Bacillus altitudinis JYD51 and application thereof in aquaculture

Abstract

The invention discloses bacillus altitudinis JYD51 and application of the bacillus altitudinis JYD51 in aquaculture, the bacillus altitudinis JYD51 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.32164, and the preservation date is October 10, 2024. The bacillus altitudinis JYD51 can be applied to aquaculture. The JYD51 strain disclosed by the invention can be fermented by using a culture medium rich in organic matters, and a fermented product of the JYD51 strain has no toxic effect on penaeus vannamei boone and crabs and has the effects of inhibiting infection of enterocytozoon hepatopenaei to the penaeus vannamei boone and inhibiting harm of microsporidia crabs to the crabs. Meanwhile, the microbial agent prepared by compounding the JYD51 strain and the lysinibacillus sphaericus JYQ7 can effectively and cooperatively control the harm of enterocytozoon hepatopenaei and microsporidia crabs, purify the water body environment for growth of the shrimps and the crabs, improve the survival rate of the shrimps and the crabs in aquaculture and improve the product quality.

Description

Technical Field

[0001] The invention belongs to the field of microbial biotechnology, and specifically relates to a strain of Bacillus subtilis JYD51 and application thereof in aquaculture. Background Art

[0002] Enterocytozoon hepatopenaei (EHP) belongs to the Microsporidia family and the Enterocytozoon genus. The mature spore size is about (0.9±0.3)μm×(1.8±0.3)μm and is oval. As an obligate intracellular parasite, EHP mainly parasitizes the epithelial cells of the hepatopancreas of shrimp. It is highly contagious and is one of the important pathogens affecting global shrimp farming production. It can infect a variety of economically farmed shrimps, such as Penaeus vannamei, P. carincauda and P. monodon. In pond farming, it was found that EHP-infected shrimp had no obvious clinical symptoms in the early stage, and the feeding was normal, without large-scale deaths. After 2-3 months, the diseased shrimp showed slow growth or even stopped growing, and the size difference was obvious. When the infection is severe, the shrimp's intestines become inflamed, the liver and pancreas become atrophic, soft, and darker in color, and a few shrimp may have white stools, which eventually leads to the death of the shrimp. Currently, the infection caused by EHP has seriously harmed the shrimp farming industry.

[0003] The crab currently farmed is mainly the Chinese mitten crab. The microsporidia Endoreticulatuseriocheir sp.nov of the Chinese mitten crab is a single-cell eukaryotic organism that lives an obligate intracellular parasite. The individual is extremely small and oval in shape, with a long diameter of 1.7±0.2μm and a short diameter of 1.0±0.2μm. It has one nucleus that occupies the center of the body, located between the polar membrane layer and the post-vacuole, surrounded by 2 or 3 layers of endoplasmic reticulum and 78 turns of polar tubes. The microsporidia is a new aquatic pathogen that causes hepatopancreatic necrosis of the Chinese mitten crab. After the host is infected with the microsporidia, its disease resistance becomes weak and its growth and development is slow. River crab hepatopancreatic necrosis syndrome is commonly known as water-shrinking disease. Its typical symptoms are gradual atrophy and necrosis of the river crab's hepatopancreas, and the color gradually changes from normal golden yellow to grayish white. The appendages become empty and weak, and "hydration" symptoms appear. When encountering sudden changes in the environment or during transportation, they are prone to stress reactions and die in large numbers. Even if they survive, their fatness is significantly reduced and they have no edible value. In recent years, the disease has become explosively prevalent, causing huge losses to the crab farming industry.

