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

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%.