Rice bacterial blight bacteria phage E12-2 and application thereof

Abstract

The invention discloses Xanthomonas oryzae phage E12-2 and application thereof, and belongs to the technical field of microorganisms, the phage is obtained by performing multiple rounds of directed evolution on Xanthomonas oryzae with phage resistance, is named as Xanthomonas oryzae phage E12-2, is preserved in China Center for Type Culture Collection on March 17, 2025, and is preserved in China Center for Type Culture Collection on March 17, 2025. The strain is named as Xanthomonas oryzae pv. Oryzae, is preserved in No. 299 of eight road, Wuchang District, Wuhan City, Hubei Province, has a preservation number of CCTCC NO: M 2025486, can be used for cracking common Xanthomonas oryzae pv. Oryzae and resistant strains thereof, is wide in host spectrum and relatively strong in cracking property, and has a wide application prospect in prevention and treatment of Xanthomonas oryzae pv. Oryzae.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a phage E12-2 of bacterial blight pathogen of rice and an application thereof. Background Art

[0002] Rice is an important food and cash crop. Bacterial blight of rice is a significant bacterial disease caused by Xanthomonas oryzae pv. oryzae. The pathogen infects rice plants through stomata or wounds. The primary symptom of bacterial blight is the formation of water-soaked spots on rice leaves, which gradually turn yellow or white, eventually leading to leaf death. In severe cases, the disease can spread to the entire plant, resulting in sterile rice grains, misshapen panicles, or loose grains, leading to reduced or even complete crop failure.

[0003] Rice bacterial leaf blight is particularly serious under high temperature and humid climatic conditions. The disease can occur in different growth stages of rice, but is most serious from jointing to filling stage. The disease often occurs on both sides of rivers, low-lying and easily flooded areas and rice fields, especially after heavy rain, when the moist environment of rice fields is conducive to the spread of pathogens. At present, the prevention and control methods of rice bacterial leaf blight mainly include farmland management, chemical control and controlling the spread of the disease by breeding disease-resistant varieties. For example, Chinese patent document with publication number CN118556706A discloses a fungicide for preventing and controlling rice bacterial leaf blight, wherein the active ingredients of the fungicide are fluopicolide, copper gluconate and zhongshengmycin mixed in a mass ratio of 1:1-9:1; Chinese patent document with publication number CN107535504A discloses 1,3,4-thiadiazole compounds. The application of the compounds in preventing and controlling rice bacterial blight is discussed. The corresponding 1,3,4-thiadiazole compounds have a significant inhibitory effect on the activity of the hpa1 promoter and can be better applied to the prevention and control of rice bacterial blight. The Chinese patent document with publication number CN115521888A discloses a biofungicide that can effectively prevent and control rice bacterial blight. The active ingredient of the biofungicide is Bacillus velezensis Bv-303, and the deposit number is CGMCC No. 23395.

[0004] However, the widespread use of fungicides based on antibiotics and chemical pesticides has led to drug resistance and ecological safety concerns. Biocontrol methods that exploit interactions between biological species are environmentally friendly, highly specific, and offer long-lasting control, making them a key research focus. Bacteriophages, as an emerging biopesticide, hold broad application prospects. They possess high host specificity, infecting and lysing only specific bacterial hosts, thus avoiding impacts on non-target organisms and reducing environmental pollution and ecological risks. On the other hand, this host specificity limits phages to specific pathogens, which in some cases limits their widespread application in diverse disease control. Furthermore, with long-term phage use, bacteria may gradually develop drug resistance through mutations and modifications to their receptors, thereby compromising the control efficacy of phages. Therefore, overcoming the host specificity limitations of phages and improving their resistance to drug-resistant strains remain crucial topics in current phage application research. Summary of the Invention

[0005] The present invention provides a phage for bacterial blight pathogen of rice. The phage is phage E12-2 for bacterial blight pathogen of rice, which is obtained by multiple rounds of directed evolution using bacterial blight pathogen of rice with phage resistance. The phage can lyse common bacterial blight pathogen of rice and its resistant strains, and has broad application prospects in the prevention and control of bacterial blight pathogen of rice.

