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The first complete genome sequence characterization for a myoviridal bacteriophage, [Phi]Ea21-4, infecting Erwinia amylovora, Erwinia pyrifoliae, and Pantoea agglomerans strains is described. The findings identified five [Phi]Ea21-4 genes coding for virion structural proteins, including the major capsid protein.

Lytic bacteriophages are in development as biological control agents for the prevention of fire blight disease caused by E rwinia 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 P antoea agglomerans with real-time polymerase chain reaction primers developed to detect E . amylovora phages belonging to the M yoviridae and P odoviridae families. P antoea agglomerans, an orchard epiphyte, is easily infected by E rwinia 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 P odoviridae 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 E rwinia spp. phages. [ABSTRACT FROM AUTHOR]

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 Erwinia pyrifoliae, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.