analysis on plasmid pEA29 of isolates of Erwinia amylovora responsible of an outbreak in Central Europe

A collection of 127 strains of Erwinia amylovora, the causative agent of fire blight, was tested by PCR amplification of a fragment of the plasmid pEA29. A variability in the length of the DNA fragment obtained was observed after digestion by MspI and Sau3A restriction enzymes. Strains were distributed into three groups according to the length of the DNA product. Most of the strains analysed were placed into two groups. Thirteen strains were clustered into a third group which was linked with the geographical origin of strains: they were all isolates from recently reported outbreaks of fire blight in Austria and in southern Bavaria in Germany. The variation in the length of the amplified fragment is probably due to an insertion into this fragment.     

Molecular characterization of Erwinia amylovora strains from different host plants through RFLP analysis and sequencing of hrpN and dspA/E genes

A total of 73 Erwinia amylovora strains obtained from 13 Maloideae host species and from Rubus spp., and isolated from different geographic areas, were assessed using RFLP and DNA sequencing analysis of the 3' hrp N gene and/or of a fragment of 1341 bp of the dsp A/E region. An Erwinia pyrifoliae strain, used as outgroup, was checked in the same way. For the three strains isolated from Rubus spp. and for one strain from Amelanchier sp., RFLP analysis of the hrp N gene using the Rsa I enzyme yielded a PCR product 60 bp smaller than that of all the other strains. Sequence analysis of the gene revealed this was due to the absence of a 60 bp fragment in the noncoding region downstream of the gene. The strain PD 2915, isolated from Amelanchier sp. grown in Canada, showed five same-sense substitutions and one missense substitution at position 868 of the hrp N gene, converting aspartic acid into asparagine. Also, restriction analysis of a fragment of 613 bp of the dsp A/E region with Cfo I revealed an RFLP pattern suitable for differentiating the E. amylovora strains isolated from Rubus spp. and Amelanchier sp. from all the others. In the dsp A/E coding region, the four strains showed 13–14 missense point mutations, in some cases yielding drastic amino acid substitutions. In addition, partial sequencing of the dsp A/E region of PD 2915 from Amelanchier sp. indicated a higher similarity to E. amylovora strains isolated from Rubus spp. than towards strains from other Maloideae hosts. The E. pyrifoliae strain showed 23 single nucleotide substitutions along the hrp N gene and 88% of nucleotide identity with E. amylovora strains in the portion of dsp A/E region. Artificial inoculations on immature pear fruits and young shoots of Maloideae and Ruboideae showed a restricted pathogenicity for the strains from Rubus and Amelanchier , with the latter inciting blight symptoms only on Amelanchier .