Characterisation of naturally occurring <Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains lacking the common plasmid pEA29 and their detection with real-time PCR

Erwinia amylovora, the causal agent of fire blight, carries the common plasmid pEA29 of 29 kb. To screen for occurrence of natural strains without plasmid pEA29, we applied PCR analysis with primers from the plasmid and the chromosomal ams region. In addition, a described TaqMan probe from pEA29 and newly designed primers from the ams-region were used for identification by real-time PCR. One strain isolated in Iran, one strain from Spain and two strains from Egypt lacked plasmid pEA29. From a recent screening series in southern Germany, in 123 E. amylovora strains from necrotic fire blight host plants, one strain was found without the common plasmid. The strains without pEA29 were virulent in assays with immature pears and on apple seedlings, but showed a reduced growth level in minimal medium without amino acids and thiamine. Transposon-labelled pEA29 was transformed into the plasmid-free strains resulting in restoration of this growth deficiency. The plasmid was stably maintained in these E. amylovora cells. The newly designed chromosomal primers for conventional and for real-time PCR identified E. amylovora strains in field samples lacking pEA29. These variants are apparently rare, but were detected in isolates from different regions in the world with fire blight.     

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.     

Streptomycin resistance in Clavibacter michiganensis subsp. michiganensis strains from Chile is related to an rpsL gene mutation

Streptomycin has been used for decades in Chile to control Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of tomato bacterial canker. The aim of this work was to evaluate streptomycin resistance and to analyse the presence of resistance-related genes in Cmm strains from Chile. A collection of 25 Cmm strains isolated from different localities in central Chile between 1996 and 2015 was analysed. Minimum inhibitory concentration (MIC) of streptomycin was determined. A search of streptomycin resistance-related genes was carried out in Cmm genomes, and the presence of these genes was studied in all Chilean strains using PCR and sequencing techniques. MIC results showed that four of 25 strains were highly sensitive to streptomycin, with MIC values <2 μg mL⁻¹. The remaining 21 strains possessed MIC of streptomycin ≥100 μg mL⁻¹. The strB gene, encoding an aminoglycoside 6-phosphotransferase that inactivates streptomycin, was detected in all Chilean strains, including sensitive and resistant strains. In the 21 resistant strains, a mutation in codon 43 of the rpsL gene was determined, conferring high streptomycin resistance. Interestingly, the four streptomycin-sensitive Cmm strains did not possess this mutation. This study proposes that the continuous use of streptomycin leads to emergence of resistant Cmm strains, challenging researchers to look for novel alternatives to control this plant pathogenic bacterium.     

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.     

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.     

Biological control of fire blight of apple and pear with antagonistic Lactobacillus plantarum

Lactic acid bacteria (LAB) can be a source of biological control agents (BCA) of fire blight disease. Several species of LAB are inhabitants of plants and are currently used as biopreservatives of food because of their antagonistic properties against bacteria, and are considered as generally safe. Candidates to BCA were selected from a large collection of LAB strains obtained from plant environments. Strains were first chosen based on the consistency of the suppressive effect against E. amylovora infections in detached plant organs (flowers, fruits and leaves). Lactobacillus plantarum strains PC40, PM411, TC54 and TC92 were effective against E. amylovora in most of the experiments performed. Besides, strains PM411, TC54 and TC92 had strong antagonistic activity against E. amylovora and also other target bacteria, and presented genes involved in plantaricin biosynthesis (plnJ, plnK, plnL, plnR and plnEF). The strains efficiently colonized pear and apple flowers; they maintained stable populations for at least 1 week under high RH conditions, and survived at low RH conditions. They were effective in preventing fire blight on pear flowers, fruits and leaves, as well as in whole plants and in a semi-field blossom assay. The present study confirms the potential of certain strains of L. plantarum to be used as active ingredient of microbial biopesticides for fire blight control that could be eventually extended to other plant bacterial diseases.     

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.     

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.     

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.     

Acibenzolar-S-methyl Induces the Accumulation of Defense-related Enzymes in Apple and Protects from Fire Blight

Acibenzolar-S-methyl (Novartis) is a chemical inducer of systemic acquired resistance in several annual plants. The ability of this novel chemical to induce resistance was studied in a perennial plant (apple) affected by fire blight caused by Erwinia amylovora. Acibenzolar-S-methyl (100 and 200mg/l active ingredient) protected Golden Delicious seedlings, scions and trees from artificial infection when applied before inoculation. The protection of apple seedlings was similar to the protection obtained with the standard for fire blight control, plantomycin (100mg/l streptomycin sulfate), applied immediately before inoculation. The mean levels of control in scions in the greenhouse and in trees in orchards were approximately 69% and 50%, respectively. The protection of apple seedlings was constantly associated with the activation of two families of defense-related enzymes, peroxidases and -1,3-glucanases. Accumulation of both enzymes was induced locally in treated leaves and systemically, especially for -1,3-glucanases, in upper untreated leaves, and was sustained for at least 17 days. These results suggest that acibenzolar-S-methyl promotes induced systemic resistance in apple by increasing defense-related compounds. This chemical could provide a new approach of control of fire blight but its practical use needs further investigation.     

