Phenotypic and genetic diversity of Erwinia amylovora: the causal agent of fire blight

Erwinia amylovora is a polyphagous bacterium causing fire blight on apple, pear and over 130 other plant species belonging mainly to the Rosaceae family. Although E. amylovora is regarded as a very homogenous species, the particular strains can differ in pathogenic ability as far as their host range is concerned (e.g. those originating from Rubus or Maloidae plants) as well as by the extent of the disease they cause. It was found that strains originating from North America are generally more genetically heterogeneous than those from Europe. Diversity of E. amylovora is also related to streptomycin resistance as a result of its application to control of fire blight. The level of genetic heterogeneity of E. amylovora is so low (comparative genome analysis revealed a similarity of over 99% for the two genomes tested) that standard DNA-based techniques fail in detection of intra-species variability. Amplified fragment length polymorphism was found to be most useful for differentiation of strains of fire blight causal agent as well as techniques ensuing release of pan-genome sequences of two E. amylovora strains: multi-locus variable number of tandem repeats analysis and clustered regularly interspaced short palindrome repeats.     

Characterization of Further Erwinia amylovora Strains and the Application of the API 20E System in Diagnosis

The former phenotypic study of Erwinia amylovora (VANTOMME et al. 1982) was extended with a collection of 54 Erwinia amylovora strains from a broad plant and geographic origin. From the 85 phenotypic features studied, 72 (85%) were present in at least 90% of the strains. Only 49 (58%) of the features were shared by all strains. Thirty-eight strains were also examined by the API 20E system. The API 20E code numbers for E. amylovora are unique and, combined with an immature, (green) pear test, may be used for an accurate identification of Erwinia amylovora.     

Comparative genomic analysis of Erwinia amylovora reveals novel insights in phylogenetic arrangement,plasmid diversity,and streptomycin resistance

Erwinia amylovora is a destructive pathogen of Rosaceous plants and an economic concern worldwide. Herein, we report 93 new E. amylovora genomes from North America, Europe, the Mediterranean, and New Zealand. This new genomic information demonstrates the existence of three primary clades of Amygdaloideae (apple and pear) infecting E. amylovora and suggests all three independently originate from North America. The comprehensive sequencing also identified and confirmed the presence of 7 novel plasmids ranging in size from 2.9 to 34.7 kbp. While the function of the novel plasmids is unknown, the plasmids pEAR27, pEAR28, and pEAR35 encoded for type IV secretion systems. The strA-strB gene pair and the K43R point mutation at codon 43 of the rpsL gene have been previously documented to confer streptomycin resistance. Of the sequenced isolates, rpsL-based streptomycin resistance was more common and was found with the highest frequency in the Western North American clade.     

Hypersensitive response and acyl‐homoserine lactone production of the fire blight antagonists Erwinia tasmaniensis and Erwinia billingiae

Fire blight caused by the Gram‐negative bacterium Erwinia amylovora can be controlled by antagonistic microorganisms. We characterized epiphytic bacteria isolated from healthy apple and pear trees in Australia, named Erwinia tasmaniensis, and the epiphytic bacterium Erwinia billingiae from England for physiological properties, interaction with plants and interference with growth of E. amylovora. They reduced symptom formation by the fire blight pathogen on immature pears and the colonization of apple flowers. In contrast to E. billingiae, E. tasmaniensis strains induced a hypersensitive response in tobacco leaves and synthesized levan in the presence of sucrose. With consensus primers deduced from lsc as well as hrpL, hrcC and hrcR of the hrp region of E. amylovora and of related bacteria, these genes were successfully amplified from E. tasmaniensis DNA and alignment of the encoded proteins to other Erwinia species supported a role for environmental fitness of the epiphytic bacterium. Unlike E. tasmaniensis, the epiphytic bacterium E. billingiae produced an acyl‐homoserine lactone for bacterial cell‐to‐cell communication. Their competition with the growth of E. amylovora may be involved in controlling fire blight.     

