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About 60 water samples were collected from geographically different water sources in Kenya and China during the years 2014 and 2015. These samples were assayed for the presence of lytic bacteriophages against R. solanacearum strain G as the host. Approximately 30% of the tested water samples (18 samples) produced lytic plaques on assay plates. Finally, we obtained 12 lytic phages that were virulent to all three field isolates. The lytic phages had different characteristics, as summarized in Table 1. In addition to R. solanacearum strain G, all the phages were found to be able to lyse the other three bacterial isolates (PS-X4-1, PS-X10-2, and PS-13-1), showing that they might have relatively wide spectra against R. solanacearum. Adsorption kinetics curves and one-step growth curves of all the 12 phages are presented in Supplementary Figure S1 and Supplementary Figure S2, respectively. As presented in Figure 1, TEM analysis showed that two of the selected phages (P-PSG-1 and P-PSG-7) belong to family Siphoviridae, while P-PSG-6 did not have a tail and spikes; therefore, it belongs to family Cystoviridae.
Phage name Isolate source Diameter#
(cm)Optimal MOI Latent period*
(min)Burst size*
(ratio)P-PSG-1 Zhengdian, Hubei
Province, China0.4 10–3 / 10.5 P-PSG-2 Zhengdian, Hubei
Province, China0.6 10–2 / 15.5 P-PSG-3 East-Lake, Hubei
Province, China0.5 10–3 20 1200 P-PSG-4 East-Lake, Hubei
Province, China0.5 10–3 20–30 65.8 P-PSG-5 Qingshan Lake, Hubei
Province, China0.5 10–4 / 2.7 P-PSG-6 BuHe, Anhui
Province, China0.1 10–4 15 191.4 P-PSG-7 River Sare,
Migori, Kenya0.5 10–5 90 200 P-PSG-8 River Migori,
Migori, Kenya0.5 10–5 50–70 53.0 P-PSG-9 Stella Well,
Kisumu, Kenya0.4 10–3 70 330 P-PSG-10 River Rianyago,
Migori, Kenya0.4 10–4 70 633.3 P-PSG-11 River Nyambira,
Migori, Kenya0.4 10–4 80 124.8 P-PSG-12 Mwamogesha Spring,
Migori, Kenya0.4 10–5 10–20 198.6 Note: #, diameter of plaque; *, latent period and burst size are derived from adsorption kinetics curve and one-step growth curves. Table 1. Partial characterization of the 12 bacteriophages
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The 12 phages could be roughly classified into four types based on the curves shown in Figure 2. P-PSG-3 and P-PSG-4 belong to the first group. These two phages inhibited growth of the host bacteria immediately after infection. The OD600 decreased to baseline within 2.5 h post-infection (Figure 2A). The second group consists of six phages (P-PSG-1, P-PSG-8 to P-PSG-12). The OD600 increased to the maximum within the first 1 h, then remained stable for about 1 h, and finally decreased to baseline within 6 h post-infection by these phages (Figure 2B). The third group comprises P-PSG-2 and P-PSG-7. The OD600 increased to the maximum within the first 3h, and then gradually decreased to baseline more than 12 h post-infection (Figure 2C). The last group consists of P-PSG-5 and P-PSG-6. The OD600 increased to the maximum within the first 5 h, and then gradually decreased, following the same trend as the group comprising P-PSG-2 and P-PSG-7 (Figure 2D). The OD600 of the bacterial control uninfected with any bacteriophages increased gradually within the first 8 h to the maximum (about 0.65) and was still stable after 12 h when the experiment was terminated (Figure 2F).
Figure 2. Growth curves of R. solanacearum strain G uninfected (F), or after infection with different bacteriophages (A–D), or the phage cocktail P1 (E). Inocula (250 μL) of R. solanacearum G cells of mid-exponential phase (1010 CFU/mL) were mixed with 250 μL of solutions of the 12 lytic phages alone or in combination at a MOI of 1, then 500 μL of CPG liquid medium was added into the mixture. The absorption of the final mixtures at 600 nm (OD600) was monitored continuously with a micro-plate reader at 28 °C, over 12 h.
