Molecular Characterization of Natural Erwinia pyrifoliae Strains Deficient in Hypersensitive Response

From necrotic tissue of a Nashi pear tree, 24 Erwinia pyrifoliae strains, found to be identical by pulsed-field gel electrophoresis analysis, were isolated. Thirteen strains were not virulent on immature pears and did not induce a hypersensitive response in tobacco leaves. The defective gene hrpL was complemented with intact genes from E. pyrifoliae and Erwinia amylovora.     

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

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

Discrimination and Detection of Erwinia amylovora and Erwinia pyrifoliae with a Single Primer Set

Erwinia amylovora and Erwinia pyrifoliae cause fire blight and black-shoot blight, respectively, in apples and pears. E. pyrifoliae is less pathogenic and has a narrower host range than that of E. amylovora. Fire blight and black-shoot blight exhibit similar symptoms, making it difficult to distinguish one bacterial disease from the other. Molecular tools that differentiate fire blight from black-shoot blight could guide in the implementation of appropriate management strategies to control both diseases. In this study, a primer set was developed to detect and distinguish E. amylovora from E. pyrifoliae by conventional polymerase chain reaction (PCR). The primers produced amplicons of different sizes that were specific to each bacterial species. PCR products from E. amylovora and E. pyrifoliae cells at concentrations of 10⁴ cfu/ml and 10⁷ cfu/ml, respectively, were amplified, which demonstrated sufficient primer detection sensitivity. This primer set provides a simple molecular tool to distinguish between two types of bacterial diseases with similar symptoms.     

Isolation and characterization of Hfr strains of Erwinia amylovora

Hfr strains (Hfr 159 and its derivatives, Hfr 160 and Hfr 161) were constructed from Erwinia amylovora ICPB EA178 by introducing an Escherichia coli F'his+ plasmid and then selecting for integration of F'his+ after treatment with acridine orange. The Hfr strains were relatively stable upon repeated transfers on nonselective media. Interrupted mating experiments and analyses of inheritance of unselected markers showed that his+ is transferred by Hfr 159 as the proximal marker at a relatively high frequency (about 5 x 10(-4) recombinants per input donor cell), followed by ilv+, orn+, arg+, pro+, rbs+, met+, trp+, leu+, ser+, and thr+ (not necessarily in that precise order). The donor strains, previously constructed in E. amylovora by integration of F'lac+ from E. coli transfer cys+ as the proximal marker followed by ser+. Further analysis of one of those earlier donor strains, Hfr99, showed that ser+ is followed by arg+, orn+, met+, pro+, leu+, ilv+, rbs+, his+, trp+, and thr+ (not necessarily in that precise order). Thus, the Hfr strains constructed by integration of F'his+ are different, in terms of origin and direction of transfer, from those derived from integration of F'lac+. The applicability of these Hfr strains to mapping the genes on the E. amylovora chromosome is indicated.     

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.     

Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages phiEa1 and phiEa7 and 3 novel phages named phiEa100, phiEa125, and phiEa116C, were identified based on differences in genome size and restriction fragment pattern. phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages phiEa100, phiEa7, and phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. phiEa116C contained an approximately 75-kb genome. phiEa1, phiEa7, phiEa100, phiEa125, and phiEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. phiEa116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10(5) CFU.     

Genetic characterization of Erwinia amylovora strains by amplified fragment length polymorphism

