Instability of short-sequence DNA repeats of pear pathogenic Erwinia strains from Japan and Erwinia amylovora fruit tree and raspberry strains

An array of short-sequence DNA repeats (SSRs) occurs in the plasmid pEA29 of the fire blight pathogen Erwinia amylovora. A large number of "fruit tree" strains, mainly from Central and Western Europe, were screened for their SSR numbers, and the analyses were extended to five raspberry strains from North America and six pear pathogenic Erwinia strains from Japan. The repeat ATTACAGA present in all E. amylovorastrains was found to be reiterated 3 to 15 times. The Japanese strains contained the major repeat sequence GGATTCTG, which was reiterated 16 to 24 times. ATTACAGG, which resembles the SSR of E. amylovora, was reiterated two or three times. In a novel approach, sequencing gels were used to visualize the rare occurrence of shorter arrays (down to three repeats) in E. amylovoraand the Japanese Erwinia strains. Changes in the repeat numbers in E. amylovora were observed repeatedly when the bacteria had been exposed to stress conditions. The repeat structures of homo- and heteroduplices of PCR-amplified repeats were also analyzed by cleavage of annealed molecules with the single-strand-specific endonuclease from bacteriophage T4. Not only heteroduplexes, but also homoduplexes showed non-matching regions in the SSRs, which could arise from transient formation of loops due to strand slippage during the assays.     

Following spread of fire blight in Western, Central and Southern Europe by molecular differentiation of Erwinia amylovora strains with PFGE analysis

Fire blight has been detected recently in several areas of northern Spain and north-eastern Italy. To follow spread of the disease within Europe, more than 120 Erwinia amylovora strains isolated from 1957-1900 in England, France, Germany, The Netherlands, Belgium, Poland, Italy and Spain were assayed using pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA after XbaI digestion. Pattern types Pt1 and Pt4 were found for strains from England. Pt1 was also found in central Europe and eastern France, Pt4 in western France. Pt2 appeared first in Egypt, from where strains with this pattern disseminated northwards as far as into the Balkans. Pt3 was typical for northern France and Belgium. Strains from Spain displayed the pattern types Pt3 and Pt4. In Italy, Pt2 was found in the south-eastern areas, Pt3 in the north-eastern areas, and Pt1 was found very recently in orchards adjacent to the Austrian border, together with Pt3. Despite barely controlled trade with fire blight host plants and associated plant products within Europe, the PFGE patterns of the E. amylovora isolates were ordered indicating sequential spread. On the other hand, the appearance of Pt3 in northern Italy and central Spain can be explained by the import of contaminated plants by nurseries.     

Characterization of Bacillus strains from apple and pear trees in South Africa antagonistic to Erwinia amylovora

In order to find reasons for the absence of fire blight in most countries of the Southern hemisphere, bark samples from apple and pear trees in orchards of the Western Cape region in South Africa were extracted for bacteria which could be antagonistic to Erwinia amylovora. Screening was done in the late growth season and mainly Gram-positive bacteria were isolated. Approximately half of them produced growth inhibition zones on a lawn of E. amylovora. Most isolates were classified as Bacillus megaterium by microbiological assays and in API 50 test systems. They were visualized in the light microscope as non-motile large rods. These strains may not be responsible for the absence of fire blight in orchards, but they may indicate unfavourable climatic conditions for Gram-negative bacteria including E. amylovora. They may reduce the ability of E. amylovora to establish fire blight and could also be useful for application in biological disease control.     

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.     

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.     

Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein

Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme.     

Transfer of episome F'lac+ and chromosomal trp+ genes from Erwinia amylovora to Salmonella typhimurium.

