Characterization and Multiplicity of Infection: Xanthomonas Oryzae Bacteriophages XOPW and XOP2 for Biofilm Control

Xanthomonas oryzae is a bacterial pathogen that causes leaf blight disease (BLB), a devastating rice disease that is globally epidemic and causes significant crop losses of 50%. This bacterium shows resistance to antibiotics and chemicals because of the misuse of chemicals and antibiotics, so bacteriophages are used as an alternative treatment against X. Oryzae. Bacteriophages are considered biocontrol agents because they target specific bacterial strains and lyse bacteria rapidly, controlling infection. For the first time, the current study was designed to isolate and characterize the specific lytic bacteriophage against X. oryzae from Hazara Division, Shinkiari, KPK Province, Pakistan, the major cause of BLB in rice, as well as evaluate the efficiency of bacteriophages in terms of multiplicity of infection for controlling X. Oryzae infection. In this study, all isolated phages showed a very narrow host range; XOPW and XOP2 infect only X. oryzae. All phages showed excellent stability at pH 6–8 while phage treatment was for 1 hour. Heat treatment results showed that even a 1-hour exposure at 80 o C has a significant effect on phage stability, and phages become completely inactivated. Phages tolerated 60 o C heat treatment to varying degrees, resulting in a 2–3 log reduction in titer. Similarly, heat treatment at 50 o C had a minor effect on phage titer. Phages suspended in SM buffer stored at 4 o C showed a minor reduction in titer even after three months of storage. According to the findings, isolated phages were causing bacterial lysis by forming transparent plaques. The MOI studies were conducted at MOI 0.001, 0.01, 0.1, 1, 10, and 100, and the spectrophotometric technique was used to record the net decrease in the bacterial population. Phages XOP2 and XOPW showed diverse levels of efficacy, with bacterial growth being significantly suppressed at various MOIs. The isolated bacteriophages were highly effective at reducing the growth of bacteria. At MOI 1, 10, and 100, a slow but moderate decline in bacterial growth over time was seen in comparison to other MOI levels. 1, 10, and 100, bacterial growth was comparatively unchanged until 20 hours, remaining nearly parallel to the control. The XOPW and XOP2 phage effectively reduced the optical density (OD) of a 4-day-old biofilm in a dose-dependent and time-dependent manner. At all-time points (6, 8, and 24 hours), the higher concentration of phage (MOI 1) consistently resulted in a lower OD compared to the lower concentration (MOI 0.1). The antibacterial activity of all phages was sustained, despite some partial resistance, indicating their potential for therapeutic use. In this pressing situation, employing phages as a therapeutic tool to combat antibiotic-resistant X. oryzae provides another approach.