Multisite field evaluation of bacteriophages for fire blight management: incorporation of UVR protectants, and impact on the apple flower microbiome.

Fire blight, a disease of pome fruits caused by the bacterium Erwinia amylovora, has become increasingly difficult to manage after the emergence of streptomycin-resistant strains. Alternative antibiotics and copper are available; however, these chemicals have use restrictions in some countries and also can carry risks of phytotoxicity. Therefore, there is growing interest in biological-based management options, with bacteriophage (phages) showing promise, as these naturally occurring pathogens of bacteria are easy to isolate and grow. However, there are several technical challenges regarding the implementation of phage biocontrol in the field as the viral molecules suffer from ultraviolet radiation (UVR) degradation and can die off rapidly in the absence of the host bacterium. In this work we assessed the efficacy of Erwinia phages and a commercial phage product for blossom blight control in the field across multiple locations in the eastern United States. In these tests, disease control ranged from 0.0 to 82.7%, and addition of a UVR protectant only resulted in significantly increased disease control in 2 of 12 tests. We also analyzed microbial community population changes in response to phage application. Changes in bacterial community diversity metrics over time were not detected, however relative abundances of target taxa were temporarily reduced after phage applications, indicating that these phage applications did not have deleterious effects on the flower microbiome. We have demonstrated that biological control of fire blight with phages is achievable, but a better understanding of phage:pathogen dynamics is required to optimize disease control efficacy.

Function and Application of the CRISPR-Cas System in the Plant Pathogen Erwinia amylovora.

Phage-based biocontrol is an emerging method for managing the plant pathogen Erwinia amylovora. Control of E. amylovora in North America is achieved chiefly through the application of streptomycin and has led to the development of streptomycin resistance. Resistant E. amylovora can be tracked through the analysis of CRISPR spacer sequences. An alternative to antibiotics are bacterial viruses, known as phages, which lyse their hosts during replication to control the bacterial population. Endogenous CRISPR-Cas systems act as phage resistance mechanisms however, preliminary genomic analysis suggests this activity is limited in E. amylovora. This leaves the functionality of the CRISPR-Cas system, any clade-based differences, and the impact which this system may have on phage-based biocontrol in question. In this study, the CRISPR arrays from 127 newly available genomic sequences of E. amylovora were analyzed through a novel bioinformatic pipeline. Through this, the Eastern and Western North American clades were shown to be incompatible with the current PCR-based approaches for tracking E. amylovora given the size and composition of their CRISPR arrays. Two artificial CRISPR arrays were designed to investigate the functionality of the CRISPR-Cas system in E. amylovora. This system was capable of curing a targeted plasmid and providing phage resistance but was not the source of phage resistance observed within the controls. This suggests that while the CRISPR-Cas system is an important defense mechanism for invasive plasmids, an as yet unidentified mechanism is the primary source of phage resistance in E. amylovora. IMPORTANCE Erwinia amylovora is an economically significant agricultural pathogen found throughout the world. In North America, E. amylovora has developed streptomycin resistance and therefore alternative treatments using phages have received increased attention. In this study, we analyzed recently published genomes to determine that two significant groups of E. amylovora are poorly identified using the current, CRISPR-based tracking methods. We also showed that the CRISPR-Cas system and an unidentified mechanism work together to provide a significant degree of resistance against one of the phages proposed for phage-based biocontrol.

Comparative genomic analysis of Erwinia amylovora reveals novel insights in phylogenetic arrangement, plasmid diversity, and streptomycin resistance.

Erwinia amylovora is a destructive pathogen of Rosaceous plants and an economic concern worldwide. Herein, we report 93 new E. amylovora genomes from North America, Europe, the Mediterranean, and New Zealand. This new genomic information demonstrates the existence of three primary clades of Amygdaloideae (apple and pear) infecting E. amylovora and suggests all three independently originate from North America. The comprehensive sequencing also identified and confirmed the presence of 7 novel plasmids ranging in size from 2.9 to 34.7 kbp. While the function of the novel plasmids is unknown, the plasmids pEAR27, pEAR28, and pEAR35 encoded for type IV secretion systems. The strA-strB gene pair and the K43R point mutation at codon 43 of the rpsL gene have been previously documented to confer streptomycin resistance. Of the sequenced isolates, rpsL-based streptomycin resistance was more common and was found with the highest frequency in the Western North American clade.

Molecular Profile of Phage Infection: A Novel Approach for the Characterization of Erwinia Phages through qPCR.

Due to the emergence of antibiotic resistance, phage-mediated biocontrol has become an attractive alternative for pathogen management in agriculture. While the infection characteristics of many phages can be adequately described using plaque assays and optical density, the results from phages of the apple pathogen Erwinia amylovora have low reproducibility with these techniques. Using quantitative real-time PCR (qPCR), the stage of the lytic cycle was determined through a combination of chloroform-based sampling, centrifugation, and DNase treatment. Monitoring the transition of phage genomes through the lytic cycle generates a molecular profile from which phage infection characteristics such as adsorption rate and burst size can be determined. To our knowledge, this is the first report of qPCR being used to determine these infection parameters. The characteristics of four different genera of Erwinia phages were determined. The phage ΦEa461A1 was able to adsorb at a rate up to 6.6 times faster than ΦEa35-70 and ΦEa9-2. The low enrichment titer of ΦEa92 was shown to be due to the absence of lysis. The ΦEa461A1 and ΦEa214 phages had the highest productivity, with burst sizes of 57 virions in 38 min and 185 virions in 98 min, respectively, suggesting these genera would make stronger candidates for the phage-mediated biocontrol of E. amylovora.

Rapid identification of Erwinia amylovora and Pseudomonas syringae species and characterization of E. amylovora streptomycin resistance using quantitative PCR assays.

Erwinia amylovora and Pseudomonas syringae are bacterial phytopathogens responsible for considerable yield losses in commercial pome fruit production. The pathogens, if left untreated, can compromise tree health and economically impact entire commercial fruit productions. Historically, the choice of effective control methods has been limited. The use of antibiotics was proposed as an effective control method. The identification of these pathogens and screening for the presence of antibiotic resistance is paramount in the adoption and implementation of disease control methods. Molecular tests have been developed and accepted for identification and characterization of these disease-causing organisms. We improved existing molecular tests by developing methods that are equal or superior in robustness for identifying E. amylovora or P. syringae while being faster to execute. In addition, the real-time PCR-based detection method for E. amylovora provided complementary information on the susceptibility or resistance to streptomycin of individual isolates. Finally, we describe a methodology and results that compare the aggressiveness of the different bacterial isolates on four apple cultivars. We show that bacterial isolates exhibit different behaviors when brought into contact with various apple varieties and that the hierarchical clustering of symptom severity indicates a population structure, suggesting a genetic basis for host cultivar specificity.

Taxonomic reassessment of N4-like viruses using comparative genomics and proteomics suggests a new subfamily - "Enquartavirinae".

The GenBank database currently contains sequence data for 33 N4-like viruses, with only one, Escherichia phage N4, being formally recognized by the ICTV. The genus N4likevirus is uniquely characterized by that fact that its members possess an extremely large, virion-associated RNA polymerase. Using a variety of proteomic, genomic and phylogenetic tools, we have demonstrated that the N4-like phages are not monophyletic and that N4 is actually a genomic orphan. We propose to create four new genera: "G7cvirus" (consisting of phages G7C, IME11, KBNP21, vB_EcoP_PhAPEC5, vB_EcoP_PhAPEC7, Bp4, EC1-UPM and pSb-1), "Lit1virus" (LIT1, PA26 and vB_PaeP_C2-10_Ab09), "Sp58virus" (SP058 and SP076), and "Dss3virus" (DSS3φ2 and EE36φ1). We propose that coliphage N4, the members of "G7cvirus", Erwinia phage Ea9-2, and Achromobacter phage JWAlpha should be considered members of the same subfamily, which we tentatively call the "Enquartavirinae".

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.

Host exopolysaccharide quantity and composition impact Erwinia amylovora bacteriophage pathogenesis.

Erwinia amylovora bacteriophages (phages) belonging to the Myoviridae and Podoviridae families demonstrated a preference for either high-exopolysaccharide-producing (HEP) or low-exopolysaccharide-producing (LEP) bacterial hosts when grown on artificial medium without or with sugar supplementation. Myoviridae phages produced clear plaques on LEP hosts and turbid plaques on HEP hosts. The reverse preference was demonstrated by most Podoviridae phages, where clear plaques were seen on HEP hosts. Efficiency of plating (EOP) was determined by comparing phage growth on the original isolation host to the that on the LEP or HEP host. Nine of 10 Myoviridae phages showed highest EOPs on LEP hosts, and 8 of 11 Podoviridae phages had highest EOPs on HEP hosts. Increasing the production of EPS on sugar-supplemented medium or decreasing production by knocking out the synthesis of amylovoran or levan, the two EPSs produced by E. amylovora, indicated that these components play crucial roles in phage infection. Amylovoran was virtually essential for proliferation of most Podoviridae phages when phage population growth was compared to the wild type. Decreased levan production resulted in a significant reduction of progeny from phages in the Myoviridae family. Thus, Podoviridae phages are adapted to hosts that produce high levels of exopolysaccharides and are dependent on host-produced amylovoran for pathogenesis. Myoviridae phages are adapted to hosts that produce lower levels of exopolysaccharides and host-produced levan.

ECOPHAGE: Combating Antimicrobial Resistance Using Bacteriophages for Eco-Sustainable Agriculture and Food Systems.

The focus of this meeting was to discuss the suitability of using bacteriophages as alternative antimicrobials in the agrifood sector. Following a One Health approach, the workshop explored the possibilities of implementing phage application strategies in the agriculture, animal husbandry, aquaculture, and food production sectors. Therefore, the meeting had gathered phage researchers, representatives of the agrifood industry, and policymakers to debate the advantages and potential shortcomings of using bacteriophages as alternatives to traditional antimicrobials and chemical pesticides. Industry delegates showed the latest objectives and demands from consumers. Representatives of regulatory agencies (European Medicines Agency (EMA) and Spanish Agency of Medicines and Health Products (AEMPS)) presented an update of new regulatory aspects that will impact and support the approval and implementation of phage application strategies across the different sectors.

Population Dynamics between Erwinia amylovora, Pantoea agglomerans and Bacteriophages: Exploiting Synergy and Competition to Improve Phage Cocktail Efficacy.

Bacteriophages are viruses capable of recognizing with high specificity, propagating inside of, and destroying their bacterial hosts. The phage lytic life cycle makes phages attractive as tools to selectively kill pathogenic bacteria with minimal impact on the surrounding microbiome. To effectively harness the potential of phages in therapy, it is critical to understand the phage-host dynamics and how these interactions can change in complex populations. Our model examined the interactions between the plant pathogen Erwinia amylovora, the antagonistic epiphyte Pantoea agglomerans, and the bacteriophages that infect and kill both species. P. agglomerans strains are used as a phage carrier; their role is to deliver and propagate the bacteriophages on the plant surface prior to the arrival of the pathogen. Using liquid cultures, the populations of the pathogen, carrier, and phages were tracked over time with quantitative real-time PCR. The jumbo Myoviridae phage ϕEa35-70 synergized with both the Myoviridae ϕEa21-4 and Podoviridae ϕEa46-1-A1 and was most effective in combination at reducing E. amylovora growth over 24 h. Phage ϕEa35-70, however, also reduced the growth of P. agglomerans. Phage cocktails of ϕEa21-4, ϕEa46-1-A1, and ϕEa35-70 at multiplicities of infections (MOIs) of 10, 1, and 0.01, respectively, no longer inhibited growth of P. agglomerans. When this cocktail was grown with P. agglomerans for 8 h prior to pathogen introduction, pathogen growth was reduced by over four log units over 24 h. These findings present a novel approach to study complex phage-host dynamics that can be exploited to create more effective phage-based therapies.

Draft Genome Sequences of Hazelnut-Associated Pseudomonas Strains Isolated in Ontario, Canada.

Two Pseudomonas strains (H346-M and H346-S) were isolated from hazelnut trees showing symptoms of shoot dieback and necrosis. The draft genome sequences of H346-M and H346-S consist of 66 and 51 contigs, respectively, with total sizes of 5,693,988 and 5,889,925 bp and 4,885 and 5,045 protein-coding sequences, respectively.

Complete genome of the broad-host-range Erwinia amylovora phage phiEa21-4 and its relationship to Salmonella phage felix O1.

