Phage for lysing nurkholderia gladioli and use thereof

Abstract

The present invention relates to the technical field of bacteriophages, and provides a bacteriophage for lysing Burkholderia gladioli and a use of the bacteriophage. The provided bacteriophage for lys

Description

A bacteriophage for lysing Burkholderia gladiolus and its application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on June 8, 2023, with application number 2023106808478 and invention name “A phage for lysing Burkholderia gladiolus and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The invention belongs to the technical field of bacteriophages, and in particular relates to a bacteriophage for lysing Burkholderia gladiolus and an application thereof. Background Art

[0003] Burkholderia gladioli is a Gram-negative, short, non-spore-forming bacterium with an optimal growth temperature of 37°C and an optimal pH of 5-6. It is widely distributed in nature, and soil, plants, and other natural environments, as well as animals, can be contaminated by Burkholderia gladioli. Food contamination is also possible, with rice and onions potentially contaminated. Although many Burkholderia gladioli species are environmentally nontoxic, some species may cause disease in animals and plants. Four pathogenic variants of this species have been identified. Among them, B. gladioli pv. cocovenenans produces the toxin fumonisin, which has been found in fermented grains, spoiled Tremella fuciformis, and Wood Ear Mushroom. The other three variants are pathogenic to plants.

[0004] Burkholderia gladioli pv. cocovenenans is a highly lethal foodborne pathogen with an incubation period typically ranging from 30 minutes to 12 hours, sometimes up to 1-2 days. Poisoning caused by this strain has distinct regional characteristics, with reports prevalent in Guangxi, the mountainous areas of Yunnan and Guizhou, and Northeast China. This is related to the local residents' habit of consuming foods such as Tremella fuciformis (tremella), wood ear mushrooms (myrrh), diaopingjiangba (baked cakes), and fermented rice noodles. In October 2020, nine residents of Jixi City, Heilongjiang Province, were poisoned and died after consuming sour soup, a local specialty contaminated with Burkholderia gladioli, garnering widespread public attention. Fumaric acid produced by this bacterium is the primary cause of severe poisoning and death, with the optimal temperature for its production being 26-28°C. Fumonisic acid is a small molecule fatty acid with extremely strong heat resistance. Boiling it in 100°C water or steaming it in a pressure cooker cannot destroy its toxicity. It can cause poisoning after eating and can cause serious damage to important organs such as the liver, kidneys, heart, and brain.

[0005] Burkholderia gladioli poisoning rates exceed 50%, with a mortality rate exceeding 80%. However, there are no reported drugs for the prevention and treatment of this infection. Therefore, controlling this bacterium and its toxins is a pressing challenge for the food industry.

[0006] Bacteriophages are specialized viruses that specifically infect microorganisms such as bacteria, fungi, and actinomycetes. Their high specificity allows them to target specific pathogens without disrupting the normal microbial ecosystem. Phages infect and lyse host bacteria, releasing a large number of progeny phages that multiply exponentially within the host, effectively disinfecting bacteria in a short period of time. Compared to physical and chemical control methods, phages are used in food control to combat bacteria, offering advantages such as rapid action, strong efficacy, and minimal side effects. These advantages hold great promise for the application of phages in bacterial control.

[0007] Summary of the Invention

[0008] In view of this, the object of the present invention is to provide a phage strain that specifically lyses gladiolus Burkholderia, which can effectively inhibit gladiolus Burkholderia, has no lysis effect on non-gladiolus Burkholderia, and does not contain drug resistance genes and virulence genes, and is safe and reliable.

[0009] Another object of the present invention is to provide a lysate and a preparation comprising gladiolus Burkholderia phage and use thereof in preventing and controlling gladiolus Burkholderia.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a bacteriophage, which is vB_BglM_WTB and has a deposit number of CCTCC NO: M 2023525.

[0012] In the present invention, the full length of the phage genome is 68541 bp and does not contain virulence genes and drug resistance genes.

[0013] In the present invention, the pH value at which the phage activity is stable is 3-11, and the tolerable temperature is 25°C-65°C.

[0014] The present invention also provides a lysis solution, which is the lysis solution of the bacteriophage.

[0015] The present invention also provides a preparation comprising the above-mentioned bacteriophage or the above-mentioned lysate.

[0016] The present invention also provides a use of the bacteriophage, the lysate or the preparation in preventing and controlling Burkholderia gladiolus.

