Rice bacterial blight bacteriophage E12-2 and application thereof

Abstract

The application discloses a bacteriophage E12-2 of Xanthomonas oryzae and application thereof, and belongs to the technical field of microorganisms. The bacteriophage is obtained by using Xanthomonas oryzae with bacteriophage resistance for multiple rounds of directional evolution, is named Xanthomonas oryzae phage E12-2, and is preserved in the China Center for Type Culture Collection on March 17, 2025, has a preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and a preservation number of CCTCC NO: M 2025486. The bacteriophage can lyse common Xanthomonas oryzae and resistant strains thereof, has a wide host spectrum, strong lytic activity, and a wide application prospect in the prevention and treatment of Xanthomonas oryzae diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the rice bacterial blight phage E12-2 and its applications. Background Technology

[0002] Rice is an important food and economic crop. Bacterial blight of rice is a significant bacterial disease caused by Xanthomonas oryzae pv. oryzae. This pathogen enters the rice plant through stomata or wounds, causing infection. The main symptoms of rice bacterial blight are water-soaked spots on the leaves, which gradually turn yellow or white, eventually leading to leaf death. In severe cases, the disease spreads to the entire plant, causing rice sterility, panicle malformation, or unfilled grains, resulting in reduced yield or even total crop failure.

[0003] Rice bacterial blight is particularly severe under hot and humid conditions. This disease can occur at different growth stages of rice, but is most severe during the jointing to grain-filling stage. It commonly occurs along riverbanks, in low-lying and flood-prone areas, and in rice paddies, especially after heavy rains, when the moist environment of the paddies facilitates the spread of the pathogen. Currently, the main control methods for rice bacterial blight include farmland management, chemical control, and breeding resistant varieties to control the spread of the disease. For example, Chinese patent document CN118556706A discloses a fungicide for controlling rice bacterial blight, in which the active ingredient is a mixture of fluopyram, copper gluconate, and streptomycin in a mass ratio of 1:1 to 9:1. Chinese patent document CN107535504A discloses a 1,3,4-thiadiazole compound. The application of this compound in the control of rice bacterial blight shows that the corresponding 1,3,4-thiadiazole compounds have a significant inhibitory effect on the hpa1 promoter activity, and can be better applied to the control of rice bacterial blight. Chinese patent document with publication number CN115521888A discloses a biological fungicide that can effectively control rice bacterial blight. The active ingredient of this biological fungicide is Bacillus velezensis Bv-303, and the accession number is CGMCC No.23395.

[0004] However, the widespread use of fungicides with antibiotics and chemical pesticides as active ingredients leads to problems of drug resistance and ecological safety. In contrast, biological control methods utilizing interspecies relationships offer advantages such as environmental friendliness, high specificity, and long-lasting control effects, making biological control a key research focus. Bacteriophages, as an emerging type of biological pesticide, have broad application prospects. They possess high host specificity, meaning they infect and lyse only specific bacterial hosts, thus avoiding impacts on non-target organisms and reducing environmental pollution and ecological risks. On the other hand, the host specificity of bacteriophages limits their application to certain pathogens, which in some cases restricts their widespread use in controlling diverse diseases. Furthermore, with long-term use of bacteriophages, bacteria may gradually develop drug resistance through mutation and modification of their receptors, thereby affecting the control efficacy of bacteriophages. Therefore, overcoming the host specificity limitations of bacteriophages and improving their resistance to drug-resistant strains remains an important issue in current bacteriophage application research. Summary of the Invention

[0005] This invention provides a bacteriophage of rice bacterial blight, specifically bacteriophage E12-2, which was obtained through multiple rounds of directed evolution using rice bacterial blight pathogens with phage resistance. This bacteriophage is capable of lysing common rice bacterial blight pathogens and their resistant strains, and has broad application prospects in the prevention and control of rice bacterial blight.

[0006] The specific technical solution adopted is as follows:

[0007] The bacteriophage E12-2 of rice bacterial blight pathogen, named Xanthomonas oryzae phage E12-2, was deposited on March 17, 2025, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC NO:M 2025486.

[0008] The rice bacterial blight phage E12-2 has an icosahedral head and a non-contractile long tail, and belongs to the genus Xipdecavirus.

[0009] This invention utilizes rice bacterial blight pathogens resistant to phages as the host bacterium, and through multiple rounds of directed evolution, successfully obtained rice bacterial blight phage E12-2, which can overcome bacterial resistance and possesses broad-spectrum antibacterial activity. One-step growth curves show that the phage has a latency period of approximately 40 minutes to infect the rice bacterial blight resistant bacterium N1R, with a lysis rate of approximately 300 PFU / infected cell, indicating strong lytic activity.

[0010] The present invention also provides the use of the aforementioned rice bacterial blight phage E12-2 in the preparation of a bacteriostatic agent for rice bacterial blight.