The first complete genome sequence for a myoviridal bacteriophage, PhiEa21-4, infecting Erwinia amylovora, Erwinia pyrifoliae, and Pantoea agglomerans strains has been determined. The unique sequence of this terminally redundant, circularly permuted genome is 84,576 bp. The PhiEa21-4 genome has a GC content of 43.8% and contains 117 putative protein-coding genes and 26 tRNA genes. PhiEa21-4 is the first phage in which a precisely conserved rho-independent terminator has been found dispersed throughout the genome, with 24 copies in all. Also notable in the PhiEa21-4 genome are the presence of tRNAs with six- and nine-base anticodon loops, the absence of a small packaging terminase subunit, and the presence of nadV, a principle component of the NAD(+) salvage pathway, which has been found in only a few phage genomes to date. PhiEa21-4 is the first reported Felix O1-like phage genome; 56% of the predicted PhiEa21-4 proteins share homology with those of the Salmonella phage. Apart from this similarity to Felix O1, the PhiEa21-4 genome appears to be substantially different, both globally and locally, from previously reported sequences. A total of 43 of the 117 genes are unique to PhiEa21-4, and 32 of the Felix O1-like genes do not appear in any phage genome sequences other than PhiEa21-4 and Felix O1. N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight analysis resulted in the identification of five PhiEa21-4 genes coding for virion structural proteins, including the major capsid protein.

Erwinia amylovora: modern methods for detection and differentiation.

Erwinia amylovora is the causative agent of fire blight, a very destructive disease of numerous members of the rosaceae. The primary route of infection for host species, including commercially grown apple and pear, is the newly opened blossom. Susceptibility of flowers to infection for only a few days creates narrow window for infection. Not surprisingly, the risk of disease is related to E. amylovora population size. As a result, methods that supply quick, accurate and sensitive quantification of the pathogen population are important tools for determining the need for and the efficacy of disease control intervention. Plating samples and assessing colony-forming units constitutes an accurate and sensitive but slow method. Endpoint PCR is quick and sensitive but is not particularly amenable to quantification. We describe a real-time PCR procedure that provides all requirements. This method is based on chromosomal genes rather than on the pEa29 plasmid and so can be used to measure isolates that have been cured of the plasmid. The method has been used very successfully in directly quantify whole E. amylovora cells, in a variety of tissues from the orchard environment.

Host Range of Bacteriophages Against a World-Wide Collection of Erwinia amylovora Determined Using a Quantitative PCR Assay.

Erwinia amylovora is a globally devastating pathogen of apple, pear, and other Rosaceous plants. The use of lytic bacteriophages for disease management continues to garner attention as a possible supplement or alternative to antibiotics. A quantitative productive host range was established for 10 Erwinia phages using 106 wild type global isolates of E.amylovora, and the closely related Erwiniapyrifoliae, to investigate the potential regional efficacy of these phages within a biopesticide. Each host was individually infected with each of the 10 Erwinia phages and phage production after 8 h incubation was measured using quantitative real time PCR (qPCR) in conjunction with a standardized plasmid. PCR amplicons for all phages used in the study were incorporated into a single plasmid, allowing standardized quantification of the phage genome copy number after the infection process. Nine of the tested phages exhibited a broad host range, replicating their genomes by at least one log in over 88% of tested hosts. Also, every Amygdaloideae infecting E. amylovora host was able to increase at least one phage by three logs. Bacterial hosts isolated in western North America were less susceptible to most phages, as the mean genomic titre produced dropped by nearly two logs, and this phenomenon was strongly correlated to the amount of exopolysaccharide produced by the host. This method of host range analysis is faster and requires less effort than traditional plaque assay techniques, and the resulting quantitative data highlight subtle differences in phage host preference not observable with typical plaque-based host range assays. These quantitative host range data will be useful to determine which phages should be incorporated into a phage-mediated biocontrol formulation to be tested for regional and universal control of E. amylovora.

Duplex real-time polymerase chain reaction reveals competition between Erwinia amylovora and E. pyrifoliae on pear blossoms.

Erwinia amylovora and E. pyrifoliae are the causative agents of fire blight and Asian pear blight, respectively. The pathogens are closely related, with overlapping host ranges. Data are unavailable on the current distribution of E. pyrifoliae and on the interaction between the two species when they are present together on the same host. In this study, a duplex real-time polymerase chain reaction (PCR) protocol was developed to monitor the population dynamics of E. amylovora and E. pyrifoliae on the surface of Bartlett pear blossoms. Bacterial cells washed from blossoms were used directly as the PCR template without DNA extraction. Primers and a probe based on the E. amylovora levansucrase gene detected all E. amylovora strains. All E. pyrifoliae strains, including the Japanese Erwinia strains previously described as E. amylovora, were detected with a primer and probe combination based on the E. pyrifoliae hrpW gene. Disease development and severity were not significantly different in blossoms inoculated with individual Erwinia species or with a mixture of the two species. However, E. amylovora grew to greater population sizes than did E. pyrifoliae in both single species inoculations and in mixtures, suggesting that E. amylovora has a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae.

Framing the Future with Bacteriophages in Agriculture.

The ability of agriculture to continually provide food to a growing world population is of crucial importance. Bacterial diseases of plants and animals have continually reduced production since the advent of crop cultivation and animal husbandry practices. Antibiotics have been used extensively to mitigate these losses. The rise of antimicrobial resistant (AMR) bacteria, however, together with consumers’ calls for antibiotic-free products, presents problems that threaten sustainable agriculture. Bacteriophages (phages) are proposed as bacterial population control alternatives to antibiotics. Their unique properties make them highly promising but challenging antimicrobials. The use of phages in agriculture also presents a number of unique challenges. This mini-review summarizes recent development and perspectives of phages used as antimicrobial agents in plant and animal agriculture at the farm level. The main pathogens and their adjoining phage therapies are discussed.

Absence of lysogeny in wild populations of Erwinia amylovora and Pantoea agglomerans.

Lytic bacteriophages are in development as biological control agents for the prevention of fire blight disease caused by Erwinia amylovora. Temperate phages should be excluded as biologicals since lysogeny produces the dual risks of host resistance to phage attack and the transduction of virulence determinants between bacteria. The extent of lysogeny was estimated in wild populations of E. amylovora and Pantoea agglomerans with real-time polymerase chain reaction primers developed to detect E. amylovora phages belonging to the Myoviridae and Podoviridae families. Pantoea agglomerans, an orchard epiphyte, is easily infected by Erwinia spp. phages, and it serves as a carrier in the development of the phage-mediated biological control agent. Screening of 161 E. amylovora isolates from 16 distinct geographical areas in North America, Europe, North Africa and New Zealand and 82 P. agglomerans isolates from southern Ontario, Canada showed that none possessed prophage. Unstable phage resistant clones or lysogens were produced under laboratory conditions. Additionally, a stable lysogen was recovered from infection of bacterial isolate Ea110R with Podoviridae phage ΦEa35-20. These laboratory observations suggested that while lysogeny is possible in E. amylovora, it is rare or absent in natural populations, and there is a minimal risk associated with lysogenic conversion and transduction by Erwinia spp. phages.

Complete Genome Sequence of Erwinia amylovora Bacteriophage vB_EamM_Ea35-70.

The complete genome of an Erwinia amylovora bacteriophage, vB_EamM_Ea35-70 (Ea35-70), is 271,084 bp, encodes 318 putative proteins, and contains one tRNA. Comparative analysis with other Myoviridae genomes suggests that Ea35-70 is related to the Phikzlikevirus genus within the family Myoviridae, since 26% of Ea35-70 proteins share homology to proteins in Pseudomonas phage φKZ.

Silencing of the host factor eIF(iso)4E gene confers plum pox virus resistance in plum.

Plum pox virus (PPV) causes the most economically-devastating viral disease in Prunus species. Unfortunately, few natural resistance genes are available for the control of PPV. Recessive resistance to some potyviruses is associated with mutations of eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. In this study, we used an RNA silencing approach to manipulate the expression of eIF4E and eIF(iso)4E towards the development of PPV resistance in Prunus species. The eIF4E and eIF(iso)4E genes were cloned from plum (Prunus domestica L.). The sequence identity between plum eIF4E and eIF(iso)4E coding sequences is 60.4% at the nucleotide level and 52.1% at the amino acid level. Quantitative real-time RT-PCR analysis showed that these two genes have a similar expression pattern in different tissues. Transgenes allowing the production of hairpin RNAs of plum eIF4E or eIF(iso)4E were introduced into plum via Agrobacterium-mediated transformation. Gene expression analysis confirmed specific reduced expression of eIF4E or eIF(iso)4E in the transgenic lines and this was associated with the accumulation of siRNAs. Transgenic plants were challenged with PPV-D strain and resistance was evaluated by measuring the concentration of viral RNA. Eighty-two percent of the eIF(iso)4E silenced transgenic plants were resistant to PPV, while eIF4E silenced transgenic plants did not show PPV resistance. Physical interaction between PPV-VPg and plum eIF(iso)4E was confirmed. In contrast, no PPV-VPg/eIF4E interaction was observed. These results indicate that eIF(iso)4E is involved in PPV infection in plum, and that silencing of eIF(iso)4E expression can lead to PPV resistance in Prunus species.