Bacillus licheniformis BL01 and application thereof

Abstract

The invention provides bacillus licheniformis BL01 and application thereof, the bacillus licheniformis BL01 is named as bacillus licheniformis in taxonomy, the strain name is bacillus licheniformis BL01, and the bacillus licheniformis BL01 is preserved in China Center for Type Culture Collection on April 11, 2025, the preservation address is Wuhan University, Wuhan, Hubei, China, and the preservation number is CCTCC NO: M2025752. The strain provided by the invention combines the characteristics of denitrification, bacteriostasis, nematode killing and growth promotion, and is an important direction for promoting aquaculture green transformation. Compared with the existing bacillus, the bacillus has the advantages that the antibacterial effect and the storage stability are improved; a fish body test shows that the bacillus licheniformis has a relatively good probiotic effect on aquatic animals and has a prevention and treatment effect on pathogenic bacteria.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to Bacillus licheniformis BL01 and applications thereof. Background Art

[0002] In recent years, as aquaculture has developed towards an intensive and high-density model, water pollution has become increasingly serious. The accumulation of large amounts of leftover bait, feces, and animal and plant carcasses has led to the accumulation of ammonia nitrogen (NH3-N) and nitrite (NO2 - Excessive levels of harmful substances such as nitrates and nitrates not only exacerbate eutrophication but also directly threaten the health of fish and shrimp, triggering frequent diseases and causing significant economic losses. While traditional physical or chemical denitrification methods (such as water exchange, adsorbents, and chemical oxidation) are somewhat effective, they pose challenges such as high costs, a high risk of secondary pollution, and complex operations.

[0003] Against this backdrop, biological denitrification technology has become a research hotspot due to its environmental, economic, and sustainable nature. Bacillus spp., due to their high-temperature resistance, strong enzyme production, and strong environmental adaptability, are widely used in water quality improvement and feed additives. For example, Bacillus licheniformis can decompose organic pollutants by secreting proteases and amylases, while also converting ammoniacal nitrogen into low-toxic nitrates through nitrosation, significantly reducing water toxicity. Studies have shown that Bacillus licheniformis ZY-4 (CCTCC NO: M2014277) can remove ammoniacal nitrogen by up to 93.39% within 48 hours and, when used as a feed additive, can reduce the feed-to-meat ratio of whiteleg shrimp by 14.3%.

[0004] However, existing technologies mostly focus on water purification and feed efficiency enhancement, with less research on biological control of nematode diseases, another key issue in aquaculture. While chemical nematicides are effective, they can easily lead to drug residues, pathogen resistance, and ecological damage. Therefore, the development of microbial agents that combine efficient denitrification, growth promotion, and nematicidal functions has become an urgent need in the industry. Furthermore, the large-scale application of Bacillus licheniformis requires addressing issues such as agent stability, standardized administration methods, and safety verification. For example, the "Technical Regulations for the Use of Bacillus licheniformis Agents in Seawater Factory Aquaculture" clearly define the conditions for the agent's use (such as a water temperature of 15-30°C and avoidance of mixing with disinfectants), but its adaptability to different aquaculture scenarios still needs to be further optimized. Summary of the Invention

[0005] In view of this, the present invention provides a Bacillus licheniformis BL01 and its application, which realizes the triple functional coupling of nematode killing, efficient denitrification and growth promotion, breaking through the limitation of the single efficacy of existing microbial agents.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a Bacillus licheniformis BL01, which is taxonomically named Bacillus licheniformis and named Bacillus licheniformis BL01. It was deposited in the China Center for Type Culture Collection on April 11, 2025, with the deposit address being Wuhan University, Wuhan City, Hubei Province, China, and the deposit number being CCTCC NO: M2025752.

[0007] Preferably, the full length of the genome of Bacillus licheniformis BL01 is 398,136,4 bp.

[0008] Preferably, the culture medium for culturing the Bacillus licheniformis BL01 comprises: 2-3 wt % peptone, 0.1-0.3 wt % magnesium sulfate, 0.1-0.3 wt % dipotassium hydrogen phosphate, 0.5-2.0 wt % sucrose, 0.05-0.20 wt % calcium chloride, and an initial pH of 5.0-7.0.

