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In total, 11 bacteriophage strains were successfully isolated; seven (Pectobacterium phages Wc1, Wc2, Wc6, Wc7, Wc8, Wc9, and Wc10) from water samples and four (Pectobacterium phages Wc3, Wc4, Wc5, and Wc5r) from soil samples. All purified phages formed small lytic plaques of approximately 1 mm in diameter on P. carotovorum lawn on LB agar after 24 hours incubation at 28℃ (supplementary Figure S2).
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Transmission electron microscopy revealed that the isolated phages belong to two different groups of phages. The first group, including Wc2, Wc3, Wc4, Wc5, Wc7, Wcp9, and Wc10, produced virions with a contractile tail and an isometric capsid. Virions in the second group, including Wc1, Wc5r, Wc6, and Wc9, had a long, non-contractile tail and an isometric capsid (Fig. 1, Table 1).
Table 1. Partial characterization of 11 phages displaying antibacterial activity against plant-pathogenic Pectobacterium carotovorum
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Lytic curves generated on the basis of the experimental data revealed some differences in bacterial lysis dynamics (Fig. 2). The OD600 in wells containing phages Wc1, Wc2, and Wc8, increased until approximately 1.5 h, and then decreased to the baseline, whereas in wells containing the other phages, the OD600 increased until approximately 0.5 h and then decreased gradually to the baseline after 1 h, but gradually increased again after 10 h of incubation. Lysis was more effective in microplate wells containing the phage cocktail (Fig. 2C). No increase in turbidity was recorded in the wells during the course of the experiment. In the control experiment, exponential bacterial growth was observed (Fig. 2D).
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The burst sizes of the phages ranged from 69 to 196 PFUs per infectious cycle, with latency periods ranging from 1 to 1.5 h in LB broth at 28℃ (Table 1).
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All 11 phage strains exhibited antibacterial activity against 20 strains of P. carotovorum available in our lab. The phages did not infect the two P. fluorescens strains, and only one phage, Wc5r, showed lytic activity against P. atrosepticum (Table 1). Wc5r could also infect two phage-resistant strains of P. carotovorum (supplementary Figure S1).
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During phage amplification, some bacteria developed resistance against phage infection. Two phage-resistant P. carotovorum strains were isolated from flask cultures inoculated with phages Wc3 and Wc8. The two phage-resistant strains KPM01R1 and KPM01R2 were found to be resistant to ten of the isolated bacteriophages, but remained susceptible to Wc5r.
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The experimental data showed that the isolated phages can effectively control potato soft rot caused by P. carotovorum. Application of phages to potato slices at 1.0 × 109 PFUs/mL before inoculation with the bacterial pathogen resulted in a reduction of disease symptom development by more than 50% (Fig. 3). Disease symptoms on potato slices were more effectively suppressed when the phage cocktail was used; up to 100% symptom prevention was observed. When the phages were applied after bacterial inoculation, treatment was more effective if phages were applied 1 or 2 hpi than at later time points, as shown in Fig. 4. Similar results were obtained in whole potato tubers (Fig. 3B).
Figure 3. Phage efficacy/therapy test results. A Prevention of soft rot development on potato slices by individual phage strains. B Phage treatment of whole tubers, results of soft rot prevention by bacteriophages (phage cocktail), negative control (buffer applied to wounded potato tuber), and positive control (bacteria plus phage buffer).
Figure 4. Efficacy tests of bacteriophages in preventing spread of P. carotovorum on the surface of potato slices. A Phages were applied once before inoculation of bacteria on the surface of each slice. B Potato slices were treated with individual phages or phage cocktail once at different times post bacterial infection and incubated at 28 ℃. Results were recorded after 24 h and are based on the percentage increase in the diameter of the infected area on each potato slice.