Causal Agent Of Fire Blight Research Articles

Advancements in Bacteriophages for the Fire Blight Pathogen Erwinia amylovora.

Erwinia amylovora, the causative agent of fire blight, causes significant economic losses for farmers worldwide by inflicting severe damage to the production and quality of plants in the Rosaceae family. Historically, fire blight control has primarily relied on the application of copper compounds and antibiotics, such as streptomycin. However, the emergence of antibiotic-resistant strains and growing environmental concerns have highlighted the need for alternative control methods. Recently, there has been a growing interest in adopting bacteriophages (phages) as a biological control strategy. Phages have demonstrated efficacy against the bacterial plant pathogen E. amylovora, including strains that have developed antibiotic resistance. The advantages of phage therapy includes its minimal impact on microbial community equilibrium, the lack of a detrimental impact on plants and beneficial microorganisms, and its capacity to eradicate drug-resistant bacteria. This review addresses recent advances in the isolation and characterization of E. amylovora phages, including their morphology, host range, lysis exertion, genomic characterization, and lysis mechanisms. Furthermore, this review evaluates the environmental tolerance of E. amylovora phages. Despite their potential, E. amylovora phages face certain challenges in practical applications, including stability issues and the risk of lysogenic conversion. This comprehensive review examines the latest developments in the application of phages for controlling fire blight and highlights the potential of E. amylovora phages in plant protection strategies.

Synthesis of aspartic acid and tyrosine by the fire blight pathogen Erwinia amylovora is not required for proliferation on apple flower stigmas or virulence in fruitlets.

The Gram-negative bacterium Erwinia amylovora (Ea) is the causal agent of fire blight, a devastating disease of apples and pears. In the fire blight disease cycle, Ea grows in different plant tissues, each presenting a distinct nutrient environment. Here, we investigate the ability of aspartate and tyrosine double auxotroph Ea lines to proliferate on apple flower stigma surfaces representing the epiphytic growth stage of Ea and in developing fruitlets representing one endophytic growth stage of Ea. Heterologous complementation studies in an Escherichia coli aspartate and tyrosine auxotroph verify that Ea aspartate aminotransferase (AspC) and tyrosine aminotransferase (TyrB) act as aspartate and tyrosine amino transferases. Growth analysis reveals that Ea aspC tyrB mutants multiply to near-wild-type levels on apple flower stigmas and immature fruitlets. Ea AspC and TyrB are reciprocally complementing for aspartate and tyrosine synthesis in Ec and in Ea. Ea aspC and tyrB mutants obtain sufficient aspartate and tyrosine to support multiplication on stigma surfaces and virulence in immature fruitlets.

Genetic characterization of historic Norwegian Erwinia amylovora isolates by SSR-genotyping

AbstractErwinia amylovora, the causative agent of fire blight of pome fruits and other rosaceous plants belongs to the group of regulated quarantine pests. The aim of this work was to characterize the populations of E. amylovora in Norway and their geographical distribution. A total of 238 E. amylovora isolates recovered from symptomatic host plants in Norway between 1986 and 2004 were genotyped by means of a short sequence repeat (SSR) marker (ATTACAGA) on plasmid pEa29. The SSR region was amplified and amplicon size determined using fluorescent labelling and rapid, automated capillary gel electrophoresis. All isolates contained the pEa29 plasmid harbouring the investigated marker. In total, ten genotypes were identified, of which two were detected only once. The number of repeats varied from 3 to 13, with 43% of the isolates containing five repeats. Of 17 isolates collected between 1986 and 1991, all but one contained five repeats, whereas more variation was observed in isolates from the period 2000 to 2004. Most of the isolates (80%) originated from Cotoneaster bullatus, hence no relationship between genotype of the isolate and host species that it was isolated from could be detected. This historic data suggests multiple introductions of E. amylovora to Norway.

Antagonistic and protective activity of Lactobacillus plantarum strain 17M against E. amylovora.