[0004] The emergence of shrimp hepatic enterocytes and crab microsporidia seriously endangers the breeding of shrimps and crabs, and domestic and foreign scholars have also conducted a lot of research on their prevention and control measures. However, due to the strict cell parasitism of these two microsporidia, their mature spore walls have an extremely hard chitin shell, and exogenous drugs are difficult to be effective. No effective therapeutic drugs have been found so far. The main control method in the aquaculture industry is to treat the bottom mud with agents and lime, and control the pollution source during the breeding process. Once the infection is found, it is difficult to treat and control the harm. Existing patents disclose some methods of controlling shrimp hepatic enterocytes using microorganisms, such as ZL201910150847.0 discloses a strain of Bacillus microbacterium, CN201910150635.2 discloses a strain of Pseudoalteromonas, and CN20230790276.3 discloses a strain of Lactobacillus plantarum. The public documents mention that these prokaryotic microbial strains have an effect on shrimp hepatic enterocytes while controlling some pathogens in aquaculture. The related Bacillus subtilis in the currently disclosed prior art is mainly used to promote plant growth and prevent and control plant root-knot nematodes. However, there are no reports on the application of Bacillus subtilis in controlling shrimp hepatocystis and crab microsporidia in aquaculture and related data, and there are no reports on the combination of Bacillus subtilis with other strains in aquaculture. Summary of the invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a strain of Bacillus subtilis JYD51, which is isolated from the bottom mud of a breeding pond infected with shrimp hepatocystis. The strain has no toxic effect on South American shrimp and crabs. At the same time, the bacterial body and metabolites of the strain of Bacillus subtilis JYD51 have the effect of degrading the cell wall of shrimp hepatocystis and inhibiting its germination in vitro; at the same time, it can inhibit the harm of crab microcystis. The bacterial agent can effectively improve the survival rate and breeding quality of shrimp and crabs, and increase the economic benefits of breeders.

[0006] The present invention also provides a bacterial agent and application of the Bacillus subtilis JYD51.

[0007] Furthermore, the present invention also provides the highland Bacillus JYD51 and the spherical lysinibacillus JYQ7 for combined use, which can inhibit shrimp hepatocystis and crab microsporidia while purifying the water environment for shrimp and crab growth, thereby improving the quality of shrimp and crab products.

[0008] Technical solution: In order to achieve the above-mentioned purpose, the strain of Bacillus altitudinis JYD51 described in the present invention is characterized in that it has been identified as Bacillus altitudinis, has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.32164 and a deposit date of October 10, 2024.

[0009] The invention discloses an application of the Bacillus subtilis JYD51 in aquaculture.

[0010] Among them, the application of the highland Bacillus JYD51 in improving the quality of aquatic products in aquaculture.

[0011] Furthermore, the highland Bacillus JYD51 is used in improving the survival rate and quality of shrimps and crabs in aquaculture.

[0012] The invention discloses an application of the Bacillus subtilis JYD51 in inhibiting hepatocellular carcinoma of shrimp in aquaculture.

[0013] The invention discloses an application of the Bacillus subtilis JYD51 in inhibiting crab microsporidia in aquaculture.

[0014] Among them, the aquaculture includes open-air pond farming and small shed farming of shrimps and crabs.

[0015] The invention contains the highland bacillus JYD51 which is fermented to prepare a bacterial agent.

[0016] Among them, the bacterial agent includes liquid bacterial agent and solid bacterial agent; the liquid bacterial agent includes fermentation liquid obtained by fermentation of JYD51 strain, and the bacterial agent is prepared from the fermentation liquid and the bacterial bodies contained in the fermentation liquid; the solid bacterial agent includes powdered or granular bacterial agent.

[0017] The bacterial agent of the present invention is used in aquaculture to inhibit hepatoenteric cysts of shrimps and microsporidia of crabs, and to ensure and promote the survival and growth of shrimps and crabs.

[0018] The highland Bacillus JYD51 and the spherical lysinibacillus JYQ7 of the present invention are used in combination to inhibit shrimp hepatoenterozoa and crab microsporidia while purifying the water environment for the growth of shrimps and crabs, improving the survival of shrimps and crabs in aquatic products and improving the quality of shrimps.

[0019] The highland Bacillus JYD51 and the spherical lysine Bacillus JYQ7 are used in combination by preparing a composite bacterial agent, wherein the composite bacterial agent is prepared by fermenting the two strains of bacteria separately, and the bacterial agents are prepared separately, and are mixed to form a composite bacterial agent when used; or after each strain of bacteria is fermented separately, the fermented products containing the fermentation liquid and bacterial bodies of the two strains are mixed to prepare the bacterial agent, which is the composite bacterial agent.

[0020] Preferably, the composite bacterial agent is prepared by fermenting two strains of bacteria separately to prepare the bacterial agent separately, and then mixing them in a 1:1 ratio when using, which is the composite bacterial agent; or after each strain of bacteria is fermented separately, the fermented products (containing fermentation liquid and bacterial bodies) of the two strains are mixed in a 1:1 ratio and then the bacterial agent is prepared by a conventional method, which is the composite bacterial agent.

[0021] The highland bacillus JYD51 of the invention controls shrimp hepatocystis and crab microsporidia in aquaculture, thereby improving the quality of shrimp and crab products.

