Newly Isolated Bacteriophages from the Podoviridae, Siphoviridae, and Myoviridae Families Have Variable Effects on Putative Novel Dickeya spp

Abstract

Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This study reports the isolation of three bacteriophages active against putative novel Dickeya spp. isolates from commercially produced infected orchids that show variable host-range profiles. Bacteriophages were isolated through enrichment from Dickeya-infected orchid tissue. Convective interaction media monolith chromatography was used to isolate bacteriophages from wastewaters, demonstrating its suitability for the isolation of infective bacteriophages from natural sources. Based on bacteriophage morphology, all isolated bacteriophages were classified as being in the order Caudovirales, belonging to three different families, Podoviridae, Myoviridae, and Siphoviridae. The presence of three different groups of bacteriophages was confirmed by analyzing the bacteriophage specificity of bacterial hosts, restriction fragment length polymorphism and plaque morphology. Bacteriophage BF25/12, the first reported Podoviridae bacteriophage effective against Dickeya spp., was selected for further characterization. Its genome sequence determined by next-generation sequencing showed limited similarity to other characterized Podoviridae bacteriophages. Interactions among the bacteriophages and Dickeya spp. were examined using transmission electron microscopy, which revealed degradation of electron-dense granules in response to bacteriophage infection in some Dickeya strains. The temperature stability of the chosen Podoviridae bacteriophage monitored over 1 year showed a substantial decrease in the survival of bacteriophages stored at -20°C over longer periods. It showed susceptibility to low pH and UV radiation but was stable in neutral and alkaline pH. Furthermore, the stability of the tested bacteriophage was also connected to the incubation medium and bacteriophage concentration at certain pH values. Finally, the emergence of bacteriophage-resistant bacterial colonies is highly connected to the concentration of bacteriophages in the bacterial environment. This is the first report on bacteriophages against Dickeya from the Podoviridae family to expand on potential bacteriophages to include in bacteriophage cocktails as biocontrol agents. Some of these bacteriophage isolates also showed activity against Dickeya solani, an aggressive strain that causes the soft rot of potatoes, which indicates their broad potential as biocontrol agents.

Keywords: Dickeya; Podoviridae; bacteriophages; convective interaction media monolith chromatography; genome sequencing; resistance development.

FIGURE 1

Representative bacteriophage plaque morphologies and restriction fragment length polymorphism profiles. Plaque morphologies for bacteriophages isolated from plant material (A) and wastewater (B,C). Bacteriophages against Dickeya spp. isolated from wastewater (D) and from diseased Phalaenopsis (E) digested with Hind II restriction enzyme. M, Gene Rule 1 kb DNA ladder.

FIGURE 2

Representative transmission electron microscopy micrographs of bacteriophages isolated from diseased Phalaenopsis leaves (A) and sewage water (B,C), all of which were only active against Dickeya spp.

FIGURE 3

Representative ultrastructure analyses of bacteria–bacteriophage interactions. (A,B) Strain UDL-4 before (A) and after (B) mixing with bacteriophage BF25/12 (arrows). (C,D) Strain UDL-3 before (C) and after (D) mixing with bacteriophage BF25/12 (arrows). (E,F) Lysed UDL-3 bacteria from the border of a plaque (E) and new bacteriophages in the cytoplasm (arrowheads) (F). G, intracellular granules; CW, cell wall; PL, plasmalemma; PS, periplasmic space.

FIGURE 4

Annotated BF25/12 bacteriophage genome. According to the annotation, the genome does not contain any genes encoding antibiotic resistance or toxins. The open reading frames coding for structural proteins, proteins involved in bacteriophage replication, and other conserved proteins are shown in yellow, and those coding for hypothetical proteins are shown in gray. Unit of the presented genome annotation is in bp.

FIGURE 5

Temperature stability with time of Dickeya spp. bacteriophage BF25/12 incubated at temperatures of +4, +28, –20 and –80°C (as indicated).

FIGURE 6

Effect of pH on stability of bacteriophage BF25/12. Stability was tested in SMG buffer and sterile demineralised water at two different bacteriophage concentrations, 10⁵ and 10³ pfu/mL. Concentration of bacteriophages presented in the graph was calculated to the level of bacteriophage stock, used for preparation of testing dilutions.

FIGURE 7

Emergence of Dickeya spp. B16 resistant to bacteriophage BF 25/12. Experiment was performed as two technical repeats, shown on graph as squares and diamonds.