2.1 RSJ2 phage preparation and purification

• An overnight culture of Ralstonia solanacearum strain RS10/3 (race 1 biovar 3) was shaken at 28 °C and 230 rpm (Bhunchoth et al., 2015). The culture was diluted 100-fold with 200 mL CPG medium containing 0.1 % (w/v) casamino acids (BD, USA), 1 % (w/v) peptone (HiMedia, India), and 0.5 % (w/v) glucose supplemented with 0.01 M MgSO₄ (Merck KGaA, Germany) in a 1000-mL flask.

• RSJ2 phage was added at a multiplicity of infection of 0.1 after the cultures reached an OD₆₀₀ (optical density of the culture measured at a wavelength of 600 nm) of 0.4–0.6. Clear culture indicated bacterial lysis after an overnight incubation.

• The culture was incubated for an hour with DNase I (Roche, Germany) and RNase A (Sigma-Aldrich, USA) at 37 °C. The cell debris was removed by centrifuging at 7,500 rpm for 45 min at 4 °C.

• The supernatant was filtered through a 0.45-µm membrane (GVS, Italy). The phages were collected through centrifugation at 19,500 rpm for 1 h at 4 °C. The phages were resuspended in SM buffer containing 50 mM Tris-HCl pH 7.5 (Merck KGaA, Germany), 100 mM NaCl (Carlo Erba, Germany), and 10 mM MgSO₄.

• The phages were purified by sucrose (Sigma-Aldrich, USA) density gradient (20–50 % sucrose in SM buffer) and subjected to ultracentrifugation at 24,000 rpm at 4 °C for 1 h in swing buckets. The phage band was extracted by puncturing the tube side wall. The phages were diluted with the experimental buffers (Table S1) before being precipitated by ultracentrifugation at 24,000 rpm at 4 °C for 1 h. The phages were resuspended in each buffer and stored at 4 °C.

2.2 Buffers with varying pH and ionic strength

Nine buffers with varying pH and ionic strength were studied for their effect on RSJ2 phage. 2-(N-Morpholino)ethanesulfonic acid or MES (Sigma-Aldrich, USA) at pH 6.0 was used for acidic buffers. Tris-HCl at pH 7.5 was used for neutral buffers. N,N-Bis(2-hydroxyethyl)glycine or Bicine at pH 8.3 was used for basic buffers. The NaCl concentration was adjusted (0, 0.1, and 0.5 M) for ionic strength varying. The buffer compositions are listed in Table S1. pH and NaCl concentration range were chosen to mimic the variation of pH and NaCl in natural soil conditions of the RSJ2 phage. The ranges were also relatively moderate, so the buffer conditions could be modified for future storage formulation.

2.3 Transmission electron microscopy (TEM)

Following the previous method, the RSJ2 phage structure and morphology were investigated using TEM. The TEM grid (EMS, USA) was cleaned with a rotary pumped coater (Quorum, UK) (Ackermann, 2012, 2009). The phages were deposited on the cleaned grid before being rinsed with deionized water and stained with a 2 % uranyl acetate solution (EMS, USA). The sample grid was examined using 80 kV TEM (Hitachi High-Tech, Japan) at magnifications of 50,000 to 100,000 times.

2.4 Plaque assay of RSJ2 phage

R. solanacearum strain RS10/3 cultured overnight at 28 °C and 230 rpm shaking was diluted to OD₆₀₀ of 0.25 with CPG medium. A 0.01 mL phage sample was added to 0.25 mL of diluted culture. After incubating for 30 min, the sample was mixed with molten 0.45 % agar (PAC, Thailand) in CPG medium and overlaid on a CPG plate containing 1.5 % agar. The number of plaques was recorded after 16–18 h of incubation at 28 °C and converted to plaque forming unit (PFU) per mL. The PFU represented the number of infectious phage particles that can initiate a clear zone or plaque. The titers of the RSJ2 phage in 9 buffers stored at 4 °C were recorded every 7 days for 57 days. The plaque assay was performed in triplicate, and the average titers were calculated and normalized to the phage titers recorded on the first day (termed Day 0) (Abedon, 2018).

2.5 AFM imaging and nanoindentation of RSJ2 phage

The substrate preparation for the phage immobilization was previously described (Sae-Ueng et al., 2020). AFM imaging and measurement were performed with a JPK NanoWizard 3 and NanoWizard ULTRA Speed 2 (Bruker, USA). The AFM micrographs were captured in a liquid milieu in non-contact modes. The nanoindentation data were collected in a liquid milieu using force spectroscopy. The calculation and analysis of the force-distance curve from nanoindentation data were described elsewhere (Ivanovska et al., 2007). A BioLever mini BL-AC40TS-C2 (Olympus, Japan) was used for this study, with the spring constant of 0.03–0.10 N/m determined by thermal tuning. The collection of the images and the stiffness data were described elsewhere (Sae-Ueng et al., 2022, 2020). The RSJ2 phage stiffness in each buffer was collected at Day 0.

