CN110129282B

CN110129282B - Vibrio alginolyticus bacteriophage, bacteriophage composition and application thereof

Info

Publication number: CN110129282B
Application number: CN201910431133.7A
Authority: CN
Inventors: 张永雨; 李成成
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2019-05-22
Filing date: 2019-05-22
Publication date: 2021-05-28
Anticipated expiration: 2039-05-22
Also published as: CN110129282A

Abstract

The invention provides a vibrio alginolyticus bacteriophage, a bacteriophage composition and application thereof, and belongs to the technical field of microbial control. The two new virulent vibrio alginolyticus bacteriophages obtained by screening have strong host specificity and have strong cracking and killing effects on vibrio alginolyticus, and simultaneously, when the two bacteriophages are mixed for use, the vibrio alginolyticus can be effectively prevented from generating resistance mutation, so that an ideal germ removal effect is achieved, and the vibrio alginolyticus bacteriophages have important application value in the aspect of preventing and controlling vibrio alginolyticus infectious diseases of marine cultured animals.

Description

Vibrio alginolyticus bacteriophage, bacteriophage composition and application thereof

Technical Field

The invention belongs to the technical field of microbial control, and particularly relates to a vibrio alginolyticus bacteriophage, a bacteriophage composition and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

The vibrio alginolyticus is a common conditional pathogen in the aquaculture fishery, can infect seawater fish and freshwater fish, is shown by skin injury and bacteremia of the fish in a large range, and can develop muscle and multi-organ edema to further cause death of the fish in severe cases. China is a big aquaculture country, and huge economic losses are caused to the aquaculture industry due to the infection of vibrio alginolyticus every year. More seriously, Vibrio alginolyticus not only infects aquatic livestock, but also causes human diseases through the transmission of aquatic products. Therefore, the problem of the infection of the vibrio alginolyticus in the aquaculture is significant for the health of human beings and the economic development.

Traditionally, antibiotics are widely used in aquaculture industry for the prevention and treatment of vibrio diseases. However, in recent years, the abuse of antibiotics in the aquaculture industry has become more serious. Excessive use of antibiotics not only pollutes the environment and harms human health, but also causes more and more drug-resistant bacteria to be generated, and finally, the antibiotic method becomes 'restraint ineligible' for treating vibrio infection. Therefore, a new safe and efficient method for treating vibrio alginolyticus infection is urgently needed to replace antibiotics, and the defects of traditional medicines are overcome.

Phage therapy is an antibiotic replacement therapy that has evolved again in recent years, with a number of advantages over antibiotics: the phage only cracks host bacteria, other beneficial flora is not damaged, the specificity is strong, and the application safety is high; the bacteriophage is a living body existing in nature, has no chemical pollution, and is an environment-friendly medicament; the phage can be autonomously replicated in host bacteria, and the industrial production cost is low. Thus, phage therapy is a promising approach for the prevention and treatment of vibrio disease.

However, phage therapy also has certain limitations, for example, pathogenic bacteria are easily resistant to infection by a single phage, and the actual therapeutic effect is affected. To solve this problem, a method of phage cocktail was proposed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the vibrio alginolyticus bacteriophage and the bacteriophage composition and the application thereof, two new virulent vibrio alginolyticus bacteriophages are obtained by screening, the host specificity is strong, the vibrio alginolyticus bacteriophage has strong cracking and killing effects, and meanwhile, when the two bacteriophages are mixed for use, the vibrio alginolyticus can be effectively prevented from generating resistance mutation, so that an ideal germ removing effect is achieved, and the vibrio alginolyticus bacteriophage and bacteriophage composition has an important application value in the aspect of preventing and controlling vibrio alginolyticus infectious diseases of marine aquaculture animals.

The invention is realized by the following technical scheme:

in a first aspect of the present invention, there is provided a vibrio alginolyticus phage, which is vibrio alginolyticus phage ValB1 MD or vibrio alginolyticus phage ValB1 HC;

wherein the Vibrio alginolyticus phage (Vibrio phase) ValB1 MD has been sent to China center for type culture Collection (address: Wuhan university in Logania and Logania, Wuhan, Hubei province) with a preservation date of 2019, 4 and 25 days and a preservation number of CCTCC M2019290.

The Vibrio alginolyticus phage (Vibrio phage) ValB1 HC is sent to China center for type culture Collection (address: Wuhan university in Logania Hainan city, Wuhan, Hubei province) and the preservation date is 2019, 4 and 25 days, and the preservation number is CCTCC M2019291.