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Based on the above lytic kinetics, bacteriophages from each of the groups were selected for mixing to produce cocktails (Supplementary Table S1). Generally, all the cocktails showed better inhibition of the growth of the host bacterial strain G than single bacteriophages. The initial phase of increasing OD600 was reduced to less than 1 h post-infection by the cocktails. The OD600 value decreased rapidly to the baseline within 4 h. Exceptionally, of all the cocktails tested, one phage cocktail, P1, consisting of P-PSG-1, P-PSG-2, P-PSG-3, P-PSG-7, P-PSG-8, and P-PSG-9, left the lowest number of live bacterial cells at 12 h post-infection (Figure 2E). Therefore, the phage cocktail P1 was chosen for the following experiments to test for efficacy in the prevention and treatment of potato wilt, as well as in the decontamination of pathogen-contaminated soil.
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Generally, two approaches could be used to prevent bacterial wilt. One is to pretreat the potato plants with the cocktail, so that the plants could be prevented from later infections by the bacteria pathogen. Another is to decontaminate pathogen-contaminated soil before planting potato plants.
We first tested whether pretreating the potato plants with the P1 phage cocktail could prevent bacterial wilt development by injecting R. solanacearum into the stem of the potato plants. Plants injected with P1 (P1 group of bacteriophages) or SW (SW group) grew normally and did not develop any symptoms of bacterial wilt. The results show that P1 is quite safe to the plants. In contrast, as shown in Table 2, all the plants developed wilt after inoculation with either R. solanacearum strain G (G group) or strain X (X group). The times to the first observation of wilt symptoms were different for different plants. Generally, the plants developed wilt within 3–11 days after inoculation of R. solanacearum G, while the times ranged from 3 to 10 days after inoculation of R. solanacearum X. However, once they showed wilt symptoms, the plants were completely dead within 3–5 days when infected with R. solanacearum G, and within 2–3 days when infected with R. solanacearum X. These results show that the virulence of R. solanacearum X is stronger than that of R. solanacearum G.
Group Plant no. Day of first
observation of wilt
after inoculationDay of complete
wilt after
inoculationGroup Plant no. Day of first
observation of wilt
after inoculationDay of complete
wilt after
inoculationG 1 5 10 X 1 4 6 2 5 9 2 5 7 3 7 11 3 6 9 4 10 15 4 7 10 5 11 16 5 10 12 P1-G 1 / / P1-X 1 / / 2 / / 2 5 10 3 / / 3 / / 4 / / 4 / / 5 10 18 5 / / G-P1-3d 1 / / X-P1-3d 1 7 13 2 / / 2 7 14 3 / / 3 5 10 4 / / 4 3 6 5 3 8 5 9 16 G-P1-6d 1 / / X-P1-6d 1 7 12 2 6 / 2 3 5 3 / / 3 10 16 4 7 / 4 5 9 5 / / 5 3 6 G-P1-9d 1 5 9 X-P1-9d 1 6 9 2 7 12 2 3 6 3 / / 3 3 6 4 9 18 4 7 9 5 / / 5 5 7 G-P1-12d 1 5 9 X-P1-12d 1 3 5 2 6 10 2 4 6 3 4 9 3 5 7 4 10 15 4 3 6 5 / / 5 9 12 G-P1-15d 1 5 9 X-P1-15d 1 5 7 2 9 14 2 9 11 3 4 8 3 3 5 4 4 9 4 7 10 5 9 14 5 3 6 Table 2. Disease progression in potato plants following inoculation of R. solanacearum under different bacteriophage treatments
Injecting P1 into the potato plant stems 2 h before inoculation with either R. solanacearum G (P1-G group) or X (P1-X group) was found to prevent the development of wilt. As shown in Table 2, the morbidities of the plants (both P1-X group and P1-G group) were reduced to 20%. These results showed that the bacteriophage cocktail P1 could prevent infection by R. solanacearum in potato plants.
P1 also showed a capability to reduce the density of live bacteria in pathogen-contaminated sterilized soils. The CFU of the bacteria in the contaminated soils decreased more than 5-fold after spraying P1 into the soils, compared with the control (Figure 3). One week post-spray, the number of live bacteria was only about 2% of those in the control soils.
Figure 3. Bioassay for the bactericidal effect of the P1 cocktail: decontamination of pathogen-contaminated soil by P1. R. solanacearum G grown in CPG liquid medium was harvested at exponential growth phase as described in the Methods. A 5-mL inoculum of cell suspension (6.0×108 CFU/mL) or SW (as a control) was added to autoclaved soil (30 g) in a 50-mL flask and mixed well. One day later, 5 mL of P1 (6.0×109 PFU/mL each) was sprayed on the surface of the control soil and the contaminated soil at a MOI of 10 (approx. 1.0×109 PFU/g soil). The soil mixtures were kept at room temperature and small portions (1 g) were collected on different days to monitor the number of GIM1.74 cells in the soils. Bacterial cell numbers were counted on CPG agar plates using the spread-plating method.