[0022] The Bacillus subtilis JYD51 strain of the present invention is isolated and screened from the bottom mud of the breeding pond of Penaeus vannamei infected with shrimp hepatoenterozoa. The strain can be preserved by any technical method for preserving Bacillus provided in the public literature of the art. The bacterial agent can be prepared by any technical method for preparing various bacterial agents provided in the public literature of the art, and the prepared bacterial agent can be a freeze-dried bacterial agent, a spray-dried bacterial agent, an extruded granular bacterial agent and a liquid bacterial agent. Among them, the bacterial content in the solid bacterial agent is 1.00×10 8 ~1.00×10 10 CFU / g, these bacterial agents are stored in a dry state. The liquid bacterial agent contains fermentation liquid and bacterial cells, and the bacterial cell content in the liquid bacterial agent reaches 1.00×10 8 ~1.00×10 10 CFU / mL. The liquid bacterial agent should be stored in a sealed container at room temperature.

[0023] The present invention also provides the application of the bacterial agent in shrimp and crab breeding ponds, and the application method is as follows: there are two methods for using the liquid bacterial agent, one method is to directly adopt the water surface broadcasting method, use 2-3L per mu, spray once a day for 3 consecutive days; the second method is to mix with feed and feed, 1L of bacterial agent is mixed with 100-150 kg of granular feed and fed.

[0024] Solid bacterial agents include freeze-dried bacterial agents, spray-dried powders and extruded granules, which are used by spreading on the water surface and treating bottom mud; the dosage for surface spraying is 1-2 kg per mu, and it is used continuously for 3 days; for bottom mud treatment, 200 grams of bacterial agent per square meter is used and stirred evenly.

[0025] The composite bacterial agent prepared by the highland Bacillus JYD51 and the spherical lysine Bacillus JYQ7 (disclosed in application number 202411109472.0) of the present invention can produce the effect of reducing the dosage and increasing the functional range. There are two methods for preparing the composite bacterial agent. One method is: the two strains of bacteria are fermented separately, and the bacterial agent is prepared separately, and the mixture is mixed in a ratio of 1:1 when used; the second method is: after the two strains of bacteria are fermented separately, the fermented products (containing fermentation liquid and bacterial bodies) of the two strains are mixed in a ratio of 1:1, and then the bacterial agent is prepared by a conventional method, that is, the composite bacterial agent.

[0026] Since the strain of the present invention is derived from the pond mud of farmed South American shrimp infected with shrimp hepatoenteric cysts, it can grow normally in the pond mud and is in the same ecological site as the dormant body of hepatoenteric cysts in the pond mud, and the two can be in close contact for a long time. Furthermore, experimental studies have also found that Bacillus subtilis JYD51 and spherical lysine Bacillus JYQ7 (disclosed in application number 202411109472.0) can grow simultaneously when mixed and cultured without affecting each other. The composite bacterial agent mixed and compounded by the two does not affect the functions of the two. For example: on the basis of reducing the dosage of the functional bacteria of Bacillus subtilis, the composite bacterial agent also has the ability to control shrimp hepatoenteric cysts and crab microsporidia; the composite bacterial agent also has the ability to purify water bodies on the basis of reducing the dosage of the functional bacteria spherical lysine Bacillus. The bacterial agent prepared by combining the JYD51 strain of the present invention with the spherical lysinibacillus JYQ7 can effectively and synergistically control the harm of shrimp hepatoenteroides and crab microsporidia, and simultaneously synergistically purify the water environment for the growth of shrimps and crabs, thereby improving the survival rate of shrimps and crabs and the product quality in aquaculture.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0028] The isolated and screened Bacillus altitudinis JYD51 of the present invention is derived from the bottom mud of the pond of the cultured white shrimp infected with shrimp hepatocytosis, can grow normally in the bottom mud of the pond, and is in the same ecological position as the dormant body of the hepatocytosis in the bottom mud of the pond, and the two can be in close contact for a long time. In the present invention, the JYD51 strain can be fermented with a medium rich in organic matter, and its fermentation product has no toxic effect on white shrimp and crabs, and can promote the growth of crabs and shrimps and increase the survival rate. The strain JYD51 has been experimentally verified to have the ability to inhibit the infection of hepatocytosis (Enterocytozoonhepatopenaei, EHP) on white shrimp and the harm of crab microsporidia to crabs. The bacterial agent prepared by using the fermentation product of JYD51 in the pond of culturing shrimp can effectively control the harm of shrimp hepatocytosis, and can control the harm of crab microsporidia in the pond of culturing crabs. In addition, the bacterial agent prepared by combining the JYD51 strain with Lysinibacillus sphaericus JYQ7 can control the harm of microsporidia to shrimps and crabs while purifying the water environment where shrimps and crabs grow, improving the quality of shrimp and crab products and increasing the economic benefits of farmers.

