A strain of Bacillus velez D34 with both biocontrol and water regulation functions and its application

Abstract

The present invention discloses a strain of Bacillus velezensis D34 with both biocontrol and water regulation functions and its applications. The D34 strain has a deposit number of CCTCC NO: M 2019457, and is deposited with the China Center for Type Culture Collection at Wuhan University in Wuhan, Hubei Province, China, on June 17, 2019. The strain has an inhibitory effect on the fish pathogen Gill mold, protects fish against infection by the pathogen Aeromonas, and effectively degrades ammonia nitrogen and nitrite, toxic substances in aquaculture water.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a strain of Bacillus velezensis D34 having both biocontrol and water regulation functions and applications thereof. Background Art

[0002] Aeromonas are opportunistic pathogens widely present in aquaculture water environments and are one of the main bacterial pathogens causing outbreaks of disease in freshwater aquaculture animals in my country. They can cause bacterial septicemia in fish, redleg disease in frogs, and mumps in turtles. In today's high-density, intensive aquaculture model, the accumulation of fish leftovers and feces not only leads to severe excess levels of toxic substances such as ammonia nitrogen and nitrite in the water, polluting water quality, but also promotes the proliferation of pathogenic Aeromonas, causing frequent disease outbreaks and significant economic losses to the aquaculture industry annually. Branchial mycoses are a serious fungal disease of fish caused by branchial mycoses infecting the gill filaments of fish. Branchial mycoses belong to the subdivision Mastigomycetes, order Saprolegniales, and genus Branchial mycoses. Two main types of pathogenic branchial mycoses have been reported: Haemobranchia and Penetrantia, which spread infection via zoospores and rely on hyphae for parasitism. After attaching to the gill epithelium of fish, branchial fungi spores continuously grow and develop into hyphae, often attached to zoosporangia. Mature zoospores within these zoosporangia break through the wall and are released into the gill lamellae, where they develop into new hyphae. These hyphae adhere closely to capillaries and block the lumen of the sinusoidal capillaries, causing swelling, congestion, and hemorrhage of the gill lamellae. Branchial fungi are not selective in their hosts and can infect grass carp, black carp, bighead carp, dace, common carp, crucian carp, bream, and yellow catfish. Dace are the most sensitive and suffer the most severe damage. Trauma to the gills of fish caused by parasites or other physical and chemical factors often leads to secondary acute infection with branchial fungi. Infected fish exhibit loss of appetite, difficulty breathing, and sudden death. The mortality rate is generally over 50%, and can reach 100% in severe cases. Due to the specific location of branchial fungi infection, the isolation and purification of the pathogen is extremely difficult, and a safe and effective drug for its control is still lacking.

[0003] In existing technologies, the main methods for aquatic disease control and water quality regulation include physical, chemical, and biological methods. Physical methods have very limited effectiveness. Chemical control methods, which widely use antibiotics and chemical agents, not only cause pathogen resistance but also lead to drug residues in aquaculture, restricting aquatic product trade and seriously affecting the sustainable development of the aquaculture industry. Biological control methods can effectively reduce drug residues and prevent the development of drug resistance, and are currently being promoted. Reported microorganisms used for biological control of aquatic animal diseases and water quality regulation mainly include photosynthetic bacteria, Bacillus, lactic acid bacteria, yeasts, and nitrifying bacteria. Bacillus is the most widely used due to its heat resistance, easy storage, and strong vitality. Summary of the Invention

[0004] The present invention aims to provide a strain of Bacillus velezensis D34 having both biological control and water regulation functions and application thereof.

[0005] To achieve the above-mentioned purpose, the present invention provides one of the technical solutions:

[0006] A strain of Bacillus velezensis D34, which has both biocontrol and water regulation functions, has been deposited with CCTCC NO: M2019457 and is deposited with the China Center for Type Culture Collection at Wuhan University in Wuhan, Hubei Province, China, on June 17, 2019. This strain has inhibitory effects on the fish pathogen Gill mold, protects fish against infection by the pathogen Aeromonas, and effectively degrades ammonia nitrogen and nitrite, toxic substances in aquaculture water.

[0007] The biological characteristics of the above-mentioned Bacillus velezensis D34 are as follows: (1): The D34 colonies cultured on the LB plate at 30°C for 24 hours are round, 4.2mm-4.6mm in diameter, milky white, smooth in surface, slightly wrinkled and bulging in the middle, opaque, and with irregular edges; after 48 hours of culture, the surface is convex, forming multi-pointed star-shaped wrinkles, and sticky strings can be seen with the naked eye when picking colonies; Gram staining is positive, the bacteria are long rod-shaped, and the size is 1.6μm×0.5μm; a layer of white biofilm can be formed on the surface after the culture solution is left to stand for 3 days; (2) D34 bacteria can utilize sucrose and glucose, and the results of nitrate (reduced), arginine and ornithine are positive, and the results of arabinol, inositol, mannitol, maltose, lactose, galactose, amino acid control and hydrogen sulfide are negative.