The subject of the study was to identify the antagonism of the Lactobacillus plantarum strain 17M against the causative agent of fire blight, the bacterium Erwinia amylovora, and to evaluate its protective capabilities on apple blossoms against this disease. For comparison 9 strains of lactic acid bacteria from the LLP "SPC of Microbiology and Virology" collection were included in the study. Strain 17M appeared to be superior in limiting the growth of the pathogen on all 3 liquid media tested. Its maximum inhibitory activity was proved on MRS Broth medium. The analysis of the secondary metabolites produced by strain 17M in liquid medium revealed that it consisted mainly of acetic acid (53.2 ± 4.3%), lactic acid (16.3 ± 2.3%) and 2,3-butanedione (14.84 ± 4.1%). The presence of other organic compounds was also detected but in a smaller amount. Study on influence of those compounds on growth of E. amylovora showed that lactic acid at concentration of 5% showed inhibitory activity but it was not toxic to apple flowers. The effectiveness of strain 17M culture liquid diluted with sterile water and added to E. amylovora inoculum at 10% or 20% was 76.7 ± 5.8% and 88.3 ± 12.6%, respectively. This study confirms the potential use of strain 17M as an active microbial agent to combat fire blight of fruit crops in Kazakhstan.

Biological control of the fire blight pathogen Erwinia amylovora using bacteriophages

AbstractBacteriophages offer unique benefits for the specific control of Erwinia amylovora, the causative agent of fire blight in pome fruit. Unfortunately, the majority of described phages were tested for efficacy in vitro only. Data from infection assays using pear slices or detached blossoms is rare and field trials are barely reported. It remains to be studied if bacteriophages could control fire blight in the open field. This review summarizes current knowledge of different phages infecting E. amylovora. In addition, the requirements for a phage to optimally function as a biological antimicrobial agent in agriculture are discussed together with the ideal setup of field trials and the formulation of phages for best results.

The fire blight prevalence on different varieties of apple trees in the main industrial zone of horticulture (south and south-east of Kazakhstan)

Apple is the most significant fruit in Kazakhstan, as well as in many other countries of the world. According to the Statistics Agency in 2020, the apple cultivation area in Kazakhstan is about 36 thousand hectares with a gross har-vest of 259.1 thousand tons. The fire blight of fruit crops, in particular apple trees, is one of the most harmful infectious diseases, rapidly spreading and cre-ating a constant threat to the apple plantations of Kazakhstan. In order to avoid the further spread of fire blight in the territory of Kazakhstan, it is very important to grow varieties of fruit trees resistant to fire blight. The article pre-sents the results on the spread of fire blight on zoned, introduced, and promis-ing apple varieties in plantations of the main industrial zone of horticulture (Turkestan, Zhambyl, Almaty regions). As a result of the bacteriological analy-sis of the selected samples, 8 bacterial isolates were extracted from the varieties such as Aport, Voskhod, Sinap Almaty, Maksat, Golden Delicious, Kon-fetnoye, Pinova, Pink Lady, which, and were like the causative agent of fire blight by morphological and cultural characteristics. Bacterial pathogenicity testing on immature apple fruit showed positive test results confirming the presence of the bacterium Erwinia amylovora, the causative agent of fire blight.

CHARACTERISTICS OF THE FIRE BLIGHT PATHOGEN ERWINIA AMYLOVORA ISOLATED FROM FRUIT CULTURES IN SOUTHEASTERN KAZAKHSTAN

One of the most dangerous and quarantine diseases of fruit crops of the Rosaceae family is fire blight which affected on plants. Its causative agent Erwinia amylovora causes necrosis of all organs of host plants. In the territory of Republic of Kazakhstan, the disease belongs to a limited number of quarantine species. A special place is given to laboratory diagnostics of E.amylovora, which is necessary for reliable detection of the pathogen. Bacteriological analyses of diseased samples of fruit crops were carried out under laboratory conditions to isolate pathogens into pure cultures. Isolating of the quarantine and causative agent of fire blight into a pure culture was carried out on King B nutrient media, Levan and Miller Schroth selective media. They were tested for pathogenicity on indicator plants of indoor geranium by hypersensitivity reaction by the infectious-infiltration method of Clement. The pathogenicity of isolated bacteria was also tested by testing on unripe pear fruits, on which the damaging bacteria caused the release of milky-white exudate. After the selected isolates of the fire blight pathogen were identified by two pairs of specific primers PEANT F/PEANT2 R and FER F/rgER2-R approved by the European and Mediterranean Plant Protection Organization. For molecular differentiation, the nucleotide sequences of 16S rRNA genes were compared

Investigating the Biocontrol Potential of the Natural Microbiota of the Apple Blossom.

Erwinia amylovora, the causative agent of fire blight, leads to important economic losses of apple and pear crops worldwide. This study aimed to investigate the potential of the resident microbiota of the apple blossom in combatting plant disease-causing organisms, with a focus on controlling fire blight. We obtained 538 isolates from sites around Canton Zurich, which we tested for activity against Pectobacterium carotovorum and E. amylovora. We also evaluated the isolates' activity against oomycete and fungal pathogens. Nine isolates showed activity against P. carotovorum, and eight of these against E. amylovora. Furthermore, 117 showed antifungal, and 161 anti-oomycete, activity. We assigned genera and in some cases species to 238 of the isolates by sequencing their 16S RNA-encoding gene. Five strains showed activity against all pathogens and were tested in a detached apple model for anti-E. amylovora activity. Of these five strains, two were able to antagonize E. amylovora, namely Bacillus velezensis #124 and Pantoea agglomerans #378. We sequenced the P. agglomerans #378 genome and analyzed it for secondary metabolite clusters using antiSMASH, revealing the presence of a putative bacteriocin cluster. We also showed that B. velezensis #124 exhibits strong activity against three different fungi and two oomycetes in vitro, suggesting a broader capacity for biocontrol. Our results showcase the protective potential of the natural apple blossom microbiota. We isolated two candidate biocontrol strains from apple blossoms, suggesting that they might persist at the most common entry point for the causative agent of fire blight. Furthermore, they are probably already part of the human diet, suggesting they might be safe for consumption, and thus are promising candidates for biocontrol applications.

Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora

Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) coupled with lateral flow test or coupled with additional amplification based on CRISPR/Cas12a resulting from the fluorescence of the Cas12a-cleaved probe. To compare the four amplification techniques, we chose the bacterial phytopathogen Erwinia amylovora (causative agent of fire blight), which has a quarantine significance in many countries and possesses a serious threat to agriculture. Three genes were chosen as the targets and primers were selected for each one (two for RPA and six for LAMP). They were functionalized by labels (biotin, fluorescein) at the 5' ends for amplicons recognition by LFT. As a result, we developed LAMP-LFT, LAMP-CRISPR/Cas, RPA-LFT, and RPA-CRISPR/Cas for E. amylovora detection. The detection limit was 104 CFU/mL for LAMP-LFT, 103 CFU/mL for LAMP-CRISPR/Cas, and 102 CFU/mL for RPA-LFT and RPA-CRISPR/Cas. The results of four developed test systems were verified by qPCR on a panel of real samples. The developed assays based on RPA, LAMP, CRISPR/Cas12a, and LFT are rapid (30-55 min), user-friendly, and highly sensitive for E. amylovora detection. All proposed detection methods can be applied to fire blight diagnosis and effective management of this disease.

Identification and Characterization of Erwinia Phage IT22: A New Bacteriophage-Based Biocontrol against Erwinia amylovora.

Erwinia amylovora is a quarantine phytopathogenic bacterium that is the causal agent of fire blight, a destructive disease responsible for killing millions of fruit-bearing plants worldwide, including apple, pear, quince, and raspberry. Efficient and sustainable control strategies for this serious bacterial disease are still lacking, and traditional methods are limited to the use of antibiotics and some basic agricultural practices. This study aimed to contribute to the development of a sustainable control strategy through the identification, characterization, and application of bacteriophages (phages) able to control fire blight on pears. Phages isolated from wastewater collected in the Apulia region (southern Italy) were characterized and evaluated as antibacterial agents to treat experimental fire blight caused by E. amylovora. Transmission electron microscopy (TEM) conducted on purified phages (named EP-IT22 for Erwinia phage IT22) showed particles with icosahedral heads of ca. 90 ± 5 nm in length and long contractile tails of 100 ± 10 nm, typical of the Myoviridae family. Whole genome sequencing (WGS), assembly, and analysis of the phage DNA generated a single contig of 174.346 bp representing a complete circular genome composed of 310 open reading frames (ORFs). EP-IT22 was found to be 98.48% identical to the Straboviridae Erwinia phage Cronus (EPC) (GenBank Acc. n° NC_055743) at the nucleotide level. EP-IT22 was found to be resistant to high temperatures (up to 60 °C) and pH values between 4 and 11, and was able to accomplish a complete lytic cycle within one hour. Furthermore, the viability-qPCR and turbidity assays showed that EP-IT22 (MOI = 1) lysed 94% of E. amylovora cells in 20 h. The antibacterial activity of EP-IT22 in planta was evaluated in E. amylovora-inoculated pear plants that remained asymptomatic 40 days post inoculation, similarly to those treated with streptomycin sulphate. This is the first description of the morphological, biological, and molecular features of EP-IT22, highlighting its promising potential for biocontrol of E. amylovora against fire blight disease.