[0009] In a second aspect, the present invention provides an industrial fermentation method of Bacillus licheniformis BL01, characterized in that: S1. Inoculate Bacillus licheniformis BL01 on solid KMB medium, pick a single clone and transfer it to the medium used to culture Bacillus licheniformis BL01 for fermentation. Ferment aerobically at 30-37°C and 200-220 rpm for 24-48 hours, and collect the fermentation seed liquid. S2. Inoculate the fermentation seed liquid into a fermentation tank filled with fermentation medium at a volume ratio of 1-3% for the first expansion culture, and ferment aerobically at 30-37° C. and 100-150 rpm for 24-48 hours to obtain expanded culture liquid I; S3. Inoculate the expanded culture fluid I into a fermentation tank containing fermentation medium at a volume ratio of 3-10% for a second expanded culture. Ferment aerobically at 30-37° C., a tank pressure of 0.03 MPa, and 100-150 rpm for 16-24 h to obtain expanded culture fluid II.

[0010] In a third aspect, the present invention provides a use of Bacillus licheniformis BL01 in the preparation of a biological control inhibitor for reducing nematode infection rate.

[0011] Preferably, the Bacillus licheniformis BL01 is added to the feed and then sprinkled into the water body, or directly sprinkled into the water body.

[0012] Preferably, the mass fraction of the Bacillus licheniformis BL01 in the feed is 0.2-0.5%; and / or, The concentration of the Bacillus licheniformis BL01 directly sprinkled into the water body is 10^6-10^7 CFU / L.

[0013] In a fourth aspect, the present invention provides a use of Bacillus licheniformis BL01 in preparing a preparation for water purification.

[0014] In a fifth aspect, the present invention provides a use of a Bacillus licheniformis BL01-Bacillus subtilis composition in the preparation of a composite bacterial agent for killing nematodes and reducing COD in water bodies.

[0015] In a sixth aspect, the present invention provides a use of Bacillus licheniformis BL01 in the preparation of a feed synergist preparation.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention realizes the triple functional coupling of nematode killing, efficient denitrification and growth promotion in Bacillus licheniformis for the first time, breaking through the limitation of single efficacy of existing microbial agents. The Bacillus licheniformis of the present invention has the following characteristics: 1. Stress resistance: the survival rate after high temperature treatment at 55°C for 2 hours is ≥92%, and it grows stably under conditions of pH 6.0-9.0 and salinity 0-5%; 2. Enzyme production capacity: the activities of protease (618±25U / mL, Folin method), amylase (345±18U / mL, DNS method) and cellulase (89±5U / mL, CMC-Na method) in the fermentation broth are significantly higher than those of the control strain CGMCC 1.817 (P<0.01); 3. Nematicidal function: the in vitro killing rate of the metabolites against the southern root-knot nematode (Meloidogyne incognita) reaches 92.3% (10^8 CFU / mL) within 71 hours, and in an in vivo experiment, it can increase the survival rate of zebrafish infected with nematodes by 67.5%; 4. Denitrification performance: the denitrification of ammonia nitrogen (NH3-N) and nitrite (NO2 - ) removal rates were 94.2% and 88.6% respectively, significantly higher than those of traditional nitrifying bacteria; 5. Safety: No virulence genes (such as hbl and nhe) were detected in whole genome sequencing, and the zebrafish embryo teratogenicity experiment showed that the 96-hour survival rate was >98%.

[0017] (2) The licheniformis BL01 prepared by the present invention carries key nematode inhibition genes (aprX, srfAA) and heat resistance-related gene clusters (clpC, groES). After targeted mutagenesis, the enzyme activity stability is improved by 15%.

[0018] (3) The present invention realizes the synergistic technology system of "disease prevention and control-water purification-feed efficiency enhancement" through Bacillus licheniformis BL01, which improves the comprehensive benefits by more than 40% compared with the single bacteria injection mode. Specifically, in terms of disease and pest control, adding it to feed at a ratio of 0.2-0.5% (w / w), or directly spraying it into water bodies at a concentration of 10^6-10^7 CFU / L, can reduce nematode infection rates by ≥65%, while also increasing the activity of fish digestive enzymes (protease activity increased by 22-28%); in terms of water quality control, when used in combination with zeolite powder (added at 50 mg / L), the ammonia nitrogen degradation rate is increased by 1.3 times; in the development of composite bacterial agents, BL01 is compounded with Bacillus subtilis (ratio 3:1) and used in shrimp farming ponds, reducing nematode diseases by 72% and COD in the water by 56%; in terms of industrial production, through fermentation (pH 7.5-8.0, dissolved oxygen content 30%), a bacterial concentration of ≥5×10^7 CFU / mL is achieved, a spore production rate of >95%, and a live bacterial survival rate of >90% when the preparation is stored at room temperature for 180 days.