[0008] The screening process of the above-mentioned Bacillus Velez subtilis D34 is as follows:

[0009] Step 1): The 57 strains of Bacillus for screening were isolated from the sediment of healthy aquaculture water bodies in a conventional manner; and the indicator pathogen used for primary and secondary screening was Aeromonas;

[0010] Step 2): The Bacillus for screening was inoculated on an LB plate, cultured at 28 ° C for 24 hours, and then transferred to LB liquid medium, cultured at 28 ° C for 24 hours, and a sterilized gun was used to draw an appropriate amount of bacterial liquid and drop it on a blank drug-sensitive paper. It was naturally dried for use to obtain a primary screening drug-sensitive paper; then, the crucian carp pathogen Aeromonas was used as an indicator bacteria, and the indicator bacteria was inoculated on an LB plate. After being cultured in a constant temperature incubator at 28 ° C for 24 hours, an appropriate amount of bacterial moss was scraped with a sterilized gun tip, added to a centrifuge tube containing 1 mL of sterile water, and evenly pipetted to prepare a bacterial suspension. 100 μL of the bacterial suspension was evenly spread on the LB plate, and the primary screening drug-sensitive paper was evenly pasted on the LB plate. After co-culture at 28 ° C for 48 hours, the biocontrol Bacillus with obvious inhibition zone was observed and screened out;

[0011] Step 3): The Bacillus that has been initially screened in step 2) is inoculated onto an LB plate and cultured at 28°C for 24 h, then transferred to LB liquid culture medium and cultured at 28°C and 150 r / min for 24 h, or cultured at 28°C overnight and then cultured at 150 r / min for 7 h; then the culture solution is centrifuged, and an appropriate amount of the supernatant after centrifugation is dropped onto a blank drug-sensitive paper sheet, dried naturally for later use, and a re-screened drug-sensitive paper sheet is obtained; the pathogenic Aeromonas from Chinese soft-shelled turtle, grass carp, and silver carp is used as an indicator bacteria to prepare a bacterial suspension and coat the LB plate, and then the re-screened drug-sensitive paper sheet is respectively pasted on the LB plates of the three pathogens mentioned above, cultured at 30°C for 48 h, and the size of the inhibition zone is observed to screen out the biocontrol Bacillus with a broad-spectrum inhibitory effect on Aeromonas.

[0012] The second technical solution provided by the present invention is to provide a microecological preparation containing Bacillus Velezii D34 spores.

[0013] The third technical solution provided by the present invention is to provide a method for culturing Bacillus velezensis D34, specifically, inoculating it into a fermentation medium for culturing, wherein the fermentation medium comprises: 5.0 g / L yeast extract powder, 15.0 g / L raw soybean powder, 10.0 g / L sodium chloride, and 8.0 g / L magnesium sulfate.

[0014] Preferably, the above culture conditions are 25-35°C and 180-220 r / min shaking culture.

[0015] Preferably, the culture conditions are: 30°C, 5% inoculum size, and a rotation speed of 220 r / min for 48 h of fermentation culture of D34 Bacillus.

[0016] The fourth technical solution provided by the present invention is the application of Bacillus Velez D34 or its fermentation product or fermentation extract, and microbial preparations containing Bacillus Velez D34 spores to the prevention and control of fish pathogens Aeromonas and gill mold in aquaculture, as well as their application in regulating aquaculture water quality.

[0017] The present invention has at least the following beneficial effects:

[0018] The present invention screened and obtained a biocontrol Bacillus D34 with antagonistic effects on Aeromonas and Branchiomycetes, had no infection effect on mammalian skin, was non-toxic to grass carp fry, and had good biosafety. The results of the optimization of the fermentation medium and fermentation conditions of the D34 bacteria showed that the highest spore count of 2.03×10 was obtained when the fermentation medium and fermentation conditions were 5.0 g / L yeast extract powder, 15.0 g / L raw soybean powder, 10.0 g / L sodium chloride, and 8.0 g / L magnesium sulfate, with a 5% inoculum size, 30°C, and a rotation speed of 220 r / min for 48 h. 9cfu / mL, with a spore formation rate of 96.50%. D34 inhibited mycelial growth of Gill mold by 61%-77% and exhibited a strong inhibitory effect on spore germination, with an inhibition zone diameter of 25 mm. It also demonstrated a significant protective effect against infection by the pathogen Aeromonas verticillioides in grass carp summer flower fry, with the 48-hour survival rate of grass carp summer flower fry in the experimental group increasing by 51.7%-53.3%. Bacillus D34 also demonstrated a moderate degradation effect on ammonia nitrogen and nitrite in aquaculture water from various sources. Large pond tests demonstrated that Bacillus D34 rapidly and persistently degraded nitrite in ponds, making it a promising candidate strain for use as a microbial water quality regulator. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of the primary screening and rescreening of Bacillus for biocontrol of aquatic animal diseases of the present invention (where A is the primary screening result, the indicator pathogen is Aeromonas OB; BD are the rescreening results, the indicator bacteria are Aeromonas TBF2, HTE, and XCP2, respectively; ① the drug-sensitive paper disc prepared from the supernatant of Bacillus using the continuous shaking culture method; ② the drug-sensitive paper disc prepared from the supernatant of Bacillus using the combined static and shaking culture method; the arrow indicates the position of the drug-sensitive paper disc of the Bacillus D34 agent);

