Rapid identification of Erwinia amylovora and Pseudomonas syringae species and characterization of E. amylovora streptomycin resistance using quantitative PCR assays.

Erwinia amylovora and Pseudomonas syringae are bacterial phytopathogens responsible for considerable yield losses in commercial pome fruit production. The pathogens, if left untreated, can compromise tree health and economically impact entire commercial fruit productions. Historically, the choice of effective control methods has been limited. The use of antibiotics was proposed as an effective control method. The identification of these pathogens and screening for the presence of antibiotic resistance is paramount in the adoption and implementation of disease control methods. Molecular tests have been developed and accepted for identification and characterization of these disease-causing organisms. We improved existing molecular tests by developing methods that are equal or superior in robustness for identifying E. amylovora or P. syringae while being faster to execute. In addition, the real-time PCR-based detection method for E. amylovora provided complementary information on the susceptibility or resistance to streptomycin of individual isolates. Finally, we describe a methodology and results that compare the aggressiveness of the different bacterial isolates on four apple cultivars. We show that bacterial isolates exhibit different behaviors when brought into contact with various apple varieties and that the hierarchical clustering of symptom severity indicates a population structure, suggesting a genetic basis for host cultivar specificity.

Microbiological Examination of Erwinia amylovora Exopolysaccharide Ooze.

Fire blight, caused by the pathogen Erwinia amylovora, is the most devastating bacterial disease of pome fruit in North America and worldwide. The primary method of dispersal for E. amylovora is through ooze, a mass of exopolysaccharides and bacterial cells that is exuded as droplets from infected host tissue. During the 2013 and 2014 field seasons, 317 ooze droplets were collected from field-inoculated apple trees. Populations of E. amylovora in ooze droplets were 108 CFU/µl on average. Ooze droplets harboring larger (>108 CFU/µl) cell populations were typically smaller in total volume and had darker coloring, such as orange, red, or dark red hues. Examination of apple host tissue at the site of emergence of ooze droplets using scanning electron microscopy revealed that ooze was not exuding through natural openings; instead, it was found on erumpent mounds and small (10-µm) tears in tissue. These observations suggested that E. amylovora-induced wounds in tissue provided the exit holes for ooze extrusion from the host. Analyses of E. amylovora populations in ooze droplets and within the stems from which ooze droplets emerged indicated that approximately 9% of the total bacterial population from infected stems is diverted to ooze. Gene expression analyses indicated that E. amylovora cells in stem sections located above ooze droplets and in ooze droplets were actively expressing critical pathogenicity genes such as hrpL, dspE, and amsK. Thus, our study identified ooze as a source of large, concentrated populations of E. amylovora that emerged from the host by rupturing host tissue. Because the cells in ooze droplets are expressing genes required for pathogenesis, they are already primed for infection should they be dispersed from ooze to new infection courts.

Characterization of a cfr-Carrying Plasmid from Porcine Escherichia coli That Closely Resembles Plasmid pEA3 from the Plant Pathogen Erwinia amylovora.

The multiresistance gene cfr was found in two porcine Escherichia coli isolates, one harboring it on the conjugative 33,885-bp plasmid pFSEC-01, the other harboring it in the chromosomal DNA. Sequence analysis of pFSEC-01 revealed that a 6,769-bp fragment containing the cfr gene bracketed by two IS26 elements was inserted into a plasmid closely related to pEA3 from the plant pathogen Erwinia amylovora, suggesting that pFSEC-01 may be transferred between different bacterial genera of both animal and plant origin.

Droplet digital PCR for absolute quantification of pathogens.

The recent advent of different digital PCR (dPCR) platforms is enabling the expansion of this technology for research and diagnostic applications worldwide. The main principle of dPCR, as in other PCR-based methods including quantitative PCR (qPCR), is the specific amplification of a nucleic acid target. The distinctive feature of dPCR is the separation of the reaction mixture into thousands to millions of partitions which is followed by a real time or end point detection of the amplification. The distribution of target sequences into partitions is described by the Poisson distribution, thus allowing accurate and absolute quantification of the target from the ratio of positive against all partitions at the end of the reaction. This omits the need to use reference materials with known target concentrations and increases the accuracy of quantification at low target concentrations compared to qPCR. dPCR has also shown higher resilience to inhibitors in a number of different types of samples. In this chapter we describe the droplet digital PCR (ddPCR) workflow for the detection and quantification of pathogens using the droplet digital Bio-Rad platform QX100. We present as an example the quantification of the quarantine plant pathogenic bacterium, Erwinia amylovora.