In a second aspect of the present invention, there is provided a Vibrio alginolyticus phage composition comprising the Vibrio alginolyticus phage (Vibrio phase) ValB1 MD and Vibrio alginolyticus phage (Vibrio phase) ValB1 HC.

In a third aspect of the invention, the application of the vibrio alginolyticus bacteriophage or the bacteriophage composition in preparing a vibrio alginolyticus bactericide is provided.

In a fourth aspect of the invention, a vibrio alginolyticus bactericide is provided, which comprises the vibrio alginolyticus bacteriophage or the bacteriophage composition.

In a fifth aspect of the invention, there is provided the use of the above vibrio alginolyticus bacteriophage, bacteriophage composition or vibrio alginolyticus fungicide in controlling vibrio infection in aquaculture.

The invention has the beneficial effects that:

the invention obtains two new virulent vibrio alginolyticus bacteriophages through screening, and the two bacteriophages have strong host specificity and have strong cracking and killing effects on vibrio alginolyticus. Meanwhile, the two phage strains are jointly applied, so that the efficient and long-acting sterilization effect can be achieved, the problem that the bacteriophage mutation resistance is easily generated by pathogenic bacteria in phage therapy is effectively avoided, and the phage strain can be widely applied to the aquaculture industry, so that the practical application value is good.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1: plate culture photograph of Vibrio alginolyticus phage ValB1 MD in example 2;

FIG. 2: a photograph of a plate culture of the Vibrio alginolyticus phage ValB1 HC in example 4;

FIG. 3: transmission electron micrograph of Vibrio alginolyticus phage ValB1 MD in example 7;

FIG. 4: a transmission electron micrograph of the Vibrio alginolyticus phage ValB1 HC in example 7;

FIG. 5: FIG. 8 shows the results of the chloroform sensitivity test of the Vibrio alginolyticus phage ValB1 MD;

FIG. 6: FIG. 8 shows the results of the chloroform sensitivity test of the Vibrio alginolyticus phage ValB1 HC;

FIG. 7: one-step growth profile of the Vibrio alginolyticus phage ValB1 MD in example 12;

FIG. 8: one-step growth profile of the Vibrio alginolyticus phage ValB1 HC in example 13;

FIG. 9: graph of the effect of the Vibrio alginolyticus phage ValB1 MD single phage infection Vibrio alginolyticus VIB 283T in example 14;

FIG. 10: the graph of the effect of the Vibrio alginolyticus bacteriophage ValB1 HC single phage infection Vibrio alginolyticus VIB 283T in example 15;

FIG. 11: the effect of the Vibrio alginolyticus bacteriophages ValB1 MD and ValB1 HC in combination with Vibrio alginolyticus VIB 283T infection in example 16 is plotted.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

As mentioned above, phage therapy has certain limitations, for example, pathogenic bacteria are susceptible to resistance mutation to infection by a single phage, affecting its actual therapeutic effect.

In view of the above, in one embodiment of the present invention, there is provided a vibrio alginolyticus phage, wherein the vibrio alginolyticus phage is vibrio alginolyticus phage ValB1 MD or vibrio alginolyticus phage ValB1 HC.

Wherein the Vibrio alginolyticus phage ValB1 MD has been sent to China center for type culture Collection (Wuhan university in Wuchan Loya, Wuhan, Hubei province) with a preservation date of 2019, 4 months and 25 days and a preservation number of CCTCC M2019290.

The biological characteristics of the ValB1 MD are as follows: ValB1 MD appeared under an electron microscope to consist of a regular hexahedral head of about 90nm in diameter and a tail 117nm in length and 25nm in width, belonging to the Myoviridae family.

The ValB1 MD of the present invention was insensitive to both 2% and 20% chloroform.

Tests prove that the ValB1 MD disclosed by the invention is specifically infected with Vibrio alginolyticus VIB 283T, and is not infected with other Vibrio to be detected.

The infection of ValB1 MD is characterized in that the incubation period of ValB1 MD infection of Vibrio alginolyticus VIB 283T is 30min, the lysis period is 3.5h, and the phage release amount is 25 PFU/cell.

The Vibrio alginolyticus phage ValB1 HC has been sent to China center for type culture Collection (Wuhan university in Wuchang Loojia mountain, Wuhan, Hubei province) with a preservation date of 2019, 4 months and 25 days and a preservation number of CCTCC M2019291.