2.6 Statistical analysis and data visualization

Statistical analysis was performed using the t-test function and exponential decay model in SciPy Python and LinearGAM in pyGAM Python. The t-test quantifies the difference between the two data sets. The ‘n.s.’ (not significant) denotes a p-value ≥ 0.05. The ‘*’ denotes 0.05 > p-value > 0.01. The ‘**’ denotes 0.01 > p-value > 0.001. The ‘***’ denotes a p-value < 0.001. An exponential decay model was fitted to the relative phage titer data (Table S2). The fitting returned the initial relative phage and the decay rate constant. The plot figures were generated by the Mathplotlib and Seaborn libraries in Python (Python Software Foundation, n.d.).

3.1 RSJ2 phage morphology

A soilborne RSJ2 (or C19) phage was isolated from a soil sample in Chiang Mai, Thailand (Bhunchoth et al., 2015). The TEM micrographs (Fig. 1.) exhibited the RSJ2 phage structure consisting of capsid or head, tail, and tail fibers. The phage head contains double-stranded genomic DNA, and the phage tail functions as a delivery pass box for the DNA. The functions of the tail fibers are not only to recognize and attach the binding sites but also to have a role in phage stability by interacting with other components (e.g., tail) (Ouyang et al., 2024). The phage components agree with the morphology of the phages in the Autographiviridae family (National Center for Biotechnology Information (NCBI), n.d.).

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Fig. 1

TEM micrographs of the RSJ2 phage. The scale bars in (A) and (B) are 100 nm. Tail and tail fibers of the RSJ2 phage were observed (B).

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3.2 RSJ2 phage infectivity in buffers with varying pH and NaCl concentration

Single-step growth experiments were conducted to understand the infection cycle of the RSJ2 phage (Fig.S1). As the number of progeny phage particles increased, the number of host cells decreased. The reduction of the host cells was observed at about four hours after being incubated with the RSJ2 phage. One infection cycle of the RSJ2 phage took about eight hours, and the average burst size was 23 PFU per infected cell. As a soilborne phage in a tropical region like Thailand, RSJ2 is habitually exposed to constantly changing soil environments, such as lowering salt concentration due to rain or increasing salt concentration due to aridity (Arunin and Pongwichian, 2015). Understanding how phage infectivity varies as a function of salts and pH allows us to decipher how phages may survive in the fields. Knowing the infectivity dependence on pH and salts provides valuable insight into developing phage storage formulation. Therefore, we investigated how RSJ2 phage infectivity varies in buffers with varying pH and NaCl, as NaCl is prominently found in soil in Thailand (Sritongon et al., 2022). RSJ2 phage was stored in buffers at varying pH (6.0, 7.5, and 8.3) and NaCl concentration (0, 0.1, and 0.5 M) at 4–10 °C. The titers representing infectivity were recorded every 7 days for 57 days (Fig. 2. and Table S2).

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Fig. 2

RSJ2 phage titers in buffers at pH 6.0 (A), 7.5 (B), and 8.3 (C) with 0, 0.1, and 0.5 M NaCl. The titers were recorded every 7 days and normalized by the “Day 0” titers. Error bars represent the standard deviations from the triplicate experiments.

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The phage titers drastically decreased in the buffers with pH 6.0 (Fig. 2A). Without NaCl, the titers decreased to about 40 % within 3 weeks. At pH 6.0, RSJ2 phage did not favor NaCl since the titers decreased to almost 0 % within 3 weeks and a month for 0.1 and 0.5 M NaCl, respectively. The decay rate constants from fitting the exponential decay model into the data also showed that the decay rates were higher in the buffer with pH 6.0 with 0.1 and 0.5 M NaCl (Fig.S2).

The phage titers gradually decreased in the buffers with pH 7.5 (Fig. 2B). Without NaCl, the titers decreased to about 40 % within 3 weeks, similar to the buffer at pH 6.0 without NaCl. However, the titers decreased to about 60 % within a month for 0.1 and 0.5 M NaCl. The results imply that 0.1 to 0.5 M helps RSJ2 phage preserve the infectivity at pH 7.5 (but not pH 6.0). The decay rates from the buffers with 0.1 and 0.5 M were also lower than those without NaCl (Fig.S2). The results further suggest that pH and NaCl are not independent factors.

At pH 8.3, the phage titers also gradually decreased (Fig. 2C). Without NaCl and at 0.1 M NaCl, the titers decreased to about 10–40 % after two months. With 0.5 M NaCl, the titer remained at 60 % after two months of storage. The data suggest that a high concentration of NaCl (0.5 M) helps retain phage infectivity at the slightly basic pH. The decay rate in this condition was also the lowest among all 9 buffer conditions (Fig.S2). Therefore, RSJ2 phage prefers the buffer at pH 8.3 with 0.5 NaCl the most. pH 6.0, slightly acidic, also harms the RSJ2 phage the most. In other cases where the titers gradually decrease, NaCl may play some role in stabilizing phages, thus preserving phage infectivity.

