In the face of rising antimicrobial resistance, food insecurity, and climate change, bacteriophages are gaining renewed attention as promising biological alternatives to antibiotics across human, animal, and plant health sectors. Their high specificity, self-replicating capacity, and biodegradability make them valuable tools for combating antimicrobial or pesticide resistance and promoting sustainable biocontrol. This review discusses commonly accepted selection criteria for therapeutic phages, such as avoiding temperate types and screening for undesirable genes, while acknowledging ongoing debates and exceptions in specific clinical or ecological contexts. An overview of phage-based applications within a One Health framework is provided, spanning human medicine, veterinary practice, aquaculture, food safety and crop protection. Particular attention is given to agricultural biocontrol, where several successful plant protection strategies are highlighted, illustrating the feasibility and diversity of phage-based approaches. Despite their potential, key challenges remain regarding phage stability, formulation, and persistence under environmental conditions. Emerging innovations such as encapsulation, carrier bacteria, and protective formulations aim to enhance field performance. Furthermore, this review emphasizes the need to assess the environmental safety of phage applications, particularly their impacts on natural ecosystems, microbial communities, and ecological functions. Finally, the regulatory and policy challenges that currently limit the large-scale deployment of phages, particularly in the European Union, where they remain evaluated under conventional chemical pesticide frameworks are discussed. The development of dedicated regulatory pathways, better adapted to the specificities of phages, is argued to be essential for supporting their integration into agroecological transition strategies and next-generation antimicrobial policies.

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 Φ Xhv ‐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 Φ Xhv ‐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.

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 virulent phages reported to date. In this study, eight novel virulent 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 with the five 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. IMPORTANCE: Ranked 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 virulent 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.

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 Φ 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. IMPORTANCE: Ranked 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.

La gestion durable des organismes pathogènes en agronomie est un défi pour la sécurité alimentaire. Ce projet de thèse vise à évaluer l’efficacité et la durabilité de l’utilisation de virus spécifiques des bactéries, les bactériophages, en tant qu'agents de biocontrôle contre les bactérioses végétales, afin de diminuer les pertes agricoles tout en respectant les objectifs de développement durable. La compréhension des interactions entre des bactériophages et la bactérie phytopathogène Xanthomonas hortorum pv. vitians (Xhv), responsable de la bactériose de la laitue, permettra d’élaborer des mélanges de phages avec des activités lytiques complémentaires et des stratégies d’infections variées. Des tests de protection sur différentes variétés de laitue seront menés en serre et en plein champ avec les autorisations nécessaires. L’apparition de mutants Xhv résistants aux bactériophages sera prise en compte, ainsi que les trade-offs que ces adaptations pourraient entraîner lors de l’interaction de Xhv avec la plante. Les effets éventuels de l’utilisation de bactériophages comme agents de biocontrôle sur les écosystèmes seront étudiés au niveau des communautés bactériennes des sols et de la phyllosphère. Ce projet ciblé servira de modèle pour changer notre perception de la place et du rôle des bactériophages dans leur utilisation en biocontrôle pour d’autres cultures.

Phage cocktails are promising biocontrol agents against bacterial plant diseases by broadening host range and limiting the emergence of resistant mutants. To date, nine lytic phages with properties suitable for biocontrol have been isolated against Xanthomonas hortorum pv. vitians , the causal agent of bacterial leaf spot of lettuce. Here, a six-phage cocktail was rationally designed based on complementary host ranges, covering 91% of tested vitians strains while maintaining strict phage specificity toward the pathovar. To design a robust biocontrol, three distinct phage infection strategies, identified by transposon insertion sequencing, were combined in a cocktail. The susceptibility determinants were involved in LPS biosynthesis, a modified O-antigen structure, and an outer membrane protein putatively linked to the type I secretion system. As these structures contribute to plant colonization and virulence, phage resistance is expected to impose substantial fitness costs. In growth-chamber experiments, the phage cocktail provided dose-dependent protection, with significant symptom reduction observed across all tested concentrations, from 17% at 106 PFU.mL-1, to 34.7% at 107 PFU.mL-1 (two applications), and up to 66% at 108 PFU.mL-1. In two independent field trials conducted across contrasting growing seasons, weekly applications consistently reduced disease severity by 30%, decreased the proportion of non-marketable lettuce heads by more than 84%, and reduced post-harvest trimming losses from 20.7% to 18.1% in summer and from 17.8% to 14.0% in autumn. These findings provide the first demonstration of a reproducible and effective phage-based biocontrol strategy against Xanthomonas hortorum pv. vitians under field conditions.