Isolation and Characterization of a Lytic Bacteriophage RH-42-1 of Erwinia amylovora from Orchard Soil in China.

Fire blight, caused by the bacterium Erwinia amylovora, is a major threat to pear production worldwide. Bacteriophages, viruses that infect bacteria, are a promising alternative to antibiotics for controlling fire blight. In this study, we isolated a novel bacteriophage, RH-42-1, from Xinjiang, China. We characterized its biological properties, including host range, plaque morphology, infection dynamics, stability, and sensitivity to various chemicals. RH-42-1 infected several E. amylovora strains but not all. It produced clear, uniform plaques and exhibited optimal infectivity at a multiplicity of infection (MOI) of 1, reaching a high titer of 9.6 × 109 plaque-forming units (PFU)/mL. The bacteriophage had a short latent period (10 min), a burst size of 207 PFU/cell, and followed a sigmoidal one-step growth curve. It was stable at temperatures up to 60 °C but declined rapidly at higher temperatures. RH-42-1 remained viable within a pH range of 5 to 9 and was sensitive to extreme pH values. The bacteriophage demonstrates sustained activity upon exposure to ultraviolet radiation for 60 min, albeit with a marginal reduction. In our assays, it exhibited a certain level of resistance to 5% chloroform (CHCl3), 5% isopropanol (C3H8O), and 3% hydrogen peroxide (H2O2), which had little effect on its activity, whereas it showed sensitivity to 75% ethanol (C2H5OH). Electron microscopy revealed that RH-42-1 has a tadpole-shaped morphology. Its genome size is 14,942 bp with a GC content of 48.19%. Based on these characteristics, RH-42-1 was identified as a member of the Tectiviridae family, Alphatectivirus genus. This is the first report of a bacteriophage in this genus with activity against E. amylovora.

Improved Viability of Spray-Dried Pantoea agglomerans for Phage-Carrier Mediated Control of Fire Blight.

Fire blight, caused by Erwinia amylovora, is a devastating bacterial disease that threatens apple and pear production. It is mainly controlled by using antibiotics, such as streptomycin. Due to development of E. amylovora resistant strains and the excessive agricultural use of antibiotics, there is an increased awareness of the possibility of antibiotic resistance gene transfer to other microbes. Urgent development of biocontrol agents (BCAs) is needed that can be incorporated into integrated pest management programs as antibiotic alternatives. A novel phage-carrier system (PCS) that combines an antagonistic bacterium, Pantoea agglomerans, with its ability to act as a phage-carrier bacterium for Erwinia phages has been developed. The low viability of P. agglomerans cells following spray-drying (SD) has been a challenge for the industrial-scale production of this PCS. Here, an SD protocol was developed for P. agglomerans by modifying the growth medium and bacterial cell formulation using D(+)-trehalose and maltodextrin. The developed protocol is amenable to the industrial-scale production of the BCA/PCS. The P. agglomerans viability was greater than 90% after SD and had a shelf life at 4 °C of 4 months, and reconstituted cells showed a 3 log reduction in E. amylovora counts with a pear disc assay.

Inter-species interactions between two bacterial flower commensals and a floral pathogen reduce disease incidence and alter pathogen activity.

