Isolation and preliminary analysis of a lytic bacteriophage infecting Agrobacterium tumefaciens to control bacterial overgrowth during plant transformation

Agrobacterium tumefaciens is responsible for causing crown gall disease in plants, which it induces by transferring genetic material from its Ti plasmid into the plant genome. This unique ability is exploited in plant genetic engineering but poses challenges, such as overgrowth. Antibiotic control of A. tumefaciens is often ineffective due to bacterial resistance and potential harm to plant tissue, highlighting the need for alternative treatments. Bacteriophages, viruses that infect bacteria with high specificity, present a promising solution. This study focused on isolating and preliminary analysis of a bacteriophage targeting A. tumefaciens. Bacteriophage WB (ΦWB) was isolated from wastewater in Cikarang, West Java, produces a clear plaque with an average diameter of 3.92 ± 0.22 mm. Cryo-TEM analysis classified ΦWB as a Myovirus , showing an icosahedral capsid (66.04 ± 2.17 nm), a neck (10.70 ± 1.25 nm), and a contractile tail (72.93 ± 2.48 nm uncontracted; 48.37 ± 3.86 nm contracted). ΦWB has a double-stranded DNA (dsDNA) genome estimated to be 46 kbp in size. Host range assays demonstrated that ΦWB has a narrow host specificity, infecting only A. tumefaciens C58 derivatives. Additionally, ΦWB exhibited an adsorption time of 18 min and lysed the majority of bacterial cells within 6 h at an MOI of 0.01. While its lytic activity is slower than some previously reported phages, the absence of phage titer data in those studies limits direct comparisons. ΦWB demonstrated resilience across a broad pH range and high temperatures, suggesting its potential as a biocontrol agent for A. tumefaciens in agricultural settings. • Discovery of a potentially new bacteriophage (ΦWB): Isolated from Cikarang wastewater. • Specific host targeting: Infects A. tumefaciens C58 derivatives, useful for controlling overgrowth. • Characterized as a Myovirus: Cryo-TEM shows an icosahedral head, neck, and contractile tail, typical of Myovirus. • High efficacy at low MOI: Strongly reduces A. tumefaciens even at low multiplicities of infection. • Biocontrol potential: Offers antibiotic-free method for A. tumefaciens in plant biotechnology.