A bacteriophage of Ralstonia solanacearum and its application in the control of tobacco bacterial wilt

Abstract

The present invention relates to the field of biotechnology, and in particular to a solanacearum phage and its application in preventing and controlling tobacco bacterial wilt. The present invention discloses a solanacearum phage and its application in preventing and controlling tobacco bacterial wilt. The solanacearum phage is named YL2, classified as Ralstonia phage, and has a preservation number of CCTCC NO: M 20242305. The phage has a wide lysis spectrum and good lysis activity against solanacearum of Solanaceae crops, has a good prevention and control effect on tobacco bacterial wilt caused by solanacearum, and can become a phage source for preventing and controlling tobacco bacterial wilt.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a solanacearum phage and application thereof in the prevention and treatment of tobacco bacterial wilt. Background Art

[0002] Tobacco bacterial wilt, caused by infection with Ralstonia pseudosolanacerum, is the largest bacterial disease in tobacco production, causing economic losses of more than 1 billion yuan to my country's tobacco agriculture every year. Tobacco bacterial wilt is a typical high-temperature and high-humidity pathogen, and is more serious in flue-cured tobacco growing areas in the middle and lower reaches of the Yangtze River, such as Hunan, Hubei, Chongqing, Jiangxi, Fujian and Anhui. In addition, it is also more serious in dryland tobacco areas in Guizhou and Yunnan. Bacterial wilt has become one of the main obstacles to the sustainable development of tobacco agriculture in the above-mentioned main tobacco-producing areas.

[0003] The prevention and control of tobacco bacterial wilt is extremely difficult. Bacterial wilt overwinters in the soil. After infecting the root system, it does not cause disease immediately, but colonizes in the xylem vessels. After a long incubation period, when the temperature and humidity meet the disease, it enters the peak period of disease. Since the time from the onset of symptoms to the death of tobacco plants is very short, it is difficult to achieve a good prevention and control effect through traditional chemical control or biological control, so it is also called plant cancer. Studies have shown that the population size of Ralstonia solanacearum in the soil is positively correlated with the incidence and severity of bacterial wilt. Reducing the population size of Ralstonia solanacearum in the soil can significantly reduce the occurrence of the disease.

[0004] Ralstonia solanacearum phage is a virus that specifically infects Ralstonia solanacearum. It has the characteristics of fast infection speed, short lysis time and strong host specificity. It can quickly reduce the population of Ralstonia solanacearum in the environment in a short period of time and has no negative impact on non-host organisms. Therefore, Ralstonia solanacearum phage is considered to be a high-quality biocontrol resource for effectively preventing and controlling bacterial wilt.

[0005] The genetic variation of Ralstonia solanacearum is frequent. The results of pan-genomic studies show that the core genome of Ralstonia solanacearum only accounts for about 42.7% of its total genes, and more than 50% of its genes come from horizontal DNA transfer or other pathways, which can easily lead to its escape from phage infection and lysis. Because of this, the mining and application of Ralstonia solanacearum phages follow the idea of ​​"from farm to laboratory, and then to farm". Therefore, providing updated Ralstonia solanacearum phages has important practical significance. Summary of the invention

[0006] In view of this, the present invention provides a bacterial wilt phage of Ralstonia solanacearum and its application in the prevention and treatment of tobacco bacterial wilt, which provides a phage source for the application of phage preparations in production to prevent and control tobacco bacterial wilt.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a bacteriophage named YL2, classified and named Ralstonia phage, which was deposited in China Center for Type Culture Collection on October 23, 2024, with the address being: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, with the deposit number being: CCTCC NO: M 20242305.

[0009] The above-mentioned bacteriophage YL2 belongs to the class Caudoviricetes, the genus Gervaiseviru, and has a polyhedral head and a short tail.

[0010] The complete genome sequence of the above-mentioned phage YL2 is 60,673 bp long.

[0011] The above phage YL2 was treated at 60℃ for 60 min, and the phage titer remained at 10 10 PFU / ml, its titer remains at 10 7 PFU / ml and above.

[0012] The present invention also provides the use of the bacteriophage in lysing Ralstonia pseudosolanacerum.

[0013] The present invention also provides application of the bacteriophage in preventing and treating bacterial wilt.

[0014] In some specific embodiments of the present invention, the bacterial wilt disease mentioned in the above application is tobacco bacterial wilt disease.

[0015] The present invention also provides a biocontrol preparation, comprising the above-mentioned bacteriophage, and acceptable adjuvants or auxiliary agents.

[0016] 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 1.4×108 PFU / ml.

[0017] In some specific embodiments of the present invention, the titer of the bacteriophage in the biocontrol agent is 10 8 PFU / ml.

[0018] The present invention also provides application of the biocontrol agent in cracking Ralstonia solanacearum.

[0019] The present invention also provides application of the biocontrol agent in preventing and controlling bacterial wilt.

[0020] In some specific embodiments of the present invention, the bacterial wilt disease mentioned in the above application is tobacco bacterial wilt disease.

