Culture medium suitable for fermentation of Ralstonia solanacearum bacteriophage, culture method and application

Abstract

The invention provides a culture medium suitable for fermentation of Ralstonia solanacearum bacteriophage, a culture method and application. The culture medium comprises thymidine and L-tryptophan. The culture medium provided by the invention can realize more effective culture and fermentation of Ralstonia solanacearum bacteriophage, improves the titer of bacteriophage fermentation liquor in a tank, and has the advantages of simple preparation method, more scientific components and suitability for industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to a culture medium and culture method suitable for Ralstonia solanacearum phage fermentation and application. BACKGROUND

[0002] Bacterial wilt caused by Ralstonia solanacearum is one of the main diseases affecting crop yields, and is a devastating soil-borne disease of Solanaceae crops such as peppers, tomatoes, and tobacco, as well as 450 species of plants such as peanuts and bananas. Ralstonia solanacearum invades the plant body from the roots, rapidly colonizes the vascular bundle, and accumulates a large amount of extracellular polysaccharides, blocking the vascular bundle of the plant and causing water transport to be blocked in the stem, resulting in wilting of the leaves and presenting the symptoms of "wilt". Currently, bacterial wilt has caused extensive damage to plants worldwide and has become an important limiting factor affecting the yield of many crops. At present, the means to prevent bacterial wilt are mostly chemical prevention, but at the same time, it also causes damage to beneficial organisms, changes the original microbial flora structure and soil physical and chemical properties, and excessive use of pesticides leads to increased resistance of plant pathogenic bacteria, serious soil pesticide residues, and also causes environmental and food pollution. This makes the prevention and control of bacterial wilt face severe challenges. Therefore, it is urgent to develop a new type of antibacterial preparation or therapy to replace chemical pesticides.

[0003] Phage (bacteriophage) is a virus that parasitizes various bacteria and is also known as a bacterial virus. It is widely distributed in nature and is considered to be the most abundant and diverse organism on Earth. It has the characteristics of strong host specificity, only infecting specific host bacteria, not infecting mammalian cells, and not harming the ecological environment. Moreover, it has the advantages of rapid reproduction and strong specificity. In the actual process of preventing plant diseases, it can effectively inhibit the proliferation of pathogenic bacteria, reduce the number of animal pathogenic bacteria in the soil, and due to its specificity, it can reduce harm to other microbial flora, maintain the balance of the soil microbial environment, and is different from chemical pesticides, meeting the requirements of green control.

[0004] Phage has special significance in preventing plant bacterial diseases due to its targeted lysis of specific strains, rapid killing of bacteria, and rapid reproduction, safety, and environmental protection. CN202410460696.X discloses a high-temperature-resistant and broad-spectrum Ralstonia solanacearum phage and its application. With the gradual deepening of research on phage preparation fermentation production, there are various problems in the industrial production of Ralstonia solanacearum phage preparations, such as expensive culture medium, low product titer, and long production cycle. Therefore, developing a low-cost, high-titer Ralstonia solanacearum phage fermentation medium formula is the key to effectively solving the current problems in the industrial production of phage.

[0005] In the prior art, TSB liquid medium or simple component medium is usually used as the culture medium of Ralstonia solanacearum phage, but a specific and high-titer culture medium for Ralstonia solanacearum phage has not been disclosed. SUMMARY

[0006] Therefore, the application provides a culture medium and a culture method and application suitable for Ralstonia solanacearum phage fermentation, and provides a Ralstonia solanacearum phage culture medium with simple preparation method and high fermentation titer.

[0007] The technical scheme of the application is implemented as follows: In a first aspect, the application provides a culture medium suitable for Ralstonia solanacearum phage fermentation, which comprises thymidine and L-tryptophan.

[0008] Fast synthesis of phage genome requires a large amount of nucleotides, and thymidine is a nucleotide additive that directly participates in phage DNA synthesis and improves replication efficiency; the main role of adding L-tryptophan is to induce conformational changes of phage tail fiber protein and expose host binding sites.

[0009] On the basis of the above technical scheme, further, in 1000 mL of the culture medium, 0.05 g to 2 g of thymidine and 0.05 g to 0.15 g of L-tryptophan are included.

[0010] On the basis of the above technical scheme, further, the culture medium further comprises tryptone, yeast extract, glutamine, magnesium chloride, potassium dihydrogen phosphate, bile salt, sodium pyruvate and trehalose.

