Bacterial strains and culture conditions
R. solanacearum strain F1C1 (obtained from Dr Suvendra Roy, Department of Molecular & Biotechnology, Tezpur University, Assam) 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.
Isolation and characterization of bacteriophages
Sampling and isolation of bacteriophage
Bacteriophages were isolated from soil samples collected from rhizosphere of different cultivars of solanaceous crops from Kanpur and Fatehpur, Uttar Pradesh, India. Soil sampling was carried out preferentially in April (summer season) and July (after precipitation). For phage enrichment, soil pool collected from various cultivars was mixed well. Five grams of soil were dissolved in 10 ml 1X phosphate-buffered saline (PBS) in 45-ml Falcon tube by vigorous shaking to release phages from soil particles, centrifuged (10,000 rpm for 10 min at 4°C), supernatant collected, and filtered through PES membrane syringe (0.45-μm pore size) (Hi Media Pvt. Ltd. India). Plaque assay was performed using double-layer agar (DLA) overlay method (Kropinski et al. 2009). Equal aliquots of log phase host bacteria R. solanacearum strain 1629 (NAIMCC-F01629) and phage filtrate (100 μl) were mixed in 0.3–0.4% soft agar and poured over a 1.5% hard agar plate in TSA media. Specificity of isolated phages was also checked against R. solanacearum strain F1C1 by employing DLA. Plate was incubated at 30±2°C for 24 to 48 h for observing plaque formation.
Bacteriophage purification and propagation
Isolated bacteriophages were purified through single successive plaque isolation and propagated by picking a well-separated plaque with the help of a sterile inoculation loop and inoculated into 200 ml TSB medium, containing overnight grown log culture of 109 host cells and incubated at 30±2°C at 140 rpm for phages stock preparation.
The phage suspension was centrifuged (10,000 rpm, 10 min, 4°C), filter sterilized, and treated with chloroform (1% v/v) to remove bacterial contamination. High titer phage purification was further conducted by using PEG-8000 method with some modification in SM buffer (50 mmol L−1 Tris-HCl at pH 7.4, 100 mmol L−1 NaCl, 10 mmol L−1 MgSO4, and 0.01% gelatin) (Yamamoto et al. 1970). Purified concentrated phages were stored in aliquots at −20°C for long-term storage. Short-term stock preparations were maintained at 4°C for further use.
Spot assay and double-layer agar (DLA) overlay assay
To check phage viability, spot assay was carried out. Briefly, phage suspension was serially diluted, and 10 μl diluted phage was spot inoculated on molten agar (0.4% agar, w/v) containing host cells of 107 CFU ml−1. Clear zones of plaques were observed after incubating the plates overnight at 30±2°C. Further, the phage titer was determined by plaque assay by employing DLA technique as described previously (Kropinski et al. 2009).
Adsorption assay, one-step growth curve
Adsorption assay and one-step growth curve were performed with some modification (Delbrück 1940). Adsorption time and burst size for phages ɸsp1 were determined. An equal amount of phages (titer 106) and bacterial suspension was taken, incubated for 5 min, diluted (1:102), and DLA performed at 5 min intervals till 30 min in order to determine the phage titer.
Temperature, pH, and UV irradiation sensitivity
Thermostability of bacteriophage was determined by incubating phage titer 106 PFU ml−1 for 10 min at 37, 45, and 55°C with intermittent shaking as per protocol of Sagar et al. (2017). For the determination of pH stability, the same titer was incubated at pH 3.0, 5.0, 7.0, and 9.0 for 20 min (Sagar et al. 2017). For UV irradiation sensitivity, phage titer 106 PFU was exposed to UV C irradiation (UV254 nm) for 5, 10, and 15 min. The treatments were followed by DLA as described previously.
Host range
Host range of phage ɸsp1 was tested by performing spot assay against R. solanacearum isolates RS1, RS2, RS3, and RS4 isolated from stem of potato plants collected from agricultural fields and R. solanacearum strain F1C1. Rhizospheric bacterial hosts such as P. aeruginosa ATCC 15442, P. aeruginosa R32 and GD2 (obtained from Department of Microbiology, CSJMU University, Kanpur) were also used to determine host specificity.
Transmission electron microscopy
Transmission electron microscope (TEM) observation to study bacteriophage morphology was performed with some modifications (Goodridge et al. 2003). High titer purified phage suspension in SM buffer was dropped on copper-coated grids (diameter, 3 mm; 300 meshes) and allowed to adsorb for 5 min. The bacteriophage particles were stained by the addition of 2% (w/v) phosphotungstic acid (PTA) for 10 s. The grid was allowed to dry for 20 min and examined under a TEM (FEI Tecnai S Twin) at 200 kv (SAIF, AIIMS, Delhi, India).
Scanning electron microscopy
Biofilm development on glass cover slip surfaces was visualized by scanning electron microscopy (SEM) with some modification (Sagar et al. 2017). Cover slip was washed gently in sterile 1X phosphate-buffered saline (PBS) to remove planktonic cells, fixed in 5% (v/v) glutaraldehyde in PBS buffer for 2 h, followed by fixing with post-fixative 1% osmium tetroxide. This was followed by dehydration steps through a graded series of 10-min ethanol immersions (30, 50, 70, 90, and 100%). Specimens were mounted on aluminum stubs and observed on SEM (SM 6490, BBAU, Lucknow, UP, India). The entire cover slip surface was examined, and images were chosen that represented the typical field of view.