Infection frequency of mature apple fruit with Erwinia amylovora deposited on pedicels and its survival in the fruit stored at low temperature

The infection frequency of mature apple fruit by Erwinia amylovora and the survival of E. amylovora in the fruit stored at low temperature were investigated. The fruit stems (pedicels) of 460 mature apple fruit were inoculated with 10⁵ or 10⁴ cfu of bioluminescent E. amylovora, tagged with lux genes. Nine days after inoculation, 43% and 27% of the fruit inoculated with 10⁵ and 10⁴ cfu, respectively, were infected. All infected fruit looked healthy. After 6 months of storage at 5°C, almost all of the 142 infected fruit had viable E. amylovora. Of the fruit containing E. amylovora internally, 19.5% had latent infections and the rest had blight symptoms. E. amylovora was not uniformly distributed in the fruit flesh, and internal brown lesions were observed where E. amylovora was densely distributed. These findings showed that mature apple fruit may be infected with E. amylovora, especially as latent infections, and act as a source for long-range dissemination.     

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.     

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.     

Characterization of theErwinia amylovora population in Israel

A collection of 205 strains ofErwinia amylovora isolated in Israel over a period of 12 years has been established. The strains were isolated from different varieties of pear, apple, loquat and quince grown in Israel, and collected from different locations in the country. They were characterized in respect to degree of virulence on several hosts and serological and molecular characters. Pathogenicity tests carried out on flowering branches of pear and apple, shoots of pears, and on trees of pear and loquat grown in containers outdoors, revealed no significant differences in the severity of blossom blight or shoot blight among the various strains. ELISA and immunofluorescence assays revealed no serotypic groups among the Israeli strains. Genomic diversity was studied by random amplified polymorphic DNA (RAPD) analysis using 24 arbitrary 10-base primers. All the strains examined (45 Israeli and 11 from Egypt, Cyprus and Greece) produced the same RAPD patterns with each of the primers used. Amplification patterns were indistinguishable from those produced by strains isolated from the neighboring countries. Results presented in this study suggest that the population ofE. amylovora in Israel is homogenous.     

Blue EaLAMP—a specific and sensitive method for visual detection of genomic Erwinia amylovora DNA

Fire blight caused by the gram-negative bacterium Erwinia amylovora is a major disease of pome fruit like apple (Malus domestica) or pear (Pyrus communis). Detection of the pathogen is hampered by low titres usually being present during initial flower infection and the brief time frame for analysis to decide upon subsequent countermeasures. Here we describe a loop-mediated isothermal amplification of DNA (LAMP) assay for genomic DNA of Erwinia amylovora, which relies on a highly specific primer design that excludes amplification from typical DNA sources of the orchard biological system and from sample handling. The assay enables the fast detection of down to approximately 20?cfu of pure Erwinia amylovora or 100?fg genomic DNA (corresponding to approximately 25 E. amyolovora equivalents) per reaction within 45?min. Fast and reliable detection of E. amylovora in orchard samples by naked eye is achieved through a visual colour change indication with hydroxynaphthol blue. The assay avoids use of complex technical devices and is thus suitable for field testing.     

Levels of fire blight (<Emphasis Type="Italic">Erwinia amylovora</Emphasis>) susceptibility of native apple,pear and quince germplasm from Lake Van Basin,Turkey

Fire blight resistance of apple, pear and quince genetic resources from Lake Van Basin (eastern Turkey) was tested using Erwinia amylovora strain Ea Van. Shoot tips of 92 native accessions (48 accessions for apple, 38 accessions for pear and 6 accessions for quince) were wounded for inoculation, and artificially inoculated with pathogenic bacteria under greenhouse conditions. The levels of resistance of accessions were classified in comparison with control varieties according to the genotype susceptibility index (GSI%) scores based on the lesion length on shoots of each genotype. Fire blight resistance of accessions consisted of five classes: resistant (R), moderately resistant (MR), moderately susceptible (MS), susceptible (S) and highly susceptible (HS). GSI% scores differed significantly among accessions from each fruit species (p < 0.01). GSI values ranged from 12.4% to 64.1% for apple genotypes, from 17.2% to 55.1% for pear genotypes, and from 17.8% to 43.4% for quince genotypes. No resistant genotypes of apple, pear and quince were observed. Seven accessions of apple, two accessions of pear and one accession of quince were MR. 25 accessions of apple, 14 accessions of pear and one accession of quince were MS. These findings indicate a considerable variation in fire blight resistance and could contribute to breeding efforts regarding fire blight resistance in apple, pear and quince.     