HrpG and HrpV proteins from the Type III secretion system of Erwinia amylovora form a stable heterodimer.

Bacterial type III secretion systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or directly into eukaryotic host cell cytoplasm. Erwinia amylovora, the main agent of rosaceous plants fireblight disease, employs an Hrp/Hrc1 T3SS to accomplish its pathogenesis. The regulatory network that controls the activation of this T3SS is largely unknown in E. amylovora. However, in Pseudomonas syringae pathovars, the HrpG/HrpV complex has been shown to directly regulate the activity of transcription factor HrpS and consequently the upregulation of the Hrp/Hrc1 T3SS related genes. In this work, we report the successful recombinant production and purification of a stable E. amylovora HrpG/HrpV complex, using pPROpET, a bicistronic expression vector. Furthermore, we present the first solution structure of this complex based on small-angle X-ray scattering data.

A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmids.

Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents.

Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot.

Here we report on the first assessment of droplet digital PCR (ddPCR) for detection and absolute quantification of two quarantine plant pathogenic bacteria that infect many species of the Rosaceae and Solanaceae families: Erwinia amylovora and Ralstonia solanacearum. An open-source R script was written for the ddPCR data analysis. Analysis of a set of samples with known health status aided the assessment and selection of different threshold settings (QuantaSoft analysis, definetherain pipeline and manual threshold), which led to optimal diagnostic specificity. The interpretation of the E. amylovora ddPCR was straightforward, and the analysis approach had little influence on the final results and the concentrations determined. The sensitivity and linear range were similar to those for real-time PCR (qPCR), for the analysis of both bacterial suspensions and plant material, making ddPCR a viable choice when both detection and quantification are desired. With the R. solanacearum ddPCR, the use of a high global threshold was necessary to exclude false-positive reactions that are sometimes observed in healthy plant material. ddPCR significantly improved the analytical sensitivity over that of qPCR, and improved the detection of low concentrations of R. solanacearum in potato tuber samples. Accurate and rapid absolute quantification of both of these bacteria in pure culture was achieved by direct ddPCR. Our data confirm the suitability of these ddPCR assays for routine detection and quantification of plant pathogens and for preparation of defined in-house reference materials with known target concentrations.

[Erwinia amylovora--the fire blight pathogen of trees in Ukraine].

Niduses of fire blight of fruit and ornamental trees have been found in the Kyiv and Vinnitsa regions of Ukraine. Pathogen Erwinia amylovora was isolated between April and October. The pathogen was often accompanied by bacteria Pseudomonas syringae pv. syringae. Artificial infection with a mixture of bacteria E. amylovora and P. syringae pv. syringae accelerates and enhances the disease process in the laboratory.

Deep sequencing revealed genome-wide single-nucleotide polymorphism and plasmid content of Erwinia amylovora strains isolated in Middle Atlas, Morocco.

Erwinia amylovora causes economic losses that affect pear and apple production in Morocco. Here, we report comparative genomics of four Moroccan E. amylovora strains with the European strain CFBP1430 and North-American strain ATCC49946. Analysis of single nucleotide polymorphisms (SNPs) revealed genetic homogeneity of Moroccan's strains and their proximity to the European strain CFBP1430. Moreover, the collected sequences allowed the assembly of a 65 kpb plasmid, which is highly similar to the plasmid pEI70 harbored by several European E. amylovora isolates. This plasmid was found in 33% of the 40 E. amylovora strains collected from several host plants in 2009 and 2010 in Morocco.

Molecular analyses of Erwinia amylovora strains isolated in Russia, Poland, Slovenia and Austria describing further spread of fire blight in Europe.