Phytotoxic material from associations betweenErwinia amylovora and pear tissue culture: Possible role in necrotic symptomatology of fireblight disease

Pear cell suspension cultures (PCSC) inoculated with virulent strains of the fireblight bacterium,Erwinia amylovora, exhibited massive necrosis within 7 days, whereas avirulentE. amylovora strains and other enterobacteria generally elicited very slight or no necrotic reactions. These results were generally repeated when pear seedlings (1–2 months old) were inoculated with these same bacterial strains. Fractions derived from massively necrotized PCSC were tested for biological activity against healthy PCSC and pear seedling cuttings. Of these fractions, the dialyzable, 70%-ethanol-soluble material—remaining after precipitation by ethanol of high-molecular-weight polysaccharides (“amylovorin”)—exclusively inhibited growth of pear callus, plasmolyzed and disintegrated pear cells in PCSC, and caused some browning of pear callus. Although cuttings of young pear seedlings placed in solutions containing this dialyzable, ethanol-soluble material generally became massively necrotized within 3 days, some residual methodological problems with this bioassay procedure must be solved. These observations suggest that some relatively small molecule(s) formed in PCSC inoculated with virulentE. amylovora can exert antagonistic biological activity against pear tissue and may play a role in the symptomatology characteristic of the fireblight disease.     

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.     

Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC‐NBS‐LRR resistance gene of Malus × robusta 5

The fire blight susceptible apple cultivar Malus × domestica Borkh. cv. ‘Gala’ was transformed with the candidate fire blight resistance gene FB_MR5 originating from the crab apple accession Malus × robusta 5 (Mr5). A total of five different transgenic lines were obtained. All transgenic lines were shown to be stably transformed and originate from different transgenic events. The transgenic lines express the FB_MR5 either driven by the constitutive CaMV 35S promoter and the ocs terminator or by its native promoter and terminator sequences. Phenotyping experiments were performed with Mr5‐virulent and Mr5‐avirulent strains of Erwinia amylovora, the causal agent of fire blight. Significantly less disease symptoms were detected on transgenic lines after inoculation with two different Mr5‐avirulent E. amylovora strains, while significantly more shoot necrosis was observed after inoculation with the Mr5‐virulent mutant strain ZYRKD3_1. The results of these experiments demonstrated the ability of a single gene isolated from the native gene pool of apple to protect a susceptible cultivar from fire blight. Furthermore, this gene is confirmed to be the resistance determinant of Mr5 as the transformed lines undergo the same gene‐for‐gene interaction in the host–pathogen relationship Mr5–E. amylovora.     

Distribution of Streptomycin Resistant Strains of Erwinia amylovora in Egypt during 1988

A survey of the occurrence of strains of Erwinia amylovora resistant to streptomycin in certain Egyptian pear orchards was earned out during April and May 1988. Twenty-two isolates out of 604 isolates collected from 11 orchards showed resistance to streptomycin. All the streptomycin resistant (Str^r) strains isolated in the present work were resistant to high levels of streptomycin with minimal inhibitory concentrations ranging from 1000 to 3000 μg/ml. The occurrence of Str^r strains in Egypt is still limited and the population of resistant strains was at relatively low level. However, such occurrence of E. amylovora with resistance to streptomycin is a potentially serious situation.     

Erwinia spp. from pome fruit trees: similarities and differences among pathogenic and non-pathogenic species

The number of described pathogenic and non-pathogenic Erwinia species associated with pome fruit trees, especially pear trees, has increased in recent years, but updated comparative information about their similarities and differences is scarce. The causal agent of the fire blight disease of rosaceous plants, Erwinia amylovora, is the most studied species of this genus. Recently described species that are pathogenic to pear trees include Erwinia pyrifoliae in Korea and Japan, Erwinia spp. in Japan, and Erwinia piriflorinigrans in Spain. E. pyrifoliae causes symptoms that are indistinguishable from those of fire blight in Asian pear trees, Erwinia spp. from Japan cause black lesions on several cultivars of pear trees, and E. piriflorinigrans causes necrosis of only pear blossoms. All these novel species share some phenotypic and genetic characteristics with E. amylovora. Non-pathogenic Erwinia species are Erwinia billingiae and Erwinia tasmaniensis that have also been described on pome fruits; however, less information is available on these species. We present an updated review on the phenotypic and molecular characteristics, habitat, pathogenicity, and epidemiology of E. amylovora, E. pyrifoliae, Erwinia spp. from Japan, E. piriflorinigrans, E. billingiae, and E. tasmaniensis. In addition, the interaction of these species with pome fruit trees is discussed.     

Identification of genes differentially expressed during interaction of resistant and susceptible apple cultivars (Malus × domestica) with Erwinia amylovora

Background  

The necrogenic enterobacterium, Erwinia amylovora is the causal agent of the fire blight (FB) disease in many Rosaceaespecies, including apple and pear. During the infection process, the bacteria induce an oxidative stress response with kinetics similar to those induced in an incompatible bacteria-plant interaction. No resistance mechanism to E. amylovora in host plants has yet been characterized, recent work has identified some molecular events which occur in resistant and/or susceptible host interaction with E. amylovora: In order to understand the mechanisms that characterize responses to FB, differentially expressed genes were identified by cDNA-AFLP analysis in resistant and susceptible apple genotypes after inoculation with E. amylovora.     

Development and evaluation of a real‐time PCR assay targeting chromosomal DNA of Erwinia amylovora

Aims:  To develop and evaluate a new and reliable real‐time PCR detection protocol on chromosomal DNA of the contagious plant pathogenic bacterium Erwinia amylovora, the causal agent of fire blight. Methods and Results:  A Taqman^® minor‐groove‐binder real‐time PCR assay targeting a hypothetical protein coding gene of Erw. amylovora has been developed. Colony PCR of 113 bacterial strains from different taxa was performed to prove specificity. Serial decimal dilutions of Erw. amylovora showed a consistent detection sensitivity of 2 bacterial units per μl. All strains of Erw. amylovora could be identified, and there were no cross‐reactions with matrices or other bacteria also testing naturally contaminated samples. Conclusions:  Rapid, reliable and sensitive detection of Erw. amylovora is important to avoid the spread of the disease within orchards, and the distribution by contaminated plant material or vectors carrying the pathogen. The selected conserved target gene allows relative quantitative detection of Erw. amylovora from different sources and host taxa. The newly developed protocol also enables the detection of recently found natural strains that lack the species‐specific plasmid pEA29, which was so far widely used as target for detection and identification of this plant pathogen by PCR. Significance and Impact of the Study:  This study demonstrates that the newly developed and evaluated real‐time assay can specifically be used for identifying all known strains of the EU quarantine plant pathogen Erw. amylovora. Low concentrations of the bacteria can be detected and relatively quantified using a different target area than other real‐time PCRs designed so far.     

Erwinia amylovora strains from outbreaks of fire blight in Spain: phenotypic characteristics

One hundred and thirty strains of Erwinia amylovora recovered from Spanish foci of fire blight from 1995 to 2000 were characterised and compared to reference strains from different sources and origins. Their rapid identification was performed by double antibody sandwich indirect (DASI) ELISA, using specific monoclonal antibodies against E. amylovora, and molecular confirmation by PCR using primers specific to the native plasmid pEA29. The Spanish strains of E. amylovora grew on different general and selective media producing typical colonies, except one of them that was deficient in levan production, whereas none of them grew on minimal agar medium with copper sulphate and low content of asparagine. All of them were susceptible to tetracycline, streptomycin, kasugamycin and oxolinic acid. Biochemical characterisation of selected strains by API 20E system revealed a great homogeneity, with 80% of the Spanish strains showing one of the two majority API 20E profiles described for E. amylovora, and the remaining strains showing minor differences. Pathogenicity on pear fruits and hypersensitivity reaction was confirmed, but a delayed reaction was observed for two Spanish strains. This is the first characterisation of a large collection of Spanish strains of E. amylovora.     

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.     

Improved recovery of Erwinia amylovora-stressed cells from pome fruit on RESC, a simple, rapid and differential medium

The bacterium Erwinia amylovora causes fire blight, a serious and widespread disease of several pome fruit and ornamental plants. The use of suitable detection tools is essential for preventing its dissemination and, according to the protocol of the European and Mediterranean Plant Protection Organization, the isolation and further identification of E. amylovora is the only conclusive test of its presence. However, bacterial growth on solid media can be hampered when the pathogen is suffering stressful conditions in pome fruit or in other habitats. Since copper is an essential micronutrient that, in E. amylovora, also increases the exopolysaccharide production in rich-nutrient media, we have designed a non-selective differential medium containing 1.5 mM CuSO₄ to improve the recovery of E. amylovora from plants under unfavorable conditions. In this new medium named Recovery Erwinia amylovora-Stressed Cells (RESC), its colonies were easily distinguished by a light yellow color and a high mucus production. The plating recovery of several E. amylovora strains in vitro and from naturally infected samples was significantly improved with respect to other media routinely employed, particularly when the pathogen was suffering stressful conditions. Thus, the recovery of stressed E. amylovora cells (after UV irradiation, nutrient deprivation, or the presence of copper ions in non-copper-complexing media) was significantly enhanced on RESC medium, and their culturability period extended. Therefore, RESC is a useful and valuable medium for the isolation of E. amylovora when adverse conditions in the natural environment are expected.     

Cellulose production,activated by cyclic di‐GMP through BcsA and BcsZ,is a virulence factor and an essential determinant of the three‐dimensional architectures of biofilms formed by Erwinia amylovora Ea1189

Bacterial biofilms are multicellular aggregates encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein and nucleic acids, which determines the architecture of the biofilm. Erwinia amylovora Ea1189 forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. The production of the EPSs amylovoran and levan is critical for the formation of a mature biofilm. In addition, cyclic dimeric GMP (c‐di‐GMP) has been reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora Ea1189. In this study, we demonstrate that cellulose is synthesized by E. amylovora Ea1189 and is a major modulator of the three‐dimensional characteristics of biofilms formed by this bacterium, and also contributes to virulence during systemic host invasion. In addition, we demonstrate that the activation of cellulose biosynthesis in E. amylovora is a c‐di‐GMP‐dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We also report that the endoglucanase BcsZ is a key player in c‐di‐GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen.     

Expression of bovine lactoferrin cDNA confers resistance to Erwinia amylovora in transgenic pear

The siderophore produced by Erwinia amylovora, the causal agent of fire blight of Maloideae, is one of the virulence factors of this bacterium. The production of siderophores enables E. amylovora to overcome the conditions of iron limitation met in plant tissue, and may also protect the bacteria against active oxygen species produced through the Fenton reaction. In this paper, we have examined the ability of an iron chelator protein, encoded by the bovine lactoferrin gene, to reduce fire blight susceptibility in pear (Pyrus communis L.). Transgenic pear clones expressing this gene controlled by the CaMV35S promoter were produced by Agrobacterium tumefaciens mediated transformation. Transformants were analysed by RT-PCR and western blot to determine lactoferrin expression levels. Most transgenic clones demonstrated significant reduction of susceptibility to fire blight in vitro and in the greenhouse when inoculated by E. amylovora. These transgenic clones also showed a significant reduction of symptoms when inoculated with two other pear bacterial pathogens : Pseudomonas syringae pv. syringae and Agrobacterium tumefaciens. Moreover, we have shown that this increase in bacterial resistance was correlated with an increase in root ferric reductase level activity and leaf iron content. Despite negative effects on the growth of a few clones, our results indicate the potential of lactoferrin gene transformation to protect pear from fire blight through increased iron chelation.     

Conventional and Real-Time PCRs for Detection of Erwinia piriflorinigrans Allow Its Distinction from the Fire Blight Pathogen,Erwinia amylovora

Erwinia piriflorinigrans is a new pathogenic species of the bacterial genus Erwinia that has been described recently in Spain. Accurate detection and identification of E. piriflorinigrans are challenging because its symptoms on pear blossoms are similar to those caused by Erwinia amylovora, the causal agent of fire blight. Moreover, these two species share phenotypic and molecular characteristics. Two specific and sensitive conventional and real-time PCR protocols were developed to identify and detect E. piriflorinigrans and to differentiate it from E. amylovora and other species of this genus. These protocols were based on sequences from plasmid pEPIR37, which is present in all strains of E. piriflorinigrans analyzed. After the stability of the plasmid was demonstrated, the specificities of the protocols were confirmed by the amplification of all E. piriflorinigrans strains tested, whereas 304 closely related pathogenic and nonpathogenic Erwinia strains and microbiota from pear trees were not amplified. In sensitivity assays, 10³ cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 10² cells/ml were detected in DNA extracted from spiked plant material by real-time PCR. The protocols developed here succeeded in detecting E. piriflorinigrans in 102 out of 564 symptomatic and asymptomatic naturally infected pear samples (flowers, cortex stem tissue, leaves, shoots, and fruitlets), in necrotic Pyracantha sp. blossoms, and in necrotic pear and apple tissues infected with both E. amylovora and E. piriflorinigrans. Therefore, these new tools can be used in epidemiological studies that will enhance our understanding of the life cycle of E. piriflorinigrans in different hosts and plant tissues and its interaction with E. amylovora.