[0017] Preferably, the application is to prevent and control Burkholderia gladioli in food.

[0018] Preferably, the food is wood ear, white fungus, shiitake mushroom, or golden needle mushroom.

[0019] Preferably, the multiplicity of infection of the phage is 1:10000-1:100.

[0020] Preferably, the bacteriophage or lysate or preparation is contacted with the Burkholderia gladiolus.

[0021] Preferably, the pH value of the contact medium is 3-11, and the contact time is at least 2 hours.

[0022] Beneficial effects of the present invention:

[0023] The present invention isolated the Burkholderia gladioli phage vB_BglM_WTB using the pathogenic Burkholderia gladioli ATCC33664 as a host strain. This phage has a strong lytic effect on pathogenic Burkholderia gladioli found in foods such as wood ear mushrooms. The phage vB_BglM_WTB specifically lyses Burkholderia gladioli, but has no lytic effect on non-gladiolus Burkholderia. Comparative genomic and phylogenetic analyses revealed that this phage has the highest similarity with Burkholderia phage Maja, at 25.7% (<50%), suggesting its formation as a new genus.

[0024] The gladiolus Burkholderia phage vB_BglM_WTB isolated by the present invention is a virulent phage isolated from nature, does not contain virulence genes and drug-resistant genes, is safe and reliable, and has good temperature tolerance and pH stability. The present invention does not perform any genetic modification on the phage.

[0025] The Burkholderia gladioli phage vB_BglM_WTB of the present invention can effectively kill Burkholderia gladioli on the surface of foods such as wood ear mushrooms. The Burkholderia gladioli phage vB_BglM_WTB has a strong lytic effect on Burkholderia gladioli in foods such as wood ear mushrooms. At 4°C, the sterilization rate reaches 99.94% after 6 hours, and the sterilization rate can still reach 99.80% after 12 hours of treatment. At 25°C, the sterilization rate reaches 99.90% after 2 hours, and the sterilization rate can still reach 99.97% after 12 hours of treatment. It can be used as an effective fungicide in foods such as wood ear mushrooms. The present invention can be used in liquid immersion, and is particularly suitable for foods that require immersion, such as wood ear mushrooms, white fungus, shiitake mushrooms, and golden needle mushrooms.

[0026] Biodeposit Information:

[0027] The Burkholderia gladioli phage vB_BglM_WTB of the present invention is deposited in the China Center for Type Culture Collection on April 12, 2023, at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCCNO: M 2023525. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is an electron micrograph of bacteriophage vB_BglM_WTB;

[0029] FIG2 is a diagram showing the lysis of bacteriophage vB_BglM_WTB against host bacteria ATCC 33664;

[0030] Figure 3 is a gene heat map of bacteriophage vB_BglM_WTB;

[0031] Figure 4 is a visualization analysis of the bacteriophage vB_BglM_WTB, BcepF1 and Maja genomes;

[0032] Figure 5 is a phylogenetic tree of the major capsid proteins of bacteriophage vB_BglM_WTB;

[0033] Figure 6 is a one-step growth curve of bacteriophage vB_BglM_WTB;

[0034] FIG7 is a schematic diagram of the acid-base stability of bacteriophage vB_BglM_WTB;

[0035] FIG8 is a schematic diagram of the thermal stability of bacteriophage vB_BglM_WTB;

[0036] Figure 9 shows the inhibitory effect of bacteriophage vB_BglM_WTB on Burkholderia gladiolus in wood ear mushrooms at low temperature of 4°C and room temperature of 25°C; Figure 9A shows the inhibitory effect of bacteriophage vB_BglM_WTB at 4°C; Figure 9B shows the inhibitory effect of bacteriophage vB_BglM_WTB at 25°C. DETAILED DESCRIPTION

[0037] The invention provides a bacteriophage vB_BglM_WTB for lysing Burkholderia gladiolus. The deposit number of the bacteriophage vB_BglM_WTB is CCTCC NO: M 2023525.

[0038] In the present invention, the bacteriophage vB_BglM_WTB was isolated from a sewage sample collected from the Huaihe Road throttling well sewage treatment plant in Hefei City and was deposited in the China Center for Type Culture Collection on April 12, 2023. It was identified that the bacteriophage vB_BglM_WTB of the present invention has a regular icosahedral head with a diameter of about 69 (± 2) nm and a tail of about 108 (± 2) nm. It belongs to the Caudoviricetes and has a titer of up to 10 9 pfu / mL or more; the phage genome of the present invention is 68541bp in length, with a GC content of 60.04%, does not contain virulence genes and drug resistance genes, and has the highest similarity with Burkholderia phage Maja, with a similarity of 25.7% (<50%), and is suggested to form a new genus.

