Biological characteristics of the bacteriophage LDT325 and its potential application against the plant pathogen Pseudomonas syringae

Abstract

Bud blight disease caused by Pseudomonas syringae is a major bacterial disease of tea plants in China. Concerns regarding the emergence of bacterial resistance to conventional copper controls have indicated the need to devise new methods of disease biocontrol. Phage-based biocontrol may be a sustainable approach to combat bacterial pathogens. In this study, a P. syringae phage was isolated from soil samples. Based on morphological characteristics, bacteriophage vB_PsS_LDT325 belongs to the Siphoviridae family; it has an icosahedral head with a diameter of 53 ± 1 nm and nonretractable tails measuring 110 ± 1 nm. The latent period and burst size of the phage were 10 min and 17 plaque-forming units (PFU)/cell, respectively. Furthermore, an analysis of the biological traits showed that the optimal multiplicity of infection (MOI) of the phage was 0.01. When the temperature exceeded 60°C, the phage titer began to decrease. The phage exhibited tolerance to a wide range of pH (3-11) and maintained relatively stable pH tolerance. It showed a high tolerance to chloroform, but was sensitive to ultraviolet (UV) light. The effects of phage LDT325 in treating P. syringae infections in vivo were evaluated using a tea plant. Plants were inoculated with 2 × 10⁷ colony-forming units (CFU)/mL P. syringae using the needle-prick method and air-dried. Subsequently, plants were inoculated with 2 × 10⁷ PFU/mL LDT325 phage. Compared with control plants, the bacterial count was reduced by 1 log10/0.5 g after 4 days in potted tea plants inoculated with the phage. These results underscore the phage as a potential antibacterial agent for controlling P. syringae.

Keywords: Pseudomonas syringae; bacterial disease; bacteriophage; biocontrol; tea bud blight.

Figure 1

Isolation and purification of the Pseudomonas syringae phage vB_PsS_LDT325. (A) Sample filtrate displays the clear lysis area on the double-layer plate overlays with suspensions of Pseudomonas syringae after incubation for 12 h, depicting lysed bacteria. (B) The phage vB_PsS_LDT325 crude filtrate. After 12 h of incubation, clear and different sized plaques were displayed on the Pseudomonas syringae lawn. (C) The purified phage vB_PsS_LDT325 displays clear and uniform patched plaques on a Pseudomonas syringae lawn after incubation for 12 h.

Figure 2

Micrograph and categorization of the phage vB_PsS_LDT325. (A,B) The transmission electron micrograph of the phage vB_PsS_LDT325 virus particles was negatively stained with 2% uranyl acetate. The scale bar represents 100 nm.

Figure 3

Biological characteristics of the phage vB_PsS_LDT325. (A) MOI of the phage vB_PsS_LDT325. Phage titers were measured at different MOIs. (B) One-step growth curve of the phage vB_PsS_LDT325. (C) The pH stability of the phage vB_PsS_LDT325. The phages were incubated in different acid–base environments for 1 h. (D) The thermal stability of the phage vB_PsS_LDT325. The phages were cultured in a water bath at different temperatures. (A–D) The phage titers were determined using the double-layer agar method and each data point represented the mean values ± standard deviations (SD) from at least three replicate experiments.

Figure 4

Determination of chloroform and ultraviolet sensitivity of phage vB_PsS_LDT325 (A) Chloroform stability: Phage vB_PsS_LDT325 was treated with chloroform (20%, vol/vol) for 12 h; (B) Ultraviolet light stability: Phage vB_PsS_LDT325 was exposed to UV light for 0, 1, 2, 3, 6, 9, 12, 15, 18 and 21 min. The data are represented as a mean of three replicates ± SD.

Figure 5

The biological control experiment of bud blight disease (A) Bacteriostasis experiment of phage in vitro. Asterisks signs indicate a statistically significant difference (p < 0.05), in which “*” (**p < 0.01) is compared with the phage-treated group, (*** p < 0.001) compared with the LB liquid medium group). (B) Bacteriostasis experiment of phage in vivo. Asterisks signs indicate a statistically significant difference (p < 0.05), in which “*” (*p < 0.05), is compared with the phage-treated group. (C) Change of lesion length on the leaves of living tea plant after inoculation with phage. Asterisks signs indicate a statistically significant difference (p < 0.05), in which “*” (**** p < 0.0001), is compared with the phage-treated and sterile water group. The data are represented as a mean of three replicates ± SD.

Figure 6

Pathological section (A) Histopathological images of plants in sterile water group. The red arrow indicates the nuclear region (HE,100×). (B) Histopathological images of the control group. The red arrow indicates the nuclear lesion area (HE,100×). (C) Histopathological images of phage treatment group. The red arrow indicates the nuclear region (HE,100×).