Biocontrol potential of bacteriophage φsp1 against bacterial wilt-causing Ralstonia solanacearum in Solanaceae crops

Background: Bacteriophages are effective biocontrol strategy as well as ecofriendly remedy for the emerging antibiotic and chemical resistance in bacterial phytopathogens such as bacterial wilt-causing Ralstonia solanacearum. One of the major challenges in the use of bacteriophage therapy for agricultural phytopathogens is maintaining their viability even during variations in pH, temperature, ultraviolet irradiation, and desiccation during field application for sustainable agriculture. Results: In this study, the isolation and characterization of phage phi sp1 for its efficacy against wilt-causing R. solanacearum performed on Solanum lycopersicum (tomato) seedlings and Solanum tuberosum (potato) tuber assay are reported. Bacteriophage was found to be viable and stable at a wide pH range (3.0-9.0) and at temperatures up to 55 degrees C. Phage phi sp1 required similar to 15 min for adsorption and completed its life cycle in 25-30 min by host cell lysis with a burst size of similar to 250-300. Phage phi sp1 eradicated 94.73% preformed R. solanacearum biofilm and inhibited biofilm formation by 73.68% as determined by the static crystal violet microtiter biofilm assay. Transmission electron microscope revealed the phage phi sp1 to be approximately 208 +/- 15 nm in size, comprising of icosahedral head (100 +/- 15 nm) and tail, as belonging to Myoviridae family. Plant bioassays showed 81.39 and 87.75% reduction in pathogen count using phages phi sp1 in potato tuber and tomato seedlings, respectively. Reversal in disease symptoms was 100% in phage-treated tuber and tomato plant (pot assay) compared to only pathogen-treated controls. Conclusion: Isolated bacteriophage phi sp1 was found to be highly host specific, effective in biofilm prevention, and capable of inhibiting bacterial wilt at low multiplicity of infection (1.0 MOI) in tomato as well as potato tuber bioassays. Phages phi sp1 were environmentally stable as they survive at variable pH and temperature. Bacteriophage phi sp1 shows a promise for development into a biocontrol formulation for the prevention of R. solanacearum bacterial wilt disease.