Broad-spectrum bacteriophage of ralstonia solanacearum and application thereof
By developing the broad-spectrum, strong cleavage activity bacterium phage YL3, the existing phage has solved the problem of narrow host range and weak cleavage ability, and effectively preventing and treating bacterium phages in various crops.
Patent Information
- Application Number
- CN202411859434.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-02-07
AI Technical Summary
The existing phages have a narrow host range and poor cleavage ability, making it difficult to effectively prevent and treat phages.
A broad-spectrum, strong cleavage bacterium phage YL3 was developed, which is a double-stranded DNA virus that can infect cleavage bacterium from crop sources including tobacco, potatoes, peppers and peanuts, and has efficient cleavage ability.
The broad-spectrum and strong cleavage activity of phage YL3 enable it to effectively prevent and treat blue wilt in a variety of crops, significantly improve the prevention and control effect, and solve the shortcomings of existing phages.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bacterial wilt prevention and treatment, and in particular to a bacterial wilt phage YL3 with broad spectrum and strong lytic activity and application thereof. Background Art
[0002] Ralstonia solanacearum (hereinafter referred to as Ralstonia solanacearum) causes serious bacterial wilt disease worldwide. Ralstonia solanacearum has a wide host range and can infect more than 200 plant species from 50 families, including tobacco, tomatoes, potatoes and peppers. Ralstonia solanacearum is a typical species complex with high variability and complex genetic diversity. Ralstonia solanacearum can be divided into Ralstonia pseudosolanacearum (Originally Asian type 1, African type 3) 、R.solanacearum (formerly American Type II) and R. syzygii subsp. indonesiensis (formerly Indonesian archipelago) 3 species.
[0003] Statistics show that the economic losses caused by bacterial wilt worldwide are about 2 billion US dollars each year, making it the second largest bacterial disease in the world. Due to the huge economic losses it causes, bacterial wilt has become an important target disease for phage therapy. The phages of Ralstonia solanacearum that have been discovered can be divided into four categories: single-stranded RNA phages, double-stranded RNA phages, single-stranded DNA phages, and double-stranded DNA phages, among which double-stranded DNA phages are the main ones. The phages used to prevent and control bacterial wilt are all virulent phages, which can lyse Ralstonia solanacearum in a shorter time, thereby achieving the purpose of quickly reducing its population. The reduction of the number of pathogenic bacteria by phages is the basis for phage therapy to work, which is of great significance to the pathogenic system that is dependent on the number of pathogenic bacteria. The expression of pathogenic characteristics of some pathogenic bacteria depends on the population size, that is, only when the population size reaches a certain threshold will the pathogenic system be activated, virulence factors be secreted, and symptoms be caused. Therefore, the bacteriophage quickly infects and lyses the host cells, which not only directly reduces the possibility of pathogenic bacteria invading the host, but also indirectly reduces their pathogenicity, thereby effectively controlling the occurrence of bacterial wilt and reducing losses due to the disease.
[0004] However, the current Ralstonia solanacearum phages have a narrow host range and weak lysis ability, so it is of great practical significance to provide a Ralstonia solanacearum phage with a broad spectrum and strong lysis activity. Summary of the invention
[0005] In view of this, the present invention provides a broad-spectrum, strong lytic activity Ralstonia solanacearum phage YL3 and its application, which has good effect in preventing and controlling bacterial wilt disease and solves the technical problems of Ralstonia solanacearum phage having a narrow host range and weak lytic ability.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a bacteriophage, which is a broad-spectrum solanacearum phage ( Ralstonia phage), named YL3, and the deposit number is CCTCC NO: M 20242306.
[0007] The above-mentioned bacteriophage YL3 is a double-stranded DNA virus, tailed bacteriophage class ( Caudoviricetes ), Brachyura phage ( Gervaiseviru ), with a polyhedral head and a short tail.
[0008] The present invention also provides a method for culturing the above-mentioned phage, comprising culturing under the condition of MOI=2.
[0009] The present invention also provides the use of the bacteriophage in lysing Ralstonia solanacearum.
[0010] The present invention also provides application of the bacteriophage in preventing and treating bacterial wilt.
[0011] The present invention also provides the use of the bacteriophage in preparing a biocontrol agent for preventing and controlling bacterial wilt.
[0012] The present invention also provides a biocontrol preparation, comprising the above-mentioned bacteriophage, and acceptable adjuvants or auxiliary agents.
[0013] In some specific embodiments of the present invention, the titer of the bacteriophage in the biocontrol agent is 0.5×10 8 PFU / ml~1.5×10 8 PFU / ml can be 0.6×10 8 PFU / ml, 0.7×10 8 PFU / ml, 0.8×10 8 PFU / ml, 0.9×10 8 PFU / ml, 1×10 8 PFU / ml, 1.1×10 8 PFU / ml, 1.2×10 8 PFU / ml, 1.3×10 8 PFU / ml or 1.4×10 8 PFU / ml.
[0014] The present invention also provides application of the biocontrol agent in cracking Ralstonia solanacearum.
[0015] The present invention also provides application of the biocontrol agent in preventing and controlling bacterial wilt.
[0016] In some specific embodiments of the present invention, the bacterial wilt disease mentioned above is at least one of tobacco bacterial wilt, potato bacterial wilt, pepper bacterial wilt, and peanut bacterial wilt.
[0017] The present invention also provides a method for preventing and controlling bacterial wilt, comprising: applying the bacteriophage or the biocontrol agent to the root of crops.
[0018] Compared with the prior art, the present invention has the following beneficial effects: The broad-spectrum Ralstonia solanacearum phage YL3 provided by the present invention has a wide host range and can lyse 34 of 38 strains of Ralstonia solanacearum from different sources, including Ralstonia solanacearum from crops such as tobacco, potato, pepper and peanut. It has strong lysis ability and a wide prevention and control range, and can more effectively solve the bacterial wilt infection occurring during crop planting, and can be used to prevent and control crop bacterial wilt. Biological Deposit Description
[0019] Biological material: Ralstonia solanacearum phage YL3, classification and nomenclature: Ralstonia phage YL3, deposited in the China Center for Type Culture Collection on October 23, 2024, the address of the collection is: Wuhan University, Wuhan, China; the collection number is CCTCC NO: M 20242306. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.
[0021] Figure 1 Shown are plaques of phage YL3 on a double-layer plate of Ralstonia solanacearum; Figure 2 Shown is the thermal stability of YL3 treated at different temperatures for 60 min; Figure 3 Shown is the pH stability of YL3 treated at different pH for 60 min; Figure 4 Shown is the appearance of bacteriophage YL3 observed under a transmission electron microscope; Figure 5 The MOI of YL3 is shown; Figure 6 Shown is the phylogenetic tree of YL3; Figure 7 Shown is a comparison of tobacco bacterial wilt symptoms after YL3 and control treatments. DETAILED DESCRIPTION
[0022] The present invention discloses a broad-spectrum, strong lytic activity of Ralstonia solanacearum phage YL3 and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.
[0023] The first object of the present invention is to provide a broad-spectrum Ralstonia solanacearum phage YL3.
[0024] The second object of the present invention is to provide an application of the broad-spectrum Ralstonia solanacearum phage YL3.
[0025] The above object of the present invention is achieved through the following technical solutions: A broad-spectrum Ralstonia solanacearum phage YL3, which was deposited in the China Center for Type Culture Collection on October 23, 2024, at Wuhan University, Wuhan, China, with a collection number of CCTCC NO: M 20242306.
[0026] The present invention isolated a solanacearum phage from the rhizosphere soil of tobacco bacterial wilt diseased plants in Longshan County, Xiangxi Autonomous Prefecture, Hunan Province, which can produce obvious plaques on a double-layer plate containing host bacteria and is named as phage YL3. Genomic results show that the phage YL3 is a double-stranded DNA virus, tailed phage class ( Caudoviricetes ), Brachyura phage ( Gervaiseviru ).
[0027] Through infection experiments on Ralstonia solanacearum strains from different host sources, the present invention found that phage YL3 has a wide host range and can infect 34 of the 38 strains of Ralstonia solanacearum tested, including Ralstonia solanacearum from crops such as tobacco, potato, pepper and peanut. Large plaques can be produced on double-layer Ralstonia solanacearum plates, indicating that phage YL3 can lyse a wide variety of Ralstonia solanacearum in crops, with a wide range of prevention and control, and has a wide lysis spectrum. At the same time, biological characteristics show that the titer of phage YL3 is stable within 30-60°C, and the phage titer remains at 10 after being treated at 60°C for 60 min. 10 PFU / ml, the lethal temperature of YL3 is 80℃; under the condition of pH 4-11, its titer remains at 10 7 PFU / ml or more. Therefore, the phage has a higher temperature tolerance and a wider acid-base stability, which is conducive to its adaptation to a wider range of environmental conditions.
[0028] Therefore, the present invention also provides the use of the broad-spectrum bacteriophage YL3 in preventing and controlling bacterial wilt of crops.
[0029] The present invention also provides the use of the broad-spectrum bacteriophage YL3 in preparing a biocontrol agent for preventing and controlling bacterial wilt of crops.
[0030] Preferably, the titer of bacteriophage YL3 in the preparation is ≥10 8 PFU / ml.