The biological characteristics of the ValB1 HC are as follows: under an electron microscope, the ValB1 HC appeared to consist of a polyhedral head of about 107nm in length and 70nm in width and a tail of 95nm in length, belonging to the Myoviridae family.

The ValB1 HC infection is characterized in that the incubation period of ValB1 HC infecting Vibrio alginolyticus VIB 283T is 30min, the lysis period is 3h, and the phage release amount is 30 PFU/cell.

The ValB1 HC of the present invention was insensitive to both 2% and 20% chloroform.

Experiments prove that the ValB1 HC of the invention can be infected with Vibrio alginolyticus NBRC 104587(T) and a mutant Vibrio alginolyticus VIB 283T-S6 generated by infecting Vibrio alginolyticus VIB 283T by a single phage ValB1 MD.

In another embodiment of the present invention, there is provided a Vibrio alginolyticus phage composition comprising the Vibrio alginolyticus phage ValB1 MD and the Vibrio alginolyticus phage ValB1 HC.

The titer of each of the Vibrio alginolyticus bacteriophages ValB1 MD and ValB1 HC is not less than 10⁸ PFU/ml。

When the Vibrio alginolyticus phage ValB1 MD and ValB1 HC are used in combination, the titer-to-titer ratio of the Vibrio alginolyticus phage ValB1 MD and the ValB1 HC is 1: 1; the MOI value of the infection complex number is 0.1-10, and researches show that when the MOI is 10, the growth of Vibrio alginolyticus VIB 283T can be completely inhibited, and no mutant strain is generated.

In another embodiment of the present invention, there is provided a use of the above Vibrio alginolyticus bacteriophage or bacteriophage composition in preparing a Vibrio alginolyticus bactericide.

In yet another embodiment of the present invention, there is provided a vibrio alginolyticus bactericide comprising the vibrio alginolyticus bacteriophage or the bacteriophage composition described above.

In another embodiment of the invention, the formulation of the vibrio alginolyticus bactericide is wettable powder, water dispersible granule or water suspending agent, preferably water suspending agent.

In another embodiment of the present invention, the Vibrio alginolyticus bactericide further comprises an aquaculture acceptable adjuvant selected from one or more of a dispersant, a stabilizer, a filler and a solvent. The sources and the like of the auxiliary materials acceptable in the aquaculture industry of the invention are not particularly limited, and the auxiliary materials can be generally obtained by adopting commercial products.

Wherein the dispersant is one or two of sodium tripolyphosphate and sodium pyrophosphate.

The stabilizer may be one or more selected from sodium alginate, gelatin, xanthan gum or guar gum.

The filler can be selected from one or more of chitosan, diatomite, bentonite and attapulgite.

The solvent may be selected from water (preferably deionized water).

In another embodiment of the present invention, there is provided a use of the vibrio alginolyticus bacteriophage, the bacteriophage composition or the vibrio alginolyticus fungicide described above for controlling vibrio infection in aquaculture.

In order to better explain the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to specific embodiments.

In the following examples, the formulation of the RO medium was: 1g of yeast powder, 1g of peptone, 1g of sodium acetate, 10ml of trace elements, 1000ml of deionized water and pH 7.8.

The solid culture medium is as follows: 1g of yeast powder, 1g of peptone, 1g of sodium acetate, 10ml of trace elements, 15g of agar, 1000ml of deionized water and pH 7.8.

The semi-solid culture medium is as follows: 1g of yeast powder, 1g of peptone, 1g of sodium acetate, 10ml of trace elements, 5g of agar, 1000ml of deionized water and pH 7.8.

Example 1 isolation of Vibrio alginolyticus phage ValB1 MD

An offshore water sample is taken, filtered by a sterile filter membrane of 0.22 mu m, added into Vibrio alginolyticus VIB 283T bacterial liquid in exponential growth phase according to the volume ratio of 10 percent, and subjected to shaking culture in a shaking table at 28 ℃. Sampling at 1d, 3d, 5d and 7d respectively, filtering and sterilizing the samples through a sterile filter head with the diameter of 0.22 mu m, diluting different concentration gradients by using SM Buffer respectively, mixing 1ml of diluted filtrate with 1ml of Vibrio algyrinthuusVIB 283T bacterial liquid at the logarithmic phase in a shaking table at the temperature of 28 ℃ for shaking and incubating for 30min, pouring melted and precooled RO semisolid at the temperature of about 48 ℃, mixing, pouring the mixture on an RO solid plate, and shaking uniformly by horizontal shaking until the semisolid is solidified. And (3) placing the semi-solid plate in a constant-temperature incubator at 28 ℃ for overnight culture, wherein the phage obtained by primary separation is obtained if plaques appear.