Fire blight, a bacteriosis of apple and pear, was assayed with molecular tools to associate its origin in Russia, Slovenia and south-eastern Austria with neighboring countries. The identification of all investigated strains was confirmed by MALDI-TOF mass spectroscopy except one. Independent isolation was verified by the level of amylovoran synthesis and by the number of short sequence DNA repeats in plasmid pEA29. DNA of gently lysed E. amylovora strains from Russia, Slovenia, Austria, Hungary, Italy, Spain, Croatia, Poland, Central Europe and Iran was treated with restriction enzymes XbaI and SpeI to create typical banding patterns for PFGE analysis. The pattern Pt2 indicated that most Russian E. amylovora strains were related to strains from Turkey and Iran. Strains from Slovenia exhibited patterns Pt3 and Pt2, both present in the neighboring countries. Strains were also probed for the recently described plasmid pEI70 detected in Pt1 strains from Poland and in Pt3 strains from other countries. The distribution of pattern Pt3 suggests distribution of fire blight from Belgium and the Netherlands to Central Spain and Northern Italy and then north to Carinthia. The PFGE patterns indicate that trade of plants may have introduced fire blight into southern parts of Europe proceeded by sequential spread.

Erwinia amylovora loop-mediated isothermal amplification (LAMP) assay for rapid pathogen detection and on-site diagnosis of fire blight.

Several molecular methods have been developed for the detection of Erwinia amylovora, the causal agent of fire blight in pear and apple, but none are truly applicable for on-site use in the field. We developed a fast, reliable and field applicable detection method using a novel target on the E. amylovora chromosome that we identified by applying a comparative genomic pipeline. The target coding sequences (CDSs) are both uniquely specific for and all-inclusive of E. amylovora genotypes. This avoids potential false negatives that can occur with most commonly used methods based on amplification of plasmid gene targets, which can vary among strains. Loop-mediated isothermal AMPlification (LAMP) with OptiGene Genie II chemistry and instrumentation proved to be an exceptionally rapid (under 15 min) and robust method for detecting E. amylovora in orchards, as well as simple to use in the plant diagnostic laboratory. Comparative validation results using plant samples from inoculated greenhouse trials and from natural field infections (of regional and temporal diverse origin) showed that our LAMP had an equivalent or greater performance regarding sensitivity, specificity, speed and simplicity than real-time PCR (TaqMan), other LAMP assays, immunoassays and plating, demonstrating its utility for routine testing.

Erwinia amylovora CRISPR elements provide new tools for evaluating strain diversity and for microbial source tracking.

Clustered regularly interspaced short palindromic repeats (CRISPRs) comprise a family of short DNA repeat sequences that are separated by non repetitive spacer sequences and, in combination with a suite of Cas proteins, are thought to function as an adaptive immune system against invading DNA. The number of CRISPR arrays in a bacterial chromosome is variable, and the content of each array can differ in both repeat number and in the presence or absence of specific spacers. We utilized a comparative sequence analysis of CRISPR arrays of the plant pathogen Erwinia amylovora to uncover previously unknown genetic diversity in this species. A total of 85 E. amylovora strains varying in geographic isolation (North America, Europe, New Zealand, and the Middle East), host range, plasmid content, and streptomycin sensitivity/resistance were evaluated for CRISPR array number and spacer variability. From these strains, 588 unique spacers were identified in the three CRISPR arrays present in E. amylovora, and these arrays could be categorized into 20, 17, and 2 patterns types, respectively. Analysis of the relatedness of spacer content differentiated most apple and pear strains isolated in the eastern U.S. from western U.S. strains. In addition, we identified North American strains that shared CRISPR genotypes with strains isolated on other continents. E. amylovora strains from Rubus and Indian hawthorn contained mostly unique spacers compared to apple and pear strains, while strains from loquat shared 79% of spacers with apple and pear strains. Approximately 23% of the spacers matched known sequences, with 16% targeting plasmids and 5% targeting bacteriophage. The plasmid pEU30, isolated in E. amylovora strains from the western U.S., was targeted by 55 spacers. Lastly, we used spacer patterns and content to determine that streptomycin-resistant strains of E. amylovora from Michigan were low in diversity and matched corresponding streptomycin-sensitive strains from the background population.