[0020] Figure 2 The morphological characteristics of the D34 strain of the present invention;

[0021] Figure 3 This is the 16S rDNA phylogenetic tree of the D34 strain of the present invention;

[0022] Figure 4 The results of the mouse skin sensitivity test of the D34 bacteria of the present invention are as follows;

[0023] Figure 5 The effects of different carbon sources, nitrogen sources and added inorganic salts on the viable count, spore count and conidia production rate of D34 bacteria in the present invention; Note: A: carbon source, B: nitrogen source, C: added inorganic salts; different lowercase letters indicate significant differences between different treatments ( P <0.05);

[0024] Figure 6 The effect of different temperatures, inoculation amounts and rotation speeds on the number of viable bacteria, spore count and conidia production rate of D34 bacteria in the present invention; Note: A: temperature, B: inoculation amount, C: rotation speed; different lowercase letters indicate significant differences in the number of spores and viable bacteria between different treatments ( P <0.05);

[0025] Figure 7The inhibitory effect of the D34 bacteria of the present invention on the mycelial growth of two gill mold pathogens (where A is a control of the grass carp-derived gill mold pathogen GCM19, B is the D34 bacteria and the gill mold GCM19 grown in confrontation for 3 days, C is the bighead carp-derived gill mold pathogen YM24 control, and D is the D34 bacteria and the gill mold YM24 grown in confrontation for 7 days);

[0026] Figure 8 The inhibitory effect of the D34 bacteria of the present invention on the spore germination of the grass carp-derived branchial mold pathogen GCM19 (A: sterile water control, B: D34 bacteria);

[0027] Figure 9 The results of the infection test of different concentrations of Aeromonas vernix on grass carp summer flowers by immersion (CK: control group without XCP2 bacteria; T1-T3: XCP2 bacteria immersion concentrations were 1x10 6 , 1x10 7 and 1x10 8 cfu / mL experimental group; different lowercase letters indicate significant differences in the mortality of grass carp infected with different concentrations of XCP2 bacteria at the same time (P < 0.05);

[0028] Figure 10 The protective effect of D34 bacteria on grass carp summer flowers against Aeromonas vermiformis infection (CK: control group without D34 bacteria; T1-T3: D34 bacteria treatment concentrations of 1x10 5 , 1x10 6 and 1x10 7 cfu / mL experimental group; different lowercase letters indicate significant differences in the survival rate of grass carp summer flowers treated with different concentrations of D34 at the same time (P < 0.05);

[0029] Figure 11 The figure shows the dynamic changes of dissolved oxygen and nitrite in pond A2-1 within 72 hours after spraying Bacillus D34 of the present invention. DETAILED DESCRIPTION

[0030] In order to make those skilled in the art better understand the present invention, the present invention is further described in conjunction with the specific implementation process. The parts not described in detail below are all carried out according to the existing technology in the field.

[0031] 1. Screening and identification of biocontrol Bacillus

[0032] 1. Sample Source: The 57 Bacillus strains screened in this experiment were isolated by the Hunan University of Arts and Science's Microbiology Innovation Team from sediment collected from healthy aquaculture water bodies in Changde. The indicator pathogens used for primary and secondary screening were Aeromonas pathogens isolated and identified by team members from various aquatic animals in the Changde area (Table 1).

[0033] Table 1 Sources of indicator pathogens

[0034]

[0035] 2. Preparation of Bacillus susceptibility strips: The primary screening strips were prepared by inoculating 57 previously stored Bacillus strains onto LB plates, culturing them at 28°C for 24 hours, then transferring them to LB liquid medium and shaking them at 28°C for 24 hours. Using a sterile pipette, 10 µL of the bacterial culture was then dropped onto a 6 mm diameter blank strip of susceptibility strips. The strips were then air-dried in a clean bench and used for subsequent screening. The secondary screening strips were prepared by inoculating the Bacillus strains obtained in the primary screening onto LB plates, culturing them at 28°C for 24 hours, then transferring them to LB liquid medium and using two different incubation methods (method 1: 28°C with continuous shaking at 150 rpm for 24 hours; method 2: static incubation at 28°C overnight, followed by continuous shaking at 150 rpm for 7 hours). 1 mL of each culture medium was then transferred to a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm for 5 minutes. 10 µL of the supernatant was then dropped onto a blank strip of susceptibility strips and air-dried in a clean bench for subsequent use.