[0011] The present invention also provides the application of the rice bacterial blight phage E12-2 in the prevention and control of rice bacterial blight.

[0012] The present invention also provides a bacteriostatic agent for rice bacterial blight, comprising the rice bacterial blight phage E12-2.

[0013] Furthermore, the rice bacterial blight inhibitor also includes agriculturally acceptable additives.

[0014] Furthermore, agriculturally acceptable adjuvants are selected from at least one of dispersants, stabilizers, fillers, and solvents.

[0015] The present invention also provides a method for controlling rice bacterial blight, by applying the rice bacterial blight bacterium phage E12-2 or the rice bacterial blight bacterium inhibitor.

[0016] Furthermore, the suspension of rice bacterial blight phage E12-2 or the solution of rice bacterial blight inhibitor can be sprayed onto rice seedlings.

[0017] Furthermore, in the suspension of rice bacterial blight phage E12-2 or in a solution prepared with an inhibitor of rice bacterial blight phage, the concentration of rice bacterial blight phage E12-2 was 1×10⁻⁶. 8 ~1×10 9 PFU / mL.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] (1) The rice bacterial blight phage E12-2 of the present invention can overcome bacterial resistance and broaden the host spectrum (from 17 wild-type strains to 32 strains), and has strong lysis ability with a lysis amount of about 300 PFU / infected cell. It can be used for the prevention and control of rice bacterial blight caused by rice bacterial blight.

[0020] (2) The present invention utilizes the bacteriostatic agent with rice bacterial blight phage E12-2 as the active ingredient to control rice bacterial blight. It is environmentally friendly, effective and can significantly reduce the disease index. Attached Figure Description

[0021] Figure 1 This is a plate growth diagram of the rice bacterial blight phage E12-2 of the present invention;

[0022] Figure 2 This is an electron microscope image of the rice bacterial blight phage E12-2 of the present invention;

[0023] Figure 3This is a schematic diagram of the one-step growth curve of the rice bacterial blight phage E12-2 of the present invention;

[0024] Figure 4 The lysis curves of rice bacterial blight phage E12-2 and wild-type phage NP1 against rice bacterial blight are shown in this invention. The shaded areas around the scatter points represent confidence intervals.

[0025] Figure 5 The diagram shows the control effect of rice bacterial blight phage E12-2 and wild-type phage NP1 on rice bacterial blight according to the present invention. In the diagram, a, b, c and d represent significant differences between different treatment groups at the 5% level. Detailed Implementation

[0026] To make the objectives, features, and advantages of this invention more apparent and understandable, a detailed description is provided below through specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the invention can be combined appropriately without mutual conflict.

[0027] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer. Contents not described in detail in this specification are prior art known to those skilled in the art. Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0028] Example 1: Isolation of rice bacterial blight phage E12-2

[0029] (1) Isolation of rice bacterial blight pathogens resistant to phage

[0030] S01. The rice bacterial blight pathogen N1 (Xanthomonas oryzae pv. oryzae, isolated from rice bacterial blight leaves and long-term preserved in the laboratory) was inoculated into 5 mL of NB liquid medium and cultured at 30℃ and 200 rpm / min until OD. 600 =0.6(10 8 CFU / mL);

[0031] S02 added 100 μL of wild-type bacteriophage NP1 (10 8 PFU / mL)(This phage was isolated in situ from the above pathogen N1 isolate), and continued to be cultured at 30℃ with shaking at 200 rpm / min for 48 h;

[0032] S03 was dipped into a small amount of bacterial culture and streaked onto NA plates. It was incubated at 30°C until single colonies grew. Ten different single colonies were selected and purified by streaking at least three times. The culture was then inoculated onto NB liquid medium and cultured at 30°C with shaking at 200 rpm / min until the logarithmic growth phase.

[0033] S04 Take 500 μL of bacterial culture (10 8 CFU / mL) and 10 μL wild-type phage NP1 (10 6 A mixture of PFU / mL was used to create inverted double plates, with the wild-type pathogen N1 strain as a control. The presence or absence of phage plaques was used to determine whether the strain isolated from S03 had developed phage resistance. After obtaining preliminary resistant strains, the samples were passaged at least three times, and the wild-type phage NP1 resistance was tested again.

[0034] Thus, the rice bacterial blight pathogen N1R with phage resistance was isolated and verified, and this strain was used as the host for subsequent phage evolution screening.

[0035] (2) Isolation of rice bacterial blight phage E12-2

[0036] S11 selected single clones of susceptible rice bacterial blight pathogen N1 and phage-resistant rice bacterial blight pathogen N1R, respectively, and cultured them in 5 mL of NB liquid medium for 36 h. When OD 600 When the multiplicity of infection (MOI) is 0.6, 50 μL of N1 and N1R (N1:N1R = 1:9) are transferred to 5 mL of fresh NB liquid medium. Wild-type phage NP1 is added to 5 mL of NB liquid medium at a MOI of 0.01 and cultured for 24 h.

[0037] After collecting the supernatant by centrifugation at S12 and filtering it through a 0.22 μm filter, 100 μL of the supernatant was added to a new 5 mL NB liquid medium, along with 50 μL of a mixed culture of sensitive rice bacterial blight N1 and phage-resistant rice bacterial blight N1R (N1:N1R = 1:9).

[0038] S13 followed the same pattern, iterating every 24 hours for a total of 12 rounds of culture and evolution. After each round of evolution, the phage population was serially diluted and cultured on plates containing resistant rice bacterial blight pathogen N1R to identify N1R-tolerant phage clones. The results are shown in […]. Figure 1 .

[0039] like Figure 1As shown, phagocytic plaques appeared on the plate. These plaques were purified, amplified, and preserved, and named Xanthomonas oryzae phage E12-2. They were deposited on March 17, 2025, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO:M2025486.

[0040] Example 2: Determination of the biological properties of rice bacterial blight phage E12-2

[0041] (1) Determination of the titer of rice bacterial blight phage E12-2

[0042] After the final purification in Example 1, uniformly distributed and sized phage plaques were obtained. Each plaque was pricked with a pipette tip, and the tip was then immersed in 1 mL of SM liquid medium at 4°C overnight. The next day, 0.1 mL of the supernatant and 0.1 mL of the host bacterium N1R were inoculated into 5 mL of NB liquid medium and incubated at 30°C for 6 hours. The culture was then centrifuged, filtered, and serially diluted 10-fold with SM solution until a final dilution of 10⁻⁶ was achieved. -8 Take 0.1 mL of the last three dilutions of phage and mix with 0.1 mL of the host bacterium N1R. Incubate at 30°C for 20 min and plate onto a double-layer agar plate. Observe the number of phage plaques and calculate the phage titer using the following formula:

[0043] Phage titer = number of plaques × dilution factor / sample volume;

[0044] Each dilution required three replicates, and the average of the three replicates for each dilution was used for counting. Plate count results indicated that the titer of rice bacterial blight phage E12-2 was 3 × 10⁻⁶. 8 PFU / mL.

[0045] (2) Electron microscopic observation of E12-2 bacteriophage of rice bacterial blight pathogen

[0046] Take 20 μL of rice bacterial blight phage E12-2 suspension (concentration > 10). 6 A drop of PFU / mL solution was added to a copper grid, and the sample was allowed to settle naturally for 15 minutes. Excess liquid was then absorbed from the side using filter paper. One drop of 2% phosphotungstic acid (PTA) was added to the copper grid to stain the phages for 10 minutes. The staining solution was then absorbed from the side using filter paper. After the sample dried, its morphology was observed using a transmission electron microscope. The results are shown in [Figure number missing]. Figure 2 .

[0047] like Figure 2As shown, the isolated rice bacterial blight phage E12-2 has an icosahedral head and a non-contractile long tail, belonging to the genus Xipdecavirus.

[0048] (3) Determination of the one-step growth curve of rice bacterial blight phage E12-2

[0049] Take 10 μL of rice bacterial blight phage E12-2 suspension (3 × 10⁻⁶) 8 Mix PFU / mL with 1 mL of host bacteria N1R, adsorb at room temperature for 20 min, centrifuge at 13,000 g for 30 s, and discard the supernatant; wash the precipitate twice with NB liquid medium to remove free phages that have not adsorbed host bacteria; add to 5 mL of NB liquid medium and mix thoroughly, place in a shaker at 200 rpm / min, take 50 μL samples every 10 min, centrifuge at 13,000 g for 30 s, take the supernatant to measure the titer, and plot a one-step growth curve.

[0050] The results are as follows Figure 3 As shown in the one-step growth curve, the incubation period of rice bacterial blight phage E12-2 infecting host bacterium N1R is about 40 minutes, and the lysis amount is about 300 PFU / infected cell, indicating strong lysis.

[0051] (4) Host spectrum determination of E12-2 bacteriophage of rice bacterial blight pathogen

[0052] Take 1 mL of the test bacterial strain in the logarithmic mid-phase (OD) 600 =0.6) and 5 mL of melted semi-solid NA medium were thoroughly mixed and poured onto the surface of solid NA medium to prepare a double plate. After the surface solidified, 2 μL of wild-type phage NP1 and phage E12-2 (1×10) were taken respectively. 8 A drop of PFU / mL was added to the center of each double plate, dried, and then inverted in 30°C culture medium for overnight incubation. The results were observed, photographed, and recorded.

[0053] The results are shown in Table 1. Compared with the wild-type phage NP1, the host spectrum of the evolved phage E12-2 has been significantly expanded, and the number of host strains it can infect has increased from 17 to 32, showing a wider host spectrum.

[0054] Table 1 Host spectrum of wild-type bacteriophage NP1 and bacteriophage E12-2

[0055]

[0056] + indicates that infection is permissible; - indicates that infection is not permissible.

[0057] Example 3: Rice bacterial blight phage E12-2 for the control of rice bacterial leaf blight.

[0058] (1) Lysis curves of wild-type bacteriophage NP1 and bacteriophage E12-2 under in vitro conditions

[0059] Rice bacterial blight pathogen N1 and resistant strain N1R were cultured to the pre-log phase, and 0.5 mL of the culture was added to 5 mL of NB liquid medium. The treatment groups were each given 10 μL of wild-type phage NP1 suspension (1×10⁻⁶). 8 PFU / mL) and phage E12-2 suspension (1×10⁻⁶) 8 The control group was treated with 20 μL of NB liquid culture medium (PFU / mL), and the samples were incubated in a shaker. OD values ​​were measured in 96-well plates at various time points, and lysis curves were plotted.

[0060] The results are as follows Figure 4 As shown, the rice bacterial blight phage E12-2 can inhibit the growth of strain N1 and resistant strain N1R, and effectively inhibit the generation of bacterial resistance, and can be used to control rice bacterial blight.

[0061] (2) Control effect of rice bacterial blight phage E12-2 on rice bacterial blight under in vivo conditions

[0062] The control effects of wild-type bacteriophage NP1 and bacteriophage E12-2 on rice bacterial blight were tested in a greenhouse experiment. The concentration of rice bacterial blight pathogen N1 was adjusted to OD. 600 =0.6(10 8 CFU / mL). When the rice reached the three-leaf-one-heart stage (21 days), inoculation was performed using the leaf-cutting method. 24 hours later, the treatment groups were sprayed with a concentration of 10 CFU / mL onto the rice leaves. 8 Wild-type bacteriophage NP1 and E12-2 suspensions (PFU / mL) were inoculated until the leaves were completely wetted. The control group was treated with water and pathogen N1 in the same way. Pot experiments were conducted, and approximately 15 days after leaf cutting and inoculation, the disease index (based on leaf area) was calculated according to the bacterial blight grading standard: Grade 0 (immune): no disease; Grade 1 (highly resistant): lesion area less than 1%–5% of leaf area; Grade 3 (moderately resistant): lesion area 6%–12% of leaf area; Grade 5 (moderately susceptible): lesion area 13%–25% of leaf area; Grade 7 (susceptible): lesion area 26%–50% of leaf area; Grade 9 (highly susceptible): lesion area 51%–100% of leaf area. The calculation formula is as follows: Disease index = ∑(number of diseased leaves at each grade × relative grade) / (total number of investigated leaves × highest grade value) × 100.

[0063] The results are as follows Figure 5As shown, the disease index of rice leaves treated with the suspension of rice bacterial blight phage E12-2 was significantly lower than that of wild-type phage NP1, indicating that the rice bacterial blight phage E12-2 of the present invention has certain application prospects in the prevention and control of rice bacterial blight.

[0064] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Rice bacterial blight phage E12-2, characterized in that, The accession number is CCTCC NO:M 2025486.

2. The application of the rice bacterial blight phage E12-2 according to claim 1 in the preparation of a bacteriostatic agent for rice bacterial blight.

3. The application of the rice bacterial blight phage E12-2 according to claim 1 in the prevention and control of rice bacterial blight.

4. A fungicide for inhibiting bacterial blight of rice, characterized in that, Includes the rice bacterial blight phage E12-2 as described in claim 1.

5. The antibacterial agent for rice bacterial blight according to claim 4, characterized in that, It also includes agriculturally acceptable auxiliary materials.

6. The antifungal agent for rice bacterial blight according to claim 5, characterized in that, Agriculturally acceptable adjuvants are selected from at least one of dispersants, stabilizers, fillers, and solvents.

7. A method for controlling bacterial leaf blight in rice, characterized in that, Apply the rice bacterial blight phage E12-2 as described in claim 1 or the rice bacterial blight inhibitor as described in any one of claims 4-6.

8. The method for controlling rice bacterial blight according to claim 7, characterized in that, Spray rice seedlings with a suspension of rice bacterial blight phage E12-2 or a solution of rice bacterial blight inhibitor.

9. The method for controlling rice bacterial blight according to claim 8, characterized in that, In a suspension of rice bacterial blight phage E12-2 or a solution prepared with an inhibitor of rice bacterial blight phage, the concentration of rice bacterial blight phage E12-2 is 1×10⁻⁶. 8 ~1×10 9 PFU / mL.

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