Characterization of Erwinia amylovora strains from Bulgaria by pulsed-field gel electrophoresis.

The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-field gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profile. About 82% of the strains displayed a PFGE profile identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profiles, not reported so far, were established--one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profiles, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profile in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum.

Differentiation of Erwinia amylovora and Erwinia pyrifoliae strains with single nucleotide polymorphisms and by synthesis of dihydrophenylalanine.

Fire blight has spread from North America to New Zealand, Europe, and the Mediterranean region. We were able to differentiate strains from various origins with a novel PCR method. Three Single Nucleotide Polymorphisms (SNPs) in the Erwinia amylovora genome were characteristic of isolates from North America and could distinguish them from isolates from other parts of the world. They were derived from the galE, acrB, and hrpA genes of strains Ea273 and Ea1/79. These genes were analyzed by conventional PCR (cPCR) and quantitative PCR (qPCR) with differential primer annealing temperatures. North-American E. amylovora strains were further differentiated according to their production of L: -2,5-dihydrophenylalanine (DHP) as tested by growth inhibition of the yeast Rhodotorula glutinis. E. amylovora fruit tree (Maloideae) and raspberry (rubus) strains were also differentiated by Single Strand Conformational Polymorphism analysis. Strains from the related species Erwinia pyrifoliae isolated in Korea and Japan were all DHP positive, but were differentiated from each other by SNPs in the galE gene. Differential PCR is a rapid and simple method to distinguish E. amylovora as well as E. pyrifoliae strains according to their geographical origin.

Rootstock-regulated gene expression patterns associated with fire blight resistance in apple.

BACKGROUND: Desirable apple varieties are clonally propagated by grafting vegetative scions onto rootstocks. Rootstocks influence many phenotypic traits of the scion, including resistance to pathogens such as Erwinia amylovora, which causes fire blight, the most serious bacterial disease of apple. The purpose of the present study was to quantify rootstock-mediated differences in scion fire blight susceptibility and to identify transcripts in the scion whose expression levels correlated with this response. RESULTS: Rootstock influence on scion fire blight resistance was quantified by inoculating three-year old, orchard-grown apple trees, consisting of 'Gala' scions grafted to a range of rootstocks, with E. amylovora. Disease severity was measured by the extent of shoot necrosis over time. 'Gala' scions grafted to G.30 or MM.111 rootstocks showed the lowest rates of necrosis, while 'Gala' on M.27 and B.9 showed the highest rates of necrosis. 'Gala' scions on M.7, S.4 or M.9F56 had intermediate necrosis rates. Using an apple DNA microarray representing 55,230 unique transcripts, gene expression patterns were compared in healthy, un-inoculated, greenhouse-grown 'Gala' scions on the same series of rootstocks. We identified 690 transcripts whose steady-state expression levels correlated with the degree of fire blight susceptibility of the scion/rootstock combinations. Transcripts known to be differentially expressed during E. amylovora infection were disproportionately represented among these transcripts. A second-generation apple microarray representing 26,000 transcripts was developed and was used to test these correlations in an orchard-grown population of trees segregating for fire blight resistance. Of the 690 transcripts originally identified using the first-generation array, 39 had expression levels that correlated with fire blight resistance in the breeding population. CONCLUSIONS: Rootstocks had significant effects on the fire blight susceptibility of 'Gala' scions, and rootstock-regulated gene expression patterns could be correlated with differences in susceptibility. The results suggest a relationship between rootstock-regulated fire blight susceptibility and sorbitol dehydrogenase, phenylpropanoid metabolism, protein processing in the endoplasmic reticulum, and endocytosis, among others. This study illustrates the utility of our rootstock-regulated gene expression data sets for candidate trait-associated gene data mining.

Erwinia amylovora novel plasmid pEI70: complete sequence, biogeography, and role in aggressiveness in the fire blight phytopathogen.

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5-92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.

Novel virulent and broad-host-range Erwinia amylovora bacteriophages reveal a high degree of mosaicism and a relationship to Enterobacteriaceae phages.

