The fire blight pathogen Erwinia amylovora requires the rpoN gene for pathogenicity in apple

RpoN is a σ⁵⁴ factor regulating essential virulence gene expression in several plant pathogenic bacteria, including Pseudomonas syringae and Pectobacterium carotovorum. In this study, we found that mutation of rpoN in the fire blight pathogen Erwinia amylovora caused a nonpathogenic phenotype. The E. amylovora rpoN Tn5 transposon mutant rpoN1250::Tn5 did not cause fire blight disease symptoms on shoots of mature apple trees. In detached immature apple fruits, the rpoN1250::Tn5 mutant failed to cause fire blight disease symptoms and grew to population levels 12 orders of magnitude lower than the wild‐type. In addition, the rpoN1250::Tn5 mutant failed to elicit a hypersensitive response when infiltrated into nonhost tobacco plant leaves, and rpoN1250::Tn5 cells failed to express HrpN protein when grown in hrp (hypersensitive response and pathogenicity)‐inducing liquid medium. A plasmid‐borne copy of the wild‐type rpoN gene complemented all the rpoN1250::Tn5 mutant phenotypes tested. The rpoN1250::Tn5 mutant was prototrophic on minimal solid and liquid media, indicating that the rpoN1250::Tn5 nonpathogenic phenotype was not caused by a defect in basic metabolism or growth. This study provides clear genetic evidence that rpoN is an essential virulence gene of E. amylovora, suggesting that rpoN has the same function in E. amylovora as in P. syringae and Pe. carotovorum.     

Protection of apple against fire blight induced by an hrpL mutant of Erwinia amylovora

A regulatory hrpL non-virulent mutant of Erwinia amylovora is effective in controlling fire blight disease when inoculated on apple seedlings simultaneously with the pathogenic parental strain. Mechanisms involved in this protective effect were investigated. The use of two marker genes, uidA and lacZ, expressed in the hrpL mutant and the pathogenic strain, respectively, allowed to localize simultaneously the two inoculated strains in plant tissue. An anti-β-glucuronidase antibody was also used to detect the hrpL mutant. Both techniques indicated that the two strains localized mainly in separate areas of the leaf tissue. In addition, leaves infiltrated with the hrpL mutant exhibited a significant increase in peroxidase activity in contrast to a hrp secretion mutant known to be less effective in the protection. It is suggested that protection obtained with the hrpL mutant relies on the physical separation between the mutant and the parental strain after co-inoculation and the rapid and sustained activation of plant defense mechanisms in reactive tissue, i.e. not invaded by the virulent strain.     

Identification of low-temperature-regulated genes in the fire blight pathogen Erwinia amylovora

Genes involved in pathogenicity of several plant pathogens were shown to be induced at relatively cold temperatures. Loci from the fire blight pathogen Erwinia amylovora (Burrill) induced at 18 degrees C were identified using the miniTn5 transposon that contains the promoterless reporter gene gusA coding for beta-glucuronidase (GUS). Certain mutants (2.7%) expressed GUS predominantly at 18 degrees C on minimal medium plates, indicating that the transposon had been inserted downstream of a putatively thermoregulated promoter. Those mutants were further screened with a quantitative GUS fluorometric assay. A total of 21 mutants were selected: 19 mutants had a transposon insertion in temperature-dependent genetic loci, with a 2.2- to 6.3-fold induction of gusA gene expression at 18 degrees C, and two mutants with impaired growth at 18 degrees C. Some of these genetic loci encoded (i) proteins implicated in flagella biosynthesis, biotin biosynthesis, multi-drug efflux, and type II secretion protein, and (ii) proteins of unknown function.     

Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA.

Erwinia amylovora, the causative agent of fire blight, was identified independently from the common plasmid pEA29 by three different PCR assays with chromosomal DNA. PCR with two primers was performed with isolated DNA and with whole cells, which were directly added to the assay mixture. The oligonucleotide primers were derived from the ams region, and the PCR product comprised the amsB gene, which is involved in exopolysaccharide synthesis. The amplified fragment of 1.6 kb was analyzed, and the sequence was found to be identical for two E. amylovora strains. The identity of the PCR products was further confirmed by restriction analysis. The 1.6-kb signal was also used for detection of the fire blight pathogen in the presence of other plant-associated bacteria and in infected plant tissue. For further identification of isolated strains, the 16S rRNA gene of E. amylovora and other plant-associated bacteria was amplified and the products were digested with the restriction enzyme HaeIII. The pattern obtained for E. amylovora was different from that of other bacteria. The sequence of the 16S rRNA gene was determined from a cloned fragment and was found to be closely related to the sequences of Escherichia coli and other Erwinia species. Finally, arbitrarily primed PCR with a 17-mer oligonucleotide derived from the sequence of transposon Tn5 produced a unique banding pattern for all E. amylovora strains investigated. These methods expand identification methods for E. amylovora, which include DNA hybridization and a PCR technique based on plasmid pEA29.     

Evaluation of old Carpathian apple cultivars as genetic resources of resistance to fire blight (Erwinia amylovora)

In the interests of re-diversifying cultivar use in apple, one of the most important breeding aims, apart from using local cultivars in breeding, is to improve resistance to fire blight (FB). At the Corvinus University of Budapest, the investigation of Hungarian cultivars found in the Carpathian Basin as genetic resources is a major part of the apple breeding program aimed at multiple resistance. The present study, initiated in the early 2000s, evaluates the FB resistance of 31 apple cultivars. Examination of the damage to shoots and flowers, and of the correlation between them, was carried out after artificial inoculation. Two dominant SCAR markers and one SSR marker were used for the genetic analysis of the cultivars giving the best results in phenotypic analysis, in order to detect quantitative trait locus alleles coding for FB resistance. Based on the results of several years of inoculation tests, the assayed cultivars exhibited a wide range of susceptibility levels on the basis of shoot necrosis and in terms of flower damage. A positive correlation (R = 0.058, p = 0.04) was demonstrated between the susceptibility found for the two plant organs. The cultivars Szabadkai szercsika and Sikulai, which gave outstanding results both in the flower and shoot tests and in the genetic analysis, could be suitable genetic resources for resistance breeding programmes. The present work confirmed the complex polygenic nature of FB resistance and the need to identify further markers in addition to those found on linkage group (LG) 3 and LG 7.     

Autoinducer-2 of the fire blight pathogen Erwinia amylovora and other plant-associated bacteria

Autoinducers are important for cellular communication of bacteria. The luxS gene has a central role in the synthesis of autoinducer-2 (AI-2). The gene was identified in a shotgun library of Erwinia amylovora and primers designed for PCR amplification from bacterial DNA. Supernatants of several Erwinia amylovora strains were assayed for AI-2 activity with a Vibrio harveyi mutant and were positive. Many other plant-associated bacteria also showed AI-2 activity such as Erwinia pyrifoliae and Erwinia tasmaniensis. The luxS genes of several bacteria were cloned, sequenced, and complemented Escherichia coli strain DH5alpha and a Salmonella typhimurium mutant, both defective in luxS, for synthesis of AI-2. Assays to detect AI-2 activity in culture supernatants of several Pseudomonas syringae pathovars failed, which may indicate the absence of AI-2 or synthesis of another type. Several reporter strains did not detect synthesis of an acyl homoserine lactone (AHL, AI-1) by Erwinia amylovora, but confirmed AHL-synthesis for Erwinia carotovora ssp. atroseptica and Pantoea stewartii.     

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.     

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.     

Genomics and current genetic understanding of Erwinia amylovora and the fire blight antagonist Pantoea vagans

The bacterial plant pathogen Erwinia amylovora causes fire blight, a major disease threat to pome fruit production worldwide with further impact on a wide-range of Rosaceae species. Important factors contributing to the development of the disease were discovered in the last decades. Comparative genomics of the genera Erwinia and Pantoea is coming into focus with the recent availability of complete genome sequences. Insights from comparative genomics now position us to answer fundamental questions regarding the evolution of E. amylovora as a successful pathogen and the critical elements for biocontrol activity of Pantoea spp. This trove of new data promises to reveal novel determinants and to understand interactive pathways for virulence, host range and ecological fitness. The ultimate aim is now to apply genomics and identify the pathogen Achilles heels and antagonist mechanisms of action as targets for designing innovative control strategies for fire blight.     

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.     

