2.1. Phage Stability Test

The differences in phage L522 viability under various physicochemical conditions (temperature, pH, UV-A, and UV-B) were assessed according to the method of Xuan et al. [12]. To determine the heat stability, phage preparation was diluted in SM buffer (100 mM NaCl, 10 mM MgSO₄, 0.01% gelatin, 50 mM Tris-HCl, pH = 7.5) at approximately 10⁷ PFU/mL, then incubated at different temperatures (4, 20, 30, 37, and 50°C) for 1 h. Samples taken at 10-min intervals were titrated using a plaque assay with bacterial strain Xoo LA1+. In the pH stability test, either 1 M HCl or 1 M NaOH was used to adjust the pH value of tryptone soya broth (TSB) to obtain solutions with a pH range of 3–11. The phage suspension (~ 10⁷ PFU/mL) was added to an equal volume of the adjusted TSB and incubated at 30°C. After 24-h incubation, the phage titer was determined. The effect of UV radiation was also studied at 311 nm (UV-B, model Philips PL-S 9W/01/2P) and 365 nm (UV-A, model Phillips Actinic BL TL-D 15W). Phage stock was diluted in SM buffer to an initial titer of ~ 10⁸ PFU/mL. A 2-mL dilution was placed on an Φ-6mm petri dish and exposed to UV radiation from a distance of 30 cm for 1 h. Sampling was carried out every 10 min and then titrated. The experiment was conducted in triplicate.

2.2. Bacterial Challenge Test

To evaluate the inhibitory activity of phage L522 against Xoo, the bacterial challenge test was conducted. The culture of Xoo strain LA1+ was incubated at 30°C with shaking at 150 rpm in TSB medium. Until the absorbance at 600 nm reached approximately 0.1 (~ 6 × 10⁷ CFU/mL), a volume of the phage suspension was added at a multiplicity of infection (MOI) of 0.01, 0.1, or 1.0. The sample without phage was used as the control. Mixtures were incubated as described above. Sampling was carried out periodically to determine the OD₆₀₀, which indicated the changes in bacterial growth by phage lysis. The experiment was performed in triplicate.

2.3. Preparation of Phage Suspension for the In Vivo Trial

The culture of Xoo strain LA1+ was incubated at 30°C with shaking at 200 rpm in TSB medium. Until the absorbance at 600 nm reached approximately 0.1, a volume of the phage stock was added at an MOI of 0.1. After 8 h shaking as above condition, the mixture became clear and was then distributed into 2-mL centrifuge tubes and centrifuged at 9,727 × g for 5 min at 4°C. The supernatant was passed through 0.22-μm filter membranes to obtain the final phage suspension.

2.4. In Vivo Trial of Phage in Rice

The in vivo efficacy of phage L522 to control BLB was assessed at Southern Research Center for Plant Protection in Long An province, Vietnam. Jasmin 85 rice variety provided by Cuu Long Delta Rice Research Institute was used. After pre-germinated at room temperature for 48 h, 5 seeds were sown in each 17.5 × 12 cm plastic pot containing 1 kg of soil. Then rice seedlings were pruned to 3 plants per pot. After 45 days of sowing, the experiment was conducted in an arrangement of completely randomized design at environmental temperature with three replications per treatment. Rice plants were infected with the Xoo L024 from microbial-type culture collection in the laboratory, by wound-inducing and spraying bacterial suspension at a concentration of approximately 10⁸ CFU/mL. At day 1 and day 5 after the bacterial infection, four treatments were sprayed phages at different titers such as ~10⁶, 10⁷, 10⁸, 10⁹ PFU/mL, respectively. Another treatment was sprayed a popular commercial pesticide in Vietnam (Starner 20WP) according to the manufacturer’s instruction. The control was infected plants sprayed with sterile water. The plants were observed daily. On day 7 after the second treatment with phage, disease rate (%) and disease incidence (%) were measured as following equations:

n₁, n₃, n₅, n₇: Corresponding to the number of leaves whose diseased area was <1%, 5%, 25%, 50%; n9: The number of leaves whose diseased area was >50%. N: total number of leaves in the treatment.

Curative ratio (%) was calculated as following equation:

DI stands for Disease Incidence. The treated plant in this equation indicated plants treated with phage or pesticide in each test. The above assessment was based on the national technical regulation on surveillance methods of rice pests [13]. Statistical analysis was performed using one-way analysis of variance by IBM SPSS Statistics 20 Software, and the significant differences between means were determined by Duncan’s multiple range test at P ≤ 0.05.

3.1. Effect of Temperature, pH, and UV Radiation on the Viability of Phage

As shown in Figure 1a, the temperature stability of phage L522 was stable at all tested temperatures during 1-h incubation. Even at 50°C, the phage concentration was similar to the initial value (P > 0.05). The same results were found with several published Xanthomonas phages [6]. In addition, the titer of the phage remained unchanged over a wide pH range of 4 to 11 as presented in Figure 1b. It is clarified that phage L522 is more resistant to acidic environment than phage ΦXOF4, the titer of which was affected by pH below 6 after 1-h exposure [14]. The phage titer only sharply reduced at pH 3. While temperature and environment acidification are the main factors limiting phage activity [15], the high tolerance of phage L522 contributed information to devise suitable application strategies in rice farming.

Figure 1: Stability of the phage L522 under various conditions including temperatures (a), pHs (b), and ultraviolet (UV)-A and UV-B exposure (c). Error bars indicate 95% confidence intervals for the averaged values (n = 3). [Click here to view]

Figure 1c presents that the phage viability was gradually less affected by UV-A and UV-B light during 1-h exposure. Viable phage counts slightly declined and reached by ~ 0.5 log 10 units at the end of the experiment under UV-B. However, no significant changes in phage titer were recorded under UV-A radiation. Natural sunlight UV level (UV-A and UV-B) is one of the stability issues when applied in both greenhouse and field conditions. Efforts are being made to prolong the viability of phages on the rice leaf surface, including preventing daylight exposure or improving phage formulation [16].

3.2. Inactivation of Xoo in Broth by Phage

The inhibition of phage L522 on Xoo growth in TSB medium was evaluated at three MOI (0.01, 0.1, 1.0). From the initial concentration (OD₆₀₀ ~ 0.1), the growth of host culture was inhibited post-infection (p.i.) by phage and the mixture became clear at 6 h p.i. Figure 2 shows that the host OD₆₀₀ began to decrease at different times depending on the MOI. In detail, while the OD₆₀₀ of the mixture with MOI of 0.1 and 1.0 started to decline at 2 h p.i, the OD₆₀₀ of host culture with an MOI of 0.01 remained at about 0.1 until 4 h p.i. It is proved that the higher phage concentration resulted in a faster inhibitory effect at the beginning. Growth inhibition was maintained for approximately 45 h by phage infection, whereas the control culture without phage grew sharply and entered the stationary phase at around 30 h (OD₆₀₀ ~ 2). After 45 h p.i, there were gradual increases of OD₆₀₀ in all three phage treatments disregarding MOI that reflected the growth of phage-resistant bacteria. The OD₆₀₀ returned to ~ 0.1 after 55 h. There was no significant difference in the inhibition time among different MOIs. The results of the in vitro test revealed that L522 phage is reasonable to be studied further in the in vivo trial.

Figure 2: Changes in optical density at 600 nm during inactivation of Xoo LA1+ by L522 in tryptone soya broth medium at an multiplicity of infection of 0.01, 0.1, or 1.0. The sample without phage was used as the control. Error bars indicate 95% confidence intervals for the averaged values (n = 3). [Click here to view]

3.3. In Vivo Efficacy of Phage in Rice

The concentration of phage suspension prepared for this experiment was approximately 10⁹ PFU/mL. This phage preparation was then diluted to desired concentrations. In the planta pot experiment, treatments at four phage titers (10⁶, 10⁷, 10⁸, 10⁹ PFU/mL) and a commercial pesticide (Starner 20WP), were applied twice at day 1 and day 5 after the bacterial inoculation. On day 7 after the second treatment, the tests showed significant differences [Figure 3 and Table 1]. Compared to the control, rice leaves treated with phage L522 and the pesticide were obviously less damaged. Table 1 shows that the lowest disease rate and disease incidence of BLB occurred when applying the phage concentration of 10⁹ PFU/mL. With the highest phage titer, the disease rate decreased by approximately 9%, whereas that of the pesticide treatment did not significantly differ from the control plants. Moreover, the efficacy of these two treatments (the phage concentration of 10⁹ PFU/mL and the pesticide) in reducing the disease incidence was equivalent. The disease severity and extent declined to one-half of the control. These results may induce the similarity in the curative ratio of phage preparation at 10⁹ PFU/mL (44.7%) and the pesticide application (45.7%). Further, the in vivo efficacy to control BLB decreased at lower phage concentrations (10⁶, 10⁷, 10⁸ PFU/mL). As shown in Table 1, the phage titer at 10⁸ PFU/mL is considered as a second option of treatment following 10⁹ PFU/mL, when its curative ratio was obviously higher than other phage treatments (10⁶ and 10⁷ PFU/mL). The active chemical of Starner 20WP is oxolinic acid, one of the antibiotics most commonly used against plant pathogen bacteria worldwide [17]. However, the gradual decrease in oxolinic acid efficacy due to resistant strains was recorded during 2009 – 2014 in Israel [18]. The antibiotic resistance could spread to the surrounding environment via plasmid-mediated quinolone resistance genes [19]. As comparable efficacy to Starner 20WP, the phage L522 application is suggested as a more environmental-friendly alternative to control BLB.

Table 1: In vivo efficacy to control bacterial leaf blight in rice by Starner 20WP treatment and phage treatments at various concentrations (10⁶, 10⁷, 10⁸, 10⁹ PFU/mL) at day 12 after Xoo L024 infection.

Figure 3: Differences in the lesion length of rice leaves by treatment with Starner 20WP and phage at various concentrations (106, 107, 108, 109 PFU/mL) at day 12 after Xoo L024 infection. In the control, bacterial infected plants were sprayed with sterile water. [Click here to view]