A diverse set of 24 novel phages infecting the fire blight pathogen Erwinia amylovora was isolated from fruit production environments in Switzerland. Based on initial screening, four phages (L1, M7, S6, and Y2) with broad host ranges were selected for detailed characterization and genome sequencing. Phage L1 is a member of the Podoviridae, with a 39.3-kbp genome featuring invariable genome ends with direct terminal repeats. Phage S6, another podovirus, was also found to possess direct terminal repeats but has a larger genome (74.7 kbp), and the virus particle exhibits a complex tail fiber structure. Phages M7 and Y2 both belong to the Myoviridae family and feature long, contractile tails and genomes of 84.7 kbp (M7) and 56.6 kbp (Y2), respectively, with direct terminal repeats. The architecture of all four phage genomes is typical for tailed phages, i.e., organized into function-specific gene clusters. All four phages completely lack genes or functions associated with lysogeny control, which correlates well with their broad host ranges and indicates strictly lytic (virulent) lifestyles without the possibility for host lysogenization. Comparative genomics revealed that M7 is similar to E. amylovora virus ΦEa21-4, whereas L1, S6, and Y2 are unrelated to any other E. amylovora phage. Instead, they feature similarities to enterobacterial viruses T7, N4, and ΦEcoM-GJ1. In a series of laboratory experiments, we provide proof of concept that specific two-phage cocktails offer the potential for biocontrol of the pathogen.

Molecular and physiological properties of bacteriophages from North America and Germany affecting the fire blight pathogen Erwinia amylovora.

For possible control of fire blight affecting apple and pear trees, we characterized Erwinia amylovora phages from North America and Germany. The genome size determined by electron microscopy (EM) was confirmed by sequence data and major coat proteins were identified from gel bands by mass spectroscopy. By their morphology from EM data, φEa1h and φEa100 were assigned to the Podoviridae and φEa104 and φEa116 to the Myoviridae. Host ranges were essentially confined to E. amylovora, strains of the species Erwinia pyrifoliae, E. billingiae and even Pantoea stewartii were partially sensitive. The phages φEa1h and φEa100 were dependent on the amylovoran capsule of E. amylovora, φEa104 and φEa116 were not. The Myoviridae efficiently lysed their hosts and protected apple flowers significantly better than the Podoviridae against E. amylovora and should be preferred in biocontrol experiments. We have also isolated and partially characterized E. amylovora phages from apple orchards in Germany. They belong to the Podoviridae or Myoviridae with a host range similar to the phages isolated in North America. In EM measurements, the genome sizes of the Podoviridae were smaller than the genomes of the Myoviridae from North America and from Germany, which differed from each other in corresponding nucleotide sequences.

Diversity, evolution, and functionality of clustered regularly interspaced short palindromic repeat (CRISPR) regions in the fire blight pathogen Erwinia amylovora.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system confers acquired heritable immunity against mobile nucleic acid elements in prokaryotes, limiting phage infection and horizontal gene transfer of plasmids. In CRISPR arrays, characteristic repeats are interspersed with similarly sized nonrepetitive spacers derived from transmissible genetic elements and acquired when the cell is challenged with foreign DNA. New spacers are added sequentially and the number and type of CRISPR units can differ among strains, providing a record of phage/plasmid exposure within a species and giving a valuable typing tool. The aim of this work was to investigate CRISPR diversity in the highly homogeneous species Erwinia amylovora, the causal agent of fire blight. A total of 18 CRISPR genotypes were defined within a collection of 37 cosmopolitan strains. Strains from Spiraeoideae plants clustered in three major groups: groups II and III were composed exclusively of bacteria originating from the United States, whereas group I generally contained strains of more recent dissemination obtained in Europe, New Zealand, and the Middle East. Strains from Rosoideae and Indian hawthorn (Rhaphiolepis indica) clustered separately and displayed a higher intrinsic diversity than that of isolates from Spiraeoideae plants. Reciprocal exclusion was generally observed between plasmid content and cognate spacer sequences, supporting the role of the CRISPR/Cas system in protecting against foreign DNA elements. However, in several group III strains, retention of plasmid pEU30 is inconsistent with a functional CRISPR/Cas system.

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.