Biofilm assay
Effect of phage treatment on biofilm inhibition was determined using crystal violet biofilm microtiter assay (Umrao et al. 2020). Briefly, log phase 106 CFU ml−1 R. solanacearum strain 1629 was simultaneously treated by phage ɸsp1 for biofilm formation assay, while 24-h preformed biofilms were phage treated at 1.0 multiplicity of infection (MOI) for biofilm eradication assay. Post 48 h incubation, the biofilm was washed with 1X PBS and stained with (1% w/v) crystal violet for 20 min. Excess stain was removed, plates washed with PBS, and dimethyl sulfoxide (DMSO) was used to solubilize crystal violet-stained biofilm. Results were evaluated by using spectrophotometry (Thermo Scientific Multiscan EX, USA).
Plant bioassay
Plant bioassay study was carried out, using Solanum lycopersicum (tomato seedlings and plants) and in S. tuberosum (potato tuber slices).
Solanum lycopersicum (tomato) seedling assay
Variety S-22 of tomato seeds were selected and sterilized with 70% ethanol, grown on sterile wet cotton bed on plastic tray (Singh et al. 2018). The tray was covered up to maintain humidity and accessibility of light for seed germination in the month of November. Seedling started appearing from the 5th day onwards. Tomato seedlings of 4–5 cm in height with two cotyledon leaves were used for pathogenicity test. The experiment was designed for 2.0-ml microcentrifuge tubes with phosphate buffer containing pathogen of 108 CFU ml−1 for pathogenicity by root inoculation method (Singh et al. 2018). Phage ɸsp1 (1.0 MOI) was used for biocontrol, and untreated tomato seedlings in phosphate buffer were used as control. The experiment was performed in triplicates. The seedlings were observed daily, and disease parameters included wilting of stem and leaves. Fresh weight of the infected, treated, and control seedlings was measured at 72 h post-inoculation. Spectrophotometric reading of cell suspensions in microcentrifuge tubes containing the tomato seedling was taken at 620 nm (Thermo Scientific Multiscan EX, USA).
Solanum lycopersicum (tomato) pot assay
Plant bioassay study was undertaken to check bacteriophage biocontrol efficiency and persistence of phage by using soil-drenching method in greenhouse setup in pots containing 250 g per pot (soil, sand, coconut fiber in 20:4:1). Tomato plants (variety S-22) of 5–6-cm length in triplicate were used for the experiment. The base of plant’s stem was scratched by a sterilized needle, and plants were inoculated by 20 ml of 108 CFU ml−1 R. solanacearum strain 1629 (8×106 CFU g−1) and treated with phage ɸsp1 (1.0 MOI) poured around tomato seedlings in the soil. Disease symptoms of tomato plants were recorded twice in a week by using wilting grade scale according to Kempe and Sequerie (1983). Grade 1, 25% plant leaves wilted; grade 2, 26–50% of plant leaves wilted; grade 3, 51–75% plant leaves wilted; grade 4, 76% or more plant leaves wilted and stem collapses; and grade 5, death of plants (Kempe and Sequeira 1983). All experiments were performed in triplicates.
Solanum tuberosum (potato) tuber slices assay
The experiment was designed for direct inoculation method on potato tuber slices to check phage biocontrol efficiency against R. solanacearum strain 1629 as described previously (Champoiseau et al. 2009). Surface-sterilized (70% ethanol) potato tuber was pieced into slices (4.0 × 3.5 × 0.6 cm3) and inoculated with 108 CFU ml−1 of host R. solanacearum strain 1629 for pathogenicity control. Host + phage ɸsp1 (1.0 MOI), phage ɸsp1 (1.0 MOI), and uninoculated tuber slices were also incubated at 28°C under daily observation. The experiment was performed in triplicate. Diseased symptoms which included vascular browning, bacterial ooze, and tuber necrosis were recorded daily up to day 10 post-incubation. Grading scale used for qualitative measure of tuber disease symptoms was as follows: grade 0, no symptoms; grade 1, yellow discoloration, no ooze, and no necrosis; grade 2, brown discoloration, no ooze, softening of tissue at center of lesion but hard to scoop or pick with inoculating loop; grade 3, brown discoloration with ring formation, whitish ooze from tuber, necrotic tissue softening which is easy to scoop; and grade 4, dark brown-blackish discoloration within the ringed lesion, copious whitish ooze from lesion, total tissue necrosis which is easy to scoop.
Standard plate count was performed at day 10 by plating (100 μl) of (0.1 g ml−1) infected inoculated tuber slices and control tubers. Colony count was recorded after plating for 48 h at 28°C incubation. Cell density of infected tuber tissue solution (0.1 g ml−1) was also quantified by spectrophotometry (A620nm) (Thermo Scientific Multiscan EX, USA). The presence of R. solanacearum was confirmed by simple staining with crystal violet using bright field microscopy.
Ethics approval and consent to participate
Not applicable.
Statistical analysis
Statistical analysis was done using Student’s t test. All experiments were repeated at least twice in triplicates. p≤ 0.05 was considered as biologically significant.