[0031] The present invention also provides use of any of the above-mentioned preparations in preventing and treating bacterial wilt of crops.
[0032] Preferably, the crop bacterial wilt is one or more of tobacco bacterial wilt, potato bacterial wilt, pepper bacterial wilt and peanut bacterial wilt.
[0033] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.
[0034] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.
[0035] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.
[0036] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.
[0037] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.
[0038] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.
[0039] NB medium: tryptone 10 g, beef extract 3 g, glucose 10 g, NaCl 5 g, 1000 ml ddH2O.
[0040] SM buffer: 50 mM Tris-CL, pH=7.5, 100 mM NaCl, 10 mM MgSO4, 0.01% gelatin solution.
[0041] The present invention will be further described below in conjunction with the embodiments. Example 1: Isolation and purification of bacteriophage
[0042] 3 ml of sterile water was added to 1 g of rhizosphere soil sample from tobacco fields in Longshan County, Xiangxi Autonomous Prefecture, and the slurry was vortexed and homogenized. The slurry was allowed to stand for 30 min, and then centrifuged at 13000 r / m for 8 min at room temperature. The supernatant was collected and sterilized by filtration with a 0.22 μm filter. The filtrate and the RS-12 solanacearum solution provided in Table 1 were mixed in equal proportions and added to a triangular flask containing 20 mL of NB liquid culture medium. The mixture was cultured overnight at 30°C and 170 r / m. 10 mL of the co-culture solution was centrifuged, the supernatant was sterilized by filtration with a 0.22 μm aqueous filter, and gradiently diluted to 10 -2 ~10 -4 Then, 100 μL of Ralstonia solanacearum RS-12 and 100 μL of phage gradient dilution were taken, vortexed and mixed, and then spread on double-layer plates and cultured at 30°C for 12 h. The plaques were picked and added to 1.6 mL of NB liquid medium, followed by inoculation of 20 μL of Ralstonia solanacearum RS-12, and cultured at 30°C and 170 r / m until the turbid bacterial solution became clear and filamentous lysates could be observed. The bacterial solution was centrifuged at 13,000 r / m for 7 min, the supernatant was filtered through a 0.22 μm filter, and gradiently diluted to 10 -8 ~10 -9 , take 50 μL of the host bacteria and phage dilutions, vortex mix, spread on double-layer plates, and culture at 30°C until plaques appear. Repeat the above operation 4 times and name the purified phage YL3 ( Figure 1 ).
[0043] Example 2: Host range determination of bacteriophage YL3
[0044] 38 strains of R. solanacearum isolated from tobacco, pepper, potato and peanut were streaked and activated, and single colonies were picked and inoculated into 5 mL NB liquid medium at 30°C and 170 r / m for overnight shaking. The overnight cultured R. solanacearum strains and phage culture medium were inoculated into 10 mL NB at an equal ratio of 0.5% (V / V), and the control was made without phage culture medium. After co-culture at 30°C and 180 r / m for 6 h, the OD of the culture medium of the treatment group and the control group was measured. 600 . According to the OD of the culture medium after lysis 600 The ratio of the mean to CK defined the lysis efficiency (+: lysis efficiency ≤40%; ++: lysis efficiency 40%-80%; +++: lysis efficiency ≥80%). The lysis activity of bacteriophage YL3 against different Ralstonia solanacearum strains is shown in Table 1 .
[0045] Table 1: Lysis spectrum and lytic activity of bacteriophage YL3
[0046] Note: +++: plaques are obviously bright; ++: plaques are obvious; +: plaques are lighter.
[0047] The results showed that YL3 could lyse 34 of the 38 tested R. solanacearum strains, with a lysis rate of 89.47%, and had strong lysis activity against 26 of them, indicating that YL3 has a wide lysis spectrum and high lysis activity against R. solanacearum isolated from tobacco, potato, pepper and peanut.
[0048] Example 3: Determination of biological properties of bacteriophage YL3
[0049] ① Temperature tolerance of YL3: Prepare the bacterial culture medium and phage YL3 culture medium as described above. The phage was placed in a water bath at 30℃, 37℃, 50℃, 60℃, 70℃, and 80℃ for 60 min, and each temperature gradient was repeated 3 times. The titer was determined by the double-layer plate method ( Figure 2 The results showed that YL3 had good thermal stability in the range of 30-60℃, and the lethal temperature was 80℃.
[0050] ② YL3 tolerance to acid and alkali: Prepare the culture medium of Ralstonia solanacearum and phage YL3 according to the above method. Adjust the pH of water to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0 (990 μL) with NaOH or HCl, then add 10 μL of phage, and incubate in 30℃ water bath for 60 min. Repeat 3 times for each treatment, and determine the titer by double-layer plate method ( Figure 3 The results showed that the activity of YL3 remained stable in the pH range of 4-11, indicating that YL3 has good tolerance to the above pH.
[0051] ③ Observe the appearance of the phage using a transmission electron microscope: Prepare the phage YL3 culture solution and concentrate it according to the above method. Drop 10 μL of YL3 on a copper grid and precipitate it for 1 min. After absorbing the floating liquid, cover the phage liquid surface with 10 μL of phosphotungstic acid staining solution for 1 min. After absorbing the excess liquid, dry it at room temperature for 10 min. The results of transmission electron microscopy showed that the head of phage YL3 was polyhedral, about 50 nm wide, and the tail was short, about 30 nm long. According to the classification standards of the International Committee on Taxonomy of Viruses (ICCV), it was identified as Brachyuraviridae ( Figure 4 ).
[0052] Example 4: Optimal multiplicity of infection (MOI) of bacteriophage YL3
[0053] The optimal infection multiplicity of phage YL3 was determined according to Wei’s method (2017) and the OD of the co-culture solution was measured using a microplate reader (TECAN spark) from 0 to 12 h. 600 The results showed that the optimal infection multiplicity of phage YL3 was 2. The lysis curve of phage against RS-12 was determined according to the optimal MOI ratio. It was found that the OD of the co-culture solution within 4 h after adding phage YL3 was 2. 600 It dropped below 0.074 at 5 h, briefly rebounded to 0.135 at 5 h, and dropped again to 0.053 1 h later. The OD of the control (RS-12) 600 It continued to increase within 12 hours, reaching 0.429 at 3 hours and 1.128 at 12 hours. The experimental results showed that when MOI=2, phage YL3 strain can produce more progeny phages ( Figure 5 ). Example 5: Genome sequencing and phylogenetic analysis of bacteriophage
[0054] The genomic nucleic acid of bacteriophage YL3 was extracted according to the method of Beijing Tiangen DP-315 viral genomic DNA extraction kit and sent to Wuhan Fraser Gene Information Co., Ltd. for sequencing. The genome sequence of YL3 was compared with the existing phage sequences in the NCBI database, and the phylogenetic tree was established using MEGA 10.0 with Pseudomonas phage Itty13 as the outgroup. It was found that YL3 and Ralstonia PhageAhaGv was on the same branch, thus YL3 was identified as a short-tailed phage ( Figure 6 ). Example 6: Potted plant experiment of bacteriophage YL3 to control tobacco bacterial wilt
[0055] The pot experiment was conducted with two treatments, three replicates per treatment, and 16 tobacco seedlings per replicate. 600Adjust to 0.1, and inoculate 10 mL of Ralstonia solanacearum solution per tobacco seedling. Prepare bacteriophage YL3 culture solution according to the above method and dilute it to 1×10 8 PFU / ml, 50 mL of phage was inoculated per tobacco seedling. The greenhouse temperature was set at 30°C, and the light / dark period was 12 h / 12 h. 14 days after inoculation, the disease level of each tobacco seedling was investigated plant by plant according to the national standard GB / T 23222-2008, and the disease index and control effect were calculated. The results are shown in Table 2. Figure 7 shown.
[0056] Table 2: Potted control effect of bacteriophage YL3 on tobacco bacterial wilt
[0057] The results showed that bacteriophage YL3 had a good control effect on bacterial wilt, with a control efficiency of 93.98%, which was a good control effect.
[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A bacteriophage, characterized in that The deposit number is CCTCC NO: M 20242306.
2. Use of the bacteriophage as claimed in claim 1 in lysing Ralstonia solanacearum.
3. Use of the bacteriophage as claimed in claim 1 in preventing and treating bacterial wilt.
4. Use of the bacteriophage according to claim 1 in the preparation of a biocontrol agent for preventing and controlling bacterial wilt.
5. A biocontrol agent, characterized in that: The invention comprises the bacteriophage according to claim 1, and acceptable auxiliary materials or additives.
6. The biocontrol agent according to claim 5, characterized in that The titer of the phage is 0.5×10 8 PFU / ml~1.5×10 8 PFU / ml.
7. Use of the biocontrol agent according to claim 5 or 6 in lysing Ralstonia solanacearum.
8. Use of the biocontrol agent according to claim 5 or 6 in preventing and controlling bacterial wilt.
9. The use according to claim 3, 4 or 8, characterized in that The bacterial wilt disease is at least one of tobacco bacterial wilt disease, potato bacterial wilt disease, pepper bacterial wilt disease, and peanut bacterial wilt disease.
10. A method for preventing and controlling bacterial wilt, characterized in that: include: Apply the bacteriophage according to claim 1 or the biological control agent according to claim 5 or 6 to the roots of crops.