[0011] Tryptone supplies amino acids and short peptides; yeast extract provides nitrogen source, growth factors and nucleotides; glutamine provides high-quality nitrogen source; MgCl2 promotes phage adsorption; potassium phosphate dibasic provides phosphorus source and buffer; bile salt can inhibit miscellaneous bacteria; sodium pyruvate can provide additional carbon source and enhance host energy metabolism; and trehalose can stabilize phage coat protein and reduce shear force damage.

[0012] On the basis of the above technical scheme, further, in 1000 mL of the culture medium, 16 g to 20 g of tryptone, 2.5 g to 5 g of yeast extract, 1.0 g to 1.5 g of glutamine, 0.8 g to 1 g of MgCl2, 2.0 g to 2.5 g of potassium phosphate dibasic, 0.1 g to 0.4 g of bile salt, 0.30 g to 0.6 g of sodium pyruvate and 5 g to 12 g of trehalose are included.

[0013] On the basis of the above technical scheme, further, the pancreatin, yeast extract, L-tryptophan, magnesium chloride, potassium dihydrogen phosphate, bile salt, sodium pyruvate and trehalose are dissolved in deionized water or distilled water, the pH is adjusted to 7.0±0.3, sterilized at 121℃ for 20min, then thymidine and glutamine are dissolved in deionized water or distilled water, and added after filtering with a filter membrane of 0.22 μm.

[0014] On the basis of the above technical scheme, further, in 1000 mL of the culture medium, thymidine 0.13g, L-tryptophan 0.125g, pancreatin 18g, yeast extract 4g, glutamine 1.35g, MgCl20.9g, potassium phosphate dibasic 2.3g, bile salt 0.35g, sodium pyruvate 0.55g and trehalose 10g are included.

[0015] On the basis of the above technical scheme, further, the culture medium includes liquid medium or solid medium, and the solvent is deionized water or distilled water.

[0016] On the basis of the above technical scheme, further, when the culture medium is solid medium, the culture medium further includes agar powder.

[0017] On the basis of the above technical scheme, further, thymidine and / or L-tryptophan are added as components of the feed when the OD value of the host bacteria reaches 0.25-0.3.

[0018] The culture medium components of the present application can be used in the way of separate feed for bacteriophage fermentation, so that the infection and proliferation efficiency is improved. In the process of bacteriophage fermentation, the supplement of nucleic acid or its precursor can improve the bacteriophage titer by optimizing the replication efficiency, stability of bacteriophage genome and host metabolic environment. The rapid synthesis of bacteriophage genome requires a large amount of nucleotides, and the supplement of nucleotides can directly alleviate the shortage of endogenous nucleotides in the host, accelerate the synthesis of bacteriophage genes, but excessive nucleotides will inhibit the growth of the host, so the feed after the host bacteria reach the appropriate OD value has significance for improving the bacteriophage titer.

[0019] On the basis of the above technical scheme, further, glutamine, MgCl2 and / or trehalose are added as components of the feed when the OD value of the host bacteria reaches 0.25-0.3.

[0020] When the OD value of the host bacteria reaches 0.25-0.3, the feed components are added, the bacteriophage is inoculated, and fermentation culture is carried out.

[0021] When thymidine, L-tryptophan, glutamine, MgCl2 and / or trehalose are added as components of the feed, sterilization treatment is required.

[0022] In a second aspect, the present application provides a method for culturing a Ralstonia solanacearum bacteriophage, inoculating the Ralstonia solanacearum bacteriophage into the culture medium, and then culturing.

[0023] Further, when the Ralstonia solanacearum bacteriophage is inoculated, the multiplicity of infection (MOI value) of the Ralstonia solanacearum bacteriophage and the Ralstonia solanacearum is 1000:1.

[0024] In a third aspect, the present application provides a Ralstonia solanacearum bacteriophage culture solution obtained by the above-mentioned culturing method.

[0025] In a fourth aspect, the present application provides application of the culture medium in culturing the Ralstonia solanacearum bacteriophage and / or the Ralstonia solanacearum, or in bacterial counting and bacteriophage counting of the Ralstonia solanacearum.

[0026] Compared with the prior art, the present application has the following beneficial effects: (1) The culture medium of the present application can realize more effective fermentation of the Ralstonia solanacearum bacteriophage, improve the titer of the bacteriophage fermentation broth, has the advantages of simple preparation method, more scientific components, and suitability for industrial production.