[0029] The present invention also provides a biological control method for controlling the damage of shrimp hepatocystis and crab microsporidia in aquaculture. At the same time, the bacterial agent of the present invention can be produced by a general fermentation method, has the advantages of low production cost and convenient use, is suitable for the cultivation of various shrimps and crabs in aquaculture, and has important significance for increasing the economic benefits of farmers. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a colony photo of the strain JYQ7 of the present invention;

[0031] Figure 2 This is a microscope picture of the bacterial cell JYQ7 of the present invention;

[0032] Figure 3 This is a microscope photograph of the spores of the strain JYQ7 of the present invention.

[0033] Figure 4 Phylogenetic tree image for identification of strain JYQ7 of the present invention DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0035] The raw and auxiliary materials, reagents, instruments, etc. used in the present invention are all well known in the art, and other experimental equipment well known in the art can be applied to the implementation of the present invention in the following manners.

[0036] Among them, the spherical lysine spore-forming bacteria JYQ7 strain is the applicant's previously disclosed patent CN118853488A and has been publicly deposited.

[0037] Enterocytozoon hepatopenaei (EHP) and Endoreticulatus eriocheir sp.nov (nov) were directly isolated and extracted from infected hosts of farmed shrimp and crabs and identified and confirmed.

[0038] Example 1

[0039] 1. Control the isolation of hepatocellular carcinoma strains:

[0040] Collect the mud from the ponds of Penaeus penaeus infected with hepatoenterozoa, bring it back to the laboratory by keeping the temperature in an ice bottle, dilute and separate it with sterile saline immediately, culture it in a 30℃ biochemical incubator, select the growing colonies for streaking purification, select the positive bacteria after Gram staining of the purified strains and number them. Inoculate these numbered purified strains with ordinary broth medium and culture them on a slant for 5 days, place them in a 4℃ refrigerator for 7 days, observe them under a microscope, and select the spore-producing strains for reserve.

[0041] 2. Preparation of bacterial suspension:

[0042] The retained spore-forming strains were inoculated into ordinary broth medium and cultured on a slant for 5 days. The cells were washed with sterile water to prepare a cell suspension. The OD of each strain suspension was measured using a spectrophotometer. 600 The quantitative OD value (OD 600The same strain suspension was used to control hepatocellular carcinoma.

[0043] 3. Screening of strains for controlling shrimp hepatocellular carcinoma

[0044] The crude extract of hepatocystis spores was isolated from diseased shrimp infected with hepatocystis, and the oval dormant spores of hepatocystis were observed under a microscope by staining method, and the spore suspension was diluted to 20 / ml for artificial infection experiment to confirm the parasitic nature of the isolated hepatocystis.

[0045] 4. Two methods are used to screen strains for controlling hepatocellular carcinoma:

[0046] The first method is a preliminary screening: the bacterial suspension obtained in step (2) and the shrimp hepatocystis suspension obtained in step (3) are mixed in a volume ratio of 1:1 and then dripped onto a glass slide. The glass slide is placed in a moisturizing box and placed in a constant temperature box at 25°C for 10 hours. The same volume of bacterial suspension and sporocyst suspension are used as controls. After 10 hours, slides are taken and stained every 2 hours to observe the status of the sporocysts. The strains that can degrade hepatocystis within 20 hours are selected to obtain 3 strains.

[0047] The second method is rescreening: the three strains obtained in the initial screening are made into bacterial suspensions according to the method of step (2), 6 culture tanks are taken and each is injected with 5L of water, and 100 young white shrimps with a body length of 4.0±0.3cm and a body weight of 1.5±0.3g are placed. Among them, 4 culture tanks are injected with 1mL of spore suspension (20 / ml) every day to artificially infect shrimp hepatocystis. After one week of infection, each strain is inoculated into the infected culture tank, and each tank is inoculated with 1mL of bacterial suspension (OD 600 0.7), inoculated for 3 consecutive days, set the same shrimp hepatoenteric cytozoon infected without bacterial suspension as control 1, the shrimp hepatoenteric cytozoon not infected with bacterial suspension only as control 2, blank control 3. Observe the growth of shrimp every day, record the number of deaths, and start sampling and testing when the shrimp in the culture tank of control 1 are sick and dead. The sampling method is to randomly take 10 shrimps from each culture tank. The detection method adopts the real-time fluorescence quantitative PCR detection method commonly used in the art to detect the EHP gene. The test results show that the DNA copy numbers of shrimp hepatoenteric cytozoon after treatment with the three strains are: 10115 copies / mg, 2546 copies / mg, and 68 copies / mg respectively; the lowest copy number of DNA of shrimp hepatoenteric cytozoon in the strain JYD51 treatment group is 68 copies / mg, which has a significant inhibitory effect on shrimp hepatoenteric cytozoon.

[0048] Example 2

[0049] Identification of strains

[0050] like Figure 1As shown, the colony morphology of strain JYD51 on NA plate is round, milky white, non-transparent, with raised surface and neat edges; the microscopic picture of strain JYD51 is shown in Figure 2 As shown, the bacteria are Gram-positive, rod-shaped (0.2–0.64 μm × 0.6–1.0 μm), with rounded ends and elongated spores ( Figure 3 ).

[0051] The genomic DNA of strain JYD51 was used as a template and the gyrB gene sequence was used for PCR amplification and comparison. The gyrB gene sequence is shown in SEQ ID NO.1. The results showed that strain JYD51 had 99% sequence similarity with Bacillus altitudinis. Sequences with strong correlation with the target sequence were selected from the BLAST results to construct a phylogenetic tree ( Figure 4 ). Combining the colony characteristics and physiological and biochemical test results, the common bacterial system identification manual and Bergey's bacterial identification manual were consulted, indicating that JYD51 was Bacillus altitudinis. The strain JYD51 was sent to the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number: CGMCC No.32164, and the deposit date is October 10, 2024.

[0052] Example 3

[0053] Experiment on the control of artificial infection of hepatocellular carcinoma

[0054] The spore suspension (20 / ml) of hepatocystis prepared according to the method of Example 1 was used to artificially infect whiteleg shrimp. 5L of water was injected into the culture tank, and 100 healthy juvenile shrimps were injected with the following size: body length 4.0±0.3cm, weight 1.5±0.3g. Each group was injected with 1mL of spore suspension every day. After 1 week, 10 shrimps were randomly selected and observed under a microscope to detect whether they were infected. After confirming the infection, the bacterial suspension prepared by JYD51 was inoculated in three culture tanks in parallel, and each tank was inoculated with 1mL of bacterial suspension (OD 600 0.7), inoculated for 3 consecutive days, set the shrimp infected with hepatobiliary cytozoon without bacterial suspension as control 1, the shrimp not infected with hepatobiliary cytozoon only inoculated with bacterial suspension as control 2, and blank control 3. Observe the growth of shrimp every day, record the number of deaths, and find that 12 shrimps died in control 1. Each parallel treatment, including the control treatment, randomly sampled 10 shrimps to detect the EHP gene using real-time fluorescence quantitative PCR. The test results showed that the DNA copy number of EHP in control 1 was 31,300 copies / mg; that of the treatment group was 48 copies / mg. EHP was significantly inhibited in the treatment group and was basically controlled.

[0055] The same method as above was used to conduct an experiment on controlling the microsporidia of Chinese mitten crabs artificially infected with the above mentioned crabs. The experiment found that the microsporidia of Chinese mitten crabs in the treatment group were significantly inhibited and basically controlled.

[0056] Example 4

[0057] Preparation of liquid inoculant of strain JYD51 and its application in crab breeding ponds

[0058] The strain JYD51 was fermented using conventional broth medium to obtain the fermentation broth and the bacteria contained in the fermentation broth. The bacterial content of the fermentation broth was 6.92×10 8 cfu / mL, pH is 6.1, the fermentation liquid is adjusted to pH 4.5 with citric acid, that is, the strain JYD51 liquid bacterial agent is prepared, and the spraying method is combined with the feeding method to be used in the crab breeding pond infected with Chinese mitten crab microsporidia. Specifically, in Fuhu Village, Mingzuling Street, Huaihe Town, Xuyi County, Huai'an City, Jiangsu Province, the crab breeding area is about 60 acres, and the average water depth is about 0.8 meters. Before use, three crabs were randomly sampled for real-time fluorescence quantitative PCR detection of the DNA copy number of Chinese mitten crab microsporidia, which was 10060 copies / mg. The method of using the bacterial agent is 1 liter per acre, spraying continuously for 3 days, and feeding with 1 liter of bacterial agent mixed with 100 kg of conventional crab breeding feed. After 7 days, crabs were randomly taken for testing, and the test result was that the DNA copy number of microsporidia was 85 copies / mg. The test results show that microsporidia are basically controlled.

[0059] Example 5

[0060] Application of strain JYD51 in the culture of white shrimp

[0061] The liquid bacterial agent prepared by the method of Example 4 was used in a small shed breeding pond of white shrimp by a combination of spraying and feeding. The use time is May 30, 2024, and the address is: Group 8, Dangjia Village, Fengli Town, Rudong County, Nantong City, Jiangsu Province: the area of ​​the small shed is 10 meters * 40 meters. The average water depth is 0.9 meters. Before use, the shrimp growth was uneven and the feeding was reduced compared to normal conditions. Samples were taken in two breeding sheds, and the DNA copy number of enterocytosis (EHP) was detected by real-time fluorescence quantitative PCR. The DNA copy number of EHP in the first shed was 3515 copies / mg, and acute hepatopancreatic necrosis (EMS) was negative; the EHP in the second shed was 2151 copies / mg, and EMS was negative. The method of using the bacterial agent is the same as in Example 4, which is 1 liter of bacterial agent per mu (the bacterial content is 6.92×10 8cfu / mL, pH 4.5), and sprayed continuously for 3 days. At the same time, 1 liter of bacterial agent was mixed with 100 kg of conventional white shrimp feed for feeding. After 7 days, shrimp bodies were randomly taken for testing. The test results showed that the DNA copy number of EHP in the first shed was 18 copies / mg; the DNA copy number of EHP in the second shed was 38 copies / mg. The test results showed that EHP was basically under control.

[0062] Example 6

[0063] Effect of compound bacterial agent prepared by Bacillus subtilis JYD51 and Bacillus sphaeroides JYQ7

[0064] The JYD51 liquid bacterial agent (the bacterial cell content of the fermentation liquid is 6.92×10 8 cfu / mL, pH 4.5) and JYQ7 liquid inoculum (the bacterial content of the fermentation broth was 8.31×10 8 cfu / mL, pH 4.5), the two are mixed in a volume ratio of 1:1 to form a composite microbial agent, and the combination of spraying and feeding is used in a small shed culture pond of Penaeus vannamei. Xiashe Xinqiao Village, Xin'an Town, Deqing County, Huzhou City, Zhejiang Province, has a culture area of ​​5 mu and an average water depth of 0.6 meters. 3 kg of microbial agent was used. Before use, the uneven growth of shrimp bodies was investigated, and 20 shrimps were randomly taken to measure the length of 4.6-6.2 cm. The feeding was reduced compared to the normal state. The hydrogen sulfide content in the water was 55.16 mg / ml, and the ammonia nitrogen was 198 mg / ml. The water was turbid. The DNA copy number of enterocytosis (EHP) detected by real-time fluorescence quantitative PCR was 672000 copies / mg, and acute hepatopancreatic necrosis (EMS) was negative. The method of using the microbial agent is the same as in Example 4, 1 liter per mu, sprayed continuously for 3 days, and 100 kg of conventional Penaeus vannamei culture feed was mixed with 1 liter of microbial agent and fed. Shrimp were randomly sampled for testing after 5 and 7 days. The results showed that the DNA copy number of EHP was 15 copies / mg and 10 copies / mg respectively. On the 7th day, the hydrogen sulfide in the water was 0.57 mg / ml and the ammonia nitrogen was 2.57 mg / ml. The shrimp body length was 5.4-6.2 cm and the growth was uniform. The water was relatively clear.

[0065] From the above test results, it can be seen that EHP is significantly inhibited, and hydrogen sulfide and ammonia nitrogen in the water body are significantly reduced. The use of Bacillus subtilis JYD51 in the composite bacterial agent is reduced by about 50%, and it also has the ability to control shrimp hepatocystis and crab microsporidia. At the same time, the use of Bacillus sphericalus JYQ7 is reduced by about 50%, and it also has the ability to purify the water body.