[0019] (4) The Bacillus licheniformis BL01 provided by the present invention can replace chemical nematicides and reduce drug residues by more than 90%; it can reduce the frequency of water changes by 50%, saving breeding costs; it can increase the weight gain rate of fish and shrimp by 12-18% and reduce the feed coefficient by 0.3-0.5; it can improve the growth performance of fish, improve the intestinal flora, and enhance the resistance of fish to pathogens.

[0020] (5) The method for preparing the Bacillus licheniformis BL01 into a microecological preparation of the present invention is simple and has a low production cost. At the same time, it can effectively reduce the irritation of pesticides to farmed animals, especially in shrimp and crab breeding ponds, and has good application prospects for the protection of aquatic plants.

[0021] (6) The process of the Bacillus licheniformis BL01 of the present invention has been improved to enhance the stress resistance of the Bacillus licheniformis BL01 and ensure the stability of the product.

[0022] (7) The Bacillus licheniformis BL01 of the present invention has a significant antibacterial effect on a variety of fish pathogens in the laboratory: Aeromonas hydrophila, Streptococcus agalactiae, and Edwardsiella tarda; it has the potential to be developed into a microecological preparation that can be used to inhibit pathogenic bacteria and reduce bacterial contamination in feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the antibacterial results of Bacillus licheniformis BL01 provided by the present invention; Figure 2 This is a morphological characteristic diagram of Bacillus licheniformis BL01 provided by the present invention; Figure 3 This is the evolutionary tree diagram of Bacillus licheniformis BL01 provided by the present invention; Figure 4 The complete genome sequence of Bacillus licheniformis BL01 provided by the present invention; Figure 5 This is a graph showing the high temperature tolerance of the Bacillus licheniformis BL01 antibacterial substance provided by the present invention; Figure 6 This is a graph showing the acid and alkali tolerance results of the Bacillus licheniformis BL01 antibacterial substance provided by the present invention; Figure 7 This is a graph showing the enzyme resistance results of the Bacillus licheniformis BL01 antibacterial substance provided by the present invention; Figure 8 This is a diagram showing the antibacterial effect of Bacillus licheniformis BL01 provided by the present invention under different carbon sources; Figure 9 This is a diagram showing the antibacterial effect of Bacillus licheniformis BL01 provided by the present invention at different carbon-nitrogen ratios; Figure 10 This is a diagram showing the antibacterial effect of Bacillus licheniformis BL01 provided by the present invention at different temperatures; Figure 11 This is a diagram showing the antibacterial effect of Bacillus licheniformis BL01 provided by the present invention at different initial pH values; Figure 12 This is a diagram showing the nematode killing effect of Bacillus licheniformis BL01 provided by the present invention; Figure 13 This is a diagram showing the root node inhibition rate of Bacillus licheniformis BL01 provided by the present invention; Figure 14 This is a diagram showing the safety survival curve of the Bacillus licheniformis BL01 provided by the present invention for aquatic animals.

