Amylovora Strains Research Articles

Effects of Consecutive Streptomycin and Kasugamycin Applications on Epiphytic Bacteria in the Apple Phyllosphere.

Antibiotic applications are essential for fire blight management in the eastern United States. Recently, streptomycin-resistant Erwinia amylovora strains were found in New York. There are growing concerns that streptomycin resistance may develop from postbloom streptomycin applications in local orchards. Our goal was to investigate the impacts of increasing streptomycin and kasugamycin applications on bacterial epiphyte community composition and antibiotic resistance in the phyllosphere of 'Idared' apple plantings in 2014 and 2015. Rinsate samples from leaves treated with 0, 3, 5, and 10 applications of streptomycin and kasugamycin were collected to isolate, enumerate, and identify epiphytic bacterial species. The majority of isolated epiphytic bacteria were identified as Pantoea agglomerans and fluorescent Pseudomonas spp., whereas E. amylovora was rarely found. Overall, postbloom streptomycin use did not result in an increased recovery of streptomycin-resistant E. amylovora. However, other streptomycin-resistant epiphytes (P. agglomerans and Pseudomonas spp.) did increase with increasing streptomycin applications. Increasing kasugamycin applications reduced the overall number and percentage of streptomycin-resistant epiphytes in the phyllosphere, which has important implications regarding the use of kasugamycin in orchards where streptomycin resistance is a concern.

Characterization of the incompatible interaction between Erwinia tracheiphila and non-host tobacco (Nicotiana tabacum)

Bacterial wilt is one of the most destructive diseases affecting a wide range of crops in the Cucurbits family including muskmelon (Cucumis melo), cucumber (Cucumis sativus), and squash (Cucubita pepo). The disease is caused by Erwinia tracheiphila, a Gram-negative and xylem-inhabiting species of Erwinia, which pathogenic mechanism is poorly understood. Many Gram-negative phytobacteria induce hypersensitive response (HR) in non-host plants, an immunity reaction triggered by pathogen recognition. With some exemptions, Erwinia species—notably E. amylovora, the causative agent of fire blight of rosaceous crops, and the reclassified soft rot pathogens, Pectobacterium and Dickeya species (formerly E. carotovora and E. chrysanthemi)—have been known to elicit HR in tobacco. However, concerning its pathogenic mechanism, the elicitation of classic HR has not been reported for some less-studied Erwinia species including E. tracheiphila. We characterized the induction of HR by the bacterial wilt pathogen in tobacco (Nicotiana tabacum cultivar ‘Xanthi’) using visual and physiological methods. We surveyed 21 E. tracheiphila strains and found that all of them elicited programmed cell death. Three strains (HCa1-5, UnisCu1-1, and MISpSq) fluorescently labeled with GFP could be visualized in the infiltrated leaves. We aligned the sequences of their HR-inducing protein, harpin (HrpN), predicted the secondary structures, and located the position of putative HR elicitors. We discovered differences between Cucurbita and Cucumis strains and found a close association of E. tracheiphila HrpN with those of Pantoea sp., Erwinia piriflorinigrans, and Erwinia pyrifoliae. Pre-infiltration of tobacco leaves with a lower cell population prevented HR following a subsequent challenge at the same area with HR-inducing levels of inoculum. The selected strains induced leaf conductivity levels similar to the HR-inducing E. amylovora strain E9, and their populations in the leaves decreased days after infiltration. Our results indicate that E. tracheiphila induces a classic HR in tobacco just like other HR-inducing Erwinia species.

Characterization of Erwinia amylovora strains from Hungary

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.

Unusual non-fluorescent broad spectrum siderophore activity (SID EGYII) by Pseudomonas aeruginosa strain EGYII DSM 101801 and a new insight towards simple siderophore bioassay.