[0021] Compared with the prior art, the beneficial effects of the present invention include:

[0022] (1) The present invention separates and purifies a tobacco bacterial wilt bacteriophage YL2, which has a wide lysis spectrum and good lysis activity against bacterial wilt pathogens prevalent in the tobacco growing areas of northwestern Hunan, providing a high-quality phage source for the use of phages to control bacterial wilt in tobacco growing areas of northwestern Hunan;

[0023] (2) After the bacteriophage YL2 provided by the present invention was treated at 60°C for 60 min, the phage titer remained at 10 10 PFU / ml, its titer remains at 10 7 PFU / ml or more, so 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;

[0024] (3) The present invention directly uses bacteriophage YL2 to prevent and control tobacco bacterial wilt. The application is carried out by root irrigation, which can significantly reduce the incidence rate and disease index, and has a good control effect on tobacco bacterial wilt.

[0025] Biological Deposit Description

[0026] Biological material: Ralstonia phage YL2, classification name: Ralstonia phage, deposited in the China Center for Type Culture Collection on October 23, 2024, the address of the collection center is: Wuhan University, Wuhan, China; the collection number is CCTCCNO: M 20242305. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] Figure 1 is the plaque image of bacteriophage YL2;

[0029] Figure 2 The appearance characteristics of bacteriophage YL2 observed by transmission electron microscopy;

[0030] Figure 3 Shown is the phylogenetic tree of bacteriophage YL2;

[0031] Figure 4 Shown is the temperature stability diagram of bacteriophage YL2;

[0032] Figure 5 Shown is the pH stability diagram of bacteriophage YL2;

[0033] Figure 6 Shown is a potted plant picture of bacteriophage YL2 controlling tobacco bacterial wilt. DETAILED DESCRIPTION

[0034] The present invention discloses a strain of Ralstonia solanacearum phage and its application in the prevention and treatment of tobacco bacterial wilt. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed 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.

[0035] The present invention isolated and identified a bacterial wilt bacteria phage YL2 from the rhizosphere soil of tobacco bacterial wilt diseased plants in the northwestern Hunan tobacco area. The phage has a wide lysis spectrum and good lysis activity against the bacterial wilt pathogen prevalent in the northwestern Hunan tobacco area, and has good application prospects for the prevention and control of tobacco bacterial wilt in the area. It was deposited in the China Center for Type Culture Collection on October 23, 2024, with the address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, and the preservation number is: CCTCC NO: M 20242305.

[0036] Furthermore, phage YL2 belongs to the order Caudovirales, genus Brachyura, with a regular polyhedral head of about 50 nm wide and a tail of about 30 nm long.

[0037] Furthermore, the phage YL2 titer remained at 10 after being treated at 60°C for 60 min. 10 PFU / ml, its titer remains at 10 7 PFU / ml and above.

[0038] The present invention also provides application of the above-mentioned Ralstonia solanacearum bacteriophage in preventing and controlling tobacco bacterial wilt disease.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In the following examples, the solid culture medium is NB, and its formula is (tryptone 10 g, beef extract 3 g, glucose 10 g, NaCl 5 g, 1000 ml ddH2O):

[0046] In the following examples, the SM buffer formula is (50 mM Tris-CL, pH=7.5, 100 mM NaCl, 10 mM MgSO4, 0.01% gelatin solution):

[0047] In the following examples, the specific operation steps of the double-layer plate method are as follows: in a sterile operating table, take an appropriate amount of phage suspension and an equal volume of Ralstonia solanacearum liquid (OD600 = 0.1) and vortexed to mix, then mixed the phage and Ralstonia solanacearum mixture with the solid culture medium, poured onto a solidified solid plate after mixing, and placed in a 30 ℃ constant temperature incubator for 16 h.

[0048] The present invention will be further described below in conjunction with the embodiments.

[0049] Example 1: Isolation and purification of bacteriophage YL2

[0050] Using Ralstonia solanacearum isolated from Xiangxi Autonomous Prefecture as the host, the modified double-layer agar method was used to isolate virulent phages from tobacco root soil: 1 g of soil was added to 10 ml of sterile water and vortexed to mix, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was filtered with a 0.22 μm bacterial filter. An equal volume of NB medium and 0.3% host bacterial solution (V / V) were added to the filtrate, and co-cultured at 30°C for 12 h. The culture solution was collected, centrifuged and filtered again, and the filtrate was diluted 1000 times with SM buffer. The dilution was mixed with an equal volume of Ralstonia solanacearum solution (OD 600 =1.0) to make the upper plate, and culture at 37°C until plaques appear. Repeat the above process for more than 5 times to complete the purification of phage ( Figure 1 ).