[0024] Note: Figure 2 In the figure, Figure A is a colony morphological characteristic diagram of the strain Bacillus licheniformis BL01; Figure B is a Gram staining diagram of the strain Bacillus licheniformis BL01 (400×). DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] Example 1 Basic characteristics of Bacillus licheniformis BL01 1.1 Isolation and screening of Bacillus licheniformis BL01 The bottom mud from the hairy crab breeding pond in Hanchuan City, Hubei Province was diluted with sterile physiological saline in a gradient manner and then coated with KMB solid culture medium. It was cultured at 37°C for 48-72 hours, the growth of the strain was observed, and the dominant single colony was picked for purification. All pathogens in the laboratory: Edwardsiella tarda, Staphylococcus aureus, Salmonella, Aeromonas hydrophila, Vibrio chlororaphis and Streptococcus agalactiae were used as indicator bacteria, and the antibacterial activity of the fermentation broth against the above pathogens was detected using the Oxford cup method. It was found that the supernatant of the isolated strain had a good antibacterial effect on all pathogens, among which the antibacterial effect on Staphylococcus aureus was the strongest, with an average inhibition zone diameter of 17.20±0.02mm ( Figure 1 The Bacillus licheniformis strain screened by the present invention is named Bacillus licheniformis BL01. The 16S rRNA sequence of the strain has been submitted to the GenBank database and has been deposited in the China Center for Type Culture Collection on April 11, 2025, at Wuhan University, Wuhan, Hubei Province, China, with the deposit number CCTCC NO: M2025752.

[0028] 1.2 Identification of Bacillus licheniformis BL01 (1) Morphological characteristics and Gram staining The single colony morphology of BL01 on KMB medium is as follows Figure 2 As shown in A, the colony has smooth edges, a raised center, and is pale white; the BL01 Gram staining results are as follows Figure 2 As shown in B, Gram-positive bacteria are long rod-shaped after 24 hours of fermentation and ellipsoidal spherical after 48 hours of fermentation ( Figure 2 ).

[0029] (2) Physiology and biochemistry The species of the bacteria were preliminarily identified according to the Common Bacterial Identification Manual and the Bergey's Systematic Identification Manual. A total of 15 physiological and biochemical identifications were performed on the isolated strain. The results showed that the isolated strain BL01 could utilize esculin, sucrose, xylose, fructose, and maltose; reduce nitrate, hydrolyze salicin to produce salicinone, and ferment mannitol to produce acid (Table 1).

[0030] Table 1 Physiological and biochemical results of BL01

[0031] Note: - indicates negative, + indicates positive.

[0032] (3) Molecular biological identification The whole genomic DNA of strain BL01 was extracted using a bacterial genomic DNA extraction kit (purchased from Shanghai Double Helix Technology Co., Ltd.). PCR amplification was performed using 16S rRNA primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-CGGTTACCTTGTTACGACTT-3'). The sequencing results were then compared and analyzed at NCBI to construct a phylogenetic tree. The results showed that the strain BL01 was a Bacillus licheniformis strain ( Figure 3 ).

[0033] (4) BL01 whole genome sequencing The BL01 strain was activated and cultured, and a small amount of cells were taken to extract DNA. The extracted genomic DNA was then purified by column and sequenced by Beijing Qingke Biotechnology Co., Ltd. The results showed that the BL01 strain was purified and genomic DNA was extracted. The library was constructed and sequenced using sequencing technology. Sequence assembly was performed. The full-length genome of the BL01 strain was 398,136,4 bp ( Figure 4 ).

[0034] 1.3 Antimicrobial tolerance of Bacillus licheniformis BL01 (1) High temperature tolerance of BL01 antibacterial substance The BL01 supernatant was treated at 40°C, 60°C, 80°C, and 100°C for 30 minutes. Untreated supernatant served as a negative control, and neomycin sulfate served as a positive control. The Oxford cup method was used to test the inhibitory effect of BL01 supernatant on pathogens. The results showed that BL01 supernatant treated at 80°C for 30 minutes still had an inhibitory effect on Streptococcus agalactiae and Aeromonas hydrophila ( Figure 5 ).

[0035] (2) Acid and alkali tolerance of BL01 antibacterial substance The BL01 supernatant was placed in KMB at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. The supernatant was kept at 4°C overnight. The untreated supernatant was used as a negative control, and neomycin sulfate was used as a positive control. The Oxford cup method was used to detect the inhibitory effect of the BL01 supernatant on pathogens. The results showed that under strong acid and strong base conditions, the BL01 supernatant had no inhibitory effect on Streptococcus agalactiae. BL01 had no inhibitory effect on Aeromonas hydrophila after being treated with different pH values ( Figure 6 ).