AIMS: Erwinia amylovora is one of the most important pathogens of pear and apple and is subject to strict quarantine regulations worldwide, although its patterns of dispersal are largely unknown. Previous attempts to fingerprint E. amylovora strains by molecular techniques have detected very little polymorphism because of the high genetic homogeneity of this bacterium. Our aim was to establish and test a typing method to quantify genetic diversity among strains of this plant pathogen. METHODS AND RESULTS: Twenty-two strains from different hosts and geographical locations were examined by PCR fingerprinting with four primers and by amplified fragment length polymorphism (AFLP) with four selected combinations of primers with a single base extension. PCR fingerprinting revealed little polymorphism producing the same amplification patterns for 17 strains, while the combined AFLP patterns yielded 78 polymorphic bands (34% of total bands) and allowed the differentiation of all but two strains. Clustering of strains in the resulting dendrogram was not correlated with host, year or country of isolation, and questions previous genealogies based on PFGE patterns. CONCLUSIONS: The AFLP technique allowed the detection of an unprecedented number of genetic markers in E. amylovora and proved to be the most useful tool so far for discriminating among strains of this pathogen. The results obtained in this study strongly suggest the occurrence of multiple introductions of the pathogen in Spain and other European countries. SIGNIFICANCE AND IMPACT OF THE STUDY: A major limitation in understanding the ecology of fire blight is the lack of typing techniques with a high power of discrimination. This study demonstrates the high resolution and the usefulness of the AFLP technique to differentiate among E. amylovora strains.     

Molecular characterization of a protease secreted by Erwinia amylovora.

A protease with a molecular mass of 48 kDa is secreted by the fire blight pathogen Erwinia amylovora in minimal medium. We characterized this activity as a metalloprotease, since the enzyme was inhibited by EDTA and o -phenanthroline. A gene cluster was determined to encode four genes connected to protease expression, including a structural gene (prtA) and three genes (prtD, prtE, prtF) for secretion of the protease, which are transcribed in the same direction. The organization of the protease gene cluster in E. amylovora is different from that in other Gram-negative bacteria, such as Erwinia chrysanthemi, Pseudomonas aeruginosa and Serratia marcescens. On the basis of the conservative motif of metalloproteases, PrtA was identified to be a member of the metzincin subfamily of zinc-binding metalloproteases, and was confirmed to be the 48 kDa protease on gels by sequencing of tryptic peptide fragments derived from the protein. The protease is apparently secreted into the external medium through the type I secretion pathway via PrtD, PrtE and PrtF which share more than 90% identity with the secretion apparatus for lipase of S. marcescens. A protease mutant was created by Tn 5 -insertions, and the mutation localized in the prtD gene. The lack of protease reduced colonization of an E. amylovora secretion mutant labelled with the gene for the green fluorescent protein (gfp) in the parenchyma of apple leaves.     

Evidence of Greater Competitive Fitness of Erwinia amylovora over E. pyrifoliae in Korean Isolates

Erwinia amylovora and E. pyrifoliae are the causative agents of destructive diseases in both apple and pear trees viz. fire blight and black shoot blight, respectively. Since the introduction of fire blight in Korea in 2015, the occurrence of both pathogens has been independently reported. The co-incidence of these diseases is highly probable given the co-existence of their pathogenic bacteria in the same trees or orchards in a city/district. Hence, this study evaluated whether both diseases occurred in neighboring orchards and whether they occurred together in a single orchard. The competition and virulence of the two pathogens was compared using growth rates in vitro and in planta. Importantly, E amylovora showed significantly higher colony numbers than E. pyrifoliae when they were co-cultured in liquid media and co-inoculated into immature apple fruits and seedlings. In a comparison of the usage of major carbon sources, which are abundant in immature apple fruits and seedlings, E. amylovora also showed better growth rates than E. pyrifoliae. In virulence assays, including motility and a hypersensitive response (HR), E. amylovora demonstrated a larger diameter of travel from the inoculation site than E. pyrifoliae in both swarming and swimming motilities. E. amylovora elicited a HR in tobacco leaves when diluted from 1:1 to 1:16 but E. pyrifoliae does not elicit a HR when diluted at 1:16. Therefore, E. amylovora was concluded to have a greater competitive fitness than E. pyrifoliae.     

Molecular characterization of Lactobacillus casei strains

Abstract The monoclonal antibody LA7 was raised against the species-specific Borrelia burgdorferi lipoprotein P22 (= IPLA7), which induces antibody formation in patients with Lyme arthritis. It is composed of 194 amino acids with a calculated molecular mass of 21.8 kDa. Its gene on the linear chromosome is 582 nucleotides in length. The aim of this study was to localize the protein P22 by immune electron microscopy. Immunolabeling of Borrelia burgdorferi with LA7 and an anti-mouse immunogold conjugate proved that P22 is an outer membrane protein. This finding was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the outer envelope fraction, which contained 99% of the P22 proteins.     

