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.     

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.     

Real-time PCR, a method fit for detection and quantification of Erwinia amylovora

Fire blight, a devastating disease of pome fruit trees continues to pose threat to agricultural production. Detection of its causative agent, bacterium Erwinia amylovora, is usually straightforward in symptomatic samples. Methods with increased sensitivity however, are sometimes needed for detection of E. amylovora and real-time PCR assays have been shown to have required sensitivity and reliability. Here we summarize our previous results on real-time PCR detection of fire blight and present new, fast and sensitive real-time PCR assay based on amsC gene performed on SmartCycler^? instrument. The setting is optimal for analysis of small number of samples in the laboratory or for on-site detection. Many advantages of real-time PCR assays warrant their use in detection and diagnosis of E. amylovora, particularly in detection of low concentrations of target bacteria e.g. in testing for latent infections. It is to be expected that the use of real-time PCR will increase in both diagnostics and in research, as a tool for target detection and quantification as well as for gene expression analysis.     

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.     

In planta recovery of Erwinia amylovora viable but nonculturable cells

Little is known about the survival mechanisms of Erwinia amylovora outside its hosts. It has been demonstrated that it enters the viable but nonculturable state (VBNC) when exposed to different types of stress. In the VBNC state, bacterial cells remain viable but unable to grow on the solid general media where they usually do, and are thus undetectable by conventional culture-dependent methods. In this work, we have evaluated the recovery of E. amylovora VBNC cells by passage through pear plantlets, in comparison with other recovery methods commonly used for this pathogen: incubation in KB broth and inoculation of immature fruits. VBNC cells were obtained by exposure of bacterial cells to different types of stress (oligotrophy, nutrient deprivation and chlorine), and recovery assays were performed at 26°C. In all cases, the recovery of VBNC cells was more effective in plantlets than in liquid KB or immature fruits. In fact, when cells were exposed to chlorine for more than 30 min, only passage through host plant gave positive result, enabling recovery of E. amylovora cells few days after inoculation of plants. These results suggest a higher effectiveness of in planta recovery than those performed with liquid KB or detached fruits. Our results support the hypothesis of the VBNC state being part of the E. amylovora life cycle. The potential existence of this physiological state in nature should be taken in consideration in epidemiological studies of fire blight, with the aim to optimize the management and control of this disease.     

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.     

Medfly Ceratitis capitata as Potential Vector for Fire Blight Pathogen Erwinia amylovora: Survival and Transmission

Monitoring the ability of bacterial plant pathogens to survive in insects is required for elucidating unknown aspects of their epidemiology and for designing appropriate control strategies. Erwinia amylovora is a plant pathogenic bacterium that causes fire blight, a devastating disease in apple and pear commercial orchards. Studies on fire blight spread by insects have mainly focused on pollinating agents, such as honeybees. However, the Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae), one of the most damaging fruit pests worldwide, is also common in pome fruit orchards. The main objective of the study was to investigate whether E. amylovora can survive and be transmitted by the medfly. Our experimental results show: i) E. amylovora can survive for at least 8 days inside the digestive tract of the medfly and until 28 days on its external surface, and ii) medflies are able to transmit the bacteria from inoculated apples to both detached shoots and pear plants, being the pathogen recovered from lesions in both cases. This is the first report on E. amylovora internalization and survival in/on C. capitata, as well as the experimental transmission of the fire blight pathogen by this insect. Our results suggest that medfly can act as a potential vector for E. amylovora, and expand our knowledge on the possible role of these and other insects in its life cycle.     

Floral traits affecting fire blight infection and management

Erwinia amylovora, the causative agent of fire blight, colonizes primarily the flowers of the sub-family Maloideae. Commercially important fruit tree species such as apple (Malus domestica) and pear (Pyrus communis) are also affected by the disease. Epiphytic bacterial populations develop on the stigma, from where the pathogen colonizes the hypanthium, aided by moisture. Under favorable conditions, nectar provides a rich medium for growth, which allows bacterial invasion of tissues through the stomata of the nectary. The paper reviews various floral traits that may play a role in the onset and progression of the infection. Flower age, stigma morphology and longevity, the size of epiphytic bacterial population, morphology of the hypanthium, anatomy of the nectary, dynamics of nectar secretion, as well as the volume, concentration and composition of the nectar are discussed in detail, comparing traits of susceptible versus tolerant apple and pear cultivars. Management programs, aiming at the suppression of E. amylovora on floral parts by antibiotics, chemical compounds, natural substances or biological control agents, are also discussed.     

Interference of Copper Sulphate in Growth of Erwinia amylovora

To understand the toxicity of copper salts on Erwinia amylovora, which are used in the control of fire blight, bacterial growth and cell metabolism was assayed with copper sulphate in the presence or absence of complex-forming compounds such as various amino acids or citrate. In minimal medium without amino acids copper sulphate strongly interfered with the growth of E. amylovora. A concentration of 15 μm CuSO₄ resulted in about 50% growth inhibition. In contrast to a strong effect of streptomycin, copper ions barely killed the cells when incubated in minimal medium for 1 h. The addition of 4 g asparagine per litre relieved a‘bacteriostatic’effect of copper ions and allowed growth of the bacteria at 2 mm CuSO₄. Other amino acids had a similar effect in the protection of E. amylovora against copper ions. This was in contrast to glycine betain, which was unable to suppress growth inhibition by CuSO₄. Presumably, the free ammonium groups of amino acids participated in the protective effect. The addition of citrate, exceeding the amount of copper-ions, was also protective. Bioluminescence of E. amylovora cells was expressed via a constitutive promoter from the lux-operon of Vibrio fischeri. The light emission is dependent on active cell metabolism. In a novel approach to determine the immediate response of E. amylovora after the addition of copper sulphate, the change of bioluminescence was determined. Addition of copper ions to MM3 medium strongly affected the bioluminescence, but no change in light production was noticed, when citrate or asparagine were present in addition to copper sulphate. A decrease of bioluminescence to 50% was observed for 50 μm CuSO₄ in the absence of amino acids.     