[0027] (2) The present application finds an important factor affecting the titer of the fermentation bacteriophage, i.e., feeding the components in the culture medium of the present application before inoculating the bacteriophage can improve the titer of the fermentation broth, which affects the cost of industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0029] Fig. 1 The titer detection results of the Ralstonia solanacearum bacteriophage RSP2037001 in Example 1; Fig. 2 The titer detection results of the Ralstonia solanacearum bacteriophage RSP2037002 in Example 1; Fig. 3 The titer detection results of the Ralstonia solanacearum bacteriophage RSP2037003 in Example 1. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0031] The reagents and materials used in the present application are all ordinary commercially available products, wherein: Thymidine is purchased from China Pharmaceutical, with the product number C226740050; L-tryptophan is purchased from China Pharmaceutical, with the product number LJ62508100G; Tryptone is purchased from Angel Yeast, with the product number FP318; Yeast extract is purchased from Angel Yeast, with the product number FM885; Glutamine is purchased from China Pharmaceutical, with the product number HYSH3033603; Magnesium chloride is purchased from China Pharmaceutical, with the product number C447150025; Potassium dihydrogen phosphate is purchased from China Pharmaceutical, with the product number 10017618; Bile salt is purchased from China Pharmaceutical, with the product number 69005062; Sodium pyruvate is purchased from China Pharmaceutical, with the product number 30169138; Trehalose is purchased from China Pharmaceutical, with the product number 63011536; TSB medium is purchased from BD, with the product number 211825.

[0032] The Ralstonia solanacearum phage involved in the experiments of the present application includes: The Ralstonia solanacearum phage with the number RSP2037001 is named Ralstonia solanacearum phage RSP2307001. The preservation unit of the Ralstonia solanacearum phage RSP2307001 is China Center for Type Culture Collection, with the address of Wuhan University, Luojia Hill, Wuchang, Wuhan, Hubei, China, the postcode of 430072, the preservation date of December 6, 2023, the preservation number of CCTCC NO:M 20232474, and the host bacteria RS520.

[0033] RSP2307002, Ralstonia solanacearum phage RSP2307002, deposited with the China Center for Type Culture Collection, located at Wuhan University, Luojia Hill, Wuchang, Wuhan, Hubei, China, with the address of 430072, on March 15, 2024, with the accession number of CCTCC NO: M 2024499, and the host bacteria of RS520; RSP2307003, Ralstonia solanacearum phage RSP2307003, deposited with the China Center for Type Culture Collection, located at Wuhan University, Luojia Hill, Wuchang, Wuhan, Hubei, China, with the address of 430072, on March 15, 2024, with the accession number of CCTCC NO: M 2024500, and the host bacteria of RS520.

[0034] In the following specific embodiments, Ralstonia solanacearum RS520 is derived from pathogenic bacteria isolated in Yunnan tobacco planting areas, and Ralstonia solanacearum RS784 is derived from pathogenic bacteria isolated in Guizhou tobacco planting areas.

[0035] In the following specific embodiments, the method for detecting titer is: (1) Prepare a sufficient number of TSA (or LB solid) plates in advance.

[0036] (2) Pick a single colony or scrape the slope from the host bacteria plate into a certain volume of TSB (or LB) medium, and incubate overnight in an incubator at an appropriate temperature to obtain pure bacterial solution.

[0037] (3) Take the phage sample to be tested, make 10-fold serial dilution, take 10 5 , 10 6 , 10 7 dilution of phage diluent 100 μL respectively, and 300 μL of the above prepared fresh Ralstonia solanacearum, take 1 mL of the sample to be tested with appropriate concentration in a 10 mL centrifuge tube, add 300 μL of host bacteria solution respectively, and stand for 10-15 min, (4) Add 5-10 mL of TSB semi-solid medium (or LB semi-solid medium, i.e. 100 mL medium added with 0.7 g of agar powder to prepare sterilization) with temperature not higher than 45℃ into a centrifugal tube, pour immediately onto a TSA (or LB solid) medium plate, shake to mix and then stand to solidify.

[0038] (5) Take out the plate after overnight culture in a 30℃ constant temperature incubator, count the phage plaques on the plate and calculate the phage concentration.

[0039] Example 1 The present example provides a liquid medium suitable for Ralstonia solanacearum phage fermentation, and the preparation method comprises the following steps: (1) Take the following raw materials: 18 g of tryptone, 4 g of yeast extract, 0.9 g of MgCl2, 2.3 g of potassium phosphate dibasic, 0.125 g of L-tryptophan, 0.35 g of bile salt, 0.55 g of sodium pyruvate, and 10 g of trehalose dissolved in deionized water, and add deionized water to 999 mL; (2) Adjust the pH value and sterilize: adjust the pH to 6.7 and sterilize at 121℃ for 20 min; (3) Add thymidine and glutamine: dissolve 0.13 g of thymidine and 1.35 g of glutamine in 1 mL of deionized water, filter with a 0.22 μm filter membrane, and then add.

