Characterization of putatively lytic bacteriophages able to infect Xanthomonas citri subsp. citri and identification of novel putative exopolysaccharide depolymerases.

Abstract

Asiatic citrus canker (ACC), caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (X. citri), leads to substantial economic losses in the global citrus industry, necessitating sustainable alternatives to conventional copper-based bactericides. In this study, we isolated and sequenced 72 putatively lytic bacteriophages (including 65 previously uncharacterized isolates from São Paulo, Brazil) and characterized their host range, stability and biocontrol potential. Genomic analysis revealed highly successful but low-diversity phage genomic signatures; 70 isolates shared ~95% DNA similarity and were closely related to the Japanese phage CP2, mirroring the clonal nature of the endemic X. citri population. These phages primarily belong to the Autographiviridae family, with the exception of the Schitoviridae isolate XacP77. Using HHsearch and AlphaFold structural modelling, we identified conserved tail-fibre genes predicted to encode putative exopolysaccharide depolymerases with structural homology to carbohydrate-binding modules (CBMs), which may facilitate the degradation of the bacterial xanthan gum capsule during infection. While the phages exhibited robust stability across a wide pH range (4-11) and temperatures up to 55 °C, they were highly sensitive to UV exposure, reaching total inactivation after 160 s. Greenhouse assays demonstrated that treatment with phage P27 reduced ACC lesion production by 60%, pointing to the potential of these viruses and their candidate CBM-containing proteins as components of a sustainable biocontrol development within integrated pest management strategies for X. citri.