[0051] Example 2: Whole genome sequencing and analysis of bacteriophage YL2

[0052] The purified 50 ml phage YL2 stock solution was sent to Wuhan Fraser Gene Information Co., Ltd. for whole genome sequencing. The sequencing platform was Illumina NovaSeq. A5-MiSeq and SPAdes were used for de novo assembly to obtain the phage whole genome sequence. The full length of the YL2 genome was 60,770 bp, the G+C content was 64.86%, and there were 73 open reading frames.

[0053] Example 3: Appearance and morphological characteristics of bacteriophage YL2

[0054] Prepare the culture medium of bacteriophage YL2 and concentrate it according to the above method. Take 10 μl of YL1 and precipitate it on the copper mesh for 1 min. Use filter paper to absorb the floating liquid. Then add 10 μl of phosphotungstic acid staining solution to the copper mesh to cover the phage liquid surface for 1 min. Use filter paper to absorb the excess liquid and dry it at room temperature for 10 min. Use electron microscope to detect the image (100~120 kV). The transmission electron microscopy observation results show that the head of bacteriophage YL1 is polyhedral, about 50 nm wide, and the tail is short, about 30 nm long. According to the classification standards of the International Committee on Taxonomy of Viruses (ICCV), it was identified as Brachyurae ( Figure 2 ).

[0055] Example 4: Phylogenetic analysis of bacteriophage YL2

[0056] The genome sequence of YL2 was compared with the existing phage sequences in the NCBI database, and a phylogenetic tree was constructed using MEGA 10.0 with Pseudomonas phage Itty13 as the outgroup. It was found that YL1 and Ralstonia Phage AhaGv were on the same branch, and thus it was identified as a short-tailed phage ( Figure 3 ).

[0057] Example 5: Thermal stability test of bacteriophage YL2

[0058] 1 ml of phage culture solution was added to a 2 ml EP tube, and the tube was placed in a water bath at different temperatures (30°C, 37°C, 50°C, 60°C, 70°C, and 80°C) for 1 hour, then taken out and cooled to room temperature. The number of plaques was calculated by the double-layer plate method, and the titer of phages after treatment at different temperatures was compared. The results showed that the titer of YL2 was stable in the range of 30-60°C, but after 1 hour of water bath at 70°C, the titer of YL2 decreased significantly, and no plaques were detected at 80°C and above, indicating that its inactivation temperature was 80°C ( Figure 4 ).

[0059] Example 6: pH stability test of bacteriophage YL2

[0060] Take 10 μl of phage culture solution and add 990 μl of SM buffer with different pH values ​​(2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 11.0), incubate in water bath at the optimal temperature for 1 hour, and then determine the phage titer after different pH treatments by double-layer plate method. The titer of phage YL2 was stable at 10 in the pH range of 4-11. 8 PFU / ml, it decreased significantly when pH < 4 and pH > 11, and no plaques were detected at pH = 3 and pH = 12 ( Figure 5 ).

[0061] Example 7: Bacteriophage YL2 controls tobacco bacterial wilt

[0062] Yunyan 87 (four-leaf stage) was transplanted into the seedling medium. During the experiment, the greenhouse temperature was maintained at 30°C and the soil moisture was maintained at 95%. PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1000 ml ddH2O) was used to adjust the pathogen OD 600 =0.1, phage titer = 10 8PFU / ml, each tobacco plant was first inoculated with 10 ml of Ralstonia solanacearum suspension, and then inoculated with 50 ml of phage suspension. Each treatment had 12 tobacco plants, and 3 biological replicates were set. At the early stage and peak stage of the disease, the disease classification was investigated and the incidence rate was calculated according to the "Grading and Investigation Methods for Tobacco Diseases and Insects" (GB / T 23222-2008), and the disease index and relative control effect were calculated.

[0063] The control group showed wilt symptoms on the 5th day after inoculation with Ralstonia solanacearum, and the control group entered the peak period of disease on the 14th day after inoculation. At this time, only some tobacco plants in the treatment group inoculated with bacteriophage were observed to wilt. On the 24th day after inoculation, the incidence and disease index of bacterial wilt in the control group were significantly higher than those in the treatment group inoculated with bacteriophage. The survey and statistical results showed that the prevention effect of YL2 on tobacco bacterial wilt was 76.39±8.37%, indicating that inoculation with bacteriophage YL2 can effectively reduce the occurrence of bacterial wilt (Table 1, Figure 6 ).

[0064] Table 1: Evaluation of the efficacy of bacteriophage against bacterial wilt in potted plants (surveyed 24 days after inoculation)

[0065]

[0066] 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 20242305.

2. Use of the bacteriophage according to claim 1 in lysing Ralstonia pseudosolanacerum.

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. The biocontrol agent according to claim 6, characterized in that The titer of the phage is 10 8 PFU / ml.

8. Use of the biocontrol agent according to any one of claims 5 to 7 in lysing Ralstonia solanacearum.

9. Use of the biocontrol agent according to any one of claims 5 to 7 in controlling bacterial wilt.

10. The use according to claim 9, characterized in that The bacterial wilt disease is tobacco bacterial wilt disease.

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