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.     

Survival and Possible Spread of Erwinia amylovora and Related Plant-Pathogenic Bacteria Exposed to Environmental Stress Conditions

The fire blight pathogen Erwinia amylovora was assayed for survival under unfavourable conditions such as on nitrocellulose filters, in non‐host plants as well as in inoculated mature apples and in infested apple stem sections. In a sterile dry environment, an E. amylovora EPS (exopolysaccharide) mutant, and to a lesser extent its parental wild‐type strain decreased within 3 weeks to a low titre. However, under moist conditions the decrease of viable cells occurred only partially for both strains. Very low cell titres were recovered after application of E. amylovora onto the surface of tobacco leaves, whereas infiltration into the leaves produced lesions (hypersensitive response, HR), in which the bacteria survived in significant amounts. A similar effect was found for the necrotic zones of HR in tobacco leaves caused by E. pyrifoliae, by Pseudomonas syringae pathovars and HR‐deficient E. amylovora mutants or mutants deficient in EPS synthesis and disease‐specific genes. During 7 years of storage, the viability of E. amylovora in wood sections from fire blight‐infested apple trees declined to a low titre. In tissue of mature apples, E. amylovora cells slowly dispersed and could still be recovered after several weeks of storage at room temperature. A minimal risk of accidental dissemination of E. amylovora apart from infested host plants can experimentally not be excluded, but other data confirm a very low incidence of any long distance distribution.     

Genomics of iron acquisition in the plant pathogen <Emphasis Type="Italic">Erwinia amylovora</Emphasis>: insights in the biosynthetic pathway of the siderophore desferrioxamine E

Genomics has clarified the biosynthetic pathway for desferrioxamine E critical for iron acquisition in the enterobacterial fire blight pathogen Erwinia amylovora. Evidence for each of the individual steps and the role of desferrioxamine E biosynthesis in pathogen virulence and cell protection from host defenses is presented. Using comparative genomics, it can be concluded that desferrioxamine biosynthesis is ancestral within the genera Erwinia and Pantoea.     

Comparative genomics of Spiraeoideae‐infecting Erwinia amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low‐virulence strain

Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae‐infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations (‘Widely Prevalent’ clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low‐virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.     

Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu²⁺ was inoculated with 10⁷ CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.     

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.     

Detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> by novel chromosomal polymerase chain reaction primers

A new sensitive and specific method for the detection of Erwinia amylovora was developed. The method is based on the detection of a chromosomal DNA sequence specific for this bacterial species and enables detection of E. amylovora pathogenic strains, including recent isolates that lack plasmid pEA29 and thus cannot be detected by the previously popular PCR methods based on the detection of this plasmid. A species-specific random amplified polymorphic DNA (RAPD) marker was identified, cloned, and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. The E. amylovora specific sequence, 1269 bp long, was amplified in polymerase chain reaction and detected with electrophoresis in agarose gel stained with ethidium bromide. Amplification with other bacterial species did not produce any PCR product detectable by electrophoresis. Matching of the E. amylovora specific sequence to chromosomal DNA was confirmed by computer analysis of the E. amylovora genome. A consistent sensitivity limit of the method was 3 CFU/reaction, and in some cases it was possible to detect 0.6 CFU/reaction. Due to its high sensitivity and specificity, our method of E. amylovora detection is currently the most reliable, taking into account that the reliability of PCR methods based on plasmid pEA29 has been compromised by the isolation of pathogenic E. amylovora strains that lack this plasmid.     

Erwinia amylovora catalases KatA and KatG are virulence factors and delay the starvation‐induced viable but non‐culturable (VBNC) response

The life cycle of the plant pathogen Erwinia amylovora comprises periods inside and outside the host in which it faces oxidative stress caused by hydrogen peroxide (H₂O₂) and other compounds. The sources of this stress are plant defences, other microorganisms and/or exposure to starvation or other environmental challenges. However, the functional roles of H₂O₂‐neutralizing enzymes, such as catalases, during plant–pathogen interactions and/or under starvation conditions in phytopathogens of the family Erwiniaceae or closely related families have not yet been investigated. In this work, the contribution of E. amylovora catalases KatA and KatG to virulence and survival in non‐host environments was determined using catalase gene mutants and expression, as well as catalase activity analyses. The participation of E. amylovora exopolysaccharides (EPSs) in oxidative stress protection was also investigated. Our study revealed the following: (i) a different growth phase regulation of each catalase, with an induction by H₂O₂ and host tissues; (ii) the significant role of E. amylovora catalases as virulence and survival factors during plant–pathogen interactions; (iii) the induction of EPSs by H₂O₂ despite the fact that apparently they do not contribute to protection against this compound; and (iv) the participation of both catalases in the detoxification of the starvation‐induced intracellular oxidative stress, favouring the maintenance of culturability, and hence delaying the development of the viable but non‐culturable (VBNC) response.