Characteristics and whole-genome analysis of a novel Pseudomonas syringae pv. tomato bacteriophage D6 isolated from a karst cave

Abstract

Pseudomonas syringae is a gram-negative plant pathogen that infects plants such as tomato and poses a threat to global crop production. In this study, a novel lytic phage infecting P. syringae pv. tomato DC3000, named phage D6, was isolated and characterized from sediments in a karst cave. The latent period of phage D6 was found to be 60 min, with a burst size of 16 plaque-forming units per cell. Phage D6 was stable at temperatures between 4 and 40 °C but lost infectivity when heated to 70 °C. Its infectivity was unaffected at pH 6-10 but became inactivated at pH ≤ 5 or ≥ 12. The genome of phage D6 is a linear double-stranded DNA of 307,402 bp with a G + C content of 48.43%. There is a codon preference between phage D6 and its host, and the translation of phage D6 gene may not be entirely dependent on the tRNA library provided by the host. A total of 410 open reading frames (ORFs) and 14 tRNAs were predicted in its genome, with 92 ORFs encoding proteins with predicted functions. Phage D6 showed low genomic similarity to known phage genomes in the GenBank and Viral sequence databases. Genomic and phylogenetic analyses revealed that phage D6 is a novel phage. The tomato plants were first injected with phage D6, and subsequently with Pst DC3000, using the foliar spraying and root drenching inoculum approach. Results obtained after 14 days indicated that phage D6 inoculation decreased P. syringae-induced symptoms in tomato leaves and inhibited the pathogen's growth in the leaves. The amount of Pst DC3000 was reduced by 150- and 263-fold, respectively. In conclusion, the lytic phage D6 identified in this study belongs to a novel phage within the Caudoviricetes class and has potential for use in biological control of plant diseases.

Keywords: Caudoviricetes; Pseudomonas syringae; Bacteriophage; Biocontrol; Karst cave; Plant pathogen; Whole genome analysis.

Fig. 1

Morphology of phage D6. a Phage D6 plaques. Bar = 5 mm. b Morphology of phage D6 as revealed by transmission electron microscopy. Bar = 100 nm

Fig. 2

The optimal MOI (a) and one step growth curve (b) of phage D6. Data are presented as the average of triplicate experiments, and the error bars indicate the standard deviation

Fig. 3

Thermal (a), pH (b), chloroform (c), and UV (d) stability of phage D6. Data are presented as the average of triplicate experiments, and the error bars indicate the standard deviation. *: significant difference (p < 0.05)

Fig. 4

Adsorption rate (a) and bacteriostasis effect in vitro (b) of phage D6. Values are means from three independent experiments. Error bars indicate standard deviation

Fig. 5

Genome map of phage D6. The genome of phage D6 was depicted in circular. The first and second circles from the outside to the inside are the ORFs on the positive and negative strands. The different colors represent different gene functions. The third circle represents the GC-Skew value. The fourth circle indicates the GC content

Fig. 6

Neighbor-joining phylogenetic tree was constructed based on terminase large subunit. The bootstrap values are shown at the nodes. The red star represents phage D6 isolated in this study. The reference sequences were collected from the NCBI database. The tree was constructed based on the MUSCLE alignment of MEGA 7.0. The bootstrap values were based on 1000 replicates

Fig. 7

Pairwise comparisons of the whole genomes of Pseudomonas phage D6 and phage Psa21 was visualized using Easyfig. The arrows are the encoded proteins and the gray line connecting the two represents the similarity between the two, with darker shades of gray representing higher similarity

Fig. 8

Comparative proteomic phylogenetic analysis of phage D6 and similar phages performed using VipTree. The red star represents Pseudomonas phage D6 isolated in this study. The phylogenetic branches marked in red are represented as phage sequence not found in the Virus-Host Database. Additional genomes were downloaded from the NCBI GenBank database and manually added to the analysis. Left line represents virus family, right line represents host group

Fig. 9

Heatmap of genome sequence similarity (right) and alignment metrics (left) between phage D6 and its eight most closely related phages generated by VIRIDIC. The red number represents phage D6 isolated in this study

Fig. 10

Biocontrol evaluation of phage D6 against Pst DC3000 in planta. After 24 h of inoculation with 10 mL of phage D6 (2.3 × 10⁶ PFU/mL) by the FS method (a) and RD method (b), tomato plants were infected with 10 mL of Pst DC3000 culutre (Pst + D6 group). The leaf symptoms were observed. The tomato plants were treated with phage D6, Pst DC3000 (OD₆₀₀ = 0.2), or magnesium chloride solution (10 mM) (CK group) were used as controls. (c) After 14 d of treatment, the top, middle, and bottom leaves of the plants were collected to detect the amounts of Pst DC3000 bacteria. The experiments were performed in triplicates and the data are shown as the mean ± SD. *: significant difference (p < 0.05)