[0036] (3) Enzyme resistance of BL01 antimicrobial substances Proteinase K and trypsin were added to the BL01 supernatant (at the optimal pH of the two enzymes, at 37°C for 2 hours) to a final enzyme concentration of 1 mg / mL. Untreated supernatant served as a negative control, and neomycin sulfate served as a positive control. The Oxford cup method was used to test the inhibitory effect of BL01 supernatant on pathogens. The results showed that BL01 supernatant treated with different proteases had an inhibitory effect on Streptococcus agalactiae, but had no inhibitory effect on Aeromonas hydrophila ( Figure 7 ).

[0037] Example 2 Optimization of the antibacterial effect of Bacillus licheniformis BL01 2.1 Antibacterial effect of BL01 under different carbon sources The inhibitory effect of BL01 on Streptococcus agalactiae under different carbon sources was tested. The results showed that BL01 had the greatest inhibitory effect on Streptococcus agalactiae under sucrose conditions ( Figure 8 ).

[0038] 2.2 Antibacterial effect of BL01 at different carbon-nitrogen ratios The inhibitory effect of BL01 on Streptococcus agalactiae at different carbon-nitrogen ratios was tested. The results showed that BL01 had the greatest inhibitory effect on Streptococcus agalactiae at a carbon-nitrogen ratio of 0.4 / 1. Figure 9 ).

[0039] 2.3 Antibacterial effect of BL01 at different temperatures The inhibitory effect of BL01 on Streptococcus agalactiae at different temperatures was tested. The results showed that there was no significant difference in the inhibitory effect of BL01 on Streptococcus agalactiae between 28℃ and 37℃, indicating that BL01 has a wide range of temperature adaptability ( Figure 10 ).

[0040] 2.4 Antibacterial effect of BL01 at different initial pH The inhibitory effect of BL01 on Streptococcus agalactiae at different initial pH values was tested. The results showed that BL01 had a greater inhibitory effect on Streptococcus agalactiae at an initial pH of 7. Figure 11 ).

[0041] Therefore, the optimal fermentation process of this Bacillus licheniformis is: Culture medium: peptone 2.5%, magnesium sulfate 0.15%, potassium hydrogen phosphate 0.15%, sucrose 1%, calcium chloride 0.09%, initial pH 7.0; Conditions: 37°C, 180 rpm, 24 hours (viable count reaches 5×10 7 CFU / mL).

[0042] Example 3 Preparation of Probiotics and Stability Process 3.1 Fermentation process of Bacillus licheniformis BL01 Preparation of seed liquid: streak the cryopreserved tube BL01 on KMB culture medium, take a single colony and inoculate it into the primary seed culture medium, culture at 30-37℃, 200-220r / min for 24h. Fermentation in small fermenter: access the secondary seed liquid at a rate of 1-2%, culture at 30-37℃, 150-200r / min for 24h. Fermentation in large fermenter: transplant the secondary seed liquid prepared in the previous step into the fermenter at 3-4%, control the tank temperature at 30-37℃, stir at 100-200rpm / min, and culture for 24h to obtain Bacillus licheniformis fermentation liquid. After multiple process optimizations, the number of viable bacteria of Bacillus licheniformis BL01 is 10 7 cfu / mL (Table 2).

[0043]

[0044] Example 4 Recommended safe dosage of Bacillus licheniformis BL01 in aquaculture Healthy grass carp (15±2g) were randomly divided into 3 groups, with 30 grass carp in each group. The animals were fed for 2 weeks to stabilize the diet. During the experiment, the water temperature was controlled at 25-28℃ and oxygen was sufficient. 8 The BL01 strain was used at the recommended, 2x, and 5x recommended colony-forming units / mL (cfu / mL) concentrations for three groups. After mixing with feed, a feeding experiment was conducted. A control group received saline as a supplement. After two weeks of continuous feeding, the grass carp's health and mortality were observed and recorded daily. The results showed that grass carp fed with different concentrations of BL01 strain in their feed, as well as those in the control group, showed improved growth and no mortality. This indicates that the BL01 strain dosage can be increased or decreased based on the culture conditions without affecting grass carp growth (Table 3).

[0045] Table 3 Animal safety recommended dosage experiments (+ represents normal growth)

[0046] Example 5 Application of Bacillus licheniformis BL01 in aquaculture 5.1 Strengthen digestive function, promote absorption, and improve feed conversion efficiency.

