Unveiling Host Interactions and Evolutionary Constraints of a Novel Bacteriophage Infecting Xanthomonas hortorum pv....

Due to limitations in disease management strategies and the impact of climate change, phytopathogenic bacteria are threatening global crop production. Xanthomonas hortorum pv. vitians, the causal agent of bacterial leaf spot of lettuce, remains difficult to manage due to the lack of efficient treatment options. As an alternative, phage-based biocontrol offers a promising solution, but its long-term efficacy depends on a thorough understanding of phage-host interactions and the potential development of bacterial resistance. In this study, we isolated and characterised the lytic bacteriophage ΦXhv-1, which defines a novel genus within the class Caudoviricetes. Using transposon insertion sequencing, we identified 36 bacterial genes essential for phage susceptibility, primarily involved in lipopolysaccharide biosynthesis and surface polysaccharide modifications. Targeted mutagenesis and fluorescence microscopy confirmed that ΦXhv-1 adsorbs onto specific residues of the LPS O-antigen side chains. Phage-resistant mutants exhibited a decreased motility in vitro and a significant reduction in virulence in planta. These findings reveal a strong evolutionary trade-off between phage resistance and bacterial fitness, suggesting that resistance emergence in the field may be naturally constrained. This study provides new insights into X. hortorum pv. vitians-phage interactions and supports the development of sustainable phage-based biocontrol strategies against bacterial leaf spot of lettuce.