Exopolysaccharides amylovoran and levan contribute to sliding motility in the fire blight pathogen Erwinia amylovora.

Erwinia amylovora, the causative agent of fire blight, uses flagella-based motilities to translocate to host plant natural openings; however, little is known about how this bacterium migrates systemically in the apoplast. Here, we reveal a novel surface motility mechanism, defined as sliding, in E. amylovora. Deletion of flagella assembly genes did not affect this movement, whereas deletion of biosynthesis genes for the exopolysaccharides (EPSs) amylovoran and levan resulted in non-sliding phenotypes. Since EPS production generates osmotic pressure that potentially powers sliding, we validated this mechanism by demonstrating that water potential positively contributes to sliding. In addition, no sliding was observed when the water potential of the surface was lower than -0.5MPa. Sliding is a passive motility mechanism. We further show that the force of gravity plays a critical role in directing E. amylovora sliding on unconfined surfaces but has a negligible effect when cells are sliding in confined microcapillaries, in which EPS-dependent osmotic pressure acts as the main force. Although amylovoran and levan are both required for sliding, we demonstrate that they exhibit different roles in bacterial communities. In summary, our study provides fundamental knowledge for a better understanding of mechanisms that drive bacterial sliding motility.

Spontaneous Resistance of Erwinia amylovora Against Bacteriophage Y2 Affects Infectivity of Multiple Phages

Broad application of antibiotics gave rise to increasing numbers of antibiotic resistant bacteria. Therefore, effective alternatives are currently investigated. Bacteriophages, natural predators of bacteria, could work as such an alternative. Although phages can be highly effective at eliminating specific bacteria, phage resistance can be observed after application. The nature of this resistance, however, can differ depending on the phage. Exposing Erwinia amylovora CFBP 1430, the causative agent of fire blight, to the different phages Bue1, L1, S2, S6, or M7 led to transient resistance. The bacteria reversed to a phage sensitive state after the phage was eliminated. When wild type bacteria were incubated with Y2, permanently resistant colonies (1430Y2R) formed spontaneously. In addition, 1430Y2R revealed cross-resistance against other phages (Bue1) or lowered the efficiency of plating (L1, S2, and S6). Pull down experiments revealed that Y2 is no longer able to bind to the mutant suggesting mutation or masking of the Y2 receptor. Other phages tested were still able to bind to 1430Y2R. Bue1 was observed to still adsorb to the mutant, but no host lysis was found. These findings indicated that, in addition to the alterations of the Y2 receptor, the 1430Y2R mutant might block phage attack at different stage of infection. Whole genome sequencing of 1430Y2R revealed a deletion in the gene with the locus tag EAMY_2231. The gene, which encodes a putative galactosyltransferase, was truncated due to the resulting frameshift. The mutant 1430Y2R was monitored for potential defects or fitness loss. Weaker growth was observed in LB medium compared to the wild type but not in minimal medium. Strain 1430Y2R was still highly virulent in blossoms even though amylovoran production was observed to be reduced. Additionally, LPS structures were analyzed and were clearly shown to be altered in the mutant. Complementation of the truncated EAMY_2231 in trans restored the wild type phenotype. The truncation of EAMY_2231 can therefore be associated with manifold modifications in 1430Y2R, which can affect different phages simultaneously.

PM 7/20 (3) Erwinia amylovora

PM 7/20 (3) <i>Erwinia amylovora</i>

Bacteriophage S6 requires bacterial cellulose for Erwinia amylovora infection.

Bacteriophages are highly selective in targeting bacteria. This selectivity relies on the specific adsorption of phages to the host cell surface. In this study, a Tn5 transposon mutant library of Erwinia amylovora, the causative agent of fire blight, was screened to identify bacterial receptors required for infection by the podovirus S6. Phage S6 was unable to infect mutants with defects in the bacterial cellulose synthase operon (bcs). The Bcs complex produces and secretes bacterial cellulose, an extracellular polysaccharide associated with bacterial biofilms. Deletion of the bcs operon or associated genes (bcsA, bcsC and bcsZ) verified the crucial role of bacterial cellulose for S6 infection. Application of the cellulose binding dye Congo Red blocked infection by S6. We demonstrate that infective S6 virions degraded cellulose and that Gp95, a phage-encoded cellulase, is involved to catalyse the reaction. In planta S6 did not significantly inhibit fire blight symptom development. Moreover, deletion of bcs genes in E. amylovora did not affect bacterial virulence in blossom infections, indicating that sole application of cellulose targeting phages is less appropriate to biologically control E. amylovora. The interplay between cellulose synthesis, host cell infection and maintenance of the host cell population is discussed.

Draft Genome Sequences of Four Streptomycin-Sensitive Erwinia amylovora Strains Isolated from Commercial Apple Orchards in Ohio.

ABSTRACTErwinia amylovora is the causative agent of fire blight, a devastating disease of apples and pears worldwide. Here, we report draft genome sequences of four streptomycin-sensitive strains of E. amylovora that were isolated from diseased apple trees in Ohio.

First report of Erwina amylovora in Tuscany, Italy

2-years-old plants of Pyrus communis showing symptoms of fire blight disease were sampled in an orchard in Tuscany (Italy) during Autumn 2020. Plants were obtained the previous spring from a commercial nursery located in a region where the disease is present since 1994. The collected material was processed in the lab in order to verify the presence of the bacterium Erwinia amylovora, the causal agent of fire blight. Pure isolates showing white mucoid colonies and levan producers on Levan medium were putatively assimilated to E. amylovora. DNA was extracted from the cultures and analysed with three molecular assays, including duplex PCR of the 29-Kb plasmid pEA29 and the ams chromosomal region, sequencing of the 16S rDNA and recA gene regions, two real-time PCR assays on symptomatic plant tissues. All tests confirmed the presence of E. amylovora. Symptomatic and surrounding plants were removed and immediately destroyed according to the regional phytosanitary protocol. This outcome poses a serious threat for fruit orchards in the area.

Epidemiology of Fire Blight in Fruit Crops in Kazakhstan

A survey on 13 regions of Kazakhstan was carried out to identify foci of fire blight, the extent of its spread, the range of affected crops, and the identification of infection reserves. The disease was mainly concentrated in the main fruit growing zone.at the south and southeast of Kazakhstan. Symptoms of fire blight were characteristic of two bacterial diseases: namely, necrosis, by Pseudomonas syringae Van. Hall. and fire blight by Erwinia amylovora (Burrill) Winslow et al. The authors performed bacteriological analyses to isolate and identify the causative agent of fire blight from various fruit and wild cultures of the Rosaceae family, using classical bacteriological and molecular genetic methods. Two types of bacteria were isolated from the samples affected by the disease, namely E. amylovora, the causative agent of fire blight, and P. syringae, the causative agent of bacterial necrosis. The results of the studies on the identification of bacterial species E. amylovora and P. syringae were confirmed by Swiss scientists from the Agroscope research center based on an immuno-chromatographic test and by Russian scientists at the All-Russian Research Institute of Plant Quarantine using enzyme-linked immunosorbent assay, FLASH polymerase chain reaction, and polymerase chain reaction in real-time.

Characterization of the biocontrol activity of three bacterial isolates against the phytopathogen Erwinia amylovora.

Antibiotics are sprayed on apple and pear orchards to control, among other pathogens, the bacterium Erwinia amylovora, the causative agent of fire blight. As with many other pathogens, we observe the emergence of antibiotic‐resistant strains of E. amylovora. Consequently, growers are looking for alternative solutions to combat fire blight. To find alternatives to antibiotics against this pathogen, we have previously isolated three bacterial strains with antagonistic and extracellular activity against E. amylovora, both in vitro and in planta, corresponding to three different bacterial genera: Here, we identified the inhibitory mode of action of each of the three isolates against E. amylovora. Isolate Bacillus amyloliquefaciens subsp. plantarum (now B. velezensis) FL50S produces several secondary metabolites including surfactins, iturins, and fengycins. Specifically, we identified oxydifficidin as the most active against E. amylovora S435. Pseudomonas poae FL10F produces an active extracellular compound against E. amylovora S435 that can be attributed to white‐line‐inducing principle (WLIP), a cyclic lipopeptide belonging to the viscosin subfamily (massetolide E, F, L, or viscosin). Pantoea agglomerans NY60 has a direct cell‐to‐cell antagonistic effect against E. amylovora S435. By screening mutants of this strain generated by random transposon insertion with decreased antagonist activity against strain S435, we identified several defective transposants. Of particular interest was a mutant in a gene coding for a Major Facilitator Superfamily (MFS) transporter corresponding to a transmembrane protein predicted to be involved in the extracytoplasmic localization of griseoluteic acid, an intermediate in the biosynthesis of the broad‐spectrum phenazine antibiotic d‐alanylgriseoluteic acid.

Physicochemical Characterization of Crithmum maritimum L. and Daucus carota subsp. gummifer (Syme) Hook.fil. and Their Antimicrobial Activity against Apple Tree and Grapevine Phytopathogens

Crithmum maritimum and Daucus carota subsp. gummifer are two species of the Apiaceae family that share multiple characteristics: both are halophitic, live on cliffs in the same geographic habitats, and are edible. While C. maritimum is rich in essential oils and flavonoids, D. carota is rich in terpenes and a gum producer. In the work presented herein, the biomass of these two wild plants and the bioactive compounds present in their extracts have been studied by elemental and thermal analysis, infrared spectroscopy, and gas chromatography-mass spectroscopy. To explore their bioactivities, both their hydroalcoholic extracts and their major constituents (apiole in C. maritimum and geranyl acetate in D. carota), either alone or in combination with chitosan oligomers, were assayed in vitro against bacterial and fungal pathogens that affect apple trees (Malus domestica) and grapevine (Vitis vinifera). Remarkable inhibition was observed against Erwinia amylovora, the causal agent of fire blight in apple; Xylophilus ampelinus [syn. Erwinia vitivora], the causal agent of bacterial blight of grapevine; and Diplodia seriata, a virulent pathogen of grapevines that also causes canker, leaf spot and fruit rot of apple. In view of their effectiveness against these three phytopathogens, a potential application of these two medicinal plants in organic farming may be envisaged.

Genetic characterization of Erwinia amylovora isolates detected in the wild walnut-fruit forest of South Kyrgyzstan

Erwinia amylovora, the causal agent of fire blight, was first reported in Kyrgyz orchards in 2008 and, within a few years, the disease has spread throughout most of the northern part of the country. As fire blight expanded and penetrated new areas, it also reached the unique ecosystem constituted by the Arslonbob wild walnut–fruit forests in South Kyrgyzstan. Here, we present the results of field surveys in the forests and in the nearby orchards and gardens of the Jalal-Abad region during the 2019 season. Further, we use genetic profiling of the CRISPR regions to compare the genotypes of the obtained isolates to those of previous outbreaks in the north of the country and in nearby Kazakhstan. While fire blight was found in twelve of the twenty-six sites investigated in and around the Arslonbob walnut–fruit forest, it was mostly located on domesticated varieties of quince, apple or pear in gardens, orchards or nurseries. Plants in the forest and especially endangered wild species were not affected by the disease, but their proximity to the current infection herds is cause of major concern and calls for immediate protective action. Genetic analysis suggests that the main introduction route for fire blight to Central Asia was import of contaminated plant material from already affected countries in the Eurasian continent, which probably occurred on multiple independent instances. Nonetheless, indications of natural spread of the disease were identified between the regions of South Kazakhstan and North Kyrgyzstan.