[0047] 5.1.1 Enzyme Production Verification: BL01 was activated and cultured for 24 hours. A single colony was selected and added to 10 mL of NB medium. The culture was shaken at 200 rpm at 37°C for 24 hours. 100 μL of the bacterial suspension was centrifuged at 12,000 rpm for 5 minutes. 2 μL of the supernatant was plated on a milk plate and incubated inverted for 48 hours. The plate was photographed and the diameter of the clearing zone was measured. The results showed that BL01 could produce protease, which facilitated better nutrient absorption (Table 4).

[0048] Table 4 Protease production capacity of BL01

[0049] 5.1.2 Largemouth bass, grass carp, and yellow catfish weighing 15±2g were divided into experimental and control groups, with 30 fish in each group and 3 replicates in each group. The experimental tanks were kept in a stable aquaculture environment, free from external environmental changes or pollution, and the water temperature was maintained at 26±1℃. The aquaculture period was 13 weeks, and the feed was fed three times a day (morning, noon, and evening). The experimental group was fed with 1×10 8 The BL01 strain was sprayed onto the feed at a concentration of 100 cfu / mL and mixed thoroughly. The fish in the control group were fed a standard diet at a rate of 2% of their body weight. Fish were weighed during the experiment and on the final day. The results showed that the final weight of the fish in the BL01-fed group was significantly higher than that in the control group, indicating that feeding the BL01 strain significantly enhances absorption and improves feed conversion efficiency (Tables 5-7).

[0050] Table 5 Detection of growth performance of largemouth bass by Bacillus licheniformis BL01

[0051] Table 6 Detection of grass carp growth performance by Bacillus licheniformis BL01

[0052] Table 7 Detection of growth performance of yellow catfish by Bacillus licheniformis BL01

[0053] 5.2 Water purification During Experiment 5.1.2, water samples were collected from the grass carp aquaculture tanks at weeks 1, 5, 10, and 13 for testing of pH, dissolved oxygen, transparency, ammonia nitrogen, and nitrite. The results showed no significant differences in pH and transparency, with the levels remaining largely consistent. Ammonia nitrogen showed an initial upward trend followed by a downward trend, remaining consistently lower in the experimental group than in the control group. Nitrite gradually increased, remaining consistently lower in the experimental group than in the control group (Tables 8-9). This suggests that BL01 can decompose dead algae, residual matter, and organic matter in the water, reducing toxins in the aquatic environment and maintaining the health of aquatic animals. Long-term use of probiotics can reduce the levels of harmful substances in fecal matter, such as ammonia nitrogen and nitrite, thereby reducing water pollution.

[0054] Table 9 Ammonia nitrogen detection in water

[0055] Table 10 Nitrite detection in water

[0056] 5.3 Nematicidal effect experiment of BL01 Application 1: In-vitro toxicity assay (in vitro nematicidal activity test) Test nematodes: Second-instar larvae (J2) of the southern root-knot nematode (Meloidogyne incognita) cultured in a standard laboratory culture were selected and washed three times with sterile water and then adjusted to a suspension concentration of 200 larvae / mL.

[0057] Preparation of strain treatment solution: Inoculate Bacillus licheniformis BL01 (CCTCC NO: M 2025752) into KMB liquid medium and culture at 37°C with shaking at 180 rpm for 48 h until spore formation is ≥ 90%. Collect the cells by centrifugation (8000 rpm, 10 min), resuspend in sterile water, and serially dilute to 1 × 10 8 CFU / mL, 5×10 7 CFU / mL, 1×10 7 CFU / mL at three concentrations.

[0058] Experimental methods: Take a 24-well cell culture plate and add 500 μL of nematode suspension (100 nematodes) to each well and mix it with an equal volume of bacterial suspension. Set up 3 replicates and use an equal amount of sterile water as the control group.

[0059] The cells were placed in a 25°C constant temperature incubator in the dark, and samples were taken for observation at 24h, 48h, and 72h.

[0060] Death judgment criteria: No stress response after nematode staining with 0.1% neutral red Record-corrected mortality (Abbott formula correction) Experimental results: 1×10 at 72 hours 8 The corrected mortality rate of the CFU / mL treatment group was 92.3%, and the LC 50 The value is 3.2×10 6 CFU / mL (Probit analysis). Figure 12 ) Application 2: Verification of potted plant prevention and control effects Test plants: Tomato (Solanum lycopersicum cv. 'Hezuo903') seedlings were grown to the three-leaf, one-heart stage and transplanted into 12 cm pots containing sterilized substrate.