[0006] The specific technical solutions adopted are as follows:

[0007] The rice bacterial blight pathogen phage E12-2, named Xanthomonas oryzae phage E12-2, was deposited in the China Center for Type Culture Collection on March 17, 2025, with the deposit address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number being CCTCC NO: M 2025486.

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

[0009] By using the phage-resistant bacterial blight pathogen of rice as a host, this study conducted multiple rounds of directed evolution to successfully isolate E12-2, a bacterial blight phage that overcomes bacterial resistance and exhibits broad-spectrum antimicrobial activity. A one-step growth curve showed that this phage had an incubation period of approximately 40 minutes when infecting N1R-resistant strains of the pathogen, and a lytic capacity of approximately 300 PFU per infected cell, demonstrating strong lytic activity.

[0010] The present invention also provides use of the bacterial blight pathogen phage E12-2 in preparing a bacterial blight pathogen antibacterial agent.

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

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

[0013] Furthermore, the bacterial blight inhibitor for rice also includes agriculturally acceptable auxiliary materials.

[0014] Furthermore, the agriculturally acceptable adjuvant is selected from at least one of a dispersant, a stabilizer, a filler and a solvent.

[0015] The present invention also provides a method for preventing and treating rice bacterial blight, which uses the rice bacterial blight pathogen phage E12-2 or the rice bacterial blight pathogen antibacterial agent.

[0016] Furthermore, a suspension of bacterial blight pathogen phage E12-2 or a bacterial blight pathogen antibacterial agent is made into a solution and then sprayed on the rice seedlings.

[0017] Furthermore, in the solution prepared from the suspension of bacterial blight pathogen phage E12-2 or the bacterial blight pathogen antibacterial agent, the concentration of bacterial blight pathogen phage E12-2 is 1×10 8 ~1×10 9 PFU / mL.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

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

[0020] (2) The present invention uses an antibacterial agent containing the bacterial blight pathogen phage E12-2 of rice as an effective ingredient to prevent and treat rice bacterial blight, which is environmentally friendly, has significant effects, and can significantly reduce the disease index. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0023] Figure 3Schematic diagram of the one-step growth curve of the bacterial blight pathogen E12-2 of the present invention;

[0024] Figure 4 Figure 5 is the lysis curve of bacterial blight pathogen phage E12-2 and wild-type phage NP1 against bacterial blight pathogen of oryzae. The shaded area around the scattered points represents the confidence interval.

[0025] Figure 5 This is a graph showing the control effects of the bacterial blight pathogen phage E12-2 and the wild-type phage NP1 on rice bacterial blight. In the graph, a, b, c and d represent significant differences between different treatment groups at the 5% level. DETAILED DESCRIPTION

[0026] In order to make the objects, features and advantages of the present invention more clearly understood, a detailed description is given below using specific embodiments. In the following description, many specific details are set forth to fully understand the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in the various embodiments of the present invention can be combined accordingly without conflicting with each other.

[0027] The procedures for the following examples, in which specific conditions are not specified, generally follow conventional conditions or those recommended by the manufacturer. Any material not described in detail in this specification belongs to the prior art known to those skilled in the art. The experimental materials used in the following examples, unless otherwise specified, can be purchased from conventional biochemical reagent companies.

[0028] Example 1 Isolation of Bacteriophage E12-2 from Xanthomonas oryzae

[0029] (1) Isolation of phage-resistant rice bacterial blight pathogen

[0030] S01: Xanthomonas oryzae pv. oryzae N1 (isolated from rice bacterial blight leaves and stored in the laboratory for a long time) was inoculated into 5 mL of NB liquid medium and cultured at 30°C and 200 rpm / min until OD 600 =0.6(10 8 CFU / mL);

[0031] S02 Add 100 μL of wild-type phage NP1 (10 8 PFU / mL) (the phage was isolated in situ from the pathogen N1 isolate mentioned above), and cultured at 30°C, 200 rpm / min with shaking for 48 h;

[0032] S03 was streaked with a small amount of bacterial liquid onto a NA plate and cultured at 30°C until a single colony grew. Ten different single colonies were selected and streaked for purification at least three times, and then inoculated into NB liquid medium. Cultured at 30°C with shaking at 200 rpm / min until the logarithmic growth phase;

[0033] S04 took 500 μL bacterial solution (10 8 CFU / mL) and 10 μL wild-type phage NP1 (10 6 PFU / mL) were mixed and poured onto two plates, with the wild-type pathogen strain N1 as a control. The presence of phage plaques was used to determine whether the strain isolated in S03 had phage resistance. After obtaining a preliminary resistant strain, it was necessary to repeatedly passage it at least three times and then test it again for resistance to wild-type phage NP1.

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

[0035] (2) Isolation of Bacteriophage E12-2 from Xanthomonas oryzae

[0036] S11 Single clones of sensitive rice bacterial blight pathogen N1 and phage-resistant rice bacterial blight pathogen N1R were picked and cultured in 5 mL NB liquid medium for 36 h. 600 =0.6, transfer N1 and N1R (50 μL in total) at the same time (N1:N1R=1:9 ratio) into 5 mL of fresh NB liquid medium, and add wild-type phage NP1 at a multiplicity of infection (MOI) of 0.01 into 5 mL of NB liquid medium, and culture for 24 h;

[0037] S12 was centrifuged and the supernatant was collected and filtered through a 0.22 μm filter. 100 μL was added to 5 mL of fresh NB liquid medium, and 50 μL of a mixed culture of sensitive rice leaf blight pathogen N1 and phage-resistant rice leaf blight pathogen N1R was added at the same time (N1:N1R = 1:9 ratio).

[0038] S13 was repeated in this way, with an iteration every 24 hours for a total of 12 rounds of culture evolution. After each round of evolution, the phage population was serially diluted and cultured on plates containing N1R-resistant rice leaf blight pathogen to identify N1R-tolerant phage clones. The results are shown in Figure 1 .

[0039] like Figure 1As shown, eroded plaques appeared on the plate, and the plaques were purified, amplified, and preserved, and named as Xanthomonas oryzae phage E12-2. It was deposited in the China Center for Type Culture Collection on March 17, 2025, with the deposit address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number being CCTCC NO: M2025486.

[0040] Example 2 Determination of Biological Properties of Bacteriophage E12-2 of Bacteriophage Bacteriophage of Rice

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

[0042] After the final purification in Example 1, evenly distributed and uniformly sized plaques were obtained. A single plaque was pierced with a pipette tip and the pipette tip was 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 bacteria N1R liquid were inoculated into 5 mL of NB liquid medium, cultured at 30°C for 6 h, and then centrifuged and filtered. The solution was diluted 10-fold with SM liquid until it was finally diluted to 10. -8 , pipette 0.1mL of the last three dilutions of phage dilution solution and mix with 0.1mL of host bacteria N1R, incubate at 30℃ for 20min, and spread on double-layer agar plates. Observe the number of phage plaques and calculate the phage titer according to the following formula:

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

[0044] Three parallel samples were made for each dilution. The average of the three parallel samples was taken for the count. The plate count results showed that the titer of the rice bacterial blight phage E12-2 was 3×10 8 PFU / mL.

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

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

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

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

[0049] Take 10 μL of rice bacterial blight bacteriophage E12-2 suspension (3×10 8 The precipitate (100 μg / mL) was mixed with 1 mL of host bacteria N1R and adsorbed at room temperature for 20 min. The mixture was centrifuged at 13,000 g for 30 s and the supernatant was discarded. The precipitate was washed twice with NB liquid medium to remove free phage that had not adsorbed the host bacteria. The precipitate was added to 5 mL of NB liquid medium and mixed thoroughly. The precipitate was placed on a shaker at 200 rpm / min. 50 μL of the precipitate was sampled every 10 min and centrifuged at 13,000 g for 30 s. The supernatant was used to measure the titer and draw a one-step growth curve.

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

[0051] (4) Determination of the host spectrum of phage E12-2 against bacterial blight pathogen

[0052] Take 1 mL of the bacterial strain to be tested in the middle logarithmic growth 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 form a double plate. After the surface solidified, 2 μL of wild-type phage NP1 and phage E12-2 (1×10 8 PFU / mL) was dropped onto the center of each double plate, dried, and then inverted in 30°C culture medium for overnight culture. 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 was significantly expanded, and the number of host strains it could infect increased from 17 to 32, showing a wider host spectrum characteristic.

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

[0055]

[0056] + means it can be infected; - means it cannot be infected

[0057] Example 3: Use of Bacteriophage E12-2 of Bacteriophage Pathogen to Control Rice White Leaf Disease

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

[0059] The rice bacterial blight pathogen N1 and the resistant strain N1R were cultured to the early logarithmic phase, and 0.5 mL of culture was added to 5 mL of NB liquid medium; 10 μL of wild-type phage NP1 suspension (1×10 8 PFU / mL) and phage E12-2 suspension (1×10 8 PFU / mL). The control group was added with 20 μL of NB liquid culture medium and cultured on a shaker. Samples were taken at various time points in a 96-well plate, OD values were measured, and lysis curves were drawn.

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

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

[0062] The control effect of wild-type phage NP1 and phage E12-2 on rice bacterial blight was 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 plants grew to the three-leaf and one-heart stage (21 days), the leaves were cut and inoculated. 24 hours later, the treatment groups were sprayed with 10 8 A suspension of wild-type phage NP1 and phage E12-2 at a concentration of 500 PFU / mL was inoculated until the leaves were completely wet. A control group was treated with water and pathogen N1 in the same manner and potted. Approximately 15 days after leaf inoculation, the disease index (measured on leaves) was calculated according to the bacterial blight grading standard: 0 (immune): no disease; 1 (highly resistant): lesion area less than 1%-5% of leaf area; 3 (moderately resistant): lesion area 6%-12% of leaf area; 5 (moderately susceptible): lesion area 13%-25% of leaf area; 7 (susceptible): lesion area 26%-50% of leaf area; and 9 (highly susceptible): lesion area 51%-100% of leaf area. The calculation formula is as follows: Disease index = ∑ (number of diseased leaves in each grade × relative grade) / (total number of leaves surveyed × highest grade value) × 100.

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

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

Claims

1. Bacteriophage E12-2 of bacterial blight pathogen of rice, characterized in that The accession number is CCTCCNO:M 2025486.

2. Use of the phage E12-2 against bacterial blight pathogen of rice according to claim 1 in the preparation of an antibacterial agent against bacterial blight pathogen of rice.

3. Use of the phage E12-2 against bacterial blight of rice according to claim 1 in preventing and controlling bacterial blight of rice.

4. A bacteriostatic agent for bacterial blight of rice, characterized in that: The invention comprises the phage E12-2 of bacterial blight pathogen of rice according to claim 1.

5. The antibacterial agent for bacterial blight of rice according to claim 4, characterized in that Agriculturally acceptable adjuvants are also included.

6. The antibacterial agent for bacterial blight of rice according to claim 5, characterized in that The agriculturally acceptable adjuvant is selected from at least one of a dispersant, a stabilizer, a filler and a solvent.

7. A method for preventing and treating rice bacterial blight, characterized in that: Use the rice bacterial blight pathogen phage E12-2 according to claim 1 or the rice bacterial blight pathogen antibacterial agent according to any one of claims 4 to 6.

8. The method for preventing and controlling rice bacterial blight according to claim 7, characterized in that: A suspension of bacterial blight pathogen phage E12-2 or a bacterial blight pathogen antibacterial agent is prepared into a solution and then sprayed on the rice seedlings.

9. The method for preventing and controlling rice bacterial blight according to claim 8, characterized in that: In the solution prepared from the suspension of bacterial blight pathogen phage E12-2 or the bacterial blight pathogen antibacterial agent, the concentration of bacterial blight pathogen phage E12-2 is 1×10 8 ~1×10 9 PFU / mL.

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