Causal Agent Of Fire Blight Research Articles

Fire Blight Symptomatic Shoots and the Presence of Erwinia amylovora in Asymptomatic Apple Budwood.

Erwinia amylovora, the causal agent of fire blight, causes considerable economic losses in young apple plantings in New York on a yearly basis. Nurseries make efforts to only use clean budwood for propagation, which is essential, but E. amylovora may be present in trees that appear to have no apparent fire blight symptoms at the time of collection. We hypothesized that the use of infected budwood, especially by commercial nursery operations, could be the cause, in part, of fire blight outbreaks that often occur in young apple plantings in New York. Our goal was to investigate the presence of E. amylovora in asymptomatic budwood from nursery source plantings as it relates to trees with fire blight symptoms. From 2012 to 2015, apple budwood was collected from two commercial budwood source plantings of 'Gala' and 'Topaz' at increasing distances from visually symptomatic trees. From these collections, internal contents of apple buds were analyzed for the presence of E. amylovora. E. amylovora was detected in asymptomatic budwood in trees more than 20 m from trees with fire blight symptoms. In some seasons, there were significant (P ≤ 0.05) differences in the incidence of E. amylovora in asymptomatic budwood collected from symptomatic trees and those up to 20 m from them. In 2014 and 2015, the mean E. amylovora CFU per gram recovered from budwood in both the Gala and Topaz plantings were significantly lower in budwood collected 20 m from symptomatic trees. Further investigation of individual bud dissections revealed that E. amylovora was within the tissue beneath the bud scales containing the meristem. Results from the study highlight the shortcomings of current budwood collection practices and the need to better understand the factors that lead to the presence of E. amylovora in bud tissues to ensure the production of pathogen-free apple trees.

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

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

Characterization of Erwinia amylovora strains from Hungary

Erwinia amylovora is the causative agent of fire blight, which is a destructive bacterial disease of rosaceous plants. In Hungary Erwinia amylovora (Burrill) Winslow et al. was first detected in 1996. Since the appearance of fire blight, E. amylovora samples have been collected from different host plats from various geographic locations. A motif of eight nucleotides (ATTACAGA) is repeated 3–15 times in the PstI fragment of the pEa29 plasmid in Erwinia amylovora strains, and represents a valuable tool for strain classification. The number of short-sequence DNA repeats in plasmid pEa29 of 30 Hungarian isolates were determined by PCR assays and they ranged from five to ten. The SSR test is suitable for distinguishing the individual strains between the E. amylovora isolates. The examined isolates showed high pathogenicity on immature pear fruits. Several biochemical techniques, such as miniaturized API 20E, were applied on the samples. Differences were also revealed in microbiological assays like levan formation and colony morphology on semi-selective media. Examining the Hungarian Erwinia amylovora population by molecular analysis we can draw the conclusion that the population consists of different strains, which shows great diversity. E. amylovora is a widespread pathogen in Hungary, which is supported by the 30 strains isolated from various host plants from many parts of the country. The phenotypic diversity-evaluation of the E. amylovora strains showed, that they differ metabolically, like other plant pathogenic bacteria as reported by several authors. This is the first report on the diversity of E. amylovora strains isolated from Hungary.

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

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

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

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

Phylogenetic analysis of PR genes in some pome fruit species with the emphasis on transcriptional analysis and ROS response under Erwinia amylovora inoculation in apple.

Attempts were made to identify eight pathogenesis related (PR) genes (i.e., PR-1a, PR3-ch1, PR3-Ch2, PR3-Ch3, PR3-Ch4, PR3-Ch5, PR-5 and PR-8) from 27 genotypes of apple, quince and pear, which are induced in response to inoculation with the pathogen Erwinia amylovora, the causal agent of fire blight. Totally, 32 PR genes of different families were obtained, excepting PR3-Ch2 (amplified only in apple) and PR3-Ch4 (amplified only in apple and pear), the others were successfully amplified in all the genotypes of apple, quince and pear. Evolutionary, the genes of each family exhibited significant homology with each other, as the corresponded phylogenetic neighbor-joining-based dendrograms were taken into consideration. Meanwhile, according to the expression assay, it was deduced that the pathogen activity can significantly affect the expression levels of some selected PR genes of PR3-Ch2, PR3-Ch4, PR3-Ch5 and particularly Cat I in both resistant (MM-111) and semi-susceptible (MM-106) apple rootstocks. Lastly, it was concluded that the pathogen E. amylovora is able to stimulate ROS response, particularly using generation of hydrogen peroxide (H2O2) in both aforementioned apple rootstock.

Structural insights into the pathology ofErwinia amylovora, the causative agent of Fire Blight

Structural insights into the pathology of<i>Erwinia amylovora</i>, the causative agent of Fire Blight

Protection of Erwinia amylovora bacteriophage Y2 from UV-induced damage by natural compounds.

