Isolation of a complementary bacteriophage from a phage-resistant mutant expands control strategies against...

Bacterial resistance to bacteriophages represents a major limitation for the durable use of phage-based biocontrol strategies in agriculture. Here, we used a phage-resistance derivative of Xanthomonas campestris pv. campestris (Xcc) as a selective host to isolate an additional phage with activity against resistant bacteria. A spontaneous mutant, designated Xcc 8004R, was isolated following exposure of Xcc 8004 to phage X1. Xcc 8004R exhibited a growth profile comparable to that of the wild-type strain, but differed in colony appearance, X1 adsorption efficiency, and motility phenotypes. Whole-genome resequencing identified mutations in genes annotated as encoding a lipopolysaccharide (LPS) core biosynthesis protein, PilY1, N-acetylmuramoyl-L-alanine amidase, and IS1404 transposase, and a conserved hypothetical protein. Using Xcc 8004R as the isolation host, we recovered a bacteriophage, vB_Xcc_GYRb1 (GYRb1), from agricultural soil. GYRb1 infected both Xcc 8004 and Xcc 8004R and exhibited different adsorption kinetics on the two strains. Transmission electron microscopy showed an icosahedral head and a long, non-contractile tail. Genome sequencing revealed a circularly assembled 91,478-bp double-stranded DNA genome encoding 128 predicted open reading frames and two tRNAs. No recognizable antibiotic resistance or bacterial virulence genes were detected. Phylogenomic, ANI, and gene-sharing network analyses suggest that GYRb1 is highly divergent from currently available related phages and may represent a candidate genus-level lineage within the class Caudoviricetes. GYRb1 suppressed the growth of Xcc 8004 and Xcc 8004R in vitro and reduced disease severity in cabbage leaf. A cocktail containing GYRb1 and X1 reduced OD₆₀₀-based bacterial regrowth compared with single-phage treatments in vitro. Together, these findings support the feasibility of using phage-resistant bacterial mutants as selective hosts to obtain complementary candidate phages, while highlighting the need for receptor identification, resistance-frequency analysis, lysogeny assessment, and broader host-range testing before practical application.