[0066] In addition, in order to verify whether Bacillus subtilis JYD51 and Bacillus sphaeroides JYQ7 can synergistically enhance the efficacy of controlling the damage of hepatoenteric zoanthids to shrimps and crabs, enhance the purification of the water environment for shrimp and crab growth, and improve the quality of shrimp products, a similar breeding environment to Example 6 was adopted. Experimental Group 1 was a JYD51 liquid bacterial agent prepared by the method of Example 4 (the bacterial content of the fermentation liquid was 6.92×10 8 cfu / mL, pH 4.5) and JYQ7 liquid inoculum (the bacterial content of the fermentation broth was 8.31×10 8 cfu / mL, pH 4.5), and the two were mixed in a volume ratio of 1:1 to form a composite bacterial agent; Experimental group 2 was a single JYD51 liquid bacterial agent (the bacterial cell content of the fermentation liquid was 6.92×10 8 cfu / mL, pH 4.5), Example 3 is a single JYQ7 liquid inoculum (the bacterial content of the fermentation broth is 8.31×10 8 cfu / mL, pH 4.5) The method of using the bacterial agent is the same as in Example 4, which is 1 liter per mu, sprayed continuously for 3 days, and 1 liter of the bacterial agent is mixed with 100 kg of conventional white shrimp aquaculture feed for feeding. After 3 days, the shrimp bodies are randomly taken to detect the EHP gene and the content of hydrogen sulfide and ammonia nitrogen in the water. The results show that compared with the use of the same volume of JYD51 liquid bacterial agent alone, the composite bacterial agent enhances the liver and intestine cytozoon of shrimp, and the prevention effect is increased by 10-30%, while the use of JYQ7 liquid bacterial agent alone has no effect on EHP; compared with the use of the same volume of JYQ7 liquid bacterial agent alone, the composite bacterial agent improves the purification ability of the water body, and the degradation rate of hydrogen sulfide and ammonia nitrogen is increased by 20-30%, while the use of JYD51 liquid bacterial agent alone has almost no effect on the purification of the water body, indicating that the two can synergistically enhance the effect of controlling the harm of liver and intestine cytozoon to shrimp, synergistically enhance the purification of the water environment for shrimp growth, and improve the quality of shrimp products.

Claims

1. A strain of Bacillus subtilis JYD51, characterized in that: It has been identified as Bacillusaltitudinis and has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration. The deposit number is: CGMCC No.32164 and the deposit date is October 10, 2024.

2. Use of the Bacillus subtilis JYD51 according to claim 1 in aquaculture.

3. The use according to claim 2, wherein the Bacillus subtilis JYD51 is used to inhibit shrimp hepatocystis and crab microsporidia in aquaculture.

4. The use according to claim 2, characterized in that: The highland Bacillus JYD51 is used in improving the survival rate and quality of shrimps and crabs in aquaculture.

5. The use according to claim 4, characterized in that: The aquaculture preferably includes open-air pond culture and small shed culture of shrimps and crabs.

6. A bacterial agent prepared by fermentation containing the Bacillus subtilis JYD51 according to claim 1.

7. The bacterial agent according to claim 6, characterized in that The bacterial agent includes a liquid bacterial agent and a solid bacterial agent; the liquid bacterial agent includes a fermentation liquid obtained by fermenting the JYD51 strain, and the bacterial agent is prepared from the fermentation liquid and the bacterial bodies contained in the fermentation liquid; the solid bacterial agent includes a powdered or granular bacterial agent.

8. Use of the bacterial agent according to claim 6 in aquaculture for inhibiting hepatoenteric zoanthids of shrimps and microsporidia of crabs to promote the growth of shrimps and crabs.

9. An application of the Bacillus subtilis JYD51 described in claim 1 in combination with the lysinibacillus sphaeroides JYQ7 in inhibiting shrimp hepatocellular carcinoma and crab microsporidia, purifying the water environment for shrimp and crab growth, and improving the survival rate and quality of shrimp and crab.

10. The use according to claim 9, characterized in that: The highland Bacillus JYD51 and the spherical lysine Bacillus JYQ7 are used in combination by preparing a composite bacterial agent. The composite bacterial agent is preferably prepared by fermenting the two strains of bacteria separately, and then mixing them to form a composite bacterial agent when used; or after each strain of bacteria is fermented separately, the fermented products containing the fermentation liquid and bacterial bodies of the two strains are mixed to prepare the bacterial agent, that is, the composite bacterial agent.

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