[0040] Example 2 The present example provides a liquid medium suitable for Ralstonia solanacearum phage fermentation, and the preparation method comprises the following steps: (1) Take the following raw materials: 20 g of tryptone, 5 g of yeast extract, 1 g of MgCl2, 2.0 g of potassium phosphate dibasic, 0.05 g of L-tryptophan, 0.1 g of bile salt, 0.30 g of sodium pyruvate, and 5 g of trehalose dissolved in deionized water, and add distilled water to 999 mL; (2) Adjust the pH value and sterilize: adjust the pH to 7.0 and sterilize at 121℃ for 20 min; (3) Add thymidine and glutamine: dissolve 2 g of thymidine and 1.5 g of glutamine in 1 mL of distilled water, filter with a 0.22 μm filter membrane, and then add.

[0041] Example 3 The present example provides a liquid medium suitable for Ralstonia solanacearum phage fermentation, and the preparation method comprises the following steps: (1) Take the following raw materials: 16 g of tryptone, 2.5 g of yeast extract, 0.8 g of MgCl2, 2.5 g of potassium phosphate, 0.15 g of L-tryptophan, 0.4 g of bile salt, 0.6 g of sodium pyruvate, 12 g of trehalose dissolved in deionized water, and add deionized water to 999 mL; (2) Adjust the pH value and sterilize: adjust the pH to 7.3, and sterilize at 121°C for 20 min; (3) Add thymidine and glutamine: dissolve 0.05 g of thymidine and 1.0 g of glutamine in 1 mL of deionized water, filter with a 0.22 μm filter membrane, and then add.

[0042] Example 4 The present embodiment provides a solid culture medium suitable for Ralstonia solanacearum bacteriophage fermentation. The difference between the present embodiment and Example 1 is that step (1) further includes 18 g of agar powder.

[0043] The obtained medium is cooled to 60°C and poured into a plate to obtain a solid culture medium, which is used for counting Ralstonia solanacearum. A conventional product TSB solid culture medium on the market is used as the lower layer for a control experiment. The bacterial liquid of Ralstonia solanacearum RS520 and RS784 is plated and counted, and the specific results are shown in Table 1.

[0044] Table 1 Influence of different culture media on Ralstonia solanacearum

[0045] From the above results, it can be seen that the bacterial count of Ralstonia solanacearum obtained by using the culture medium of the present application and using the TSB solid culture medium is not much different. It is proved that the culture medium of the present application can be used as a solid culture medium for bacterial counting of Ralstonia solanacearum.

[0046] Example 5 The present embodiment provides a culture method of Ralstonia solanacearum bacteriophage, comprising the following steps: (1) Three strains of Ralstonia solanacearum host bacteria RS520 and their corresponding Ralstonia solanacearum bacteriophages RSP2037001, RSP2307002 and RSP2307003 are inoculated into 1000 mL of the culture medium prepared in Example 1 according to a multiplicity of infection (MOI) of 1000:1, and are also inoculated into a conventional product TSB culture medium on the market.

[0047] (2) Culture at a fermentation temperature of 30°C, a rotation speed of 180 rpm, and aeration of 0.35 vvm for 17 h.

[0048] Example 6 The present embodiment provides a method for expanding culture of Ralstonia solanacearum bacteriophage, comprising the following steps: (1) According to the preparation method of Example 1, 100 L of culture was prepared in a 300 L fermenter, including tryptone 1800 g, yeast extract 400 g, MgCl2 90 g, K2HPO4 230 g, L-tryptophan 12.5 g, bile salt 35 g, sodium pyruvate 55 g, trehalose 1000 g, thymidine 13 g, glutamine 135 g.

[0049] (2) The Ralstonia solanacearum host strain RS520 and its corresponding Ralstonia solanacearum bacteriophage RSP2307002 were inoculated in the fermenter at a multiplicity of infection of 1000:1 for expansion culture, and cultured at 37°C, 180 rpm, and 0.35 vvm aeration for 17 h.

[0050] (3) After the tank was removed, the fermentation broth was detected for titer using the double-layer plate method.