Present study highlights an unusual non-fluorescent hydroxamate broad spectrum siderophore (SID EGYII) activity from Pseudomonas aeruginosa strain EGYII DSM 101801, a soil bacterial isolate, along with simple low cost effective siderophore bioassay. Detection of SID EGYII activity qualitatively was proved by masking this activity against Erwinia amylovora strain EGY1 DSM 101800, an indicator strain, in well-cut diffusion assay containing 100 µM FeCl3. SID EGYII activity was expressed quantitatively as arbitrary units [Siderophore arbitrary units (SAU)] 380 SAU/mL against E. amylovora strain EGY1 DSM 101800. Maximal SID EGYII activity was achieved upon growing P. aeruginosa strain EGYII DSM 101801 in PYB broth at 180 rpm for 24 h. SID EGYII displayed a broad spectrum antimicrobial activity against some human pathogens (i.e., Gram-positive bacteria, Gram-negative bacteria and yeasts) and a fireblight plant pathogen. Interestingly, transformants of Escherichia coli JM109 (DE3)pSID/EGYII harboring P. aeruginosa strain EGYII DSM 101801 plasmid demonstrated a perceivable antimicrobial activity against E. amylovora strain EGY1 DSM 101800. The broad spectrum antimicrobial activity of the unusual non-fluorescent SID EGYII would underpin its high potential in targeting bacterial pathogens posing probable threats to human health and agricultural economy. The present simple low cost effective bioassay is a new insight towards an alternative to the expensive cumbersome siderophore Chrome Azurol S assay.

Antagonistic potential of Pseudomonas graminis 49M against Erwinia amylovora, the causal agent of fire blight.

In a previous study (Mikiciński et al. in Eur J Plant Pathol, doi:10.1007/s10658-015-0837-y, 2015), we described the characterization of novel strain 49M of Pseudomonasgraminis, isolated from the phyllosphere of apple trees in Poland showing a good protective activity against fire blight on different organs of host plants. We now report investigations to clarify the basis for this activity. Strain 49M was found to produce siderophores on a medium containing complex CAS-Fe3+ and HDTMA, but was not able to produce N-acyl homoserine lactones (AHLs). Moreover, it formed a biofilm on polystyrene and polyvinyl chloride (PVC) surfaces. Strain 49M gave a positive reaction in PCR with primers complementary to gacA, the regulatory gene influencing the production of several secondary metabolites including antibiotics. The genes prnD (encoding pyrrolnitrin), pltC, pltB (pyoluteorin), phlD (2,4-diacetyl-phloroglucinol) and phzC as well as phzD (and their homologs phzF and phzA encoding phenazine), described for antagonistic fluorescent pseudomonads, however, were not detected. Research into the biotic relationship between strain 49M and Erwiniaamylovora strain Ea659 on five microbiological media showed that this strain clearly inhibited the growth of the pathogen on King’s B and nutrient agar with glycerol media, to a very small extent on nutrient agar with sucrose, and not at all on Luria–Bertani agar. On medium 925, strain 49M even stimulated E. amylovora growth. The addition of ferric chloride to King’s B resulted in the loss of its inhibitory ability. Testing the survival of 49M in vitro showed its resistance to drought, greater than that of E. amylovora.

The in planta proteome of wild type strains of the fire blight pathogen, Erwinia amylovora