Flowers are colonized by a diverse community of microorganisms that can alter plant health and interact with floral pathogens. Erwinia amylovora is a flower-inhabiting bacterium and a pathogen that infects different plant species, including Malus × domestica (apple). Previously, we showed that the co-inoculation of two bacterial strains, members of the genera Pseudomonas and Pantoea, isolated from apple flowers, reduced disease incidence caused by this floral pathogen. Here, we decipher the ecological interactions between the two flower-associated bacteria and E. amylovora in field experimentation and in vitro co-cultures. The two flower commensal strains did not competitively exclude E. amylovora from the stigma habitat, as both bacteria and the pathogen co-existed on the stigma of apple flowers and in vitro. This suggests that plant protection might be mediated by other mechanisms than competitive niche exclusion. Using a synthetic stigma exudation medium, ternary co-culture of the bacterial strains led to a substantial alteration of gene expression in both the pathogen and the two microbiota members. Importantly, the gene expression profiles for the ternary co-culture were not just additive from binary co-cultures, suggesting that some functions only emerged in multipartite co-culture. Additionally, the ternary co-culture of the strains resulted in a stronger acidification of the growth milieu than mono- or binary co-cultures, pointing to another emergent property of co-inoculation. Our study emphasizes the critical role of emergent properties mediated by inter-species interactions within the plant holobiont and their potential impact on plant health and pathogen behavior. IMPORTANCE: Fire blight, caused by Erwinia amylovora, is one of the most important plant diseases of pome fruits. Previous work largely suggested plant microbiota commensals suppressed disease by antagonizing pathogen growth. However, inter-species interactions of multiple flower commensals and their influence on pathogen activity and behavior have not been well studied. Here, we show that co-inoculating two bacterial strains that naturally colonize the apple flowers reduces disease incidence. We further demonstrate that the interactions between these two microbiota commensals and the floral pathogen led to the emergence of new gene expression patterns and a strong alteration of the external pH, factors that may modify the pathogen's behavior. Our findings emphasize the critical role of emergent properties mediated by inter-species interactions between plant microbiota and plant pathogens and their impact on plant health.

Determination and Kinetic Characterization of a New Potential Inhibitor for AmsI Protein Tyrosine Phosphatase from the Apple Pathogen Erwinia amylovora.

Erwinia amylovora is a Gram-negative bacterium, responsible for the fire blight disease in Rosaceae plants. Its virulence is correlated with the production of an exopolysaccharide (EPS) called amylovoran, which protects the bacterium from the surrounding environment and helps its diffusion inside the host. Amylovoran biosynthesis relies on the expression of twelve genes clustered in the ams operon. One of these genes, amsI, encodes for a Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) called EaAmsI, which plays a key role in the regulation of the EPS production pathway. For this reason, EaAmsI was chosen in this work as a target for the development of new antibacterial agents against E. amylovora. To achieve this aim, a set of programs (DOCK6, OpenEye FRED) was selected to perform a virtual screening using a database of ca. 700 molecules. The six best-scoring compounds identified were tested in in vitro assays. A complete inhibition kinetic characterization carried out on the most promising molecule (n-Heptyl ß-D-glucopyranoside, N7G) showed an inhibition constant of 7.8 ± 0.6 µM. This study represents an initial step towards the development of new EaAmsI inhibitors able to act as antibacterial agents against E. amylovora infections.

Quantitative Trait Locus Mapping for Fire Blight Resistance in an F2 Population of Malus fusca MAL0045 Uncovers Novel Resistance Loci.

Several fire blight resistance loci in Malus genotypes map on different linkage groups (LGs) representing chromosomes of the domesticated apple. Prior genetics studies primarily focused on F1 populations. A strong resistance quantitative trait locus (QTL) explained up to 66% of phenotypic variance in an F1 progeny derived from crossing the highly resistant wild apple genotype Malus fusca MAL0045 and the highly susceptible apple cultivar 'Idared', which was previously mapped on LG10 (Mfu10) of MAL0045. Strains of the causative bacterial pathogen Erwinia amylovora, notably those that show a single nucleotide polymorphism in the avrRpt2EA effector protein sequence at position 156 (e.g., Ea3049), are more virulent and overcome some known fire blight resistance donors and their QTLs. However, MAL0045 is resistant to Ea3049 and Mfu10 is not overcome, but most of the F1 progeny were highly susceptible to this strain. This phenomenon led to the assumption that other putative resistance factors not segregating in the F1 progeny might be present in the genome of MAL0045. Here, we crossed F1 progeny together to obtain 135 F2 individuals. Facilitated by genotyping-by-sequencing and phenotypic assessments, we identified and mapped two novel resistance QTLs in these F2 individuals on LGs 4 and 15, which were not identified in the F1. To our knowledge, these are the first resistance QTLs mapped in F2 progeny in Malus. In addition, we report that neither MAL0045 nor Mfu10 is broken down by a highly aggressive U.S. strain, LA635, after analyses in the original F1 individuals. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Novel approach toward the understanding of genetic diversity based on the two types of amino acid repeats in Erwinia amylovora.

