Isolation and characterization of novel phages for control of the phytopathogen Pseudomonas marginalis

Abstract

The current study provides the first detailed characterization of two novel bacteriophages, DG23 and RG24, that infect Pseudomonas marginalis, a causative agent of soft rot in potato and other vegetable crops. The phages were assessed for environmental stability, genetic characteristics, and biocontrol efficacy. Both DG23 and RG24 showed broad tolerance throughout a wide pH range (3-9), with RG24 still viable at pH 11, while DG23 was more sensitive to extreme pH conditions. Thermal stability assay demonstrated that both phages remained infectious up to 45 °C, but activity decreased dramatically at higher temperatures, with total inactivation at 75 °C. Phage viability reduced considerably under UV irradiation (254 nm), with DG23 demonstrating better resistance than RG24. Whole-genome sequencing revealed that both phages are lytic, with no integrase, pathogenicity, or antibiotic resistance genes, ensuring biosafety for prospective agricultural uses. Comparative genomic analysis indicated a 99% average nucleotide identity (ANI) between DG23 and RG24, showing they are the same species, but both were genetically distinct from their nearest relative, Pseudomonas phage XD2 (ANI 92%). In addition, comparative proteomic and phylogenetic analyses revealed that DG23 and RG24 form a distinct clade within the class Caudoviricetes, separate from other related phages. Biocontrol experiments showed that both phages efficiently inhibited potato soft rot when used individually, but when combined, disease severity was decreased by more than 80%, demonstrating the higher efficiency of phage cocktails. These data suggest that DG23 and RG24 are promising, safe, and effective candidates for phage-based biocontrol of soft rot caused by P. marginalis. KEY POINTS: • Novel phages DG23 and RG24 lyse Pseudomonas marginalis and lack virulence genes. • Phages show stability under broad pH, temperature, and UV conditions. • Cocktail treatment reduces potato soft rot severity by more than 80%.

Keywords: Pseudomonas marginalis; Biocontrol; Phage therapy; Soft rot disease.

Fig. 1

TEM micrographs of Pseudomonas phages DG23 and RG24 negatively stained with 2% (w/v) phosphotungstic acid (pH 7.2). A Phage DG23 exhibits a hexagonal head (72.6 × 71.46 nm) and a long tail of approximately 148 nm. B Phage RG24 displays a larger hexagonal head (128 × 94) and a remarkably long tail of approximately 132 nm. Images were obtained at a direct magnification of 60,000x

Fig. 2

Environmental stability of Pseudomonas phages DG23 and RG24. A Stability under different pH conditions (2–12) for 1 h. B Thermal stability across a range of temperatures (25–75 °C) for 1 h. C Stability under UV irradiation at 254 nm over time intervals (0–60 min). Data represent the mean of three independent replicates. Error bars indicate the standard deviation. **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 3

The genome completeness metrics of sequenced phages. A Pseudomonas phage DG23 B Pseudomonas phage RG24

Fig. 4

Genome representation of Pseudomonas phages DG23 (A) and RG24 (B). ORFs are shown as colored arrows indicating transcriptional direction, with functional categories represented by distinct colors: lysis (red), replication (green), regulation (yellow), assembly (cyan), immune (purple), hypothetical proteins (light blue), and others as indicated in the legend. The inner rings display GC content (blue) and GC skew (green for positive, purple for negative), highlighting shifts that correspond to replication origins and termini. Gene annotations are labeled around the outer circle, with structural, replication, and lysis modules

Fig. 5

The distribution of predicted gene functions in the genomes of Pseudomonas phages A DG23 and B RG24, as determined by automated annotation pipelines. Functional categories are represented by distinct colors and arranged vertically for clarity

Fig. 6

Comparative genomic analysis of isolated Pseudomonas phages. A Heatmap showing the ANI values among the isolated phages (DG23 and RG24 marked with asterisks) and other Pseudomonas genomes. Color intensity represents the degree of genomic similarity, with darker blue shades indicating higher ANI values (up to 100%) and lighter blue shades indicating lower similarity (down to 40%). B Whole-genome alignment of Pseudomonas phages DG23, RG24, and XD2 (GenBank accession no. PQ288046). Genomes are represented as linear maps, with predicted protein-coding genes shown as colored arrows. Gene colors indicate homologous gene clusters shared among genomes. Ribbons connect homologous regions, with ribbon color intensity representing pairwise nucleotide identity according to the scale shown (range: 92–100%). Crossed ribbons indicate genomic inversions. Genome sizes are indicated by the scale bars (10 kb)

Fig. 7

Proteomic analysis of the isolated Pseudomonas phages. A Phylogenetic tree based on terminase gene sequence comparisons among the Pseudomonas phages DG23 and RG24 and other closely related phages. Bootstrap values exceeding 75% (based on 1000 replicates) are represented as bubbles on the tree. B Global proteomic tree showing the placement of DG23 and RG24 (red-highlighted branch) among members of the class Caudoviricetes. The red star indicates the clade containing DG23 and RG24

Fig. 8

Effect of phages’ treatments on the occurrence of soft rot disease on potato slices inoculated with P. marginalis strain LPm33