[0036] 3. Initial screening of biocontrol Bacillus: Using Aeromonas OB, a pathogenic bacterium from crucian carp, as an indicator bacterium, inoculate it into an LB plate, place it in a constant temperature incubator at 28°C for 24 hours, scrape an appropriate amount of bacterial moss with a sterilized pipette tip, add it to a centrifuge tube containing 1 mL of sterile water, pipette and beat evenly to prepare a bacterial suspension, draw 100 µL of the bacterial suspension and evenly spread it on the LB plate, evenly stick the above-mentioned primary screening drug-sensitive paper pieces on the plate, stick 3 types of drug-sensitive paper pieces on each plate, and repeat 2 times for each drug-sensitive paper piece. After co-cultivation at 28°C for 48 hours, observe and screen the biocontrol Bacillus with obvious inhibition zones. Using Aeromonas OB from crucian carp as an indicator bacterium, 3 strains of Bacillus (D34, D38, BSL39) were preliminarily screened from 57 strains of Bacillus, which had obvious inhibitory effects on pathogens ( Figure 1 A).

[0037] 4. Rescreening of biocontrol Bacillus: Using Aeromonas pathogens from Chinese soft-shell turtle (HTE), grass carp (XCP2), and silver carp (TBF2) as indicator bacteria, bacterial suspensions were prepared and coated on LB plates. Rescreened drug-sensitive paper discs were attached to the three pathogen plates. After co-culture at 30°C for 48 hours, the size of the inhibition zone was observed to screen out biocontrol Bacillus with broad-spectrum inhibition against Aeromonas. The results of the antibacterial experiment showed that the supernatant of Bacillus D34 cultured in continuous shaking culture had a significant inhibitory effect on Aeromonas from the three different sources ( Figure 1 BD).

[0038] 5. Species identification of D34: Observe the morphology of D34 colonies using LB plates; observe the bacterial morphology using Gram staining; perform physiological and biochemical identification using bacterial microbiological identification tubes, and perform molecular biological identification using 16S rDNA sequence analysis. The species was identified based on the results of morphological observation, physiological and biochemical, and molecular biological experiments. Morphological observation results showed that D34 colonies cultured on LB plates at 30°C for 24 hours were round, 4.2mm-4.6mm in diameter, milky white, smooth in surface, slightly wrinkled and bulging in the middle, opaque, and with irregular edges ( Figure 2 -A). After 48 hours of culture, the surface bulged and formed multi-pointed star-shaped wrinkles ( Figure 2 -B), when picking the colony, sticky strings can be seen with the naked eye. Gram staining is positive, the bacteria are long rod-shaped, and the size is 1.6μm×0.5μm ( Figure 2 -C). A white biofilm will form on the surface of the culture medium after 3 days of standing. Figure 2 Physiological and biochemical identification results showed that D34 bacteria could utilize sucrose and glucose, and were positive for nitrate (reduced), arginine, and ornithine, but negative for arabitol, inositol, mannitol, maltose, lactose, galactose, amino acid control, and hydrogen sulfide (Table 2).

[0039] Table 2 Physiological and biochemical characteristics of strain D34

[0040]

[0041] The results of 16S rDNA sequence analysis of D34 strain (such as sequence 1) showed that Bacillus velezensis The 16SrDNA sequences of the two strains are homologous, with a similarity of 99.65%. The phylogenetic tree was constructed using MEGA5.2 (e.g. Figure 3 ), the results show that D34 and Bacillus velezensis strain FZB42 clustered into one branch, indicating that strain D34 and Bacillus velezensis strain FZB42, and combined with physiological, biochemical and morphological characteristics, D34 was identified as Bacillus velezinis ( Bacillus velezensis ).

[0042] 2. Biosafety Testing of D34 Bacteria

[0043] Skin sensitivity test: Healthy mice weighing approximately 18-22g (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.), two males and two females, were divided into two groups (experimental and control groups), with one male and one female in each group. The experimental group mice were secured to a wooden board with rubber bands, their abdominal hair removed, and D34 bacteria were evenly smeared from an LB culture plate using a cotton swab on their exposed skin. After 15-20 minutes of infection, the mice were released and allowed to move freely. The control group only had their abdominal hair removed and, similarly, after 15-20 minutes, the mice were released. The experimental and control groups were housed separately. During the experiment, the mice were fed a special maintenance diet and had free access to water. The experimental area, cages, and drinking utensils were cleaned and disinfected regularly. The mice were observed for three days, and their skin conditions were photographed and recorded. The results showed that neither the experimental nor the control group mice showed symptoms of infection, such as skin redness, swelling, or ulceration (e.g., Figure 4 Compared with the control group, the mice in the experimental group showed no obvious special symptoms and their feeding activities were normal, indicating that D34 bacteria have no infectious effect on mammalian skin.