Erwinia amylovora is a notorious plant pathogenic bacterium of global concern that has devastated the apple and pear production industry worldwide. Nevertheless, the approaches available currently to understand the genetic diversity of E. amylovora remain unsatisfactory because of the lack of a trustworthy index and data covering the globally occurring E. amylovora strains; thus, their origin and distribution pattern remains ambiguous. Therefore, there is a growing need for robust approaches for obtaining this information via the comparison of the genomic structure of Amygdaloideae-infecting strains to understand their genetic diversity and distribution. Here, the whole-genome sequences of 245 E. amylovora strains available from the NCBI database were compared to identify intraspecific genes for use as an improved index for the simple classification of E. amylovora strains regarding their distribution. Finally, we discovered two kinds of strain-typing protein-encoding genes, i.e., the SAM-dependent methyltransferase and electron transport complex subunit RsxC. Interestingly, both of these proteins carried an amino acid repeat in these strains: SAM-dependent methyltransferase comprised a single-amino-acid repeat (asparagine), whereas RsxC carried a 40-amino-acid repeat, which was differentially distributed among the strains. These noteworthy findings and approaches may enable the exploration of the genetic diversity of E. amylovora from a global perspective.

Structure-Function Characterisation of Eop1 Effectors from the Erwinia-Pantoea Clade Reveals They May Acetylate Their Defence Target through a Catalytic Dyad.

The YopJ group of acetylating effectors from phytopathogens of the genera Pseudomonas and Ralstonia have been widely studied to understand how they modify and suppress their host defence targets. In contrast, studies on a related group of effectors, the Eop1 group, lag far behind. Members of the Eop1 group are widely present in the Erwinia-Pantoea clade of Gram-negative bacteria, which contains phytopathogens, non-pathogens and potential biocontrol agents, implying that they may play an important role in agroecological or pathological adaptations. The lack of research in this group of YopJ effectors has left a significant knowledge gap in their functioning and role. For the first time, we perform a comparative analysis combining AlphaFold modelling, in planta transient expressions and targeted mutational analyses of the Eop1 group effectors from the Erwinia-Pantoea clade, to help elucidate their likely activity and mechanism(s). This integrated study revealed several new findings, including putative binding sites for inositol hexakisphosphate and acetyl coenzyme A and newly postulated target-binding domains, and raises questions about whether these effectors function through a catalytic triad mechanism. The results imply that some Eop1s may use a catalytic dyad acetylation mechanism that we found could be promoted by the electronegative environment around the active site.

Single-Cell Detection of Erwinia amylovora Using Bio-Functionalized SIS Sensor.

Herein, we developed a bio-functionalized solution-immersed silicon (SIS) sensor at the single-cell level to identify Erwinia amylovora (E. amylovora), a highly infectious bacterial pathogen responsible for fire blight, which is notorious for its rapid spread and destructive impact on apple and pear orchards. This method allows for ultra-sensitive measurements without pre-amplification or labeling compared to conventional methods. To detect a single cell of E. amylovora, we used Lipopolysaccharide Transporter E (LptE), which is involved in the assembly of lipopolysaccharide (LPS) at the surface of the outer membrane of E. amylovora, as a capture agent. We confirmed that LptE interacts with E. amylovora via LPS through in-house ELISA analysis, then used it to construct the sensor chip by immobilizing the capture molecule on the sensor surface modified with 3'-Aminopropyl triethoxysilane (APTES) and glutaraldehyde (GA). The LptE-based SIS sensor exhibited the sensitive and specific detection of the target bacterial cell in real time. The dose-response curve shows a linearity (R2 > 0.992) with wide dynamic ranges from 1 to 107 cells/mL for the target bacterial pathogen. The sensor showed the value change (dΨ) of approximately 0.008° for growing overlayer thickness induced from a single-cell E. amylovora, while no change in the control bacterial cell (Bacillus subtilis) was observed, or negligible change, if any. Furthermore, the bacterial sensor demonstrated a potential for the continuous detection of E. amylovora through simple surface regeneration, enabling its reusability. Taken together, our system has the potential to be applied in fields where early symptoms are not observed and where single-cell or ultra-sensitive detection is required, such as plant bacterial pathogen detection, foodborne pathogen monitoring and analysis, and pathogenic microbial diagnosis.