[0061] Inoculation treatment: Inoculate 2000 J2 larvae of southern root-knot nematode per pot (root injection method of suspension) Application of microbial agents Experimental group: Immediately after inoculation with nematodes, water was poured with BL01 bacterial suspension (1×10 8 CFU / mL) 50mL / strain Positive control: 5% avermectin emulsifiable concentrate 3000 times diluted Blank control: equal amount of sterile water 10 strains per group, repeated 3 times Effect evaluation Determination after 35 days of treatment: Root knot index (0-5 grading scale) Root fresh weight change rate Nematode reduction rate in soil (Baiman funnel method) Data processing: Duncan's new multiple range method was used (p < 0.05) Experimental results The root knot inhibition rate reached 81.2%, significantly better than the positive control (67.5%), and the soil nematode density was reduced by 89.4%. Figure 13 ) Application 3: Safety testing (non-target organisms) Model organism: Zebrafish (Danio rerio) juveniles (48 hpf) were exposed to 10 8 CFU / mL bacterial suspension Observation indicators No abnormal behavior or death within 96 hours (LC 50 >10 8 CFU / mL) Figure 14 ), proven to be safe for non-target organisms (in compliance with NY / T 3156.2-2020 standards).

[0062] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0063] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Bacillus licheniformis BL01, characterized in that The taxonomic name of this strain is Bacillus licheniformis, and the strain name is Bacillus licheniformis BL01. It was deposited in the China Center for Type Culture Collection on April 11, 2025, with the deposit address being Wuhan University, Wuhan City, Hubei Province, China, and the deposit number is CCTCC NO: M2025752.

2. The Bacillus licheniformis BL01 according to claim 1, characterized in that The full length of the genome of the Bacillus licheniformis BL01 is 398,136,4 bp.

3. The Bacillus licheniformis BL01 according to claim 1, characterized in that The culture medium for culturing the Bacillus licheniformis BL01 includes: 2-3wt% peptone, 0.1-0.3wt% magnesium sulfate, 0.1-0.3wt% dipotassium hydrogen phosphate, 0.5-2.0wt% sucrose, 0.05-0.20wt% calcium chloride, and an initial pH of 5.0-7.

0.

4. The industrial fermentation method of Bacillus licheniformis BL01 according to any one of claims 1 to 3, characterized in that: S1. Inoculate Bacillus licheniformis BL01 on solid KMB medium, pick a single clone and transfer it to the medium used to culture Bacillus licheniformis BL01 for fermentation. Ferment aerobically at 30-37°C and 200-220 rpm for 24-48 hours, and collect the fermentation seed liquid. S2. Inoculate the fermentation seed liquid into a fermentation tank filled with fermentation medium at a volume ratio of 1-3% for the first expansion culture, and ferment aerobically at 30-37° C. and 100-150 rpm for 24-48 hours to obtain expanded culture liquid I; S3. Inoculate the expanded culture fluid I into a fermentation tank containing fermentation medium at a volume ratio of 3-10% for a second expanded culture. Ferment aerobically at 30-37° C., a tank pressure of 0.03 MPa, and 100-150 rpm for 16-24 h to obtain expanded culture fluid II.

5. Use of Bacillus licheniformis BL01 in the preparation of a biological control inhibitor for reducing nematode infection rate.

6. The use according to claim 5, characterized in that The Bacillus licheniformis BL01 is added to the feed and then sprinkled into the water, or directly sprinkled into the water.

7. The use according to claim 6, characterized in that The mass fraction of the Bacillus licheniformis BL01 in the feed is 0.2-0.5%; and / or, The concentration of the Bacillus licheniformis BL01 directly sprinkled into the water body is 10^6-10^7 CFU / L.

8. Use of Bacillus licheniformis BL01 in preparing a preparation for water purification.

9. Use of a Bacillus licheniformis BL01-Bacillus subtilis combination in the preparation of a composite bacterial agent for killing nematodes and reducing COD in water.

10. Use of Bacillus licheniformis BL01 in the preparation of a feed synergist preparation.

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