Bacteriophage Cocktail Comprising Fifi044 and Fifi318 for Biocontrol of Erwinia amylovora

Abstract

Erwinia amylovora is a plant pathogen that causes fire blight on apples and pears. Bacteriophages, which are viruses that selectively infect specific species of bacteria and are harmless to animal cells, have been considered as biological control agents for the prevention of bacterial pathogens. In this study, we aimed to use bacteriophages that infect E. amylovora as biocontrol agents against fire blight. We isolated bacteriophages Fifi044 and Fifi318 infecting E. amylovora, and characterized their morphology, plaque form, and genetic diversity to use as cocktails for disease control. The stabilities of the two phages were investigated at various temperatures and pH values and under sunlight, and long-term storage experiment was conducted for a year. To evaluate whether the two phages were suitable for use in cocktail form, growth curves of E. amylovora were prepared after treating the bacterial cells with single phages and a phage cocktail. In addition, a disease control test was conducted using immature apples and in vitro cultured apple plantlets to determine the biocontrol effects of the phage cocktail. The two phages were morphologically and genetically different, and highly stable up to 50°C and pH value from 4 to 10. The phages showed synergistic effect when used as a cocktail in the inhibition of host bacterial growth and the disease control. This study demonstrated that the potential of the phage cocktail as a biocontrol agent for commercial use.

Keywords: bacteriophage; biological control agent; fire blight disease; phage cocktail.

Fig. 1

Transmission electron micrographs of Erwinia phages Fifi044 (A) and Fifi318 (B). The microscope was operated at 80-kV. Scale bars = 100 nm.

Fig. 2

Whole-genome based phylogenetic analysis of 41 Myoviridae bacteriophages infecting Erwinia amylovora. The genome sequences of 39 phages were obtained from GenBank database, and their names and accession numbers were stated in the figure. The tree was generated using VICTOR software for phages (prokaryotic viruses). The numbers above the branches represent Genome-BLAST Distance Phylogeny (GBDP) pseudo-bootstrap support values based on the conduction of replications. The origin countries of phages were indicated using different colors (United States, navy; Canada, green; Switzerland, yellow; South Korea, red; Belarus, gray; Latvia, black).

Fig. 3

Stability of Fifi044 and Fifi318 under 30–60°C (A, B), pH4–11 (C, D), and sun light condition (E, F). Different letters (a–b) on the bars indicate that the mean values differ significantly by Kruskal-Wallis test with Bonferroni-Dunn post hoc test at P < 0.05.

Fig. 4

Long-term storage tests of Fifi044 (A) and Fifi318 (B) at different temperatures. Colors ranged from black to white depending on the time when residual phages were measured. Different letters (a–g) on the bars indicate that the mean values differ significantly by Kruskal-Wallis test with Bonferroni-Dunn post hoc test at P < 0.05.

Fig. 5

Lytic activity of two bacteriophages and phage cocktail against Erwinia amylovora YKB14808. The growth curves of E. amylovora mixed with phages in different multiplicities of infection (MOI) were generated. MOI = 0.01 (A), MOI = 1 (B), MOI = 100 (C). Gray circles represent Fifi044, light gray squares represent Fifi318, and white circles represent phage cocktail. E. amylovora cultured without phages (black circle) were used as a control. Each experiment was performed three times with three technical replications. The error bars represent standard deviations.