Antonet M. Svircev

Bacteriophages of Erwinia amylovora

ABSTRACT Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and Podoviridae.

Complete Genome of the Broad-Host-Range Erwinia amylovora Phage ΦEa21-4 and Its Relationship to Salmonella Phage Felix O1

ABSTRACT The first complete genome sequence for a myoviridal bacteriophage, ΦEa21-4, 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 ΦEa21-4 genome has a GC content of 43.8% and contains 117 putative protein-coding genes and 26 tRNA genes. ΦEa21-4 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 ΦEa21-4 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. ΦEa21-4 is the first reported Felix O1-like phage genome; 56% of the predicted ΦEa21-4 proteins share homology with those of the Salmonella phage. Apart from this similarity to Felix O1, the ΦEa21-4 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 ΦEa21-4, and 32 of the Felix O1-like genes do not appear in any phage genome sequences other than ΦEa21-4 and Felix O1. N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight analysis resulted in the identification of five ΦEa21-4 genes coding for virion structural proteins, including the major capsid protein.

Differential regulation of four members of the ACC synthase gene family in plum

The regulation of ACC synthase (ACS) genes was studied in early (‘Early Golden’) and late (‘Shiro’) Japanese plum cultivars (Prunus salicina L.) in order to determine the role of this gene family in fruit ripening. Of the four Ps-ACS cDNAs isolated, two (Ps-ACS1 and -3) showed differential expression between the two cultivars. Ps-ACS1 accumulated during fruit ripening of ‘Early Golden’ (‘EG’) and ‘Shiro’ (‘SH’) in ethylene-dependent and -independent manners, respectively. Ps-ACS3a transcripts accumulated throughout fruit development and during ‘EG’ fruit ripening. Ps-ACS3b was detected only during ripening of ‘SH’ fruit. Furthermore, Ps-ACS3a transcript accumulation was negatively regulated by ethylene, whereas Ps-ACS3b was positively induced by the hormone. In both cultivars, the expression of Ps-ACS4 and -5 is under positive and negative feedback control by ethylene, respectively. Genetic analyses of ‘EG’ and ‘SH’ cultivars demonstrated that ‘EG’ is homozygous for Ps-ACS3a whereas ‘SH’ is heterozygous for Ps-ACS3 (a/b). The role of ethylene-overproducer 1-like in delaying fruit ripening by interacting with Ps-ACS proteins was also studied. The effect of the plant hormones, auxin, gibberellin, and cytokinin, in regulating ethylene production by promoting the induction of the different Ps-ACS mRNAs in plum was investigated. A model is presented in which differences in Ps-ACS alleles and gene expression between early and late plums are critical in determining the ripening behaviour of the cultivars.

Bacteriophages and the control of Erwinia carotovora subsp. carotovora

Fourteen bacteriophages of Erwinia carotovora subsp. carotovora, the causal agent of bacterial soft rot, were isolated from samples of fertilizer solutions taken from a greenhouse producing calla lily (Zantedeschia spp.). To avoid a single-host selection bias, a mixture of four bacterial hosts was used to enrich bacteriophages. Molecular characterization of the phages with restriction endonuclease digestions indicated that two distinct types had been isolated. Representatives from both molecular groups were studied by electron microscopy to determine their morphology. The phages possessed icosahedral heads with long, flexible tails. On the basis of morphotypes, the bacteriophages of E. carotovora subsp. carotovora were placed in the order Caudovirales. Limited host-range studies revealed that both phage groups would lyse only E. carotovora subsp. carotovora isolates taken from calla tubers infected with bacterial soft rot. When assayed for persistence in fertilizer solutions for calla lilies, phage titres were found to be stable in the solutions made with sterile reverse-osmosis water, but not with nonsterile tap water. Phages did not persist in preplant-treatment solutions for calla tubers. Biological control of E. carotovora subsp. carotovora by the bacteriophages was assessed in fertilizer solutions, on the surface of plugs of calla tuber tissue, and under greenhouse conditions. Phage isolates were able to significantly reduce bacterial populations in fertilizer solutions and on the surface of plugs of calla tuber tissue. The biocontrol ability of the bacteriophages was inhibited by the presence of Fe - ethylenediaminetetraacetic acid in fertilizer solutions. On plugs of tuber tissue and in greenhouse trials, phages were able to reduce the incidence of soft-rot symptoms by 50% or more.

