Genomic and phenotypic characterization of novel jumbo and small bacteriophages infecting Xanthomonas hortorum pv. vitians: Toward a phage-based biocontrol strategy

In recent years, the use of bacteriophages as biocontrol agents has gained renewed attention, especially for targeting major phytopathogenic genera like Xanthomonas. However, their potential against Xanthomonas hortorum pv. vitians, the causal agent of bacterial leaf spot of lettuce, remains largely unexplored with only two lytic phages reported to date. In this study, eight novel lytic tailed phages were isolated from wastewater samples, including three jumbophages, which are the first known to infect this pathovar. These jumbophages define a new viral genus and exhibit distinct biological characteristics, including broader host ranges, but lower burst sizes and slower lytic cycles compared to the smaller-genome phages. Together, the newly isolated phages exhibited lytic activity against nearly all X. hortorum pv. vitians strains tested while remaining highly specific to the pathovar. Genomic analyses revealed strictly lytic profiles, and absence of virulence or resistance genes, supporting their biosafety for agricultural use. Furthermore, the isolation of a phage infecting a bacterial mutant previously shown to be resistant to the virulent phage {Phi}Xhv-1 highlights the potential for designing robust phage cocktails to mitigate resistance emergence. All phages demonstrated high stability across a wide range of pH and temperature conditions, but remained highly sensitive to UV exposure. These findings provide a valuable foundation for developing phage-based biocontrol strategies against bacterial leaf spot of lettuce. IMPORTANCERanked among the leading plant-pathogenic bacterial genera, Xanthomonas species affect a wide range of crops and contribute to major agricultural losses worldwide. Among these, Xanthomonas hortorum pv. vitians, the causal agent of bacterial leaf spot of lettuce, poses a serious threat to lettuce production worldwide. Currently, no effective disease managements strategies are available to reduce this foliar hemibiotropic phytopathogen. Bacteriophage-based biocontrol has shown promising results against Xanthomonas spp. However, its application against this pathogen remains largely unexplored, with only two phages isolated to date. Successful phage application in agriculture requires careful selection and through characterization to ensure safety and efficacy. In this study, the phage diversity targeting this phytopathogen was expanded through the isolation of eight novel lytic phages, including the first jumbophages infecting this species. Their genomic and biological properties provide a valuable foundation for developing future phage-based treatment against this challenging pathogen.