Abstract:Objective Stenotrophomonas can promote the growth of plants and aquatic algae. However, limited studies have focused on the bacteriophages infecting Stenotrophomonas. In this study, we isolated bacteriophages infecting Stenotrophomonas from water and studied their biological and genomic characteristics, aiming to improve our understanding of the ecological functions of Stenotrophomonas and its bacteriophages.Methods The double-layer plate method was employed to isolate bacteriophages infecting S. indicatrix EB12 from lake water. The biological characteristics, whole genome sequence characteristics, gene functions, phylogenetic relationship, and protein structures of the isolates were analyzed.Results Transmission electron microscopy showed that two isolates Ste-X and Ste-D were short-tailed bacteriophages. The incubation period of both strains was 20 min. Ste-X and Ste-D showed the optimal multiplicities of infection being 1 000 and 10, burst sizes of 28.8 PFU/cell and 131.1 PFU/cell, temperature tolerance ranges of 20-60 °C and 20-70 °C, and pH tolerance ranges of pH 4.0-13.0 and pH 3.0-12.0, respectively. Ste-X and Ste-D were deactivated by UV irradiation for 60 min and 120 min, respectively. Their host ranges were narrow. The genomes of both strains showed the lengths of 39 429 bp, with the G+C content of 57.80% and 57.85%, respectively, and both of them contained 65 open reading frames (ORFs). The genomes of the two strains contained only three different bases. However, their homology with other bacteriophages was low, which suggested that the two strains were novel short-tailed bacteriophages. The structures of the proteins encoded by ORF34 and ORF52 were predicted. Result showed thant the proteins encoded by ORF34 were acidic, stable, and hydrophilic, containing two conserved domains, ChtBD3 and PHA00661, with the three-dimensional structures dominated by random coils. The proteins encoded by ORF52 were alkaline, unstable, hydrophilic, with no conserved domain and the three-dimensional structures dominated by α-helixes.Conclusion Two novel strains of short-tailed bacteriophages with high genome similarity but distinct biological characteristics were isolated from water.