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We further tested whether P1 could be used to treat bacterial wilt after the plants were inoculated with the pathogenic bacteria. As shown in Table 2, after inoculation of R. solanacearum G, the wilt could be controlled by early injection of P1 at 3 and 6 days post-inoculation, except for one plant in the group G-P1-3d, which developed the first sign of wilt on the same day as P1 was applied. For the more virulent strain X, all the infected plants died due to the wilt. However, earlier injection of P1 could lengthen the dying time. For example, in the group X-P1-3d, the times from the first signs of wilt to complete wilt were delayed to 4–5 days, instead of 2–3 days without the treatment.
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Group name Titer (PFU) MOI Source P-PSG-1 3.06×1010 1 Single P-PSG-1 P-PSG-2 1.84×1010 1 Single P-PSG-2 P-PSG-3 8.0×1010 1 Single P-PSG-3 P-PSG-4 9.8×1010 1 Single P-PSG-4 P-PSG-5 7.2×1010 1 Single P-PSG-5 P-PSG-6 3.0×1010 1 Single P-PSG-6 P-PSG-7 1.0×1010 1 Single P-PSG-7 P-PSG-8 1.8×1010 1 Single P-PSG-8 P-PSG-9 1.3×1010 1 Single P-PSG-9 P-PSG-10 1.42×1010 1 Single P-PSG-10 P-PSG-11 1.4×1010 1 Single P-PSG-11 P-PSG-12 2.42×1010 1 Single P-PSG-12 P-PSG-(1/2) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-2 P-PSG-(1/8) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-8 P-PSG-(3/10) 1010 1 Equivalent volume mixture of P-PSG-3, P-PSG-10 P-PSG-(6/11) 1010 1 Equivalent volume mixture of P-PSG-6, P-PSG-11 P-PSG-(1/4/7) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-4, P-PSG-7 P-PSG-(1/7/12) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-7, P-PSG-12 P-PSG-(2/9/10) 1010 1 Equivalent volume mixture of P-PSG-2, P-PSG-9, P-PSG-10 P-PSG-(3/5/12) 1010 1 Equivalent volume mixture of P-PSG-3, P-PSG-5, P-PSG-12 P-PSG-(4/8/9) 1010 1 Equivalent volume mixture of P-PSG-4, P-PSG-8, P-PSG-9 P-PSG-(5/7/9) 1010 1 Equivalent volume mixture of P-PSG-5, P-PSG-7, P-PSG-9 P-PSG-(6/8/11) 1010 1 Equivalent volume mixture of P-PSG-6, P-PSG-8, P-PSG-11 P-PSG-(1/2/7/12) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-2, P-PSG-7, P-PSG-12 P-PSG-(1/3/8/12) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-3, P-PSG-8 P-PSG-12 P-PSG-(2/3/4/5) 1010 1 Equivalent volume mixture of P-PSG-2, P-PSG-3, P-PSG-4, P-PSG-5 P-PSG-(2/4/9/11) 1010 1 Equivalent volume mixture of P-PSG-2, P-PSG-4, P-PSG-9, P-PSG-11 P-PSG-(3/5/10/12) 1010 1 Equivalent volume mixture of P-PSG-3, P-PSG-5, P-PSG-10, P-PSG-12 P-PSG-(6/8/10/12) 1010 1 Equivalent volume mixture of P-PSG-6, P-PSG-8, P-PSG-10, P-PSG-12 P-PSG-(1/2/3/4/6) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-2, P-PSG-3, P-PSG-4, P-PSG-6 P-PSG-(1/3/6/8/11) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-3, P-PSG-6, P-PSG-8, P-PSG-11 P-PSG-(2/3/4/5/8) 1010 1 Equivalent volume mixture of P-PSG-2, P-PSG-3, P-PSG-4, P-PSG-5, P-PSG-8 P-PSG-(2/3/4/6/11) 1010 1 Equivalent volume mixture of P-PSG-2, P-PSG-3, P-PSG-4, P-PSG-6, P-PSG-11 P-PSG-(2/4/5/7/9) 1010 1 Equivalent volume mixture of P-PSG-2, P-PSG-4, P-PSG-5, P-PSG-7, P-PSG-9 P-PSG-(3/6/8/10/12) 1010 1 Equivalent volume mixture of P-PSG-3, P-PSG-6, P-PSG-8, P-PSG-10, P-PSG-12 P-PSG-(4/6/7/9/11) 1010 1 Equivalent