[0051] The titer of the Ralstonia solanacearum bacteriophage RSP2307002 was 4.5 x 10 11 pfu / mL, indicating that the culture medium of the present application is also suitable for the expansion culture of Ralstonia solanacearum bacteriophage and can be used as a basis for bacteriophage production in various volumes.

[0052] Comparative Example 1 The difference between this comparative example and Example 1 is that it does not contain thymidine.

[0053] Comparative Example 2 The difference between this comparative example and Example 1 is that it does not contain L-tryptophan.

[0054] Comparative Example 3 The difference between this comparative example and Example 1 is that the thymidine is 3 g.

[0055] Comparative Example 4 The difference between this comparative example and Example 1 is that the L-tryptophan is 1 g.

[0056] Performance test 1. Effect of culture medium on bacteriophage titer The liquid culture media prepared by Examples 1 and Comparative Examples 1-4 and the conventional finished product TSB culture medium on the market were used to culture Ralstonia solanacearum bacteriophage according to the culture method described in Example 5. After the tank was removed, the fermentation broth was detected for titer using the double-layer plate method, and the titer results are shown in Table 2 and Figs. 1-3 It can be seen that the titer of each bacteriophage is significantly improved using the culture medium of the present application.

[0057] The liquid medium prepared by using the comparative examples 1-4 has a significantly decreased titer of the Ralstonia solanacearum bacteriophage. For example, RSP2037001 (Table 2), when thymidine is not contained, the dTTP pool in the host cell is insufficient, which leads to a decreased or even interrupted DNA replication of the bacteriophage, thereby decreasing the titer; when excessive thymidine is added, the host bacteria need to mobilize more enzymes and energy to process the excessive thymidine, which slows down the growth of the host bacteria and reduces the physiological activity, thereby affecting the proliferation efficiency of the bacteriophage; in another aspect, the addition of excessive thymidine can interfere with the normal metabolism of the bacteriophage, affect the synthesis and utilization of other nucleotides, and thus affect the normal growth and reproduction of the bacteriophage, thereby decreasing the titer. When L-tryptophan is not contained, the protein synthesis of the bacteriophage can be blocked, the bacteriophage particles cannot be formed in an intact and normal manner, the normal reproduction of the bacteriophage in the fermentation process is affected, the host binding site cannot be exposed, and thus the titer is decreased; when excessive L-tryptophan is added, the structural protein synthesis of the bacteriophage can be excessive or erroneous. Too many tryptophan residues in the coat protein can change the protein folding mode, leading to defects in the assembly of the bacteriophage particles (such as capsid deformation and tail filament loss), thereby reducing the infection efficiency and host recognition ability of the bacteriophage and decreasing the titer.

[0058] Table 2: Effect of different media on the titer of the bacteriophage

[0059] 2. Effect of the addition amount of thymidine and L-tryptophan on the titer of the bacteriophage in the fed-batch fermentation process Thymidine and L-tryptophan are added as components of the feed before inoculation of the bacteriophage; The host bacteria are inoculated in the medium of the application (containing no thymidine) and the conventional TSB medium on the market, respectively, 0 g, 0.05 g, 0.10 g, 0.13 g, 0.16 g, 2 g and 3 g of thymidine are added when the OD value of the host bacteria reaches 0.25-0.30, and the Ralstonia solanacearum bacteriophage is inoculated and fermented according to the method of Example 5. The host bacteria are inoculated in the medium of the application (containing no L-tryptophan) and the conventional TSB medium on the market, respectively, 0 g, 0.05 g, 0.075 g, 0.10 g, 0.125 g, 0.15 g and 1 g of L-tryptophan are added when the OD value of the host bacteria reaches xx, and the Ralstonia solanacearum bacteriophage is inoculated and fermented according to the method of Example 5. The effect of the addition of different amounts of thymidine and L-tryptophan on the titer of the bacteriophage in the fed-batch fermentation process is detected, and the results are shown in Tables 3 and 4.