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.

Isolation and characterization of eight bacteriophages infecting Erwinia amylovora and their potential as biological control agents in British Columbia, Canada

Abstract Nineteen active bacteriophages against Erwinia amylovora, the causal agent of fire blight, were collected from apple and pear orchards in the Okanagan and Fraser Valleys of British Columbia. Eight survived the isolation, purification and storage processes. Five bacteriophage isolates included in this study lysed more than 50% of the 20 E. amylovora strains tested from BC. Examination by transmission electron microscopy revealed that all eight phages belong to the order Caudovirales, the tailed phages, and included members of the families Myoviridae and Podoviridae. Bacteriophages were characterized by digestion of the phage DNA with four restriction endonucleases and two sets of PCR primers. Two novel groups, RFLP groups 7 and 8, were identified based on differences in restriction fragment patterns. Phages ΦEa1337-26 and ΦEa2345-6 reduced infection by 84% and 96%, respectively, when tested on detached pear blossoms using the epiphyte bacterium Pantoea agglomerans Eh21-5 as a carrier. In addition, bacteriophage ΦEa2345-6, applied in combination with Eh21-5, reduced infection of fire blight on apple flowers of potted apple trees by 56% and compared well with the antibiotic streptomycin.

Identification and genetic characterization of a gibberellin 2-oxidase gene that controls tree stature and reproductive growth in plum

Several dwarf plum genotypes (Prunus salicina L.), due to deficiency of unknown gibberellin (GA) signalling, were identified. A cDNA encoding GA 2-oxidase (PslGA2ox), the major gibberellin catabolic enzyme in plants, was cloned and used to screen the GA-deficient hybrids. This resulted in the identification of a dwarf plum hybrid, designated as DGO24, that exhibits a markedly elevated PslGA2ox signal. Grafting ‘Early Golden’ (EG), a commercial plum cultivar, on DGO24 (EG/D) enhanced PslGA2ox accumulation in the scion part and generated trees of compact stature. Assessment of active GAs in such trees revealed that DGO24 and EG/D accumulated relatively much lower quantities of main bioactive GAs (GA1 and GA4) than control trees (EG/M). Moreover, the physiological function of PslGA2ox was studied by determining the molecular and developmental consequences due to ectopic expression in Arabidopsis. Among several lines, two groups of homozygous transgenics that exhibited contrasting phenotypes were identified. Group-1 displayed a dwarf growth pattern typical of mutants with a GA deficiency including smaller leaves, shorter stems, and delay in the development of reproductive events. In contrast, Group-2 exhibited a ‘GA overdose’ phenotype as all the plants showed elongated growth, a typical response to GA application, even under limited GA conditions, potentially due to co-suppression of closely related Arabidopsis homologous. The studies reveal the possibility of utilizing PslGA2ox as a marker for developing size-controlling rootstocks in Prunus.

Plum pox virus in Canada : progress in research and future prospects for disease control

Plum pox virus (PPV), one of the potyviruses originally from Europe and recently found in Canada, is the causative agent of sharka in Prunoideae. The virus infects many types of ornamental as well as stone-fruit Prunus spp., such as peach, nectarine, plum, cherry, and apricot, resulting in extensive economic losses. Immediately after it was found in Canada, a PPV eradication, grower compensation, and research program funded by the Canadian federal and provincial governments was implemented. This review summarizes general information about PPV, including the physical and molecular features of the viral pathogen, symptoms of the disease, geographical distribution, and current control strategy, and highlights research progress and future prospects for control of the disease in Canada.

Host Exopolysaccharide Quantity and Composition Impact Erwinia amylovora Bacteriophage Pathogenesis

ABSTRACT 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.

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”.