[0039] In the present invention, the phage vB_BglM_WTB specifically lyses Burkholderia gladioli and has no lytic effect on non-gladiolus Burkholderia species. The phage preferably has a multiplicity of infection (MOI) of 1:10,000-1:100, with an optimal MOI of 1:10,000. It also exhibits excellent temperature tolerance and pH stability, maintaining a stable potency at pH values ​​of 3-11 and at temperatures between 25°C and 65°C.

[0040] The present invention also provides a lysate of the aforementioned phage. As an operative method, the aforementioned phage is cultured in a culture medium containing a host bacterium, and the host bacterium is removed to obtain a phage lysate. The host bacterium may be removed by filtration sterilization or centrifugation sterilization. The host bacterium is Burkholderia gladioli, and the culture medium may be TSB broth.

[0041] The present invention also provides a preparation comprising the above-mentioned bacteriophage or the above-mentioned lysate.

[0042] The preparation of the present invention may also include other antibacterial active ingredients that can inhibit or kill Burkholderia gladiolus, including but not limited to microorganisms, Chinese herbal extracts or compositions, natural compounds, chemically synthesized compounds or compositions thereof, etc.

[0043] The formulation of the present invention is not particularly limited in type and may be a liquid formulation, a solid formulation, a semisolid formulation, a gaseous formulation, or the like. Depending on the formulation type, the formulation of the present invention may also include excipients acceptable for the formulation. The present invention does not particularly limit the type of excipients, and may include, but is not limited to, one or more of a carrier, a diluent, an excipient, a preservative, a surfactant, and an antioxidant.

[0044] The present invention also provides a use of the bacteriophage, lysate or preparation in preventing and controlling Burkholderia gladiolus.

[0045] The control of the present invention includes prevention and treatment; the bacteriophage, lysate or preparation of the present invention is brought into contact with Burkholderia gladioli for control and prevention, and can be used in various forms, including but not limited to soaking, smearing, spraying, etc., applied to the surface of an object, and the surface can be a contaminated site or an expected contaminated site.

[0046] The bacteriophage, lysate or preparation of the present invention can be used for the prevention and control of Burkholderia gladiolus in food. The present invention has no special limitation on the type of food, and can be fungal foods that can be soaked, such as wood ear, white fungus, shiitake mushroom, and enoki mushroom.

[0047] When the bacteriophage, lysate or preparation of the present invention is used to prevent and control Burkholderia gladioli, it can be used under refrigeration or room temperature conditions, preferably at a refrigeration temperature of 3-8°C, and can be used at room temperature above the refrigeration temperature, preferably at a room temperature of 18-38°C, and more preferably at 23-28°C. The present invention can achieve an effective sterilization effect in as little as 2 hours of treatment, and the sterilization effect can last up to 12 hours after treatment. In the present invention, in a preferred embodiment, at 4°C, the sterilization rate reaches 99.94% in 6 hours, and after 12 hours of treatment, the sterilization rate can still reach 99.80%; at 25°C, the sterilization rate can reach 99.90% in 2 hours, and after 12 hours of treatment, the sterilization rate can still reach 99.97%.

[0048] When the bacteriophage, lysate, or formulation of the present invention is used to control Burkholderia gladioli, the pH of the contact medium is preferably 3-11. Within this pH range, the bacteriophage maintains high activity against Burkholderia gladioli. Because bacteriophage has a temperature tolerance of 25°C to 65°C, the bacteriophage, lysate, or formulation of the present invention should be stored or used at a temperature below 65°C.

[0049] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] In the following examples, unless otherwise specified, all methods are conventional.

[0051] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0052] Example 1

[0053] Isolation and identification of bacteriophages isolated from Burkholderia gladiolus

[0054] (1) Phage isolation and identification

[0055] The sewage samples used in the experiment of this invention were collected from the Huaihe Road Throttling Well Sewage Treatment Plant in Hefei City in 2022.