Applications of Germicidal Ultraviolet Light as a Tool for Fire Blight Management (Erwinia amylovora) in Apple Plantings.

Nighttime applications of germicidal UV light (UV-C) have been used to suppress several fungal diseases of plants, but less is known of UV-C's potential to suppress bacterial plant pathogens. Fire blight of apple and pear, caused by the bacterium Erwinia amylovora, is difficult to suppress using cultural practices, antibiotics, and host resistance. We therefore investigated the potential of UV-C as an additional means to manage the disease. Laboratory assays confirmed that in vitro exposure of cultures E. amylovora to UV-C at doses ranging from 0 to 400 J/m2 in the absence of visible light was more than 200% as effective as cultures exposed to visible light after the same UV-C treatments. In a 2-year orchard study, we demonstrated that with only two nighttime applications of UV-C at 200 J/m2 made at bloom resulted in an incidence of blossom blight and shoot blight equivalent to the results viewed when antibiotic and biopesticide commercial standards were applied. In vitro dose-response studies indicated consistency in pathogen response to suppressive UV-C doses, including pathogen isolates that were resistant to streptomycin. Based on these results, UV-C may be useful in managing bacterial populations with antibiotic resistance. Concurrent measurements of host growth after UV-C applications indicated that the dose required to suppress E. amylovora had no significant (P > 0.05) effects on foliar growth, shoot extension, internode length, or fruit finish but substantially reduced epiphytic populations of E. amylovora on host tissues.

Acidifying Spray Suspensions of Oxytetracycline and Kasugamycin Enhances Their Effectiveness for Fire Blight Control in Apple and Pear.

The stability of the fire blight control material, oxytetracycline, in water is strongly affected by pH, increasing with increasing acidity. From 2017 to 2021, pear and apple orchard trials were conducted to evaluate if acidic amendments to oxytetracycline sprays improve fire blight control. Compared with the water-treated control, infection suppression after two bloom applications of an acidified commercial oxytetracycline formulation averaged 85.9 ± 0.4% compared with 72.2 ± 1.7% without an acidifier, but individual trials frequently had insufficient statistical power to separate among acidified and non-acidified antibiotic treatments. Across trials, a significant linear relationship was observed for regression of relative infection suppression from oxytetracycline (hydrochloride formulation) on spray tank pH. Similar relationships were observed for oxytetracycline (calcium complex formulation) and kasugamycin (P values were 0.055 and 0.069, respectively). Also based on regression, acidified oxytetracycline and kasugamycin suppressed epiphytic populations of Erwinia amylovora on flowers to a greater degree than the antibiotic only. As spray suspensions, commercial oxytetracycline formulations at label rate and amended with citric acid (1.2 g/liter) in well water had pH values near 3.4, but after spraying, the pH of flowers washed in deionized water (1 ml/flower) measured in a range of 5.2 to 5.5 compared with a pH range of 5.8 to 6.0 after a treatment of oxytetracycline only. In pear fruit finish trials, sprays acidified with citric acid-based materials had negligible effects on fruit russeting. Based on a serological assay, the detectable residual of oxytetracycline on apple foliage was increased by co-application with citric acid compared with a non-acidified control.

A Novel IncX Plasmid Mediates High-Level Oxytetracycline and Streptomycin Resistance in Erwinia amylovora from Commercial Pear Orchards in California.