[0044] 2. Bacillus immersion challenge test on fish fry: Grass carp fry with a body length of about 1 cm (kindly donated by Xiangyun Biotechnology Co., Ltd., Changde Base) were used as test subjects. They were divided into 3 experimental groups and 1 control group, with 3 replicates in each group. The grass carp fry were randomly divided into groups and placed in plastic basins containing 5L of water, with 50 fry per basin. D34 bacteria were inoculated into 20mL of LB liquid medium and cultured overnight at 28℃ and 150r / min. The concentration of D34 bacteria was counted microscopically using a bacterial counting plate, and then an appropriate amount of bacterial solution was added to the basins, so that the final concentration of D34 bacteria was 1×10 7 cfu / mL, 1×10 8 cfu / mL and 1×10 9 Three immersion challenge groups were set up at different concentrations, with a blank control group without D34 bacteria added. The fry were observed for 15 consecutive days. During this period, they were fed an appropriate amount of food, and their feeding, activity, and survival were recorded. The results of the D34 challenge experiment showed that compared with the control group, the grass carp fry in the experimental groups showed no stress response, fed normally, and were active, with a 100% survival rate, demonstrating the safety of D34 bacteria for grass carp fry.

[0045] 3. Optimization of fermentation medium and fermentation conditions of D34 bacteria

[0046] 1. Fermentation culture and spore count of D34 bacteria: Use an inoculation loop to scrape an appropriate amount of bacterial moss directly from the LB culture plate of D34 bacteria and inoculate it into 20 / 150 mL LB liquid medium. Incubate at 28°C and 200 r / min with shaking for 16-18 h. Then transfer the inoculum to 100 / 250 mL LB fermentation medium at a volume ratio of 2% (referring to the volume ratio of bacterial liquid to fermentation medium). Incubate at 28°C and 200 r / min with shaking for 48 h. Use the gradient dilution spread plate method to dilute the fermentation broth to 10 -1 -10 -8 Calculate the concentration gradient of the fermentation broth. Spread 100 μL of the diluted solution onto an LB plate, incubate at 28°C for 48 hours, and count the total number of colonies. Simultaneously, place the fermentation broth dilutions in an 80°C water bath for 10 minutes, then spread 100 μL of each onto an LB plate. After incubation for 48 hours, count spores. Calculate the spore yield (%) as (number of spores / total number of colonies) × 100%. The viable count is the total number of colonies minus the number of spores.

[0047] 2. Fermentation Medium Optimization: Using spore count and spore rate as evaluation criteria, we first used LB (0.5% yeast extract, 1% tryptone, 1% sodium chloride) as the basal medium, with an inoculum size of 2% (volume fraction), a 100 / 250 mL volume, a culture temperature of 28°C, and a rotational speed of 200 r / min. The viable count and spore count of Bacillus in the fermentation broth were determined at 0, 18, 35, 48, 60, and 72 h, and the spore production rate was calculated to determine the optimal fermentation time. Single-factor experiments were then conducted to investigate the effects of varying carbon sources (glucose, corn starch, sucrose, and sweet potato starch), nitrogen sources (raw soybean flour, soybean meal, ammonium sulfate, and corn steep liquor), and inorganic salt additions (ferrous sulfate, dipotassium phosphate, magnesium sulfate, and manganese sulfate) on spore production by the biocontrol Bacillus species, aiming to identify the optimal carbon, nitrogen, and inorganic salt sources. Three biological replicates were used for each experiment. The results showed that, using LB as the basic medium, the spore rate reached a maximum of 93.02% at 48 h, and the number of spores produced was 5.77×10 8 cfu / mL; yeast extract powder had the highest spore count and spore rate among the screened carbon sources, reaching 5.8×10 8 cfu / mL and 89.25%, which were significantly different from the other four carbon sources ( P <0.05)( Figure 5 A); the spore number and spore rate of raw soybean flour were the highest among nitrogen sources, reaching 1.5×10 9 cfu / mL and 99.30%, which were significantly different from the other three nitrogen sources ( P <0.05)( Figure 5 B); The spore count and spore rate obtained by adding magnesium sulfate were the highest among inorganic salts, which were 1.05×10 9cfu / mL and 91.86%, which were significantly different from those of the other three inorganic salts ( P <0.05)( Figure 5 C).