Bacteriophages have regained much attention as biocontrol agents against bacterial pathogens. However, with respect to stability, phages are biomolecules and are therefore sensitive to a number of environmental influences. UV-irradiation can readily inactivate phage infectivity, which impedes their potential application in the plant phyllosphere. Therefore, phages for control of Erwinia amylovora, the causative agent of fire blight, need to be protected from UV-damage by adequate measures. We investigated the protective effect of different light-absorbing substances on phage particles exposed to UV-light. For this, natural extracts from carrot, red pepper, and beetroot, casein and soy peptone in solution, and purified substances such as astaxanthin, aromatic amino acids, and Tween 80 were prepared and tested as natural sunscreens for phage. All compounds were found to significantly increase half-life of UV-irradiated phage particles and they did not negatively affect phage viability or infectivity. Altogether, a range of readily available, natural substances are suitable as UV-protectants to prevent phage particles from UV-light damage.

First Report of Erwinia amylovora, the Causal Agent of Fire Blight, on Pear in Finland

First Report of <i>Erwinia amylovora</i>, the Causal Agent of Fire Blight, on Pear in Finland

Virulence Factors of Erwinia amylovora: A Review.

Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3′-5′)-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them.

Mating type and sexual fruiting body of Botrytis elliptica, the causal agent of fire blight in lily

Botrytis elliptica is a necrotrophic pathogen that specifically infects Lilium species. Previous records show that B. elliptica collected in the field can successfully develop apothecia in vitro, however, there are no formal descriptions of apothecia of B. elliptica. The aim of this study was to analyse the sequence of the mating type loci of B. elliptica and produce apothecia in the laboratory in order to describe their morphology. The sequences of both MAT alleles (MAT1-1 or MAT1-2) of B. elliptica were determined and compared to the sister taxa, Botrytis cinerea and Sclerotinia sclerotiorum. Two strains of each mating type were used in crosses under controlled conditions to produce apothecia. Primordium rupture from sclerotial tissue occurred 74 days after fertilization and a mature apothecium formed within 1 month after rupture. The apothecia are 7 to 12 mm in height with a disk of 3 to 4 mm in diameter and 0.5 to 1 mm in thickness. The apothecial disk is usually umbilicate, depressed to funnel and rounded in shape. The number of apothecia growing on a sclerotium was one to nine. Asci are long, cylindrical with a size of 208 × 14 μm, thin walled and bearing eight ascospores. Ascospores are hyaline in colour, ellipsoidal with rounded ends, usually 18 to 24 μm in length and 6 to 10 μm in width (mean 19.5 × 8 μm). Ascospores were infectious on lily leaves.

English

Infection by Erwinia amylovora, the causal agent of fire blight (FB) disease, induces in apple and pear host plants, the generation of reactive oxygen species (ROS). We analyzed at molecular level, the link between ROS production and susceptibility to bacterial infection. Gene expression time course showed that expression of chloroplast, mitochondrial and nuclear genes, whose transcription is redox-dependent, was down regulated or suppressed in tolerant cultivar, Harrow Sweet, in comparison with susceptible cultivar, Williams. Monitoring of oxidative burst by localization of hydrogen peroxide showed that oxidative burst was triggered faster in tolerant cultivar in response to infection. These results suggest that transcription of some redox-dependent genes of cytoplasmic organelles and nucleus, in the two cultivars, is regulated faster in the tolerant cultivar than in the susceptible one. &nbsp; Key words: Chloroplast, electron transport Chain, Erwinia amylovora, mitochondria, Pyrus communis, redox.

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.

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, 10(3) cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 10(2) 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.

Evaluation of methods for erwinia amylovora detection

ABSTRACT Phytosanitary measures to control diseases require sensitive and reliable detection methods. In case of Erwinia amylovora, the causal agent of fire blight, there are several methods developed. We tested six of them: (1) a conventional method; (2) standard PCR; (3) BIO-PCR; (4) Real-time PCR with SYBR Green; (5) Real-time PCR with TaqMan probe; and (6) Loop Mediated Isothermal Amplification (LAMP) in terms of their specificity, sensitivity and repeatability. Of the methods tested, only Real-time PCR with SYBR Green amplified non-specific fragments, which could be however identified by melting curve analysis. Real-time PCRs (both with SYBR Green and TaqMan probe) and LAMP were most sensitive and allowed to detect E. amylovora even in the samples where the concentration of cells was lower than 2 x 101 cfu/reaction. The highest repeatability was obtained using conventional method and Real-time PCRs. Of the methods used, only the LAMP technique was very insensitive to plant impurities.

The genome of theErwinia amylovoraphage PhiEaH1 reveals greater diversity and broadens the applicability of phages for the treatment of fire blight

The enterobacterium Erwinia amylovora is the causal agent of fire blight. This study presents the analysis of the complete genome of phage PhiEaH1, isolated from the soil surrounding an E. amylovora-infected apple tree in Hungary. Its genome is 218 kb in size, containing 244 ORFs. PhiEaH1 is the second E. amylovora infecting phage from the Siphoviridae family whose complete genome sequence was determined. Beside PhiEaH2, PhiEaH1 is the other active component of Erwiphage, the first bacteriophage-based pesticide on the market against E. amylovora. Comparative genome analysis in this study has revealed that PhiEaH1 not only differs from the 10 formerly sequenced E. amylovora bacteriophages belonging to other phage families, but also from PhiEaH2. Sequencing of more Siphoviridae phage genomes might reveal further diversity, providing opportunities for the development of even more effective biological control agents, phage cocktails against Erwinia fire blight disease of commercial fruit crops.