Example 2 purification of Vibrio alginolyticus phage ValB1 MD

The plaques obtained in example 1 were soaked in SM Buffer overnight, and then the soaking solution was filtered through a 0.22 μm sterile filter head and diluted with SM Buffer in a gradient manner, and double-layered plates were prepared as described in example 1, to obtain plaques uniformly dispersed on the double-layered plates. Repeating the operation for 3-5 times until the plaque size and shape on the double-layer plate obtained at the last time are consistent. The purified phage double-layer plate is shown in FIG. 1.

Example 3 isolation of Vibrio alginolyticus phage ValB1 HC

The specific implementation method is the same as that described in example 1.

Example 4 purification of Vibrio alginolyticus phage ValB1 HC

The procedure was as in example 2, and the purified phage double-layer plate was shown in FIG. 2. Plaques with uniform size and shape were formed on the plate.

Example 5 titer determination of Vibrio alginolyticus phage ValB1 MD

The phage solution filtered by a 0.22 mu m sterile filter head is diluted by different times by SM Buffer, and is mixed with logarithmic phase Vibrio alginilyticus VIB 283T bacterial liquid to prepare a double-layer plate, and the double-layer plate is cultured overnight at the constant temperature of 28 ℃ to grow the plaque. The plaques on the double-layer plate were counted and the titer was calculated by selecting the plate with the number of plaques ranging from 30 to 300. And (4) converting the number of the plaques on the plate with the corresponding dilution times to obtain the titer of the phage. Such as ValB1 MD phage liquid dilution 10^-6The double-layer plate after the fall has 100 plaques, then the titer of the ValB1 MD phage liquid is 1 x 10⁸ PFU/ml。

Example 6 titer determination of Vibrio alginolyticus phage ValB1 HC

The titer of ValB1 HC was measured in the same manner as in example 5. Experimental determination of the valency of ValB1 HC 1 x 10⁸ PFU/ml。

Example 7 Electron microscopy morphological observations of Vibrio alginolyticus bacteriophages ValB1 MD and ValB1 HC

The enriched phage liquid (about 1L) was first filtered through a 0.22 μm sterile filter, to which DNase and RNase were added at a final concentration of 2ng/L, gently mixed and allowed to stand at room temperature for 1 h. After standing, NaCl with the final concentration of 1M is added into the solution, and the solution is stood for 30min after being dissolved by light mixing. The supernatant was collected by centrifugation of the above solution (8000rpm, 30min), and PEG 8000 was added to the supernatant solution to a final concentration of 10%, and the mixture was left at 4 ℃ for 24 hours in the dark. The pellet was then collected by centrifugation at 10000g and resuspended in SM Buffer. 7ml of cesium chloride solution with a density of 1.5g/ml was added to an ultracentrifuge tube, then 5ml of the above-mentioned phage suspension resuspended in SM Buffer was added, ultra-high speed vacuum centrifugation (200000g, 4 ℃, 8h) was performed, and the pale blue phage bands obtained by centrifugation were aspirated. The Buffer was replaced with SM Buffer using a 30KD ultrafiltration tube, and the sample was negatively stained with uranyl acetate and used for transmission electron microscopy to observe phage morphology. The electron microscope morphology of ValB1 MD is shown in FIG. 3, and the electron microscope morphology of ValB1 HC is shown in FIG. 4. ValB1 MD is shown as a regular hexahedral head with a diameter of 90nm and a tail length of 117 nm. The hexahedron of ValB1 HC had a head length of 107nm, a width of 71nm, and a tail length of about 95 nm. Both belong to the family myoviridae.

Example 8 chloroform sensitive detection of Vibrio alginolyticus phage ValB1 MD

And mixing the logarithmic phase bacterial liquid with a heated and precooled semi-solid culture medium (48 ℃) to prepare a double-layer flat plate. The phage solution was used to prepare 2% and 20% chloroform solution by volume fraction, respectively, and the control group was phage solution without any treatment. After vortexing, each experimental group was allowed to stand at room temperature for 30min, and then centrifuged at 8000rpm, and 10. mu.l of the upper aqueous phase was added dropwise to the above double-layer plate. Culturing in a thermostat at 28 ℃ overnight, and observing whether the plaque size of the experimental group is different from that of the control group. The chloroform sensitivity test double-layer plate of ValB1 MD is shown in FIG. 5, the plaque size formed by the phage after chloroform treatment and the control group phage is not different, which shows that the phage is not sensitive to chloroform, and the phage has no lipid structure.

Example 9 chloroform sensitive detection of Vibrio alginolyticus phage ValB1 HC

The specific implementation method is the same as that of example 8. The chloroform sensitivity test plate of ValB1 HC is shown in FIG. 6, and the plaque size formed by the phage after chloroform treatment and the control group phage is not different, which shows that the phage is not sensitive to chloroform and the phage has no lipid structure.

Example 10 host Range detection of Vibrio alginolyticus phage ValB1 MD

In the experiment, 16 strains of vibrio stored in a laboratory are selected for phage host range detection. That is, different Vibrio species growing to logarithmic phase are mixed with RO semisolid pre-cooled by heating (about 48 ℃) to make a double-layer plate, 10 μ l of bacteriophage ValB1 MD filtered by a 0.22 μm sterile filter membrane is added dropwise after the upper layer semisolid is solidified, and the plate is placed in an incubator at 28 ℃ overnight to observe whether the plaque appears. If so, indicating that the host can be lysed by the phage ValB1 MD; if not, it indicates that the host cannot be lysed by the phage ValB1 MD. The results of the host range test of ValB1 MD are shown in Table 1, and in the tested host, ValB1 MD can only cleave Vibrio algyrinolyticus VIB 283T.

Example 11 host Range detection of Vibrio alginolyticus phage ValB1 HC

The specific implementation method is the same as that of example 9. The results of the host range test on ValB1 HC are shown in Table 1, and in the tested host, ValB1 HC can crack a total of four strains of Vibrio alismolyticus VIB 283T.

Example 12 one-step growth Curve assay of the Vibrio alginolyticus phage ValB1 MD

When Vibrio alginolyticus Vibrio algyrinolyticus VIB 283T grows to a logarithmic phase, phage ValB1 MD filtered through a 0.22 μm sterile filter is added in an amount of MOI 0.01 according to the ValB1 MD phage titer determined in examples 5 and 6, and incubated at 28 ℃ for 30min with shaking in a shaker, and then centrifuged at 8000rpm for 10min, and after cell pellets are washed with fresh RO medium in a re-suspension manner for 3 times, the RO medium is added and incubated at 28 ℃ with shaking in a shaker. Sampling every 30min from 0h, taking 1ml of sample, and directly adding glutaraldehyde (with a final concentration of 0.5%) for fixation to obtain a bacterial sample; 1ml of the sample was filtered through a 0.22 μm sterile filter head and fixed with glutaraldehyde (final concentration: 0.5%) to obtain a phage sample. And (3) fixing each group of samples in the dark by glutaraldehyde for 30min, measuring the amount of bacteria or phage by a flow cytometer, and drawing a change curve of each parameter along with time. The one-step growth curve of ValB1 MD is shown in FIG. 7. The incubation period for ValB1 MD cleavage of Vibrio alginolyticus VIB 283T is 30min, and the cleavage period is about 3.5 h.

Example 13 one-step growth Curve assay of the Vibrio alginolyticus phage ValB1 HC

The specific implementation method is the same as that of example 11. The one-step growth curve of ValB1 HC is shown in FIG. 8. The incubation period for ValB1 HC to cleave Vibrio algyrinolyticus VIB 283T was 30min, and the cleavage period was about 3 h.

Example 14 measurement of the Bactericidal Effect of Vibrio alginolyticus phage ValB1 MD

The titer of the phage ValB1 MD determined in example 5 was determined by setting 3 experimental groups by mixing phage ValB1 MD and Vibrio algyrinolyticus VIB 283T bacteria at MOI of 0.1, 1 and 10, respectively, and then shake-culturing the bacteria at 28 ℃. The control group was cultured alone with Vibrio algyrinolyticus VIB 283T. Each set of experimental groups was set in triplicate. From 0h, sampling every 1h to determine the OD of the bacterial liquid₆₀₀An absorbance value. After 12h, the samples were taken at unequal intervals up to 79 h. The growth curves of Vibrio algyrinolyticus VIB 283T bacteria under different MOI values are drawn. The bactericidal effect of ValB1 MD at different MOI values is shown in FIG. 9. As can be seen, the bactericidal effect is relatively obvious in about 6 hours at the beginning of the action of the phage ValB1 MD, but the explosion of the mutant strain is long with the time, and the bactericidal efficiency under each MOI value after 79 hours of culture is respectively: MOI 0.1, 49.5%; MOI 1, 32.7%; MOI 10, 32.7%.

Example 15 measurement of the Bactericidal Effect of Vibrio alginolyticus phage ValB1 HC

The specific implementation method is the same as that of example 12. The bactericidal effect of ValB1 HC at different MOI values is shown in FIG. 10. As can be seen, the bactericidal effect was relatively clear in the first 6 hours of the action of the phage ValB1 HC, but with time, explosive growth of the mutant occurred, and the bactericidal efficiency was as follows under the MOI values after 79 hours of cultivation: MOI 0.1, 70.5%; MOI 1, 66.3%; MOI 10, 49.5%.

Example 16 measurement of the Bactericidal Effect of the combination of Vibrio alginolyticus bacteriophages ValB1 MD and ValB1 HC

The titers of the phages ValB1 MD and ValB1 HC determined according to examples 5 and 6 were determined as follows, for ValB1 MD and ValB1 HC, at a titer of 1:1 mixing, wherein the total amount of the phage in the mixed solution is the sum of the two phage amount. 3 groups of experimental groups are set, and mixed phage and Vibrio algyrinolyticus VIB 283T bacteria are mixed according to the proportion of MOI 0.1, 1 and 10, and then the bacteria are cultured at 28 ℃. The control group was cultured alone with Vibrio alginolyticus VIB 283T. From 0h, sampling every 1h to determine the OD of the bacterial liquid₆₀₀An absorbance value. After 12h, the samples were taken at unequal intervals up to 79 h. A growth curve of Vibrio algyrinolyticus VIB 283T bacteria under different MOI values is drawn. The bactericidal effect of the combination of ValB1 MD and ValB1 HC at different MOI values is shown in FIG. 11. As can be seen, the phage ValB1 MD and ValB1 HC work together under MOI of 10, and the bactericidal efficiency is maintained at 100% when the bacterial culture time is prolonged to 79 h. The effect of thoroughly cracking pathogenic bacteria is achieved.

TABLE 1

Note: "+" indicates that the host bacterium is sensitive to the phage; "-" indicates that the host is not susceptible to phage.

It should be noted that the above examples are only used to illustrate the technical solution of the present invention and are not limited thereto. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. The vibrio alginolyticus phage composition is characterized by comprising phage ValB1 MD and phage ValB1 HC, wherein the vibrio alginolyticus phage ValB1 MD is sent to China center for type culture collection with the collection date of 2019, 4 and 25 months and the collection number of CCTCC M2019290;

the Vibrio alginolyticus phage ValB1 HC is sent to China center for type culture collection, the preservation date is 2019, 4 months and 25 days, and the preservation number is CCTCC M2019291.

2. The Vibrio alginolyticus phage composition of claim 1, wherein none of the phage ValB1 MD and ValB1 HC has a titer less than 10⁸PFU/ml。

3. The Vibrio alginolyticus phage composition of claim 1, wherein the MoI is 0.1 to 10.

4. The vibrio alginolyticus phage composition of claim 1, wherein the multiplicity of infection MOI is 10.

5. Use of the vibrio alginolyticus phage composition of claim 1 in preparing a vibrio alginolyticus bactericide.

6. A Vibrio alginolyticus bactericidal agent, wherein the Vibrio alginolyticus bactericidal agent comprises the Vibrio alginolyticus bacteriophage composition of claim 1.

7. The Vibrio alginolyticus bactericide according to claim 6, wherein the formulation of the Vibrio alginolyticus bactericide is a wettable powder, a water dispersible granule or a water suspension.

8. The Vibrio alginolyticus bactericide of claim 6, further comprising an aquaculture acceptable adjuvant.

9. The Vibrio alginolyticus fungicide of claim 8 wherein the aquaculture acceptable adjuvant is selected from one or more of a dispersant, a stabilizer, a filler and a solvent.

10. Use of a vibrio alginolyticus bacteriophage composition according to claim 1 or a vibrio alginolyticus bacteriocide according to any one of claims 6 to 9 for controlling vibrio aquaculture infection.