[0048] On this basis, L9 (3 3 ) Three-factor three-level orthogonal test to optimize the ratio of carbon source, nitrogen source and inorganic salt (results are shown in Table 4):

[0049] Table 3. Orthogonal experiment optimization of culture medium factor levels

[0050]

[0051] Table 4 Optimized ratio of carbon source, nitrogen source and inorganic salt L9(3 3 )Orthogonal test results

[0052]

[0053] Table 4 shows that K1, K2, and K3 represent the sum of the indicators at each level of each factor. R represents the range, indicating the magnitude of the factor's influence on the results; a larger R value indicates a greater impact. Based on the R values, the primary and secondary factors affecting D34 spore count are C > B > A, that is, magnesium sulfate > raw soybean powder > yeast extract powder. Based on the three K values ​​corresponding to each factor, the optimal levels for each factor are A1, B2, and C3: yeast extract powder 5.0 g / L, raw soybean powder 15.0 g / L, and magnesium sulfate 8.0 g / L.

[0054] 3. Optimization of fermentation conditions: Based on the optimization of fermentation medium, single-factor experiments were conducted with spore count as the investigation index to study the effects of different culture temperatures (25℃, 28℃, 30℃, 35℃), inoculation amounts (1%, 3%, 5%, 7%), and rotation speeds (180r / min, 200r / min, 220r / min) on Bacillus spore production. The fermentation conditions were further optimized to increase the spore count of D34. The results are as follows: Figure 6 .Depend on Figure 6 It can be seen that with the increase of temperature in the range of 25-35℃, the number of spores and the sporulation rate showed a trend of first increasing and then decreasing. When the temperature was 30℃, the number of spores and the sporulation rate reached the maximum value, which was 1.30×10 9 cfu / mL and 91.98% ( Figure 6 A). Within the range of 1-7% (volume fraction) inoculation, the number of spores and the sporulation rate showed a trend of increasing first and then decreasing with the increase of inoculation amount. When the inoculation amount was 5%, the number of spores and the sporulation rate both reached the maximum value, which were 1.49×10 9 cfu / mL and 93.14% ( Figure 6B). Within the speed range of 180-220 r / min, the number of spores and the sporulation rate both showed a gradual increase with the increase of the speed. When the speed reached 220 r / min, the number of spores and the sporulation rate both reached the highest value, specifically 2.03×10 9 cfu / mL and 96.50% ( Figure 6 C).

[0055] IV. Antibacterial test of D34 bacteria and water quality adjustment test

[0056] 1. Preparation of Bacillus D34 inoculant

[0057] (1) Activation of D34 strains: First, transfer the Bacillus D34 strain stored in the refrigerator to an LB plate and culture at 28°C for 24 h. Then, transfer an appropriate amount of strains from the D34 LB culture plate to 20 / 150 mL LB culture medium and culture at 30°C and 200 r / min for 16-18 h.

[0058] (2) Preparation of D34 bacterial agent: Transfer the above D34 bacterial solution to 400 mL / 1 L fermentation medium at a 5% inoculum volume (volume ratio of bacterial solution to fermentation liquid), culture at 30°C and 220 rpm for 48 h, spread on the plate for gradient dilution and count, and store in a refrigerator at 4°C for later use.

[0059] 2. D34 bacteria inhibition test of gill mold

[0060] (1) D34 bacteria inhibits the growth of gill mold hyphae: First, use a sterile blue tip (size 1 mL) to make 4 holes at equal intervals about 2 cm away from the center of the PDA plate. Then, use the agar transfer method to inoculate the gill mold blocks of grass carp-derived gill mold pathogen GCM19 and bighead carp-derived gill mold pathogen YM24 (isolated and stored in a 4℃ refrigerator in our laboratory) to the center of the PDA plate. Add 80 μL of D34 bacterial agent to the 4 small holes around the bacterial blocks, culture at a constant temperature of 25℃ for 3-7 days, observe and measure the growth diameter of the water mold, and use the PDA gill mold culture plate without bacterial agent as the control. The experimental group and the control group are set up with 3 replicates. The inhibition rate is calculated according to the following formula: Inhibition rate = (gill mold diameter of the control group - gill mold diameter of the experimental group) / gill mold diameter of the control group. The results are as follows: Figure 7 As shown. Figure 7 It can be seen that D34 bacteria has a good inhibitory effect on the mycelial growth of grass carp-derived branchial mold GCM19 and bighead carp-derived branchial mold YM24. When D34 bacteria grow oppositely with branchial mold GCM19 for 3 days, the inhibition rate of its mycelial growth reaches 61%, and when it grows oppositely with branchial mold YM24 for 7 days, the inhibition rate of its mycelial growth reaches 77%.