The F'lac+ episome of Escherichia coli origin was transferred by conjugation with frequencies of 10(-7) to 10(-5) from Erwinia amylovora to 14 out of 15 Salmonella typhimurium trp female parents. The chromosomal trp+ genes were transferred with frequencies of 10(-7) to 10(-6) only to one trpB and 2 trpD female parents, which have a point mutation in the 2nd and fourth structural genes, respectively, of the tryptophan operon. The transferred male trp+ genes became integrated at the selected sites of the S. tryphimurium chromosome. The resulting Trp+ hybrids were phenotypically stable, lacked a cryptic trp allele selected against in the female parent, had high genetic homology values in the tryptophan region, and showed biochemical reactions and pathogenicity typical of S. typhimurium.     

Genome‐wide patterns of latitudinal differentiation among populations of Drosophila melanogaster from North America

Understanding the genetic underpinnings of adaptive change is a fundamental but largely unresolved problem in evolutionary biology. Drosophila melanogaster, an ancestrally tropical insect that has spread to temperate regions and become cosmopolitan, offers a powerful opportunity for identifying the molecular polymorphisms underlying clinal adaptation. Here, we use genome‐wide next‐generation sequencing of DNA pools (‘pool‐seq’) from three populations collected along the North American east coast to examine patterns of latitudinal differentiation. Comparing the genomes of these populations is particularly interesting since they exhibit clinal variation in a number of important life history traits. We find extensive latitudinal differentiation, with many of the most strongly differentiated genes involved in major functional pathways such as the insulin/TOR, ecdysone, torso, EGFR, TGFβ/BMP, JAK/STAT, immunity and circadian rhythm pathways. We observe particularly strong differentiation on chromosome 3R, especially within the cosmopolitan inversion In(3R)Payne, which contains a large number of clinally varying genes. While much of the differentiation might be driven by clinal differences in the frequency of In(3R)P, we also identify genes that are likely independent of this inversion. Our results provide genome‐wide evidence consistent with pervasive spatially variable selection acting on numerous loci and pathways along the well‐known North American cline, with many candidates implicated in life history regulation and exhibiting parallel differentiation along the previously investigated Australian cline.     

Genotypic responses of scab-resistant apple cultivars/selections to two strains of Erwinia amylovora and the inheritance of resistance to fire blight

Apple cultivars and selections were tested for resistance to two strains of Erwinia amylovora following shoot tip inoculation in the greenhouse. Cv. Liberty was resistant to strain Ea 273, and Priscilla, Delicious and Coop 17 were highly resistant to strain Apple # 1. A significant genotype x strain interaction was obtained suggesting a differential host × pathogen interaction. Segregation of seedlings derived from 16 controlled crosses after fire blight inoculation indicated that resistance to fire blight is quantitatively controlled with evidence for presence of dominant additive gene effects.     

Genetic variability and interpopulational differentiation of Artemia strains from South America

Seven Artemia samples from three South American countries (Chile, Brazil, Peru) were studied by starch electrophoresis with the aim of comparing levels of genetic variation and genetic similarity to representative populations of A. franciscana (San Francisco Bay, California, USA) and A. persimilis (Buenos Aires, Argentina), which are species endemic to the New World. Based on the analysis of 22 loci, parameters measuring genetic variability were, for some populations, found to be among the highest reported for Artemia so far. The percentage of polymorphic loci varied from 31.8% (Piura, Peru; Buenos Aires) to 50% (Los Vilos and Salar de Atacama, Chile), while the observed heterozygosity varied from 0.025 (Piura) to 0.165 (Los Vilos, Chile). A dendrogram based on Nei's genetic distance (D) produced four major groups. The Argentinian form, A. persimilis; the San Francisco Bay strain together with samples from Brazil (Macau and Rio Grande do Norte) and Chile (Pichilemu and Salar de Atacama); two coastal populations from Chile (Los Vilos and Iquique) and the sample from Peru (Piura). These four groups have inter-group D values that are, in some cases, far above those normally associated with conspecific populations.     

Molecular typing of Yersinia frederiksenii strains by means of 16s rDNA and gyrB genes sequence analyses

The phenospecies Yersinia frederiksenii is known to comprise three genospecies, indistinguishable on the basis of phenotypic characteristics. In previous works, 13 strains, identified biochemically as Y. frederiksenii, were characterized using Multilocus enzyme electrophoresis and Ribotyping. In order to elucidate the phylogenetic position of these strains we performed their molecular typing by means of 16S rDNA and gyrB sequences analyses. Results demonstrated that gyrB is a better phylogenetic marker than 16S rDNA. The classification achieved by gyrB sequences analyses was in agreement with results obtained with more laborious methods. Moreover, a good phylogenetic identification could be reached also with partial gyrB sequences of only 350 bp.     

Genetic differentiation of the Fejervarya limnocharis complex from Bangladesh and other Asian countries elucidated by allozyme analyses

The present study was conducted to elucidate the genetic divergence and the phylogenetic relationships in the F. limnocharis complex from Bangladesh and other Asian countries such as Sri Lanka, Thailand, Malaysia, Taiwan and Japan by allozyme analyses. We used a total of 95 frogs of the F. limnocharis complex from these countries and F. cancrivora from the Philippines as an outgroup. Based on body size, the F. limnocharis complex from Bangladesh was divided into three distinct groups: large, medium and small types. Allozyme analyses were carried out with 28 loci encoding 20 enzymes and two blood proteins by horizontal starch-gel electrophoresis. When genetic distance was calculated, distinct divergence was found among the three types: mean genetic distance was 0.782 between the small and medium types, 1.458 between the large and medium types, and 1.520 between the large and small types. Phylogenetic trees based on genetic distance showed that all populations of Bangladesh small type strongly formed a cluster and were found to be most closely related to the Sri Lanka population; that all populations of Bangladesh large type formed a very strong cluster and were grouped with several populations from Thailand, Malaysia, Japan, and Taiwan; and that the medium type was segregated from all other groups. This may imply that each of the three types is a different species, and that the medium type is possibly an undescribed taxon.     

Genotypic variability and persistence of Legionella pneumophila PFGE patterns in 34 cooling towers from two different areas

Genotypic variability and clonal persistence are important concepts in molecular epidemiology as they facilitate the search for the source of sporadic cases or outbreaks of legionellosis. We studied the genotypic variability and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns over time (period > 6 months) in 34 positive cooling towers from two different areas. In area A, radius of 70 km, 52 indistinguishable PFGE patterns were differentiated among the 27 cooling towers. In 13 cooling towers we observed ≥ 2 PFGE patterns. Each cooling tower had its own indistinguishable Legionella PFGE pattern which was not shared with any other cooling tower. In area B, radius of 1 km, 10 indistinguishable PFGE patterns were obtained from the seven cooling towers. In four, we observed ≥ 2 PFGE patterns. Three of these 10 indistinguishable PFGE patterns were shared by more than one cooling tower. In 27 of 34 cooling towers the same PFGE pattern was recovered after 6 months to up to 5 years of follow-up. The large genotypic diversity of Legionella observed in the cooling towers aids in the investigation of community outbreaks of Legionnaires' disease. However, shared patterns in small areas may confound the epidemiological investigation. The persistence of some PFGE patterns in cooling towers makes the recovery of the Legionella isolate causing the outbreak possible over time.     

Serotypes, virulence genes, and PFGE patterns of enteropathogenic Escherichia coli isolated from Cuban pigs with diarrhea.

Thirty-six enteropathogenic Escherichia coli strains isolated from Cuban pigs with diarrhea were serotyped and screened by PCR for the presence of virulence genes. The 36 isolates belonged to 11 O serogroups and 14 O:H serotypes, with 53% of the isolates belonging to only two serotypes: O141:H- (13 isolates) and O157:H19 (6 isolates). Genes coding for STb, STa, VT2e, and LT toxins were identified in 69, 61, 53, and 6% of the isolates, respectively. The most prevalent fimbrial adhesin was F18, detected in 22 (61%) isolates. The gene encoding F6 (P987) colonization factor was identified in three (8%) isolates. None of the 36 isolates assayed contained genes encoding F4 (K88), F5 (K99), or F41. The seropathotype O141:H-:STa/STb/VT2e/F18 (13 isolates) was the most frequently detected, followed by O157:H19:VT2e/F18 (5 isolates). A genetic diversity study, carried out by pulsed-field gel electrophoresis (PFGE) of 24 representative isolates, revealed 21 distinct restriction patterns clustered in 18 groups (I-XVIII). Isolates of the same serotype were placed together in a dendrogram, but isolates of serotype O157:H19 showed a high degree of polymorphism. The results of this study demonstrate the presence in Cuba of different clusters among one of the most prevalent serotypes isolated from pigs with diarrhea. Further experiments are needed to determine whether some of these clusters have appeared recently; if so, their evolution, as well as their possible association with pathogenicity in farms should be studied.     

Molecular and genetic characterization of two pollen-expressed genes that have sequence similarity to pectate lyases of the plant pathogen Erwinia

A set of cDNAs that are expressed in tomato anthers were isolated [24]. We further characterized two of these cDNAs (LAT56 and LAT59) and their corresponding genomic clones. LAT56 and LAT59 show low levels of steady-state mRNA in immature anthers and maximal levels in mature anthers and pollen. The LAT56 and LAT59 genes are single-copy in the tomato genome, and are linked on chromosome 3, approximately 5 cM apart. Although these cDNAs did not cross-hybridize, their deduced protein sequences (P56 and P59) have 54% amino acid identity. The LAT56 and LAT59 genes each have two introns, but they are located in non-homologous positions. P56 and P59 show significant protein sequence similarity to pectate lyases of plant pathogenic bacteria. The similarity of P56 and P59 to the bacterial pectate lyases is equivalent to the homology described for different pectate lyase sequences of the genus Erwinia. We suggest that the pollen expression of LAT56 and LAT59 might relate to a requirement for pectin degradation during pollen tube growth.Abbreviations LAT, late anther tomato - bp, base pairs - MA, mature anther - PL, pectate lyase - kb, kilobase (pairs)     

Characterization of Erwinia amylovora strains from different host plants using repetitive-sequences PCR analysis, and restriction fragment length polymorphism and short-sequence DNA repeats of plasmid pEA29

AIMS: The three main aims of the study were the assessment of the genetic relationship between a deviating Erwinia amylovora strain isolated from Amelanchier sp. (Maloideae) grown in Canada and other strains from Maloideae and Rosoideae, the investigation of the variability of the PstI fragment of the pEA29 plasmid using restriction fragment length polymorphism (RFLP) analysis and the determination of the number of short-sequence DNA repeats (SSR) by DNA sequence analysis in representative strains. METHODS AND RESULTS: Ninety-three strains obtained from 12 plant genera and different geographical locations were examined by repetitive-sequences PCR using Enterobacterial Repetitive Intergenic Consensus, BOX and Repetitive Extragenic Palindromic primer sets. Upon the unweighted pair group method with arithmetic mean analysis, a deviating strain from Amelanchier sp. was analysed using amplified ribosomal DNA restriction analysis (ARDRA) analysis and the sequencing of the 16S rDNA gene. This strain showed 99% similarity to other E. amylovora strains in the 16S gene and the same banding pattern with ARDRA. The RFLP analysis of pEA29 plasmid using MspI and Sau3A restriction enzymes showed a higher variability than that previously observed and no clear-cut grouping of the strains was possible. The number of SSR units reiterated two to 12 times. The strains obtained from pear orchards showing for the first time symptoms of fire blight had a low number of SSR units. CONCLUSIONS: The strains from Maloideae exhibit a wider genetic variability than previously thought. The RFLP analysis of a fragment of the pEA29 plasmid would not seem a reliable method for typing E. amylovora strains. A low number of SSR units was observed with first epidemics of fire blight. SIGNIFICANCE AND IMPACT OF THE STUDY: The current detection techniques are mainly based on the genetic similarities observed within the strains from the cultivated tree-fruit crops. For a more reliable detection of the fire blight pathogen also in wild and ornamentals Rosaceous plants the genetic features of deviating E. amylovora strains have to be studied in detail.