Present status of bacterial blight in cotton genotypes evaluated at Busia and Siaya counties of Western Kenya

Bacterial blight (BB) incited by Xanthomonas citri subsp. malvacearum (Xcm) is an important bacterial disease occurring in all cotton growing areas throughout the world, including parts of Western Kenya, that are characterized by a hot and humid climate. The disease causes seedling blight, angular leaf spot, boll rot and black arm on petioles and branches leading to a loss of fruiting branches with yield losses of up to 35 %. Fifty- one Kenyan cotton genotypes (Gossypium hirsutum L.) were established in the two counties of Siaya and Busia famous for their cotton cultivation in the western region. The BB symptoms caused by natural Xcm infection in the field were scored for each cotton accession. In addition artificial inoculation with the same strains was done to confirm the status under controlled greenhouse conditions. The results of BB disease scoring revealed that some accessions (e.g. T 24A and T 24B) possess a reasonable level of resistance. However, most of the Kenyan genotypes surveyed showed medium to severe symptoms of BB, including KSA 81 M, the only commercially grown cultivar in the region of Western Kenya. Overall, 71 % of the genotypes showed susceptibility and 29 % were classified as either resistant or moderately resistant. There is therefore need to improve the local commercial genotypes by introducing new genetic resources with a more durable BB resistance to ensure a successful revitalization of the Kenyan cotton sector.     

Taxonomic Position of the Causal Pathogen of Bacterial Shoot Blight of Pear

Bacteriological properties and DNA-DNA homology values were compared among the pathogen causing bacterial shoot blight of pear (BSBP) isolated in 1994–1996, Erwinia amylovora isolated outside of Japan, and other Amylovora group bacteria. Bacteriological properties of BSBP strains were identical to those of E. amylovora in the majority of tests, but differed distinctively in several tests, including hydrolysis of esculin and acid production from salicin, etc. BSBP strains differed from the others in the Amylovora group in many other tests. DNA homology among the strains of BSBP ranged from 85 to 103% and from 83 to 110% among strains of E. amylovora. In contrast, the values between BSBP strains and E. amylovora strains were 55 to 81%, while those between BSBP strains and other Amylovora group strains were 42% or less. We consider, therefore, that the BSBP pathogen may well be included in E. amylovora at the species level. E. amylovora, including BSBP strains, however, can be classified into four biovars based on differences in nine tests such as growth factor requirements and crater formation on high sucrose medium. Namely, there are two biovars from Maloideae sources, one from Rubus idaeus, and one from the source of BSBP in Hokkaido. The presence of these biovars suggests a correlation with geographical, serological, and pathogenic differentiations in the species of E. amylovora. The BSBP pathogen in Hokkaido was identified as E. amylovora bv. 4 which is distinct from E. amylovora bv. 1, 2 and 3 isolated in countries outside of Japan. Received 29 July 1999/ Accepted in revised form 12 October 1999     

Novel metrics to classify fire blight resistance of 94 apple cultivars

Fire blight (Erwinia amylovora), a potentially devastating disease in apple, can cause floral, fruit and structural damage and even tree death. Most commercial apple cultivars are susceptible and the resistance/susceptibility of many modern cultivars has not been evaluated. Fire blight resistance/susceptibility is difficult to phenotype due to quantitative resistance, impacts of tree vigour and environment on susceptibility, and the erratic nature of the disease. Resistance/susceptibility levels were determined for 94 apple cultivars and important breeding parents. In 2016 and 2017, multiple actively growing shoots per tree (about three trees per cultivar) were challenged with E. amylovora Ea153n via a cut-leaf inoculation method. Proportion of current season's shoot length blighted (SLB) was calculated for each shoot. To classify cultivar responses, estimated marginal SLB means were compared to four controls, representing highly susceptible (HS) to highly resistant (HR), via Dunnett's tests. Cultivar responses ranged from HS to HR with estimated marginal SLB means of 0.001–0.995 in 2016 and 0.000–0.885 in 2017. Most cultivars demonstrated similar resistance/susceptibility levels in both years (ρ = 0.657, P < 0.0001). K-means clustering was used to classify cultivars into three resistance/susceptibility groups based on incidence, average severity (SLB), and maximum severity values (maximum SLB and age of wood infected). Sixteen cultivars were consistently moderately resistant (MR) to HR while the remainder ranged from HS to MR. An updated comparison of susceptibility of important cultivars is provided. Resistance/susceptibility information gained could be used to identify genetic loci associated with resistance/susceptibility and/or inform parental selection in apple scion breeding programmes.