[0056] Sample processing: The sewage sample was divided into 50mL centrifuge tubes and centrifuged at 10000rpm for 10min to remove larger solid particles and some bacteria; then the sample was filtered and sterilized with a 0.22μm microporous filter membrane and transferred to a clean sterile container, an appropriate amount of magnesium sulfate was added, the mixture was thoroughly stirred and allowed to stand for 15-20min; the magnesium sulfate mixture was vacuum filtered and the supernatant was discarded. The filter membrane was collected and cut into pieces and placed in a clean sterile beaker, an appropriate amount of eluent was added, and the sample was ultrasonicated for 5min. After that, the filter was placed in a centrifuge tube and centrifuged at 4000g for 2min. The supernatant was taken, filtered with a 0.45μm microporous filter and transferred to a new sterile container. The filtrate was the stock solution of bacteriophage; 500μL of bacteriophage stock solution, 500μL TSB broth medium and 50μL of logarithmic phase bacterial solution of Burkholderia gladioli were mixed evenly and cultured at 37℃ with shaking for 8h. Centrifuge the culture at 4000g for 15-20 minutes and filter through a 0.45μm microporous filter into a sterile centrifuge tube to obtain the phage lysate. Then, use ATCC 33664 as the host strain and use the spot drop method to identify the presence of phage. If plaques are present, phage is present; otherwise, no phage has been isolated and re-screening is necessary.

[0057] Purification of phage: Initially isolated phage plaques may be inconsistent in size and morphology, requiring further purification. Pick a single, independent, uniform, and clear plaque from a double-layer plate containing phage plaques. Place the plaque in 1 mL of SM solution. After incubation at 4°C overnight, centrifuge at 4000 g for 15-20 minutes. Collect the supernatant and filter it through a 0.45 μm microporous filter. Dilute the filtrate appropriately and plate it on a double-layer plate with a logarithmic phase bacterial solution of ATCC 33664. Repeat 3-4 times. When the plaques on the double-layer plate are consistent in size, morphology, and clarity, the phage is considered purified.

[0058] (2) Determination of phage titer

[0059] Dilute the purified phage solution appropriately. Take 100 μL of the last three dilutions and 100 μL of the logarithmic phase host bacterial solution and determine the phage titer using the double-layer plate method. Phage titer (pfu / mL) = number of plaques x dilution factor ÷ 0.1.

[0060] The results showed that the plaques had the same morphology, size and clarity, and had typical lytic phage characteristics. The titer of the phage could reach 10 9 pfu / mL or above.

[0061] (3) Preservation of phages

[0062] The phage proliferation liquid was mixed with polyethylene glycol 8000 (final concentration: 20%) and sodium chloride solution (final concentration: 0.5 M) in a 1:1 ratio, and the phage was concentrated at 4°C overnight. The concentrate was centrifuged at 12,000 g for 15-20 min, the supernatant was discarded, the precipitate was dissolved with SM solution, and the precipitate was mixed with 60% glycerol in a 1:1 ratio and stored at -20°C.

[0063] Example 2

[0064] Electron microscopic observation of bacteriophages

[0065] Drop the phage solution (titer 10) on the side of the copper mesh with the membrane. 9 pfu / mL) was added, and the liquid was dried with filter paper after 3-5 minutes. Then, a drop of 2% phosphotungstic acid (PTA) aqueous solution was dropped on the copper mesh for staining for 2-3 minutes. The staining liquid was absorbed with filter paper, and the phage was observed under an electron microscope. Clear phage images were selected for photography.

[0066] The results are shown in Figure 1 . The phage head is a regular icosahedron with a diameter of about 69 (±2) nm and a tail of about 108 (±2) nm. It belongs to the Caudoviricetes.

[0067] Example 3

[0068] Phage host spectrum determination

[0069] Spread TSA agar onto a sterile Petri dish and allow to dry. Add 100 μL of bacteria cultured to the logarithmic phase to 5 mL of 0.4% TSB semi-solid medium, mix well, and spread onto a dried plate. Allow to air dry to solidify the soft agar. Apply 2 μL of phage culture to the soft agar using the spotting method. Allow to air dry and incubate at 37°C for 12 hours. Results are classified as clear plaques (+) or no plaques (-).

[0070] 13 strains of Burkholderia gladiolus preserved in the laboratory were selected, of which ATCC 33664 was purchased from Beina Chuanglian Biotechnology Co., Ltd., and the remaining 12 strains were Burkholderia gladiolus isolated and identified from black fungus, enoki mushrooms, and shiitake mushrooms purchased in various vegetable markets in Hefei City from 2021 to 2022; the 10 selected strains of Vibrio parahaemolyticus were isolated from food by the Guangdong Institute of Microbiology; the remaining strains were preserved by Hefei University of Technology.