volume mixture of P-PSG-4, P-PSG-6, P-PSG-7, P-PSG-9, P-PSG-11 P-PSG-(1/2/3/4/5/6) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-2, P-PSG-3, P-PSG-4, P-PSG-5, P-PSG-6 P-PSG-(1/2/3/7/8/9)=P1 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-2, P-PSG-3, P-PSG-7, P-PSG-8, P-PSG-9 P-PSG-(2/3/6/7/9/11) 1010 1 Equivalent volume mixture of P-PSG-2, P-PSG-3, P-PSG-6, P-PSG-7, P-PSG-9, P-PSG-11 P-PSG-(3/4/5/8/9/10) 1010 1 Equivalent volume mixture of P-PSG-3, P-PSG-4, P-PSG-5, P-PSG-8, P-PSG-9, P-PSG-10 P-PSG-(4/5/6/10/11/12) 1010 1 Equivalent volume mixture of P-PSG-4, P-PSG-5, P-PSG-6, P-PSG-10, P-PSG-11, P-PSG12 P-PSG-(7/8/9/10/11/12) 1010 1 Equivalent volume mixture of P-PSG-7, P-PSG-8, P-PSG-9, P-PSG-10, P-PSG-11, P-PSG-12 P-PSG-(1/2/3/4/6/7/12) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-2, P-PSG-3, P-PSG-4, P-PSG-6, P-PSG-7, P-PSG-12 P-PSG-(1/2/3/4/6/11/12) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-2, P-PSG-3, P-PSG-4, P-PSG-6, P-PSG-11, P-PSG-12 P-PSG-(2/3/4/5/8/9/10) 1010 1 Equivalent volume mixture of P-PSG-2, P-PSG-3, P-PSG-4, P-PSG-5, P-PSG-8, P-PSG-9, P-PSG-10 P-PSG-(1/2/3/4/5/6/7/8/9/10/11/12) 1010 1 Equivalent volume mixture of P-PSG-1, P-PSG-2, P-PSG-3, P-PSG-4, P-PSG-5, P-PSG-6, P-PSG-7P-PSG-8, P-PSG-9, P-PSG-10, P-PSG-11, P-PSG-12 Host broth only 1010 (CFU) / No bacteriophages present Bacteriophage cocktail only 1010 / No host; equivalent volume mixture of P-PSG-1, P-PSG-2, P-PSG-3, P-PSG-4, P-PSG-5, P-PSG-6, P-PSG-7P-PSG-8, P-PSG-9, P-PSG-10, P-PSG-11, P-PSG-12 Table S1. Bacteriophage cocktails used in lysis kinetics analyses
Developing a bacteriophage cocktail for biocontrol of potato bacterial wilt
- Received Date: 28 March 2017
- Accepted Date: 18 October 2017
- Published Date: 16 November 2017
Abstract: Bacterial wilt is a devastating disease of potato and can cause an 80% production loss. To control wilt using bacteriophage therapy, we isolated and characterized twelve lytic bacteriophages from different water sources in Kenya and China. Based on the lytic curves of the phages with the pathogen Ralstonia solanacearum, one optimal bacteriophage cocktail, P1, containing six phage isolations was formulated and used for studying wilt prevention and treatment efficiency in potato plants growing in pots. The preliminary tests showed that the phage cocktail was very effective in preventing potato bacterial wilt by injection of the phages into the plants or decontamination of sterilized soil spiked with R. solanacearum. Eighty percent of potato plants could be protected from the bacterial wilt (caused by R. solanacearum reference strain GIM1.74 and field isolates), and the P1 cocktail could kill 98% of live bacteria spiked in the sterilized soil at one week after spraying. However, the treatment efficiencies of P1 depended on the timing of application of the phages, the susceptibility of the plants to the bacterial wilt, as well as the virulence of the bacteria infected, suggesting that it is important to apply the phage therapy as soon as possible once there are early signs of the bacterial wilt. These results provide the basis for the development of bacteriophage-based biocontrol of potato bacterial wilt as an alternative to the use of antibiotics.