[0060] Table 3: Different addition amounts of thymidine and the corresponding titer of the bacteriophage

[0061] Table 4 Different L-tryptophan addition amounts and their corresponding phage titers

[0062] As can be seen from the above experimental results, as the thymidine content per L gradually increases from 0.05 g to 0.13 g, the titer increases, but when the thymidine addition amount exceeds 0.13 g / L, the titer of the fermentation broth gradually decreases. When no thymidine is added, the titer of the tank is low, because thymidine is a required precursor for DNA synthesis, and thymidine supplementation is a key factor for ensuring replication integrity and fermentation stability: phage replication requires a large number of deoxynucleotides (including dTTP) to assemble the progeny genome. Thymidine is phosphorylated to form dTMP (thymidylic acid), which is then gradually converted to dTTP. If thymidine is lacking, the dTTP pool in the host cell is insufficient, which leads to a decrease in phage DNA replication speed or even interruption, thereby causing the titer to decrease; when excessive thymidine is added, the host bacteria need to mobilize more enzymes and energy to process the excessive thymidine, which slows down the growth rate of the host bacteria and reduces their physiological activity, thereby affecting the proliferation efficiency of the phage. The fermentation of the phage relies on the host for replication and assembly, and another reason is that excessive thymidine addition can interfere with the normal material metabolism of the phage, affecting the synthesis and utilization of other nucleotides, thereby also affecting the normal growth and reproduction of the phage, thereby causing the titer to decrease.

[0063] And in the finished TSB medium, the same effect is also present, so adding thymidine has a promoting effect on the fermentation titer of the Ralstonia solanacearum phage, but the rules need to be found and added appropriately.

[0064] The L-tryptophan addition amount also decreases as the addition amount increases to a certain value, and when the L-tryptophan addition amount in the medium of the application is 0.125 g / L, the titers of RSP2037001, RSP2307002, and RSP2307003 are optimal. When no L-tryptophan is added, the synthesis of phage proteins may be blocked, which prevents the formation of complete and normal phage particles, thereby affecting the normal reproduction of the phage during fermentation and preventing the exposure of host binding sites, thereby causing the titer to decrease; when excessive L-tryptophan is added, excessive or incorrect synthesis of phage structural proteins may occur due to the excessive L-tryptophan. Too many tryptophan residues in the coat protein may change the protein folding mode, leading to defects in phage particle assembly (such as capsid deformation and tail filament loss), thereby reducing the infection efficiency and host recognition ability of the phage, and causing the titer to decrease.

[0065] The above only describes the preferred embodiments of the application and is not intended to limit the application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A culture medium suitable for Ralstonia solanacearum bacteriophage fermentation, characterized in that, thymidine and L-tryptophan.

2. A culture medium suitable for the fermentation of a Ralstonia solanacearum bacteriophage according to claim 1, characterized in that, thymidine 0.05g-2g and L-tryptophan 0.05g-0.15g per 1000 mL of medium.

3. A culture medium suitable for the fermentation of a Ralstonia solanacearum bacteriophage according to claim 1, characterized in that, trypsin, yeast extract, glutamine, magnesium chloride, potassium dihydrogen phosphate, bile salt, sodium pyruvate and trehalose.

4. A culture medium suitable for the fermentation of a Ralstonia solanacearum bacteriophage according to claim 2, characterized in that, trypsin 16g-20g, yeast extract 2.5g-5g, glutamine 1.0g-1.5g, MgCl20.8g-1g, potassium phosphate dibasic 2.0-2.5g, bile salt 0.1g-0.4g, sodium pyruvate 0.30g-0.6g and trehalose 5g-12g per 1000 mL of medium.

5. A culture medium suitable for the fermentation of a Ralstonia solanacearum bacteriophage according to claim 1, characterized in that, thymidine 0.13g, L-tryptophan 0.125g, trypsin 18g, yeast extract 4g, glutamine 1.35g, MgCl20.9g, potassium phosphate dibasic 2.3g, bile salt 0.35g, sodium pyruvate 0.55g and trehalose 10g per 1000 mL of medium.

6. A culture medium suitable for the fermentation of a Ralstonia solanacearum bacteriophage according to claim 1, characterized in that, The medium comprises liquid medium or solid medium, and the solvent is deionized water or distilled water.

7. A culture medium suitable for the fermentation of a Ralstonia solanacearum bacteriophage according to claim 1, characterized in that, The thymidine and / or L-tryptophan are added as components of the feed when the OD value of the host bacteria reaches 0.25-0.

3.

8. A method for culturing a bacteriophage of Ralstonia solanacearum, characterized by, The culture medium as claimed in any one of claims 1-7 is inoculated with the Ralstonia solanacearum bacteriophage and then cultured.

9. The Ralstonia solanacearum bacteriophage culture solution obtained by the culture method as claimed in claim 8.

10. Use of the culture medium as claimed in any one of claims 1-7 in the culture of Ralstonia solanacearum bacteriophage and / or Ralstonia solanacearum, or in the bacterial count and bacteriophage count of Ralstonia solanacearum.

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