3.3 Host cell adsorption of the RSJ2 phage in buffers with varying pH and NaCl concentration

The plaque assay result showed that pH and NaCl concentration significantly influenced phage infectivity. However, it is unclear which infection step is affected by pH and NaCl. Plaque formation is the outcome of a successful infection, which consists of a few crucial steps. First, the phage must remain intact before interacting with the host cell. Secondly, phage must successfully bind to the binding sites on the cell membrane. Finally, the progeny phages are synthesized before lysing the cell membrane. In the plaque assay, after the adsorption (binding) step, RSJ2 phage and Rs cells proceeded in the same conditions (e.g., in a CPG medium at the same temperature). In other words, the replication and lysis occurred under the same circumstances. Therefore, the difference in the phage infectivity in 9 buffers resulted from phage adsorption or phage structure before the binding. So, the RSJ2 phage adsorption in 9 buffers was tested. The adsorption test result revealed that the phage adsorption rate was the same in 9 buffers (Table S3). The data suggested that pH and NaCl concentration did not affect the RSJ2 phage binding. Therefore, the infectivity difference was caused by the effect of the pH and NaCl concentrations on the phage structures before the binding step.

3.4 RSJ2 phage stiffness in buffers with varying pH and NaCl concentration

Phage structural components generally respond to varying pH and salts (Mylon et al., 2010; Silva et al., 2014). The collective responses of the structural components, instead of individual elements, affect the properties and functions, including infectivity. Therefore, investigating the collective response is crucial for understanding phage infection. Phage structural responses can only be observed by high-resolution microscopic techniques. A TEM is suitable for studying static structures since the samples are dried, and imaging is collected in a vacuum (Ackermann, 2012). Collecting phage data in buffers is required to capture the in situ dynamic of structural alterations. So, we collected the phage stiffness representing nanomechanical properties in buffers with varying pH and salts with AFM-based nanoindentation. This technique caresses the cantilever with a nanometer-sized tip on the particle. The tip-particle interaction results in an image of the particle (Fig. 3A). The phage capsid and tail were observed in the AFM micrograph. The tail fibers were not observed, probably because the disordering nature of the fibers prevented their immobilization on the substrate. Due to the tip convolution, the particle size must be extracted from the Z-axis profile (Fig. 3B), revealing the capsid diameter to be about 45 nm.

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Fig. 3

AFM micrograph and cross-section of the RSJ2 phage particle. (A) AFM micrograph of an RSJ2 phage particle in SM buffer. The underneath color gradient scale bar indicates the height (Z direction). The white scale bar (50 nm) indicated the lateral dimensions (X and Y directions). (B) The capsid diameter was extracted from the particle’s cross-section line profile (middle white dashed line in (A)).

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After capturing an image of a single phage, the tip was indented into the particle to probe the phage stiffness. The applied force was measured as a function of the cantilever’s vertical displacement. The force-distance curves (Fig.S3) were converted to phage stiffness. We measured the phage stiffness in buffers at varying pH (6.0, 7.5, and 8.3) and NaCl concentration (0, 0.1, and 0.5 M) at 25 °C (Fig. 4 and Table S4). The range of the overall stiffness average was 0.050 – 0.126 N/m. At pH 6.0 (Fig. 4A), the stiffness averages of the RSJ2 phage in the buffer with 0, 0.1, and 0.5 M NaCl were 0.112, 0.126, and 0.099 N/m, respectively. The statistical analysis revealed that the phage stiffness in the buffer with 0 M NaCl was not significantly different from that with NaCl at 0.1 M and 0.5 M. However, the phage stiffness in the buffer with NaCl at 0.1 M significantly differed from that with NaCl at 0.5 M.

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Fig. 4

Stiffness of the RSJ2 phage particles in the buffers with pH 6.0 (A), 7.5 (B), 8.3 (C), and varying concentrations of NaCl (0, 0.1, 0.5 M). A statistical analysis of the phage stiffness at the same pH was performed. The ‘n.s.’ (not significant) denotes a p-value ≥ 0.05. The ‘*’ denotes 0.05 > p-value > 0.01. The ‘**’ denotes 0.01 > p-value > 0.001. The ‘***’ denotes a p-value < 0.001.

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At pH 7.5 (Fig. 4B), the stiffness averages in the buffer with 0, 0.1, and 0.5 M NaCl were 0.060, 0.053, and 0.088 N/m, respectively. The phage stiffness in these buffers was lower than in the buffers at pH 6.0, implying softer phage particles at pH 7.5. The statistical analysis revealed no significant difference in the phage stiffness between the buffer with 0 M and 0.1 M NaCl. However, the phage stiffness in the buffer with NaCl at 0 and 0.1 M significantly differed from that with NaCl at 0.5 M.

Lastly, at pH 8.3 (Fig. 4C), the phage stiffness averages in the buffer with 0, 0.1, and 0.5 M NaCl were 0.050, 0.052, and 0.070 N/m, respectively. The phage stiffness in the buffers at pH 8.3 was also lower than that in the buffers at pH 6.0. The statistical analysis revealed that the stiffness of the phage in the buffer with 0 M NaCl was not significantly different from that with 0.1 M NaCl. However, the phage stiffness in the buffer with NaCl at 0 and 0.1 M significantly differed from that with NaCl at 0.5 M. The stiffness results show that the phage stiffness strongly depends on the storage buffers’ pH and NaCl concentration.