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.

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.

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.

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.

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.