A rapid lateral-flow immunoassay for phytosanitary detection of Erwinia amylovora and on-site fire blight diagnosis

Fire blight is an invasive disease caused by Erwinia amylovora that threatens pome fruit production globally. Effective implementation of phytosanitary control measures depends upon rapid, reliable pathogen detection and disease diagnosis. We developed a lateral-flow immunoassay specific for E. amylovora with a detection limit of log 5.7 CFU/ml, typical of pathogen concentrations in symptomatic plant material. The simple assay had comparable sensitivity to standard culture plating, serum agglutination and nested PCR when validated for application in a phytosanitary laboratory as a confirmatory test of cultured isolates and for first-line diagnosis of phytosanitary samples that represent the full range of commercial, ornamental and forestry host species. On-site validation in ring-trials with local plant inspectors demonstrated robust and reliable detection (compared to subsequent plating and PCR analysis). The simplicity, inspector acceptance and facilitation of expedited diagnosis (from 2 days for laboratory submitted samples to 15 min with the immunoassay), offers a valuable tool for improved phytosanitary control of fire blight.     

Autoinduction in Erwinia amylovora: evidence of an acyl-homoserine lactone signal in the fire blight pathogen

Erwinia amylovora causes fire blight disease of apple, pear, and other members of the Rosaceae. Here we present the first evidence for autoinduction in E. amylovora and a role for an N-acyl-homoserine lactone (AHL)-type signal. Two major plant virulence traits, production of extracellular polysaccharides (amylovoran and levan) and tolerance to free oxygen radicals, were controlled in a bacterial-cell-density-dependent manner. Two standard autoinducer biosensors, Agrobacterium tumefaciens NTL4 and Vibrio harveyi BB886, detected AHL in stationary-phase cultures of E. amylovora. A putative AHL synthase gene, eamI, was partially sequenced, which revealed homology with autoinducer genes from other bacterial pathogens (e.g., carI, esaI, expI, hsII, yenI, and luxI). E. amylovora was also found to carry eamR, a convergently transcribed gene with homology to luxR AHL activator genes in pathogens such as Erwinia carotovora. Heterologous expression of the Bacillus sp. strain A24 acyl-homoserine lactonase gene aiiA in E. amylovora abolished induction of AHL biosensors, impaired extracellular polysaccharide production and tolerance to hydrogen peroxide, and reduced virulence on apple leaves.     

Reduction in bacterial ooze formation on immature fruitlets after preventive treatments of Fosethyl-Al against fire blight Erwinia amylovora

Fire blight, caused by the bacterium Erwinia amylovora (Burill Winslow et al.), is a very important bacterial disease on apple and pear orchards with devastating effects in some production area and in some years. Fire blight control consists in a whole strategy of measures that should start with control measures in and around the fruit tree nurseries. Only the use of Vacciplant (Laminarin), an inducer of the self-defence mechanism, is registered in Belgium since 2009. In other European countries Fosethyl-Al has been registered for fire blight control. Recently, research trials have been done at Pcfruit research station for several years on the activity of ALiette (fosethyl-Al) against fire blight. Fosethyl-Al, also a plant defence enhancing molecule, applied preventively 3 times at a dose of 3.75 kg/ha standard orchard (3 x 3000 g a.i./ha standard orchard), showed a reduction in the host susceptibility and decreased the disease development on artificial inoculated flower clusters and shoots. Also a clear reduction in the ooze droplet formation on artificially inoculated immature fruitlets has been observed with this molecule. This reduction in the bacterial ooze formation is considered as a very important factor in the spread of the disease in the orchard.     

Partial characterization of an inhibitory strain of Erwinia herbicola with potential as a biocontrol agent for Erwinia amylovora, the fire blight pathogen

Erwinia herbicola Eh1087 isolated from apple blossom inhibits development of Erwinia amylovora in immature pear fruit and produces a broad spectrum antibiotic activity in vitro that is bactericidal for Erw. amylovora. The antibiotic activity is present in cell-free culture supernatant fluids of late log-early stationary phase cultures of Eh1087. This antibiotic activity is not inhibited by proteases, excess ferric ions or essential amino acids. It is stable to acidic and basic pH and is inactivated at high temperature. The antibiotic activity is inactivated by β-lactamase digestion.     

Isolation and Characterization of Five Erwinia amylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages Ea1 and Ea7 and 3 novel phages named Ea100, Ea125, and Ea116C, were identified based on differences in genome size and restriction fragment pattern. Ea1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages Ea100, Ea7, and Ea125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. Ea116C contained an approximately 75-kb genome. Ea1, Ea7, Ea100, Ea125, and Ea116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. Ea116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10⁵ CFU.     

The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora

The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant.