A kind of solanacearum phage with high lysis rate and its application
By screening and optimizing the isolation and fermentation conditions of RSP46 of the phage, the problems of insufficient genome information and limited host range of the phage are solved, and efficient and safe phage prevention and control effects are achieved. It is suitable for plants of the Solanaceae family Biological control.
Patent Information
- Application Number
- CN202410059586.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-16
- Publication Date
- 2024-12-13
- Estimated Expiration
- 2044-01-16
AI Technical Summary
In the prior art, the genomic information of phages of phages is lacking in detail, and the host range of different phages is limited, making it difficult to effectively prevent and control the problem of phage infection in crop planting.
A high-cleavage bacterium phage RSP46 was screened out, and the specific isolation, purification and fermentation conditions were optimized to ensure its high titer and stability, and was used to prevent and treat blue wilt from the Solanaceae plants.
This phage has high specificity, has no destructive effect on other bacterial groups in the soil, is safe and has no residue, can effectively prevent and treat bacterium wilt, and remains active under different temperatures and pH conditions, making it suitable for industrial preparation and application.
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Figure CN118086221B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, in particular to a solanacearum phage with a high lysis rate and an application thereof. Background Art
[0002] Plant bacterial wilt is a devastating soil-borne disease caused by Ralstonia solanacearum. Traditional methods to deal with bacterial wilt include physical control methods such as adsorption, high temperature and anaerobic sterilization; chemical methods such as spraying, root irrigation, fumigation, etc. can also be used to sterilize; biological control methods can also be used for treatment. Ralstonia solanacearum phage is a potential biological control resource for bacterial wilt, and its high host specificity and green safety make it a great advantage.
[0003] Application CN108642018A discloses a solanacearum phage and its application. The phage can be used to prevent or treat tomato solanacearum wilt and has the characteristics of high efficiency, non-toxicity and strong specificity.
[0004] Application CN112831475A discloses a bacteriophage of Ralstonia solanacearum and its application. The bacteriophage is applied into the soil by root irrigation. The bacteriophage inhibits soil-borne tobacco bacterial wilt, thereby preventing and controlling soil-borne tobacco bacterial wilt while avoiding damage to common microbial communities in the soil.
[0005] Application CN202310627601.4 discloses a broad-spectrum Ralstonia solanacearum phage PTY21. The Ralstonia solanacearum phage PTY21 has a wide host range and can infect 76 of 106 strains of Ralstonia solanacearum from different host sources. It also has strong lysis ability and can more effectively solve the problem of Ralstonia solanacearum infection in crop cultivation.
[0006] At present, there are still few studies on the use of bacteriophages to control bacterial wilt disease. Using bacteriophages to lyse pathogens to control bacterial wilt disease has broad application prospects. However, a large number of bacterial wilt phages still lack detailed genome information, and the host range of different bacterial wilt phages is limited. Therefore, in order to more effectively solve the problem of bacterial wilt infection in crop cultivation, the present invention provides a bacterial wilt phage with a high lysis rate and its application. Summary of the invention
[0007] In order to deal with the problem of Ralstonia solanacearum infection in crop planting, one of the purposes of the present invention is to provide a Ralstonia solanacearum bacteriophage RSP46 with a high lysis rate;
[0008] The second object of the present invention is to provide the use of Ralstonia solanacearum bacteriophage in the preparation of biological medicines for preventing and treating bacterial wilt of Solanaceae plants.
[0009] In order to achieve the above-mentioned purpose, the present invention is implemented through the following technical scheme: the applicant screened out a strain of Ralstonia solanacearum phage RSP46 from tobacco leaf samples from a tobacco planting base in Yongzhou, Hunan, and the phage was sent to the China Center for Type Culture Collection for preservation on August 14, 2023, address: Wuhan University, Wuhan, China; the preservation number is: CCTCC NO: M20231467, and the classification name is: Ralstonia solanacearum phage (Ralstonia solanacearum phage) RSP46.
[0010] Furthermore, the titer of the phage is 6.05×10 10 PFU / mL.
[0011] An application of the above-mentioned bacterial wilt phage is used for preventing and controlling bacterial wilt of Solanaceae plants.
[0012] Beneficial effects of the present invention:
[0013] 1. The high lysis rate Ralstonia solanacearum phage RSP46 has strong specificity, has no destructive effect on other bacterial flora in the soil, and will not break the microbial balance in the soil; compared with antibiotics and chemical preservatives, it has the characteristics of high specificity, no residue and safety.
[0014] 2. The high lysis rate RSP46 bacteriophage of Ralstonia solanacearum can be used to prevent and treat bacterial wilt of Solanaceae plants, and can be used for the prevention, control and treatment of plant bacterial wilt. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is a picture of bacteriophage RSP46 plaques;
[0016] Figure 2 is the titer of Ralstonia solanacearum phage at different infection multiplicity;
[0017] Figure 3 The lysis effect of bacteriophage RSP46 on Ralstonia solanacearum from different sources;
[0018] Figure 4 is the titer of RSP46 after being stored at different temperatures for different time periods;
[0019] Figure 5 This is the result of the pH stability experiment of RSP46 phage. DETAILED DESCRIPTION
[0020] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.
[0021] See also Figures 1 to 5 , the present invention provides the following technical solutions:
[0022] Embodiment 1:
[0023] Isolation and Purification of Bacteriophage RSP46 from Ralstonia solanacearum
[0024] Several samples of stems and leaves of tobacco plants suspected of bacterial wilt were collected, and 1 g of each sample was taken. After surface disinfection with 75% alcohol, it was ground in a mortar until viscous and mixed with 9 mL of sterile water. The mixture was placed on a constant temperature shaker at 30 °C and 200 r / min for 2 h, centrifuged at 6000 × g for 10 min, and the supernatant was filtered. 2 mL of the filtrate was mixed with 2 mL of 2 × TSB liquid culture medium and 200 uL of Ralstonia solanacearum culture medium in the logarithmic phase (10 8 cfu / mL) and cultured overnight at 200rpm at 30℃ to enrich phages. The sample enrichment solution was centrifuged at 6000×g for 10min, and the supernatant was sterilized by a 0.22μm microporous filter membrane to obtain a filtrate containing phages. The filtrate was gradiently diluted, and 1mL of the dilution was mixed evenly with 400uL of its host Ralstonia solanacearum solution, and allowed to stand for 15min to allow it to fully bind to the receptors on the bacterial surface. The above mixture was added to 7mL of TSB semi-solid agar medium cooled to 50℃, and immediately spread on the solidified nutrient agar bottom plate and mixed evenly. After the semi-solid agar medium solidified, it was inverted and cultured at 30℃ for 17-24h to observe the growth of phage plaques. On the double-layer plate where plaques are formed, pick up a large and transparent single plaque with a sterile pipette tip, inoculate it in 3mL TSB liquid culture medium, add 200uL of Ralstonia solanacearum liquid and mix well, culture overnight at 30℃ and 200rpm, centrifuge at 6000×g for 10min, take the supernatant and filter it through a 0.22μm microporous membrane, and observe the plaque morphology by the double-layer plate method. Repeat the operation 3-5 times to obtain plaques of uniform shape and size.
[0025] A phage was isolated, which produced a single circular plaque on the lawn of Ralstonia solanacearum with a diameter of about 2 mm. It was sent to the China Center for Type Culture Collection in 2023 for preservation, and the classification name was: Ralstonia solanacearum phage RSP46, address: Wuhan University, Wuhan, China, preservation number: CCTCC NO: M 20231467.
[0026] Embodiment 2:
[0027] Determination of the Optimal Fermentation Conditions of Bacteriophage RSP46 of Ralstonia solanacearum
[0028] A single colony of the host Ralstonia solanacearum was picked up and placed in 3mL TSB liquid culture medium, and cultured at 200rpm in a 30℃ shaker for 48h to obtain a host bacterial suspension. The bacterial suspension was transferred to 10mL TSB culture medium at a ratio of 1:100, and cultured at 30℃ and 200rpm to the early logarithmic stage. The phage RSP46 and its host Ralstonia solanacearum were diluted to a certain concentration and counted, mixed and cultured in different proportions, and TSB liquid culture medium was added to make the total volume of each tube the same. Four copies were prepared for each concentration ratio, and cultured at 200rpm for 17h at 25℃, 28℃, 32℃ and 37℃ respectively. After the culture was completed, the fermentation broth was centrifuged at 6000×g for 10min and the supernatant was collected. The supernatant was passed through a 0.22μm microporous filter membrane and counted by the double-layer plate method. Each point was cultured in duplicate tubes and the average value was taken. The condition that produced the highest phage titer was the optimal fermentation condition. The experiment was repeated 3 times.
[0029] The results are as follows Figure 2 As shown, when the concentration of bacteriophage RSP46 was 1.7×10 4 PFU / mL, host bacteria concentration is 6.5×10 7 CFU / mL, MOI was 0.00026, and the fermentation temperature was 30°C. The fermentation titer of bacteriophage RSP46 reached a maximum of 6.05×10 10 PFU / mL.
[0030] Embodiment 3:
[0031] Experiment on the Lysis Range of Bacteriophage RSP46 against Ralstonia solanacearum
[0032] The drop method was used to determine the lysis effect of RSP46 on Ralstonia solanacearum. 116 strains of Ralstonia solanacearum from different sources were prepared and inoculated in centrifuge tubes containing 2 mL of TSB. They were cultured at 30°C and 200 rpm for 24 hours to obtain bacterial suspensions of each strain. 400 μL of bacterial suspension was mixed with TSB semi-solid medium and spread on the prepared nutrient agar plate. After solidification, 5 uL was dropped with a titer of 1×10 9 PFU / mL of RSP46 phage. After drying, place at 30℃ for 24h and observe the results. Repeat the experiment three times.
[0033] The results are as follows Figure 3 As shown, phage RSP46 can lyse 98 of 116 strains of Ralstonia solanacearum from Solanaceae plants, with a lysis rate of 84.5%. The lysis rate of Ralstonia solanacearum from different plant sources tested was as high as 83%, indicating that the phage has a high lysis rate and has great application potential in preventing and controlling infections caused by Ralstonia solanacearum in Solanaceae plants.
[0034] Embodiment 4:
[0035] Temperature stability test of RSP46 bacteriophage of Ralstonia solanacearum
[0036] Take 9 mL of TSB liquid culture medium and place it in a sterile 15 mL centrifuge tube. Heat the tube in a constant temperature water bath until the temperature reaches the corresponding temperature. After the temperature is balanced, add 1 mL of 1×10 10 The pure culture solution of solanacearum phage was exposed to 4℃, 25℃, 37℃, 45℃, 60℃, 70℃ and 80℃ for 1h, 2h, 7h, 24h, 48h and 72h respectively. After the exposure, the centrifuge tube was taken out and cooled immediately, and the phage titer was determined by the double-layer plate method. Duplicate culture was performed at each point to obtain the average value, and the experiment was repeated 3 times.
[0037] The results are as follows Figure 4 As shown, the activity of R. solanacearum phage RSP46 changes little at temperatures below 37°C, and it still has a high titer after being in a 45°C water bath for 72 hours. It has good temperature stability at 4-37°C and can be stored for a long time.
[0038] Embodiment 5:
[0039] pH Stability Test of Ralstonia solanacearum Phage RSP46
[0040] Sterile water was adjusted to different pH values (1-13), filtered with a 0.22 μm microporous membrane, and 100 μL of phage fermentation broth (2×10 10 PFU / mL), mixed evenly, and allowed to stand at 25°C for 2 h before determining the phage titer.
[0041] The results are as follows Figure 5 As shown, the titer of phage RSP46 changed little under the conditions of pH 3-10, and had good pH stability.
[0042] Example 6: Experiment on the lysis range of bacteriophage RSP46 on non-host bacteria
[0043] The drop method was used to determine the lysis effect of RSP46 on non-host bacteria in the soil. 8 strains of Thiobacillus ferrooxidans, 5 strains of Thiobacillus denitrificans, 14 strains of Bacillus proteus vulgaris, 15 strains of Pseudomonas aeruginosa, 9 strains of Bacillus cereus, 8 strains of Bacillus licheniformis, 10 strains of Bacillus subtilis, and 12 strains of Klebsiella pneumoniae were prepared and inoculated into centrifuge tubes containing 2 mL of TSB, and cultured at 30°C and 200 rpm for 24 hours to obtain bacterial suspensions of each strain. Take 400 μL of bacterial suspension and mix with TSB semi-solid medium and spread on the prepared nutrient agar plate. After solidification, drop 5uL of the titer is 1×10 9 PFU / mL of RSP46 phage. After drying, place at 30℃ for 24h and observe the results. Repeat the experiment three times.
[0044] The results are shown in Table 1. Bacteriophage RSP46 was unable to lyse the 81 soil-derived non-host Ralstonia solanacearum strains tested, indicating that bacteriophage RSP46 has strong specificity and will not affect the normal soil flora. It can provide strain resources for the development of green and safe biological control agents.
[0045] Table 1 Lysis results of R. solanacearum phage RSP46
[0046]
[0047]
[0048] Note: “-” indicates no lysis; “+” indicates lysis with blurred lysis spots; “++” indicates lysis with clear lysis spots.
[0049] Embodiment 7:
[0050] Preparation of bacteriophage RSP46 by fermentation:
[0051] Pick a single colony of the host bacteria, inoculate it into a test tube containing 3mL TSB culture medium, and shake and culture it at 200rpm in a 30℃ shaker for 48h to obtain a host bacteria suspension. The bacterial suspension was transferred to 500mL TSB culture medium at a ratio of 1:50, and cultured at 30℃ and 200rpm to the early logarithmic stage and the concentration of the bacterial suspension was determined. The fermentation system was 6L, and the fermentation medium was TSB medium. Flame inoculation was used to inoculate, and phages and host bacteria were inoculated into the fermenter under the optimal fermentation conditions. Sterile air was introduced during the fermentation process, and 3‰ defoaming agent was added. The fermentation preparation time was 17h. After the fermentation was completed, all the mixed solutions of phages and host bacteria were taken out from the sampling port and placed in a sterile container, centrifuged at 6000rpm for 15min, and the supernatant was filtered into a sterile filter device through a vacuum pump to obtain phage fermentation liquid and stored at 4℃.
[0052] As shown in Table 2, after fermentation at 30°C for 17 h, the titer of bacteriophage RSP46 can reach 2.0×10 10 PFU / mL. Therefore, it is feasible to use the fermentation method for large-scale industrial preparation of bacteriophages, which provides a feasible method for the industrial production and promotion and application of the bacteriophages.
[0053] Table 2 Fermentation dynamics of Ralstonia solanacearum phage RSP46
[0054]
[0055] Embodiment 8:
[0056] Application of bacteriophage RSP46 in the prevention and control of tobacco bacterial wilt:
[0057] 120 tobacco seedlings with healthy appearance and 3-4 true leaves were selected and randomly divided into 6 groups, namely RSP46 prevention group, RSP46 treatment group, Cu(OH)2 prevention group, Cu(OH)2 treatment group, infection group and blank group. The treatments were as follows:
[0058] (1) RSP46 prevention group: phage 5.0×10 6 PFU / mL, irrigate the roots with 50mL / plant, and after 24 hours, cut once on both sides of the taproot of the tobacco seedlings with a sterilized blade and irrigate with tobacco solanacearum RS52 (1.0×10 8 cfu / mL) suspension 20mL / strain.
[0059] (2) RSP46 treatment group: A sterilized blade was used to cut once on each side of the taproot of the tobacco seedlings, and the tobacco wilt fungus RS52 (1.0×10 8 cfu / mL) suspension 20mL / strain, 12h later, phage 5.0×10 7 PFU / mL, root irrigation 50mL / plant.
[0060] (3) Cu(OH)2 prevention group: 30% Cu(OH)2 dispersant was diluted 1000 times and irrigated with 50 mL / plant. After 24 h, a sterilized blade was used to cut one cut on each side of the main root of the tobacco seedlings and then irrigated with tobacco solanacearum RS52 (1.0×10 8 cfu / mL) suspension 20mL / strain.
[0061] (4) Cu(OH)2 treatment group: A sterilized blade was used to cut one cut on each side of the taproot of the tobacco seedlings, and the tobacco wilt fungus RS52 (1.0×10 8 cfu / mL) suspension 30mL / plant, after 24h, 30% Cu(OH)2 dispersant was diluted 1000 times and irrigated with 50mL / plant.
[0062] (5) Infection group: Use a sterilized blade to cut once on each side of the main root of the tobacco seedlings, and water with tobacco wilt fungus RS52 (1.0×10 8 cfu / mL) suspension 20mL / strain.
[0063] (6) Blank group: irrigate with 50 mL of sterile water per plant. After 24 hours, make a cut on each side of the main root of the tobacco seedlings and irrigate with 20 mL of sterile water per plant.
[0064]
[0065]
[0066] The results are shown in Table 3: Bacteriophage RSP46 can effectively reduce the incidence and disease index of tobacco bacterial wilt, control the further infection of Ralstonia solanacearum, and the control effect is better than CU(OH)2 dispersant. This shows that bacteriophage has great potential in replacing chemical fungicides to control Ralstonia solanacearum, and can be developed into green and environmentally friendly biological agents to prevent and control Ralstonia solanacearum infection in Solanaceae plants.
[0067] Table 3: Effects of using bacteriophage RSP46 in tobacco cultivation
[0068]
[0069] Note: The disease index of each treatment group was calculated according to the national standard GB / T 23222-2008.
[0070] Level 0: Healthy, no diseased leaves.
[0071] Level 1: There are 1 to 5 lesions on each leaf.
[0072] Level 3: There are 6-10 spots on each leaf.
[0073] Level 5: There are 11-15 spots on each leaf.
[0074] Level 7: There are 16-20 spots on each leaf.
[0075] Level 9: Each leaf has more than 21 spots
[0076] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.
[0077] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
Claims
1. A solanacearum phage ( Ralstonia solanacearum phage )RSP46, characterized by: The applicant screened out a strain of R. solanacearum phage RSP46 from tobacco leaf samples from a tobacco planting base in Yongzhou, Hunan. The phage was sent to the China Center for Type Culture Collection for preservation on August 14, 2023. The preservation address is: Wuhan University, Wuhan, China; the preservation number is CCTCC NO: M 20231467.
2. A use of the solanacearum phage RSP46 as claimed in claim 1, characterized in that: Used to prevent and control bacterial wilt of Solanaceae plants.