[0071] Table 1 Phage host spectrum

[0072] As can be seen from Table 1, the gladiolus Burkholderia phage vB_BglM_WTB has a lytic effect on all 13 gladiolus Burkholderia strains, but has no lytic effect on non-gladiolus Burkholderia strains.

[0073] Example 4

[0074] Phage genome sequencing

[0075] After enrichment and culture of a single phage strain, genomic DNA was extracted using the phenol-chloroform-isoamyl alcohol method. The extracted DNA precipitate was dissolved in sterile water and stored at -20°C until further use. Whole-genome sequencing was performed using Illumina after the DNA solution was tested for concentration and purity.

[0076] Sequencing results showed that the genome of bacteriophage vB_BglM_WTB was 68541 bp in length and had a GC content of 60.04%.

[0077] The full genome sequences of phages with high similarity to phage vB_BglM_WTB were downloaded from the NCBI database. Using the VIRIDIC online tool, a heat map was created, as shown in Figure 3. The results show that phage vB_BglM_WTB has low similarity to other phages, with the highest similarity being 25.7% (<50%) with phage Maja. This suggests that this phage is a new species and is proposed to form a new genus.

[0078] Figure 4 shows a protein visualization analysis of phage vB_BglM_WTB. 112 ORFs were predicted, of which 39 were annotated as functional proteins and 73 as hypothetical proteins. All functional proteins can be divided into five modules, including DNA metabolism, lysis, packaging, structural, and other functional modules. Figure 5 shows a phylogenetic tree of its main capsid proteins. This phage is found on a separate branch, distantly related to other phages, and may share a common ancestor with Burkholderia phages.

[0079] Example 5

[0080] Optimal multiplicity of infection experiment

[0081] Phage propagation medium and logarithmic-phase host bacterial culture were added to TSB broth at multiplicities of infection (MOIs) of 100:1, 10:1, 1:1, 1:10, 1:100, 1:1000, 1:10000, 1:100000, and 1:1000000, ensuring the same total volume. After incubation at 37°C and 200 rpm for 8 hours, the cultures were centrifuged at 12,000 g for 10-15 minutes at 4°C, and titers were determined using the double-layer plate method. The results are shown in Table 2.

[0082] Table 2 Multiplicity of infection

[0083] The results showed that the optimal infection multiplicity of phage vB_BglM_WTB was 1:10000.

[0084] Example 6

[0085] One-step growth curve determination

[0086] Burkholderia gladioli ATCC 33664 was cultured to the early logarithmic phase, with a bacterial concentration of 10 8 cfu / mL. Add host bacteria and phage culture medium at an optimal multiplicity of infection of 1:10,000. Incubate at 37°C in a water bath for 5 minutes, centrifuge at 12,000 rpm / min for 30 seconds, and discard the supernatant. Wash the pellet twice with TSB broth. Add 30 mL of TSB broth preheated at 37°C and incubate at 37°C with shaking. Sample at 0 min, and every 10 min for the first 60 min, and every 30 min thereafter. Centrifuge at 12,000 rpm / min for 30 seconds, filter through a 0.45 μm filter, and determine the phage titer at each time point. Plot a one-step growth curve with infection time as the horizontal axis and phage titer as the vertical axis.

[0087] The results are shown in FIG6 , where 0-60 min is the latent period of the phage, 60-210 min is the lysis period of the phage, and 210-480 min is the stationary period of the phage.

[0088] Example 7

[0089] Determination of acid-base stability

[0090] Adjust the pH of TSB broth with dilute hydrochloric acid and dilute sodium hydroxide solution to prepare TSB broth with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Sterilize the TSB broths with different pH values ​​by filtering with 0.45 μm microporous filters, and then dilute the phage to 10 9 The dilutions were incubated in a water bath at 37°C for 1 h, and the titer was determined using the double-layer plate method.

[0091] The results are shown in Figure 7. vB_BglM_WTB is relatively stable in the environment of pH 3-11, and the titer is 10 8 When the pH value is higher than 11, the phage titer begins to decrease, and when the pH value reaches 12, the phage can hardly survive. The results show that the optimal pH for its growth is a neutral to alkaline environment.

[0092] Example 8

[0093] Determination of thermal stability

[0094] The phage stock solution (10 9 pfu / mL) were dispensed into EP tubes and incubated at 25°C, 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C for 60 min, respectively. After appropriate dilution with TSB broth, the titer was determined by the double-layer plate method.

[0095] The results are shown in Figure 8. As the temperature increases, the activity of the phage decreases. After 60 minutes at 65°C, the titer can still reach 1.01×10 6 pfu / mL, indicating that the phage has a certain temperature tolerance. After being exposed to 80℃ for 60min, the phage can hardly survive.

[0096] Example 9

[0097] Bactericidal effect of bacteriophage in fungus

[0098] Take some dried fungus and soak them in sterile water for 10 minutes. Weigh 0.5 (± 0.05) g of wet fungus with a size of 2 cm × 2 cm. Place the fungus of the same size in a 90°C sterile water bath for 30 minutes. Then transfer it to a clean bench and irradiate it with ultraviolet light for 2 hours, irradiating each side of the fungus for 1 hour. Soak the treated fungus in a culture solution of Burkholderia gladioli BG007 for 10 minutes. The culture solution concentration is 10 8 cfu / mL, and after air drying, the fungus was transferred into the phage stock solution (10 9 The fungus was immersed in PBS (1000 pfu / mL) for 10 min. After air-drying at room temperature, the fungus was transferred to a clean sterile culture dish. Samples were taken at 0, 2, 4, 6, 8, 10, and 12 h. The fungus was cut into pieces and resuspended in PBS. The bacterial count was determined after serial dilution.

[0099] The formula for calculating the bactericidal efficiency is: (the amount of Burkholderia in gladiolus of the control group - the amount of Burkholderia in gladiolus of the experimental group) / the amount of Burkholderia in gladiolus of the control group × 100%.

[0100] The results are shown in Figure 9. As shown in Figure 9A, at 4°C, the bacterial count of the fungus treated with phages decreased to the lowest level after 6 hours, reaching 154 cfu / mL, which was 2.6×10 5 cfu / mL (5.42log), at this time the bacteriophage vB_BglM_WTB in the fungus bactericidal rate reached 99.94%; after 12h of treatment, the bacterial count of the fungus treated with phages increased slightly, reaching 1710 cfu / mL, but it still reduced the bacterial count by 8.38×10 5As shown in Figure 9B, at 25°C, the bacterial count of the fungus treated with phages decreased to the lowest level after 2 hours, reaching only 89 cfu / mL, which was 8.5×10 lower than that of the control group. 4 cfu / mL (4.93log), with a bactericidal efficiency of 99.90%. After 12 hours of treatment, the bacterial count increased slightly to 1546 cfu / mL, but it was still reduced by 5.51×10 6 cfu / mL (6.71log).

[0101] Figures 9A and 9B show that the 25°C treatment was more effective, with bacterial counts reduced to a minimum within 2 hours of treatment, and even fewer surviving bacteria. This indicates that 25°C is more effective in disinfecting host bacteria and has a shorter duration of action. Because the number of surviving strains is small and their survival time is short, the likelihood of toxin production is reduced, enhancing the safety of foods like wood ear mushrooms.

[0102] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A bacteriophage, characterized in that: The bacteriophage is vB_BglM_WTB, and its deposit number is CCTCC NO: M 2023525.

2. The bacteriophage according to claim 1, characterized in that The phage genome has a total length of 68541 bp and does not contain virulence genes or drug resistance genes.

3. The bacteriophage according to claim 1, characterized in that The bacteriophage has a stable pH value of 3-11 and a tolerable temperature of 25°C-65°C.

4. A lysate, characterized in that: The lysate of the bacteriophage according to any one of claims 1 to 3.

5. A preparation, characterized in that It comprises the bacteriophage according to any one of claims 1 to 3 or the lysate according to claim 4.

6. Use of the bacteriophage according to any one of claims 1 to 3, the lysate according to claim 4, or the preparation according to claim 5 in preventing and controlling Burkholderia gladiolus.

7. The use according to claim 6, characterized in that: The application is to prevent and control Burkholderia gladiolus in food.

8. The use according to claim 6 or 7, characterized in that: The infection multiplicity of the phage is 1:10000-1:

100.

9. The use according to claim 6, characterized in that: The bacteriophage or lysate or preparation is contacted with the Burkholderia gladiolus.

10. The use according to claim 9, characterized in that: The pH value of the contact medium is 3-11, and the contact time is at least 2 hours.

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