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.     

An Indigenous Virulent Strain of Erwinia amylovora Lacking the Ubiquitous Plasmid pEA29

ABSTRACT An atypical strain of Erwinia amylovora was isolated near an outbreak of fire blight at a nursery in Spain in 1996. It was obtained from a Crataegus plant showing typical symptoms and was identified as E. amy-lovora by biochemical tests and enrichment-enzyme-linked immuno-sorbent assay, but not by polymerase chain reaction using primers based on the pEA29 sequence. Nevertheless, with primers from chromosomal regions, the isolate gave the expected amplification band. This strain carries one plasmid of approximately 70 kb, with no homology with the 29-kb plasmid common to all pathogenic strains, or with a large plasmid present in some E. amylovora strains. Growth of the strain in minimal medium without thiamine was slower compared with cultures in the same medium with thiamine, a characteristic typical of strains cured of the 29-kb plasmid. Nevertheless, aggressiveness assays on pear, apple, and Pyracantha plants and in immature pear fruit showed that this strain exhibited a virulence level similar to other strains containing pEA29. To the best of our knowledge, this is the first report of the isolation from naturally infected plant material of a pathogenic strain of E. amylovora without pEA29, but with a plasmid of approximately 70 kb not previously described.     

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.     

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.     

Real-time Scorpion-PCR detection and quantification of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> on pear leaves and flowers

A specific primer couple (E3–E4) amplifying a single DNA fragment of 111 bp from plasmid pEA29 was designed to identify, detect and quantify Erwinia amylovora by real-time Scorpion-PCR. Specificity of primers and probe was assessed both by means of BLAST analyses and by using genomic DNA from a large number of E. amylovora isolates and other bacteria. In Scorpion-PCR, the limit of detection was of 1 pg of total DNA and a high correlation (r = 0.999) was achieved between target DNA quantity and cycle threshold (Ct). Combining two sequential amplifications with conventional reported primers (PEANT1–PEANT2) and Scorpion primers (E3 Scorpion-E4) the detection limit was of 1 fg (nested Scorpion-PCR). Using serial dilution of the bacterial suspensions the limit of detection was 3.2 × 10⁴ CFU ml⁻¹ in Scorpion-PCR and 2.8 × 10² CFU ml⁻¹ in nested Scorpion-PCR. Real-time PCR combined with effective procedures for DNA extraction enabled the detection and the quantification of the epiphytic population of E. amylovora in the washings of flowers and leaves of artificially inoculated pear. A significant correlation (r = 0.92) was achieved between pathogen CFU on semi-selective media and the corresponding target DNA concentration evaluated by real-time PCR.     

Characterization of Erwinia amylovora Strains from Croatia

Erwinia amylovora is the causative agent of fire blight, a destructive disease of rosaceous plants. The European population can be divided into several subtypes according to differences in restriction fragment length polymorphism of the XbaI genomic DNA digest analysed with pulsed-field gel electrophoresis. This technique was also used to determine the genetic relatedness of six Croatian isolates to the E. amylovora types found in the countries surrounding Croatia. The isolates belong to the Pt2 pattern type that is characteristic of the East Mediterranean basin. All tested isolates gave essentially the same total cell protein pattern in SDS-polyacrylamide gel electrophoresis. The number of short-sequence DNA repeats in plasmid pEA29 of six isolates was determined by PCR assays and ranged from four to seven. The isolates examined showed high pathogenicity in immature pear fruits. Differences were also revealed in microbiological assays such as amylovoran synthesis, levan formation, siderophore production and colour on coliform medium.     

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.     

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.     

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.     

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.     

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.     

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.     

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene‐for‐gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’

The Gram‐negative bacterium Erwinia amylovora, causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that translocates effector proteins into plant cells that collectively function to suppress host defences and enable pathogenesis. Until now, there has only been limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. This study tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent E. amylovora strains, which are assumed to influence the host resistance response of fire blight‐resistant Malus species. The findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene‐for‐gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple cultivar Evereste and the wild species Malus floribunda 821. The results presented here provide new insights into the host–pathogen interactions between Malus sp. and E. amylovora.     

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.     

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.     

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.     

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.     

An assessment of the relative resistance of three hawthorn species to three strains of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> using three different inoculation methods

Fire blight is an important disease of hawthorn plants. In this study, the level of susceptibility of three hawthorn species (Crataegus monogyna, Crataegus laevigata, Crataegus persimilis) to the bacterium Erwinia amylovora was investigated. The results showed that all species were highly susceptible to this pathogen. In addition, the relative virulence of three different E. amylovora strains on the above species was examined. Variability among the strains was found, with strain 3 being the most virulent and strain 1 the least.     

Development and evaluation of a loop-mediated isothermal amplification assay for detection of Erwinia amylovora based on chromosomal DNA

A reliable and rapid pathogen detection protocol that utilizes loop-mediated isothermal amplification (LAMP) was developed for detection of Erwinia amylovora, the casual agent of fire blight. The six LAMP primers applied were derived from the highly conserved fragment of the chromosomally amsH gene. Despite the proposed LAMP as well as nested PCR presenting equal values of sensitivity (2?×?10¹?CFU/ml or more) for pure cultures, as compared with conventional PCR (2?×?10³?CFU/ml), both methods were together superior. The specificity assay also showed that the LAMP protocol is species-specific for detection of E. amylovora even in inter-species analysis. Meanwhile, when all 208 naturally infected samples were examined, the specificity value of LAMP was 84%, while conventional and nested PCR could detect only 59% and 73% of the whole collection. Significantly, an independent behaviour versus host plant as well as each strain origin was also observed regarding the current LAMP method as well as other two PCR-based methods. All the results, overall, indicated that the LAMP offers an interesting novel and convenient assay format for the quick and specific chromosomal detection and diagnostic tool of recognition of E. amylovora and therefore presents an alternative to PCR-based assays.