Development of the Droplet Digital PCR Method for the Detection and Quantification of Erwinia pyrifoliae

Black shoot blight disease caused by Erwinia pyrifoliae has serious impacts on quality and yield in pear production in Korea; therefore, rapid and accurate methods for its detection are needed. However, traditional detection methods require a great deal of time and fail to achieve absolute quantification. In the present study, we developed a droplet digital polymerase chain reaction (ddPCR) method for the detection and absolute quantification of E. pyrifoliae using a pair of species-specific primers. The detection range was 10³–10⁷ copies/ml (DNA templates) and cfu/ml (cell culture templates). This new method exhibited good linearity and repeatability and was validated by absolute quantification of E. pyrifoliae DNA copies from samples of artificially inoculated immature pear fruits. Here, we present the first study of ddPCR assay for the detection and quantification of E. pyrifoliae. This method has potential applications in epidemiology and for the early prediction of black shoot blight outbreaks.     

Harpin, a hypersensitive response elicitor from Erwinia amylovora, regulates ion channel activities in Arabidopsis thaliana suspension cells

HrpN, the hypersensitive response elicitor from Erwinia amylovora, stimulated K(+) outward rectifying currents in Arabidopsis thaliana suspension cells. It also decreased anion currents. These data demonstrate the ability of harpin to regulate different plasma membrane ion channels, putative components of signal transduction chains leading to defense responses and programmed cell death.     

Molecular characterization of China human rabies vaccine strains

To understand the molecular characteristics of China human rabies vaccine strains, we report the full-length genome of the aG strain and present a comprehensive analysis of this strain and almost all available lyssavirus genomes (58 strains) from GenBank (as of Jan 6, 2011). It is generally considered that the G protein plays a predominant role in determining the pathogenicity of the virus, to this end we predicted the tertiary structure of the G protein of aG strain, CTN181 strain and wild type strain HN10 based on the crystal structure of Vesicular stomatitis virus (VSV) G. The predicted RABV G structure has a similar topology to VSV G and the ectodomain can be divided into 4 distinct domains DI — DIV. By mapping the characterized mutations to this structure between China vaccine strains and their close street strains, we speculate that the G303(P-H) mutations of CTN181 and HN10 causing DII 3D change may be associated with the II attenuated virulence in both strains. Specifically, the two signature mutations (G165P and G231P) in the aG strain are withinßsheets, suggesting that both sites are of structural importance.     

Molecular characterization of Bacillus thuringiensis strains from Argentina

Bacillus thuringiensis INTA 7-3, INTA 51-3, INTA Mo9-5 and INTA Mo14-4 strains were obtained from Argentina and characterized by determination of serotype, toxicity, plasmid composition, insecticidal gene content ( cry and vip ) and the cloning of the single- vip3A gene of the INTA Mo9-5 strain. The serotype analysis identified the serovars tohokuensis and darmstadiensis for the INTA 51-3 and INTA Mo14-4 strains, respectively, whereas the INTA Mo9-5 strain was classified as "autoagglutinated". In contrast to the plasmid patterns of INTA 7-3, INTA 51-3 and INTA Mo9-5 (which were similar to B. thuringiensis HD-1 strain), strain INTA Mo14-4 showed a unique plasmid array. PCR analysis of the four strains revealed the presence of cry genes and vip3A genes. Interestingly, it was found that B. thuringiensis 4Q7 strain, which is a plasmid cured strain, contained vip3A genes indicating the presence of these insecticidal genes in the chromosome. Bioassays towards various lepidopteran species revealed that B. thuringiensis INTA Mo9-5 and INTA 7-3 strains were highly active. In particular, the mean LC(50) obtained against A. gemmatalis larvae with the INTA Mo9-5 and INTA 7-3 strains were 7 (5.7-8.6) and 6.7 (5.6-8.0) ppm, respectively. The INTA Mo14-4 strain was non-toxic and strain INTA 51-3 showed only a weak larvicidal activity.