Biosynthesis of the Antimetabolite 6‐Thioguanine in Erwinia amylovora Plays a Key Role in Fire Blight Pathogenesis

Sulfur for fire: The molecular basis for the biosynthesis of the antimetabolite 6-thioguanine (6TG) was unveiled in Erwinia amylovora, the causative agent of fire blight. Bioinformatics, heterologous pathway reconstitution in E. coli, and mutational analyses indicate that the protein YcfA mediates guanine thionation in analogy to 2-thiouridylase. Assays in planta and in cell cultures reveal for the first time a crucial role of 6TG in fire blight pathogenesis.

Characterisation of the stbD/E toxin–antitoxin system of pEP36, a plasmid of the plant pathogen Erwinia pyrifoliae

pEP36 is a plasmid ubiquitously present in Erwinia pyrifoliae, a pathogen which causes black stem blight of Asian pear. pEP36 is highly stable in its host, even in the absence of selective pressure. The plasmid is closely related to pEA29, which is widespread in E. amylovora, the causative agent of fire blight of apple and pear trees.Here we report that pEP36 possesses a functional hybrid toxin–antitoxin module, stbD/EpEP36, with the toxin showing homology to the RelE/ParE proteins and the antidote belonging to the Phd/YefM antitoxin family. Bacteria expressing the StbEpEP36 toxin arrest cell growth and enter a viable but non-culturable stage. However, they maintain their typical cell length and do not show filamentation. Pulse-chase experiments revealed that StbEpEP36 acts as a global inhibitor of protein synthesis while it does not interfere with DNA and RNA synthesis. The StbDpEP36 antitoxin is capable of neutralising StbEpEP36 toxicity. Additional experiments show that the stbD/EpEP36 module can stabilise plasmids at least 20-fold. Thus the toxin–antitoxin system may contribute to the remarkable stability of pEP36.

The status of Erwinia amylovora in the former Yugoslav Republics over the past two decades

Erwinia amylovora, the causal agent of fire blight (FB) on fruit trees and ornamental plants, rapidly spread across eastern Mediterranean countries in the early 1980s. This quarantine bacterium probably arrived in the southern parts of the former Yugoslavia (now FYR Macedonia) from Greece. Based on symptoms, and isolation and identification data, it was concluded that Erwinia amylovora was the causal agent of pear drying in Macedonia (1989). It was the first experimental confirmation of a presence of E. amylovora in the territory of the former Yugoslavia. The presence of E. amylovora was also proved in Serbia that same year. In Bosnia and Herzegovina, FB was detected during 1990. Based on an official report filed with the Federal Ministry of Agriculture in Belgrade, the presence of E. amylovora in Yugoslavia was confirmed (EPPO - Reporting Service, 1991). Therefore, the presence of the bacterium E. amylovora in the territory of Yugoslavia was officially confirmed in 1990. In Croatia, FB was first observed in villages near the border on Serbia in 1995. In Montenegro, FB was first detected in 1996. In Slovenia, FB appeared as late as in 2001. E. amylovora is now present on 10 hosts (pear, wild pear, apple, quince, medlar, mountainash, hawthorn, firethorn, cotoneaster and Japanese quince) in the territory of the former Yugoslav republics. Based on literature data, losses caused by FB in the former Yugoslav republics in the period 1989-1992 were estimated at about 12,000,000 DEM (mostly in Macedonia) and in the period 1992-1996 at 6,000,000 DEM. Total damage in a more recent epiphytotic year in Slovenia (2003) was estimated at about 474,200 EUR. Conventional and up-to-date rapid methods (PCR, ELISA and IF, BIOLOG and API System, FAME and SDS-PAGE) have been used to identify E. amylovora. Mainly preventive measures have been used to control E. amylovora in the former Yugoslav republics. Spraying with copper products has been practiced during the dormant period and in early spring. In rare cases, spraying has been done at the flowering stage (MARYBLYT), but the problem is that no synthetic bactericides are available on the market. When symptoms occur, only mechanical measures are being applied.

The influence of plant extracts on growth of Erwinia amylovora - the causal agent of fire blight

Ethanol and water extracts obtained on Soxhlet apparatus from various organs and parts (leaves, flowers, shoots, onion, bark, fruit) of 30 herbal and woody plants species were tested for growth inhibition of &lt;i&gt;Erwinia amylovora&lt;/i&gt; using agar diffusion method. Active extracts were found in 23 plant species but in 13 ofthem it was found for the first time. The highest diameter of growth inhibition zone of this bacterium was caused by extracts from &lt;i&gt;Aloe arborescens, Juglans regia, Rhus typhina, Salvia offici nalis&lt;/i&gt; and &lt;i&gt;Satureja hortensis&lt;/i&gt;. In almost all cases ethanol appeared to be a better solvent of active plant substances against &lt;i&gt;E.amylovora&lt;/i&gt; than water.