[0061] (2) D34 bacteria inhibited the germination of gill mold spores: First, prepare the gill mold spore suspension: transfer the grass carp pathogen gill mold GCM19 (isolated and stored in the laboratory in a 4℃ refrigerator) to the center of the fish blood PDA plate (formula: 2% glucose, 20% potato, 10% fresh fish blood, 2% agar powder) by agar transfer method and culture at 25℃ for 3 days. Use a sterile blue tip (specification 1mL) to punch holes on the gill mold culture plate to take the GCM19 blocks with stronger growth. Use a sterile toothpick to transfer them into a 12-well cell culture plate. Add 4mL sterile water (pH 8.0) to each well. Culture at 20℃ for 5 days to allow GCM19 to grow fully in sterile water and release spores. Then use a pipette to aspirate the culture fluid from the small holes of the culture plate. The suspension was divided into 1.5 mL centrifuge tubes and centrifuged at 3000 r / min for 5 min. The supernatant was transferred to another sterile centrifuge tube to obtain the GCM19 branchial mold spore suspension. The number of branchial mold spores was observed and counted under a phase contrast microscope. Then 200 μL of the GCM19 branchial mold spore suspension was evenly spread on a PDA plate. An Oxford cup was placed in the center of the plate and 80 μL of D34 inoculant was added. Sterile water was added to the Oxford cup as a control. Three replicates were set for the experimental group and the control group. The plates were cultured at a constant temperature of 25°C for 5 days. The size of the inhibition zone was observed and measured. The results are shown in the figure below. Figure 8 As shown. Figure 8 It can be seen that compared with the control, D34 strain had a significant inhibitory effect on the spore germination of GCM19, with the average diameter of the inhibition zone reaching 25 mm;

[0062] 3. Experiment on the protective effect of D34 bacteria on grass carp summer flowers against Aeromonas infection

[0063] (1) Expansion culture of pathogens: Inoculate grass carp-derived Aeromonas vickers XCP2 into LB liquid culture medium, culture overnight at 30°C and 150 rpm, and count the pathogen concentration using a microscopic counting plate.

[0064] (2) Source and temporary rearing of test fish fry: The test grass carp summer flower fry were purchased from the Changde base of Xiangyun Biotechnology Co., Ltd., with a number of about 2,000, with a size of 3-4 cm. They were temporarily reared in a blue plastic fish tank (size 60 cm*40 cm*35 cm) for one week. During the temporary rearing period, they were oxygenated with an air pump, fed with ultra-fine floating granular feed, and drained by siphoning every day, with 1 / 3 of the water changed;

[0065] (3) Experiment on the infection of grass carp summer flowers with Aeromonas viridis XCP2: Measure 2L of tap water aerated for more than 24 hours with a measuring cylinder and pour it into 12 circular plastic basins with a volume of 4L. Place 20 grass carp summer flowers in each basin. 6 , 1x10 7 , 1x10 8Three XCP2 concentrations were tested, with grass carp (Ctenopharyngodon spp.) soaked and infected in water at 25-28°C for 48 hours. No feeding, water changes, or aeration were performed during the infection period. The mortality of grass carp (Ctenopharyngodon spp.) fry was observed and counted after 24 and 48 hours, and the mortality rate was calculated. Figure 9 .Depend on Figure 9 Visible, in 1x10 6 -1x10 8 cfu / mL concentration range, the mortality rate of grass carp summer flower increased with the increase of Aeromonas vermiformis pathogen concentration, and the mortality rate of grass carp summer flower fry 24h and 48h between the control group and the experimental group showed significant difference (P < 0.05); at 1x10 8 The mortality rate of grass carp summer flower treated with the pathogen was the highest when immersed in the pathogen with a concentration of cfu / mL, reaching 100% within 48 hours;

[0066] (4) Test on the effect of D34 bacteria on the resistance of grass carp summer flower to Aeromonas infection: 2L of tap water aerated for more than 24 hours was measured with a measuring cylinder and poured into 12 round plastic basins with a volume of 4L. 20 grass carp summer flower were placed in each basin. At the same time, an appropriate amount of XCP2 bacterial solution was added to make the final concentration reach 1x10 8 cfu / mL. The experiment was divided into 4 groups, with 3 replicates in each group. The experimental groups were added with appropriate amounts of D34 bacteria agent, so that the final concentration of D34 bacteria in the basin was 1x10 5 , 1x10 6 , 1x10 7 cfu / mL. The survival rate of grass carp summer flowers was observed and counted after 24h and 48h with no D34 agent added as the control. Figure 10 .Depend on Figure 10 The survival rates of grass carp summer fry in the control group (CK) were 41.7% and 0% at 24 and 48 hours, respectively. In the experimental groups (T1-T3), the survival rates were 63.3%, 66.7%, and 65.0% at 24 hours, and 51.7%, 51.7%, and 53.3% at 48 hours, respectively. Compared with the control group, the survival rates of grass carp summer fry in the T1-T3 experimental groups increased by 51.7%, 51.7%, and 53.3% at 48 hours, respectively. There were no significant differences in the survival rates of grass carp summer fry among the three experimental groups (P>0.05), but there were significant differences compared with the control group (P<0.05). This suggests that Bacillus D34 has a significant protective effect on grass carp summer fry against Aeromonas verticillioides infection.