Erwinia amylovora is a Gram-negative plant pathogen that causes fire blight. This disease affects most members of the Rosaceae family including apple and pear. Here, an infection model is introduced to study proteomic changes in a highly virulent E. amylovora strain upon interaction with its host as compared to a lower virulent strain. For this purpose separate shoots of apple rootstocks were wound-infected and when infection became systemic, bacterial cells were isolated and processed for analysis in a proteomics platform combining 2-D fluorescence difference gel electrophoresis and mass spectrometry. Comparing the proteome of the isolates, significant abundance changes were observed in proteins involved in sorbitol metabolism, amylovoran production as well as in protection against plant defense mechanisms. Furthermore several proteins associated with virulence were more abundant in the higher virulent strain. Changes at the proteome level showed good accordance at the transcript level, as was verified by RT-qPCR. In conclusion, this infection model may be a valuable tool to unravel the complexity of plant-pathogen interactions and to gain insight in the molecular mechanisms associated with virulence of E. amylovora, paving the way for the development of plant-protective interventions against this detrimental disease. SignificanceDuring this research a first time investigation was performed on the proteome of E. amylovora, grown inside a susceptible host plant. This bacterium is the causal agent of fire blight, which can affect most members of the Rosaceae family including apple and pear. To do so, an artificial infection model on shoots of apple rootstocks was optimized and employed. When infection was systemic, bacterial cells were extracted from the plant tissue followed by extraction of the proteins from the bacteria. Further processing of the proteins was done by using a 2-D fluorescence difference gel electrophoresis analysis followed by mass spectrometry. By the use of two strains differing in their virulent ability, we were able to draw conclusions concerning virulence and behavior of different strains inside the host. This research provides a model to investigate plant-pathogen interactions and more importantly, we identified possible new targets for the development of novel control methods against this devastating disease.

Characterization of Streptomycin Resistance in Isolates of Erwinia amylovora in California

In surveys from 2006 to 2014, streptomycin resistance in Erwinia amylovora from pear-growing areas in California declined from very high incidence in 2006 and 2007 to very low incidence in 2013 and 2014. The majority of resistant strains were designated as moderately resistant-low (MR-L), and were almost exclusively found in Sacramento County, whereas highly resistant (HR) strains were only recovered in Sutter-Yuba and San Joaquin counties. Resistance of HR strains was associated with a mutation in codon 43 of the chromosomal rpsL gene that results in a change from lysine to arginine, the same mutation that was originally reported for resistant strains from California in the mid-1970s. MR-L strains were found to harbor the strA-strB streptomycin resistance genes on transposon Tn5393a. This transposon lacks insertion sequence IS1133 that provides a promoter for efficient expression of strA-strB, resulting in lower minimum inhibitory concentrations of MR-L strains compared with those from other locations that harbor strA-strB on Tn5393::IS1133. In contrast to previously described plasmid-mediated resistance where Tn5393 is inserted in pEa34, or pEA29, Tn5393a in MR-L strains was located on plasmid pEU30. This plasmid was first described in E. amylovora from the western United States but was not associated with streptomycin resistance determinants previously. We hypothesize that Tn5393a was introduced into an E. amylovora strain carrying pEU30 and transposed into that plasmid. This hypothesis was supported by clustered regularly interspaced short palindromic repeat (CRISPR) sequence analysis that showed that two MR-L strains share the same CRISPR1 pattern as a streptomycin-sensitive strain. With current low resistance levels in California growing regions, streptomycin could be successfully used again, but applications per season should be limited and the antibiotic should be mixed and rotated with different modes of action.

Genetic islands in pome fruit pathogenic and non-pathogenic Erwinia species and related plasmids.

New pathogenic bacteria belonging to the genus Erwinia associated with pome fruit trees (Erwinia, E. piriflorinigrans, E. uzenensis) have been increasingly described in the last years, and comparative analyses have found that all these species share several genetic characteristics. Studies at different level (whole genome comparison, virulence genes, plasmid content, etc.) show a high intraspecies homogeneity (i.e., among E. amylovora strains) and also abundant similarities appear between the different Erwinia species: presence of plasmids of similar size in the pathogenic species; high similarity in several genes associated with exopolysaccharide production and hence, with virulence, as well as in some other genes, in the chromosomes. Many genetic similarities have been observed also among some of the plasmids (and genomes) from the pathogenic species and E. tasmaniensis or E. billingiae, two epiphytic species on the same hosts. The amount of genetic material shared in this genus varies from individual genes to clusters, genomic islands and genetic material that even may constitute a whole plasmid. Recent research on evolution of erwinias point out the horizontal transfer acquisition of some genomic islands that were subsequently lost in some species and several pathogenic traits that are still present. How this common material has been obtained and is efficiently maintained in different species belonging to the same genus sharing a common ecological niche provides an idea of the origin and evolution of the pathogenic Erwinia and the interaction with non-pathogenic species present in the same niche, and the role of the genes that are conserved in all of them.

Isolation and Molecular Characterization of the Fire Blight Pathogen, Erwinia amylovora, Isolated from Apple and Pear Orchards in Egypt

A B S T R A C T Eleven Erwinia amylovora isolates, the causal agent of the fire blight disease, were isolated from apple orchards adjacent to those of pears showing fire blight disease symptoms. Initial characterization concerning morphological and biochemical traits revealed that all isolates were typical to those of E. amylovora. Furthermore, all bacterial isolates showed typical symptoms of the fire blight disease upon infection of unripe pear fruits. Molecular characterization of these isolates via PCR utilizing specific primers based on a region of plasmid pEA29 and chromosomal DNA ams-region was performed. PCR products were positive and represented the expected length 1.1 and 1.6 kb, respectively. Moreover, PCR reactions utilizing the 16S rRNA universal primers were carried out. DNA sequence of PCR products and analysis via blast and Genbank data showed that, the bacterial isolates actually belonging to E. amylovora. Moreover, there are interference between these Erwinia isolates and identified E. amylovora strains based on constructed phylogenetic analysis. To the best of our knowledge this is the first report of fire blight of apples caused by E. amylovora in Egypt.

A comparative proteome analysis reveals flagellin, chemotaxis regulated proteins and amylovoran to be involved in virulence differences between Erwinia amylovora strains

Erwinia amylovora is a Gram-negative bacterium that causes the destructive disease fire blight affecting most members of the Rosaceae family, of which apple and pear are economically the most important hosts. E. amylovora has been considered as a homogeneous species in whole, although significant differences in virulence patterns have been observed. However, the underlying causes of the differences in virulence remain to be discovered. In a first-time comparative proteomic approach using E. amylovora, 2D differential in-gel electrophoresis (DIGE) was used to identify proteins that could explain the gradual difference in virulence between four different strains. Two important proteins were identified, FliC and CheY, both involved in flagella structure, motility and chemotaxis, which were more abundant in the least virulent strain. In the highly virulent strains the protein GalF, involved in amylovoran production, was more abundant, which was consistent with the higher expression of the gene and the higher amylovoran content in this strain in vitro. Together, these results confirm the involvement of amylovoran in virulence, but also imply an indirect role of flagellin in virulence as elicitor of plant defence. Biological significanceThis research provides new insights into our current understanding of the virulence of Erwinia amylovora. This plant-pathogen is considered a homogeneous species although different strains show differences in virulence. Despite the efforts made on the genomic level which resulted in the discovery of virulence factors, the reason for the different virulence patterns between strains has not yet been identified. In our lab we used a comparative proteomic approach, which has never been published before, to identify proteins involved in these differences between strains and hereby possibly involved in virulence. Our results provide interesting insights in virulence and present us with the opportunity to glance into the proteome of E. amylovora.

Monitoring of Erwinia amylovora in Montenegro

Recent studies of Erwinia amylovora in Montenegro, conducted from 2012 to 2014, indicated that the bacterium was widespread in the northern, continental part of the country, where the most important fruit-growing regions are situated. The presence of the bacterium was confirmed on quince, pear, apple, medlar and hawthorn. Pathogenic, cultural and biochemical characteristics of E. amylovora strains sampled from pome fruit species and indigenous flora in Montenegro had been studied previously. In the present study, serological tests were used for identification of E. amylovora strains originating from pome fruit trees and indigenous plants. Monitoring of E. amylovora and collection of samples with symptoms of bacterial fire blight from different hosts and locations were performed in Montenegro from 2012 to 2014. Isolation of the bacterium on nutrient medium produced a large number of isolates, whose pathogenicity was confirmed on immature pear fruits. Twenty-seven strains of the bacterium, originating from three pome fruit species (quince, pear and apple) and one indigenous species (hawthorn) were selected for serological analyses. Two applied serological methods, ELISA and IF test, enabled rapid detection of the bacterium and simultaneous examination of a large number of samples over a short period of time. Serological analyses showed high homogeneity in antigenic structure of the studied E. amylovora strains sampled from quince, pear, apple and hawthorn from nine locations in Montenegro.

Preparatory work for the pest categorisation of three bacteria listed in the annexes of the Council directive 2000/29/EC Erwinia amylovora Winslow et al. Xylophilus ampelinus (Panagopoulos) Willems et al. Clavibacter michiganensis subsp. insidiosus(McCulloch) Davis et al

Preparatory work for the pest categorisation of three bacteria listed in the annexes of the Council directive 2000/29/EC Erwinia amylovora Winslow et al. Xylophilus ampelinus (Panagopoulos) Willems et al. Clavibacter michiganensis subsp. insidiosus(McCulloch) Davis et al

The fire blight resistance QTL of Malus fusca (Mfu10) is affected but not broken down by the highly virulent Canadian Erwinia amylovora strain E2002A

Recently, Mfu10 - the major QTL for resistance to fire blight was reported on linkage group 10 of Malus fusca which could explain about 66 % of the phenotypic variation in a population artificially inoculated with Erwinia amylovora strain Ea222_JKI. In the meantime, it had been reported that the resistance of the fire blight QTL of Malus × robusta 5 (Mr5), which could explain 80 % of phenotypic variation is strain specific and is completely overcome by the highly virulent strain Ea3049 originating from Canada (E2002A). In contrast to Mr5, the M. fusca donor of Mfu10 is only very slightly affected by this highly virulent strain. In this short communication, we prove that this particular strain is not able to overcome the resistance of M. fusca but affects Mfu10. The same F1 progenies, derived from a cross between the resistant M. fusca and the susceptible apple cultivar, Idared, used to detect Mfu10, were used for phenotyping with Ea3049. Although the mean shoot necrosis of all progenies was 62.4 %, marker-phenotype association determined by Kruskal-Wallis analysis showed that markers on LG10 correlate significantly to resistance levels with SSR markers FR481A and FRM4 having the highest K values of 37.1 and 36.7, respectively. Interval mapping and multiple QTL mapping (MQM) performed with MAP-QTL®, showed that Mfu10 could still be detected on the same position on LG10, but only explaining 41.2 % of phenotypic variation. The implications of these results are discussed.

Erwinia amylovora Enfeksiyonu Sonrası Elma, Armut ve Ayva Çeşitlerinde Konukçu Protein Miktarlarının Belirlenmesi

Fire blight disease caused by Erwinia amylovora is a destructive bacterial pathogen mainly on pears, apples and quinces from Rosaceae family. In this study, it was aimed determination of total protein amounts in different apple cultivars (Braeburn, Fuji, Gala and Golden), pear cultivars (Santa Maria and Williams) and quince cultivars (Eşme and Ekmek) in the infections of two virulent E. amylovora strains (Ea234-1 and Ea240-3) according as the time. It was taken leaf samples after leaf inoculation with E. amylovora (108 CFU ml-1) at 24th, 36th and 72nd hours. For verification of the infections, re-isolations were made from bacteria inoculated plants and the agent was identified as E. amylovora by biochemical, physiological and molecular tests. In determining the amounts of total protein and in the SDS-PAGE analyses were used Bradford and Laemmli methods, respectively, and absorbance values of protein extracts derived from the leaf samples taken, were obtained at 595 nm wavelength. According to the findings obtained; after infection of E. amylovora in the apple varieties comparing to controls, total protein concentrations at 24th hours increased and a decrease in the amount of 36th to 72nd hours and Braeburn has the highest protein content was determined. In the pear varieties, while total protein concentrations at 24th and 36th hours increased, a decrease in the amount of 72nd hour, and Santa Maria variety has the highest protein content was detected. In the quince varieties, total protein concentrations at 72th hour increased and Eşme variety has the highest protein content was identified. As a result of SDS-PAGE analysis, protein fractions which have different molecular weights were obtained. The protein bands were defined approximately 55-70 kDa and 35-55 kDa molecule weight on apple and quince varieties, respectively and also approx. 55-70 kDa in pear varieties.

COMPARATIVE MAPPING OF FIRE BLIGHT RESISTANCE IN MALUS

Fire blight, caused by enterobacterium Erwinia amylovora, is the most important bacterial disease affecting pome fruit. It can cause devastating economic losses and is reliably controlled only by the application of antibiotics, which are banned in many European countries due to environmental and sustainability issues and consumers’ mistrust. One solution could be the utilization of fire blight resistant cultivars in apple production. In 2003, we started an approach at Dresden-Pillnitz to detect different mechanisms conferring resistance to fire blight aimed at their com-bination in new cultivars. Four segregating populations were established to map QTLs for fire blight resistance. The donors used were three wild species accessions of Malus baccata, M. fusca, M. ×robusta 5 and the Pillnitz cultivar ‘Rewena’. The susceptible parent in each case was Idared. Grafted scions of each progeny were inoculated with E. amylovora strain Ea222_JKI. Average percent lesion length (PLL) of all progenies was determined. Genetic linkage maps were established using DArT- SCAR-, SNP-, and SSR-markers. Whereas in Rewena no QTL could be determined, major QTLs were detected in M. baccata on linkage group 12, in M. fusca on linkage group 10, and in M. ×robusta 5 on linkage group 3 explaining up to around 50, 85 and 85% of the phenotypic variance, respectively. The fact that all resistance QTLs are located on different linkage groups enhances the chance that different mechanisms are acting in the donors. Additionally, trees of the Idared by M. ×robusta 5 population were planted in an orchard and flowers were inoculated for phenotyping. The QTL on linkage group 3 could be con-firmed after mapping.

EVIDENCE OF A MAJOR QTL FOR FIRE BLIGHT RESISTANCE IN THE APPLE WILD SPECIES MALUS FUSCA

Fire blight is a common and very destructive disease affecting apple (Malus ×domestica) and pear (Pyrus communis) productions. Caused by enterobacterium Erwinia amylovora, its devastating economic effects ranges from losses in yield to severe damage or death of trees in the orchard, and in extreme cases loss of a whole orchard. The most effective management measure available to producers is the use of antibiotics. However, dependence on antibiotics treatment is not sustainable given risks such as the rapid selection of antibiotic-resistant Erwinia populations and environmental hazards, which have led to their application being forbidden in many countries. Therefore the planting of fire blight-resistant cultivars seems to be the most promising alternative strategy. However until now, there is no fire blight-resistant apple cultivar produced for the global market. Host resistance to fire blight in Malus is thought to be quantitatively controlled. Thus, several quantitative trait loci for resistance have been identified in some accessions of Malus spp. In this study, we explored a segregating population derived from a cross between the apple wild species Malus fusca and the Malus ×domestica cultivar ‘Idared’. F1 progenies used for mapping were artificially inoculated with E. amylovora strain Ea222 at a concentration of 109 CFU/ml in two different years. The averages of percentage lesion length (PLL) of all replicates of each genotype were used as numerical traits for statistical analysis. A Kruskal-Wallis analysis, used to determine marker-phenotype association, revealed two DArT markers (971000 and 970840) with the highest significance. QTL mapping revealed a strong QTL explaining above 60% of the phenotypic variation. This is the first report of a fire blight resistance QTL in M. fusca.

Susceptibility of apple genotypes from European genetic resources to fire blight (Erwinia amylovora)

The study was conducted in greenhouses at two locations on one-year old trees of apple cultivars and clones originating from genetic resources and breeding programs conducted in Belgium, Hungary, Germany, Poland, Sweden and Switzerland. At each location different inoculation method of trees with Erwinia amylovora was used. In Poland, at the Research Institute of Horticulture in Skierniewice (IO), the method of cutting-off shoot tips with scissors immersed in a single highly virulent strain of the inoculum (107 cfu ml−1) was applied for testing 38 genotypes. In Germany, at the Julius Kuhn-Institut in Dresden and Quedlinburg (JKI), the method of cutting-off the tips of the two youngest leaves with scissors dipped in a suspension of three highly virulent strains at concentration of 109 cfu ml−1 was used for testing 27 genotypes. Observations and measurements of fire blight development on inoculated shoots were performed at 2, 4 and 6 weeks after inoculation at IO and after 4 weeks at JKI. Each genotype was evaluated over the course of two or three seasons. Of the total 40 genotypes, 25 were tested at both locations. The final assessment based on results of all tests was either similar or the same for 17 genotypes. MR 10, Pi-As 12,53 and Pi-As 50,74 were the most resistant. ‘Free Redstar’ and ‘Reanda’, tested only at IO, as well as Pi-As 36,164, tested only at JKI, were also classified as highly resistant. The following cultivars were found to be very susceptible at both locations: ‘Blauacher Wadenswil’, ‘Bohnapfel’ and ‘Idared’. Among those tested only at IO, CRA Ma A40, CRA Ma A68, CRA Ma A70, CRA Ma B28, CRA Ma D30 and ‘Jacob Level’ appeared to be also very susceptible. No significant differences in shoot necrosis length appeared when comparing the two inoculation methods used at IO and JKI (i.e. application of single strain vs. mixture of three highly virulent E. amylovora strains, at two concentrations) on four genotypes.

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.

Evaluation of repeat sequences on plasmid pEA29 of Erwinia amylovora from Iran

The plasmid content of 38 Iranian Erwinia amylovora strains isolated from five different plant species and geographical locations from 2005 to 2013 were evaluated. With exception to the pEA29 plasmid, isolates obtained from quince and hawthorns carry the pEU72 plasmid. RFLP analysis of PstI amplified fragment of plasmid pEA29 using HpaII revealed three known fragments in all strains. Based on the observed polymorphism in the larger one that varied between 360 to 400 bp, all Iranian strains clustered into four groups. In order to determine the exact number of short-sequence DNA repeats, 18 strains were chosen for PstI fragment sequencing. About 63 % of the strains carried a repeat number of 4. The remaining 37 % harboured five, seven and eight repeats. Sometimes a correlation was seen between the RFLP patterns and SSR grouping with respect to other factors including the host plant species, geographical region or sampling year. This study reported the genetic variation of E. amylovora strains for the first time in Iran, based on enzymatic digestion of the PstI fragment of pEA29 and evaluation of the short sequence repeat units. It seems that, the latter method may be reliable for E. amylovora strains grouping.

Identification of a major quantitative trait locus for resistance to fire blight in the wild apple species Malus fusca

Fire blight, caused by the Gram-negative bacterium Erwinia amylovora, is the most important bacterial disease affecting apple (Malus × domestica) and pear (Pyrus communis) production. The use of antibiotic treatment, though effective to some degree, is forbidden or strictly regulated in many European countries, and hence an alternative means of control is essential. The planting of fire blight-resistant cultivars seems to be a highly feasible strategy. In this study, we explored a segregating population derived from a cross between the wild apple species Malus fusca and the M. × domestica cultivar Idared. F1 progenies used for mapping were artificially inoculated with Erwinia amylovora strain Ea222_JKI at a concentration of 109 cfu/ml in three different years. The averages of percentage lesion length of all replicates of each genotype were used as numerical traits for statistical analysis. A Kruskal–Wallis analysis was used to determine marker–phenotype association and revealed a linkage group with Diversity Arrays Technology (DArT) markers significantly linked with fire blight. After locating the positions of the DArT markers on the Golden Delicious genome, simple sequence repeat (SSR) markers were developed from chromosome 10 to replace the DArT markers and to determine the quantitative trait locus (QTL) region. Multiple QTL mapping (MQM) revealed a strong QTL (Mfu10) on linkage group 10 of M. fusca explaining about 65.6 % of the phenotypic variation. This is the first report on a fire blight resistance QTL of M. fusca.