Abstract
BackgroundBacteriophages 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.ResultsIn this study, the isolation and characterization of phage ɸsp1 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 °C. Phage ɸsp1 required ~15 min for adsorption and completed its life cycle in 25–30 min by host cell lysis with a burst size of ~250–300. Phage ɸsp1 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 ɸsp1 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 ɸsp1 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.ConclusionIsolated bacteriophage ɸsp1 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 ɸsp1 were environmentally stable as they survive at variable pH and temperature. Bacteriophage ɸsp1 shows a promise for development into a biocontrol formulation for the prevention of R. solanacearum bacterial wilt disease.
Highlights
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
Biofilm formation is the ability of the pathogen to form communities enclosed within an exopolymeric substance (EPS) that protects it from various stresses
Bacterial strains and culture conditions R. solanacearum strain F1C1 and R. solanacearum (NAIMCC-F01629) stock cultures were maintained at −20 °C and propagated on the tryptone soy agar (TSA) or casamino acid-peptone glucose (CPG) (Hi Media, India) at 28 to 30°C. 2,3,5-Triphenyl tetrazolium chloride (TTC) medium (Hi Media, India) was used for differentiating virulent colony from non-virulent or mutant type colonies
Summary
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. Ralstonia solanacearum has been categorized as the second most devastating pathogen causing bacterial wilt in crops of mainly the Solanaceae family worldwide (Ramesh et al 2014) It is a Gram-negative bacterial phytopathogen with a wide host range of over 200 different. Mori et al (2018) have reported the ability of R. solanacearum strain OE1-1 to form mushroom type of biofilms within intercellular spaces in tomato plant root and stem. Many biofilm factors such as sugars (ralfuranones) and extracellular nucleases contributed to R. solanacearum virulence (Minh Tran et al 2016; Mori et al 2018)
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