[0067] 4. Experiment on the effect of D34 bacteria on regulating water quality in fish farming

[0068] (1) Detection of the degradation effect of D34 bacteria on ammonia nitrogen and nitrite in aquaculture water: Water samples with excessive ammonia nitrogen or nitrite were collected from different locations and different aquaculture environments, and added to triangular flasks with a filling volume of 100 / 250 mL. D34 bacteria agent was added to the triangular flasks at a volume ratio of 1:200. The flasks were placed at room temperature. A rapid water quality test kit was used to detect the ammonia nitrogen and nitrite content in the flasks at 0 h, 24 h, and 48 h after treatment. The degradation rates of ammonia nitrogen and nitrite were calculated. The experiment was repeated for 3 groups and the average value was taken. The results are shown in Table 5:

[0069] Table 5 Degradation effect of D34 bacteria on ammonia nitrogen and nitrite in aquaculture water from different sources

[0070]

[0071] As shown in Table 5, D34 bacteria demonstrated excellent degradation effects on ammonia nitrogen and nitrite in water samples with excessive levels from various sources. Its degradation rates for ammonia nitrogen (concentration range 0.6-2.0 mg / L) ranged from 27.7-55.3% within 24 hours and reached 55.7-77.7% within 48 hours. Its degradation rates for nitrite (concentration range 0.1-1.0 mg / L) ranged from 38.7-75.0% within 24 hours and 50.0-96.3% within 48 hours.

[0072] (2) Large-pond test of D34 bacteria in situ regulation of pond water quality: A rapid water quality test kit was used to investigate the nitrite and ammonia nitrogen contents in 27 aquaculture ponds at the Changde base of Xiangyun Biological Company. A pond with seriously exceeded nitrite standards (number A2-1, mainly raising Xiangyun crucian carp No. 2 fish, with an initial nitrite concentration of 0.6 mg / L) was selected as the test pond. D34 bacteria was sprayed throughout the pond, and oxygen was added with an auxiliary aerator. Nitrite concentration and pond dissolved oxygen were used as monitoring indicators to investigate the effect of D34 bacteria on in situ regulation of aquaculture pond water quality. The results are shown in the figure. Figure 11 .Depend on Figure 11 As shown in the results, before application of the D34 inoculant, the nitrite concentration in Pond A2-1 was severely above the recommended standard, at 0.60 mg / L. However, 24 hours after application (at 10:00 AM the following day), the nitrite concentration had dropped significantly to a safe level of 0.05 mg / L, with a degradation rate of 91.7%. Nitrite concentrations remained stable at a low level of 0.05 mg / L for 52-72 hours. With the aid of an aerator, the dissolved oxygen in the pond remained above the critical dissolved oxygen value for crucian carp (0.4 mg / L) for 72 hours. In summary, Bacillus D34 demonstrated a rapid and durable nitrite degradation effect on pond nitrite.

Claims

1. A strain of Bacillus velezinis D34 with both biocontrol and water quality regulation functions is named Bacillus velezensis D34, its deposit number is CCTCC NO:M 2019457, its depository is China Center for Type Culture Collection, its deposit address is Wuhan University, Wuhan City, Hubei Province, China, and the deposit date is June 17, 2019.

2. A microbial preparation containing the Bacillus Velez subtilis D34 according to claim 1.

3. A method for cultivating Bacillus Velezii D34, characterized in that: The Bacillus velezensis D34 according to claim 1 is inoculated into a fermentation medium for cultivation, wherein the fermentation medium comprises 5.0 g / L yeast extract powder, 15.0 g / L raw soybean powder, 10.0 g / L sodium chloride, and 8.0 g / L magnesium sulfate.

4. The method according to claim 3, characterized in that The culture conditions are 25-35°C and 180-220 r / min shaking.

5. The method according to claim 4, characterized in that The culture conditions were: 30℃, 5% inoculum size, and rotation speed of 220r / min for fermentation of D34 Bacillus for 48 hours.

6. Use of the Bacillus velezensis D34 according to claim 1 and the microbial preparation according to claim 2 in the preparation of a preparation for controlling the pathogenic bacteria Aeromonas and Gill mold in fish in aquaculture, and for regulating aquaculture water quality.

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