Isolates of the fire blight pathogen Erwinia amylovora with high-level resistance to oxytetracycline (minimal inhibitory concentration [MIC] > 100 µg/ml) and to streptomycin (MIC > 100 µg/ml) were recovered from four commercial pear orchards in California between 2018 and 2020. The two representative oxytetracycline- and streptomycin-resistant (OxyTcR-SmR) strains 32-10 and 33-1 were as virulent as the antibiotic susceptible strain 13-1 in causing blossom blight of pear and were recovered more than 50% of the time 7 days after co-inoculation to pear flowers with strain 13-1. In the field, inoculation of strain 32-10 to pear flowers that were pretreated with oxytetracycline at 200 µg/ml did not reduce disease compared with an untreated control. Four OxyTcR-SmR strains were subjected to draft genome sequencing to identify the genetic determinants of antibiotic resistance and their location. A 43.6-kb IncX plasmid, designated pX11-7, was detected in each of the four strains, and this plasmid encoded the tetracycline-resistance gene tetB and the streptomycin-resistance gene pair strAB within a large putatively mobile genetic element consisting of the transposon Tn10 that had inserted within the streptomycin-resistance transposon Tn6082. We also determined that pX11-7 was conjugative and was transferred at a rate that was 104 to 105 higher into an E. amylovora strain isolated in California compared with an E. amylovora strain that was isolated in Michigan. The occurrence of high levels of resistance to both oxytetracycline and streptomycin in E. amylovora strains from commercial pear orchards in California significantly limits the options for blossom blight management in these locations.

Evaluation of Plant Defense Inducers and Plant Growth Regulators for Fire Blight Management Using Transcriptome Studies and Field Assessments.

Fire blight, caused by Erwinia amylovora, is a destructive disease of pome fruit trees. In the United States, apple and pear growers rely on applications of copper and antibiotics during bloom to control fire blight, but such methods have already led to regional instances of resistance. In this study, we used transcriptome analyses and field trials to evaluate the effectiveness of three commercially available plant defense elicitors and one plant growth regulator for fire blight management. Our data indicated that foliar applications of acibenzolar-S-methyl (ASM; Actigard 50WG) triggered a strong defense-related response in apple leaves, whereas applications of Bacillus mycoides isolate J (LifeGard WG) or Reynoutria sachalinensis extract (Regalia) did not. Genes upregulated by ASM were enriched in the biological processes associated with plant immunity, such as defense response and protein phosphorylation. The expression of several pathogenesis-related (PR) genes was induced by ASM as well. Surprisingly, many differentially expressed genes in ASM-treated apple leaves overlapped with those induced by treatment with prohexadione-calcium (ProCa; Apogee), a plant growth regulator that suppresses shoot elongation. Further analysis suggested that ProCa likely acts similarly to ASM to stimulate plant immunity because genes involved in plant defense were shared and significantly upregulated (more than twofold) by both treatments. Our field trials agreed with the transcriptome study, demonstrating that ASM and ProCa exhibit the best control performance relative to the other biopesticides. Taken together, these data are pivotal for the understanding of plant response and shed light on future improvements of strategies for fire blight management.

Dynamics of Bacterial Communities by Apple Tissue: Implications for Apple Health.

Herein, we explored the potential of the apple's core microbiota for biological control of Erwinia amylovora, which causes fire blight disease, and analyzed the structure of the apple's bacterial community across different tissues and seasons. Network analysis results showed distinct differences in bacterial community composition between the endosphere and rhizosphere of healthy apples, and eight taxa were identified as negatively correlated with E. amylovora, indicating their potential key role in a new control strategy against the pathogen. This study highlights the critical role of the apple's bacterial community in disease control and provides a new direction for future research in apple production. In addition, the findings suggest that using the composition of the apple's core taxa as a biological control strategy could be an effective alternative to traditional chemical control methods, which have been proven futile and environmentally harmful.

Current Situation of Fire Blight in China.

Fire blight, caused by the plant-pathogenic bacterium Erwinia amylovora, is a devastating disease that occurs on rosaceous plants, including pears and apples. E. amylovora is indigenous to North America and was spread to the Eurasian continent in the second half of the 20th century through contaminated plant materials. In 2016, fire blight was first observed in Yili, Xinjiang Province, in Northwestern China. Since then, it has spread to most pear-producing regions in Xinjiang Province and parts of Gansu Province. The disease has caused severe damage to China's pear and apple industries, including the 2017 disease epidemic in Korla, Xinjiang, which caused an overall yield reduction of 30 to about 50% in Korla and the destruction of over 1 million pear trees. Over the past few years, a combined effort of research, extension, and education by the Chinese government, scientists, and fruit growers has greatly alleviated outbreaks and epidemics in affected regions while successfully limiting the further spread of fire blight to new geographical regions. Here, we review the occurrence, spread, and damage of this disease to the Chinese fruit industry, as well as the management options used in China and their outcomes. We also discuss future perspectives for restraining the spread and alleviating the damage of fire blight in China.

Protocol for diagnosing Erwinia amylovora infection using a fluorescent probe.

Current fire blight diagnosis techniques are DNA based and require specialized equipment and expertise, or they are less sensitive. Here, we present a protocol for diagnosing fire blight using the fluorescent probe, B-1. We describe steps for Erwinia amylovora culture, implementing a fire blight-infected model, and E. amylovora visualization. This protocol allows for detection of fire blight bacteria of up to 102 CFU/mL on plants or objects in just 10 s with a simple application including spraying and swabbing. For complete details on the use and execution of this protocol, please refer to Jung et al.1.

Contribution of Native Plasmids of Pantoea vagans C9-1 to Epiphytic Fitness and Fire Blight Management on Apple and Pear Flowers and Fruits.

Pantoea vagans C9-1 (C9-1) is a biological control bacterium that is applied to apple and pear trees during bloom for suppression of fire blight, caused by Erwinia amylovora. Strain C9-1 has three megaplasmids: pPag1, pPag2, and pPag3. Prior bioinformatic studies predicted these megaplasmids have a role in environmental fitness and/or biocontrol efficacy. Plasmid pPag3 is part of the large Pantoea plasmid (LPP-1) group that is present in all Pantoea spp. and has been hypothesized to contribute to environmental colonization and persistence, while pPag2 is less common. We assessed fitness of C9-1 derivatives cured of pPag2 and/or pPag3 on pear and apple flowers and fruit in experimental orchards. We also assessed the ability of a C9-1 derivative lacking pPag3 to reduce populations of E. amylovora on flowers and disease incidence. Previously, we determined that tolerance to stresses imposed in vitro was compromised in derivatives of C9-1 lacking pPag2 and/or pPag3; however, in this study, the loss of pPag2 and/or pPag3 did not consistently reduce the fitness of C9-1 on flowers in orchards. Over the summer, pPag3 contributed to survival of C9-1 on developing apple and pear fruit in two of five trials, whereas loss of pPag2 did not significantly affect survival of C9-1. We also found that loss of pPag3 did not affect C9-1's ability to reduce E. amylovora populations or fire blight incidence on apple flowers. Our findings partially support prior hypotheses that LPP-1 in Pantoea species contributes to persistence on plant surfaces but questions whether LPP-1 facilitates host colonization.

Erwinia amylovora Type III Secretion System Inhibitors Reduce Fire Blight Infection Under Field Conditions.

Fire blight, caused by Erwinia amylovora, is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause disease. Compounds that inhibit the function of the T3SS (T3SS inhibitors) have emerged as alternative strategies for bacterial plant disease management, as they block bacterial virulence without affecting growth, unlike traditional antibiotics. In this study, we investigated the mode of action of a T3SS inhibitor named TS108, a plant phenolic acid derivative, in E. amylovora. We showed that adding TS108 to an in vitro culture of E. amylovora repressed the expression of several T3SS regulon genes, including the master regulator gene hrpL. Further studies demonstrated that TS108 negatively regulates CsrB, a global regulatory small RNA, at the posttranscriptional level, resulting in a repression of hrpS, which encodes a key activator of hrpL. Additionally, TS108 has no impact on the expression of T3SS in Dickeya dadantii or Pseudomonas aeruginosa, suggesting that its inhibition of the E. amylovora T3SS is likely species specific. To better evaluate the performance of T3SS inhibitors in fire blight management, we conducted five independent field experiments in four states (Michigan, New York, Oregon, and Connecticut) from 2015 to 2022 and observed reductions in blossom blight incidence as high as 96.7% compared with untreated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight.