A kind of fluorinated Velez-like bacillus and its prepared bacterial agent

Abstract

The present invention discloses a fluorine-resistant Bacillus velezensis and a bacterial agent prepared therefrom; the strain belongs to Bacillus velezensis, is named Bacillus velezensis Y500, has been deposited in the China Center for Type Culture Collection (CCTC), and its deposit number is CCTCC NO: M 2024562, and the deposit date is 2024.3.25. The optimum growth temperature of the microorganism is 15-37°C, and the optimum growth pH range is 5.0-9.0. Beneficial effect: the strain has good tolerance to fluorine element, can adsorb and accumulate fluorine element, the strain and the passage strain retaining this active strain can be applied to water defluoridation and tea defluoridation treatment, providing a research idea for improving fluorine pollution and improving tea quality.

Description

Technical Field

[0001] The invention relates to the technical field of microbial strains, and in particular to a velotetrafluoroethylene-resistant Bacillus and a bacterial agent prepared therefrom. Background Art

[0002] Bacillus velezensis is a new species of the genus Bacillus, identified only in 2005. Widely distributed in the air, soil, rivers, lakes, animal intestines, plant roots, and tissues, B. velezensis is a motile aerobic or facultatively anaerobic bacterium with flagella. It can grow in culture at a salt concentration of 12%, temperatures of 15-45°C, and a pH of 5-10. Although B. velezensis was recently discovered, research reports on it are increasing. Research both domestically and internationally has demonstrated that B. velezensis possesses antagonistic effects against plant pathogenic fungi and bacteria, making it a highly effective and exploitable new biocontrol bacterium with unlimited potential for biocontrol.

[0003] The cultivation and management of tea trees is an important part of tea garden quality. Reducing the fluoride content in tea leaves through fertilization in tea gardens is also one of the important means, which mainly includes the use of quicklime to reduce fluoride, low-concentration aluminum fertilizer, and selenium fertilizer to reduce fluoride. Chemical fertilization affects the construction of green ecological tea gardens and organic tea gardens, while biological fertilizers have obvious advantages, high cost-effectiveness, broad prospects, and are more environmentally friendly and sustainable. Although the market for microbial fertilizers is becoming increasingly mature, with a wide range of choices and diverse combinations, there are few reports on endophytic fertilizers for tea trees to reduce fluoride. Research on endophytes in tea trees is becoming more and more extensive and in-depth. By utilizing the characteristics of the tea tree's own strains, fluoride-resistant bacterial groups are screened and fluoride reduction experiments are conducted on tea trees, which has good economic and ecological value for the development of the tea industry.

[0004] Chinese patent application publication number CN117757700A discloses a Bacillus velezensis strain and its applications. The strain's Latin name is Bacillus velezensis, and its deposit number is GDMCC No. 63555. Application of the Bacillus velezensis strain and the bacterial agent prepared therefrom in the patent to the rhizosphere of plants effectively prevents and controls plant diseases and promotes plant growth, providing a new approach for agricultural production, biological control, and environmental management. However, the patent does not disclose the fluorine-resistant Bacillus velezensis strain of the present invention. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to provide a Velez-fluani-resistant Bacillus.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The first aspect of the present invention provides a Bacillus velezensis Y500 strain, which has been deposited in the China Center for Type Culture Collection (CCTC) with a deposit number of CCTCC NO: M 2024562 and a deposit date of March 25, 2024.

[0008] Beneficial Effects: The present invention provides a fluorine-resistant Bacillus velezensis Y500 strain, which exhibits milky white, round colonies with a viscous texture and a smooth, slightly convex surface. It is aerobic and survives normally within a pH range of 5.0-9.0 and a temperature range of 15-37°C. This strain exhibits excellent tolerance to fluoride, adsorbing and accumulating it. This strain, along with subcultures retaining active strains, can be used for water defluoridation and tea leaf defluoridation, providing research insights for improving fluoride pollution and enhancing tea quality.

[0009] Preferably, the 16S rDNA sequence of the strain is shown as SEQ ID NO.1.

[0010] Preferably, the strain has an optimum growth temperature of 15-37° C. and an optimum growth pH range of 5.0-9.0.

[0011] Preferably, the culture medium of the strain is LB medium, and the inoculation amount in the expanded culture is 1%.

[0012] A second aspect of the present invention provides a microbial agent comprising the aforementioned Bacillus Velezii.

[0013] Preferably, it further comprises auxiliary materials.

[0014] Preferably, the bacterial agent is in the form of a powder, granules or suspension.

[0015] The third aspect of the present invention provides the use of the above-mentioned microbial agent in the adsorption of fluorine.

[0016] Preferably, the concentration of the fluorine element is ≤600 mg / L.

[0017] Preferably, the concentration of the fluorine element is 500 mg / L.

[0018] The advantages of the present invention are:

[0019] The Bacillus velezensis Y500 strain proposed in this invention exhibits milky white, round colonies with a viscous texture and a smooth, slightly convex surface. It is aerobic and survives normally within a pH range of 5.0-9.0 and a temperature range of 15-37°C. This strain, as well as subcultures retaining this active strain, exhibits excellent tolerance to fluoride, adsorbing and accumulating it. It can be used for water defluoridation and tea leaf defluoridation, providing research insights for alleviating fluoride pollution and improving tea quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The graphs of Bacillus activity and pH value changes of LB culture medium under different pH treatments are shown;

[0021] Figure 2 The graph shows the changes in Bacillus activity and pH value of LB culture medium under different F treatment concentrations;

[0022] Figure 3 The graph shows the changes in the activity, fluoride removal rate, adsorption capacity, pH value and intracellular and extracellular fluoride content of Bacillus under different F treatment concentrations;

[0023] Figure 4 This is a graph showing the effect of Bacillus on the fluorine content in the leaves of Shucha early tea seedlings under different F treatments;

[0024] Figure 5 This is a diagram showing the effect of Bacillus on the fluoride content in the leaves of Shuchazao and Hubei population tea trees; in the figure, "*" represents p<0.05, "**" represents p<0.01, "***" represents p<0.001, and "****" represents p<0.0001. DETAILED DESCRIPTION

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

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

[0027] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0028] Example 1:

[0029] Screening of fluoride-resistant endophytes in tea plants:

[0030] Samples were collected from the tea garden of Chibi Group in Hubei Province. The leaves were washed with pure water and placed in 70% ethanol in a clean bench for 2 minutes to completely submerge them. They were then washed with sterile water 4 times and then eluted with Cl - Soak in 3% sodium hypochlorite for 3 minutes, and finally wash 3 times with sterile water. Take sterile filter paper to absorb excess water, cut the leaves into pieces with sterile scissors, put them in a sterile mortar and grind them with a small amount of sterile quartz sand. Then add 10 times the volume of phosphate buffer (pH = 7) to homogenize, let it stand for 5 minutes, and take the suspension for 10-fold serial dilution, totaling 6 concentrations. Take 100 μL of each concentration and spread it on a solid culture medium containing 20 ml LB, with an F concentration of 50 mg / L. Repeat each gradient 3 times. Seal with sealing film, invert and culture in a constant temperature incubator at 37°C in the dark. To verify the effectiveness of the plant in vitro sterilization process, take 100 μL of the sterile water used in the last wash and evenly spread it on the LB solid culture medium as a control. Culture at 37°C for one week, and the control without any colony growth is considered to be effective surface sterilization. Endophytic bacteria isolated from the 50 mg / L fluoride treatment group at different dilution gradients were inoculated onto LB solid medium containing 100 mg / L fluoride and cultured in an inverted tube in a 37°C incubator. Subsequently, the fluoride concentration was gradually increased in 50 mg / L increments while simultaneously being isolated and purified. The resulting strains were identified and stored.

[0031] The result of 16SrDNA bacterial species identification was Bacillus velezensis and it was deposited in the China Center for Type Culture Collection (CCTC) on March 25, 2024, with the deposit number CCTCC NO:M2024562, and was also named Bacillus velezensis Y500.

[0032] Its 16S rDNA sequence is shown below:

[0033] AGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGT

[0034] AACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAA

[0035] TACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACC

[0036] ACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAA

[0037] GGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC

[0038] ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAA

[0039] GTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTG

[0040] TTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAA

[0041] CCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAA

[0042] GCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGAT

[0043] GTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGT

[0044] GCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGA

[0045] GGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGA

[0046] AAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATG

[0047] AGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCA

[0048] CTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGC

[0049] CCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACC

[0050] AGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAG

[0051] TGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC

[0052] CGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGG

[0053] TGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCC

[0054] CTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAAC

[0055] CGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAAC

[0056] TCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAAT

[0057] ACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGC(SEQ ID NO.1)

[0058] Test the growth status of Bacillus velezensis under different pH values:

[0059] Adjust the pH of the LB medium using 4.1 mol / L NaOH and 10% nitric acid to 3, 4, 5, 6, 7, 8, 9, and 10, respectively. Set up three replicates, inoculate 1% of Bacillus velezensis in a flask containing 100 mL of LB liquid after sterilization, and place it in a constant temperature shaking incubator at 37°C and 150 rpm. After 5 days, measure the changes in pH and activity. Figure 1 As shown in Figure B, the growth of Bacillus Velezii is more active among pH values ​​of 5, 6, 7, 8, and 9, and the most suitable pH value for growth is 6.

[0060] The bacterial solution was centrifuged at 6000 r / 10 min and the pH value of the supernatant was measured. Figure 1 As shown in Figure A, compared with the set results, the pH values ​​of the supernatants with initial culture medium pH values ​​of 5, 6, and 7 increased significantly, while the pH values ​​of the supernatants with initial culture medium pH values ​​of 9 and 10 decreased. This shows that the growth of Bacillus Velezii can affect the pH of the culture environment to adapt to environmental changes.

[0061] Testing the minimum inhibitory concentration (MIC) and pH changes of Bacillus velezensis:

[0062] LB culture medium with different F concentrations (0, 5, 20, 50, 100, 200, 300, 400, 500, 600, 700 mg / L) was prepared with sodium fluoride and placed in a 250 mL Erlenmeyer flask. The pH of each culture medium was measured. After sterilization, the fluoride content was measured again. One ring of Bacillus velezinoff was picked and inoculated. The culture medium was placed in a constant temperature shaking incubator at 37°C and 150 rpm for 24 hours in the dark. Starting from the 8th hour of inoculation, the culture medium samples were taken every 2 hours to measure the OD 600 The changes in Figure 2 As shown. Figure 2 Figure A shows the activity changes of the strain in fluoride-containing medium for 24 hours. The activity trends of Bacillus Velezii under different F concentration treatments and at different time periods are different. Except for the strain with an F concentration of 700 mg / L, which stopped growing from the beginning to the end, the lower the F concentration, the faster the growth before 16 hours; at 18 hours, the Bacillus was in a low activity period as a whole, and then showed an overall upward trend; after 20 hours, the growth rate of Bacillus changed under different F concentration treatments, especially at 22 hours, the growth of the strains in the 0F, 5F, 20F and 50F (here 50F means 50 mg / L, and the same applies to the others) treatment groups slowed down, while the Bacillus in the 500F and 600F treatment groups grew rapidly, and the growth rate of Bacillus in the other F concentration treatment groups was also significantly accelerated.

[0063] The Bacillus velezensis culture liquids of different F treatment groups were centrifuged at 6000r / 10min in a centrifuge, and the changes in the pH value of the supernatant were measured. As shown in Figure B, in the CK group (sterile) treatment group, with the increase of F concentration, the pH value of the LB culture medium changed, but all treatment groups were in a weakly acidic condition. After the treatment with Bacillus velezensis, except for the strain containing 700mg / L F concentration, the pH of other LB culture media changed significantly, and all treatment groups were in an alkaline environment. Figure 1 It can be seen that the growth of Bacillus velez can change the pH value of LB culture medium, making the pH value of the solution around 9, which is alkaline; after the culture medium was treated with F, the pH values ​​of 0F, 5F, 20F, 50F, 100F, 200F, 300F, and 400F groups were around 8, with significant changes, while the pH values ​​of 500F and 600F groups were less than 8, with a smaller increase.

[0064] Combine Figure 1 and Figure 2 It is speculated that Bacillus Velezii has a strong adaptability to the pH value in the environment. This strain may affect the acid-base changes in the living environment through its own growth metabolism, thereby improving its tolerance to fluoride in the environment.

[0065] Testing the adsorption and accumulation ability of Bacillus Velez on F:

[0066] Set the F concentration of LB culture medium to 0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 50 mg / L, respectively. After ultrasonic dissolution, adjust the pH to 5.0 ± 0.02, and repeat each treatment 3 times. After sterile treatment, measure the actual fluoride content of different F treatment groups again. Under sterile conditions, accurately pick 1 ring of strain Y500 and inoculate it into 100 mL of sterile LB culture medium, place it in a constant temperature incubator at 37 ° C, and shake at 150 r / min for 120 hours in the dark. First take 200 μL to measure its OD 600 The remaining bacterial solution was centrifuged at 6000 r / min for 10 minutes in a 50 mL sterile centrifuge tube. 2 mL of the supernatant was taken to measure the pH change. A portion of the supernatant was mixed with TISAB buffer at a volume ratio of 1:1. The residual F concentration was measured using a fluoride ion selective electrode and then used in formula (1-1) to calculate the fluoride removal rate. The precipitated bacteria were washed three times with 0.85% NaCl and thoroughly dried at 70°C. The dry weight of the bacteria was measured and used in formula (1-2) to calculate the amount of F adsorbed by the bacteria. The results are as follows. Figure 3As shown in Figures A, B, and C, compared with the 0 mg / L F control, strain activity increased in the treatments with 5, 10, 20, and 50 mg / L F, suggesting that F may be involved in the growth and development of this strain. The amount of F adsorbed by the bacteria increased with increasing F concentration, while the fluoride removal rate in the supernatant decreased with increasing F concentration, with the overall fluoride removal rate ranging from 1% to 4%. The pH value of the supernatant increased significantly, and the culture medium shifted from slightly acidic to alkaline.

[0067]

[0068]

[0069] Where: R is the adsorption rate (%), C0 and C t are the initial actual F concentration of the supernatant and the F concentration after adsorption (mg / L), respectively; q is the adsorption amount (mg / g), V is the solution volume (mL), and m is the dry weight of the bacteria (g).

[0070] The dried bacteria in the above test were washed twice with sterile water, then vortexed with 10 mL of 0.1 M phosphate buffer (pH = 7.0) for 2 minutes and centrifuged at 6000 r / min for 10 minutes. The supernatant was mixed with TISAB buffer in a volume ratio of 1:1. The extracellular fluoride content of the strain was calculated by using a fluoride ion selective electrode and then entered into formula (1-3). The results are as follows: Figure 3 As shown in D, with the increase of F concentration, the extracellular F content of the strain gradually increased, and the change was significant compared with 0F.

[0071] The remaining precipitated bacteria were placed in an oven and dried again at 70°C. 0.1000g (accurate to 0.0001g) of completely dried bacteria were taken and placed in a 30mL nickel crucible. 1.00g of solid NaOH was added and mixed. After covering, the crucible was placed in a muffle furnace at 300°C / 30min and 600°C / 1h. After alkaline fusion digestion, the muffle furnace was turned off and cooled to room temperature. The crucible was taken out and 5.0mL of 10% dilute nitric acid solution was added to wash the inner wall of the crucible. The pH was adjusted to 8.0-9.0. The sample was transferred to a 50mL volumetric flask and fixed to volume. After mixing, it was filtered with filter paper. The filtrate was mixed with TISAB buffer at a volume ratio of 1:1. After determination with a fluoride ion selective electrode, the intracellular fluoride content was calculated by formula (1-4). The results are as follows. Figure 3 As shown in D, with the increase of F concentration, the strain's ability to accumulate F in the cell gradually increased. Combined with the changes in the extracellular and intracellular F content of the strain, under different F concentration treatments, the intracellular accumulation and extracellular attachment abilities of Bacillus velezensis to F were positively correlated with the fluoride concentration of the growth environment, and the change trends of the two were consistent.

[0072] Preparation of Bacillus Velez strain suspension:

[0073] Place Bacillus Velezii in LB medium and culture at 37℃ / 150r until OD 600 When the value is 1, centrifuge at 6000r / 10min, discard the supernatant, wash with sterile water, centrifuge again at 6000r / 10min, and dilute with sterile water to make the bacterial content of the nutrient solution or aqueous solution 2×10 -4 CFU.

[0074] Preparation and use of tea tree nutrient solution

[0075] The mother solution configuration of the nutrient solution is shown in Table 1-1. When used, it is diluted to the required concentration as shown in Table 1-2, and its pH value is adjusted with 16.8 mol / L NaOH and 20% nitric acid within the range shown in the table.

[0076] Table 1-1 Preparation of nutrient solution mother solution

[0077]

[0078]

[0079] Table 1-2 Nutrient solution dilution concentration

[0080]

[0081] Preparation of TISAB buffer solution: Place 58 g of NaCl and 68 g of Na3C6H5O7·2H2O in a beaker, add 700 mL of ultrapure water, mix well, and sonicate to dissolve. Add 57 mL of glacial acetic acid in a fume hood, mix well, and let stand for 10 min. Adjust the pH to between 5.2 and 5.3 with 16.8 mol / L NaOH. Transfer to a volumetric flask and dilute to 1 L. Cool to room temperature before use.

[0082] Determination of water-soluble fluoride content: Weigh 0.1500 g of tea sample (accurate to 0.0001 g) and place it in a 50 ml centrifuge tube. Add 20 ml of ultrapure water and place it in a 100°C water bath for 30 minutes. Take it out and cool it to room temperature. Filter it with filter paper. Add the tea sample and TISAB buffer solution in a 1:1 volume ratio and mix well in a centrifuge tube. Determine the fluoride content with a fluoride ion electrode and then enter it into formula (1-3) to calculate the final water-soluble fluoride content.

[0083]

[0084] Where: X is the final F concentration of the sample (mg / kg), A is the measured fluorine concentration of the sample (mg / L), A0 is the measured fluorine concentration of the blank solution (mg / L), V is the total volume of the sample (mL), and m is the sample mass (g).

[0085] Determination of total fluorine content: Weigh about 0.2500 g (accurate to 0.0001 g) of powdered tea leaves 1-3 and 4-6 respectively, and place the tea sample in a 30 ml nickel crucible. Add 2.5 g of solid NaOH and mix well. Cover and place in a muffle furnace and gradually increase the temperature to 300°C / 30 min, 600°C / 1 h. After alkaline dissolution, turn off the muffle furnace and wait until it cools to room temperature. Take out the crucible, add 5.0 mL of 10% volume fraction dilute nitric acid solution, wash the inner wall of the crucible, transfer the washing liquid to a beaker, adjust its pH to 8.0-9.0, then transfer the sample to a 50 mL volumetric flask and make up to volume with ultrapure water. After mixing, filter with filter paper, take the filtrate and mix it with TISAB buffer in a volume ratio of 1:1. Determine its fluorine content with a fluoride ion selective electrode and then enter it into formula (1-4) to calculate the final total fluorine content.

[0086]

[0087] Where: ω is the final F concentration of the sample (mg / kg), A is the measured fluorine concentration of the sample (mg / L), A0 is the measured fluorine concentration of the blank solution (mg / L), m is the sample mass (g), and V is the total volume of the sample extract (mL).

[0088] Hydroponic experiment

[0089] Under different F treatments, inoculation with Bacillus could affect the changes in fluorine content in tea seedling leaves. Figure 4 As shown in the A bar graph, in the groups treated with different concentrations of fluoride (0, 5, 10 mg·L -1 ), compared with the respective control groups, the water-soluble fluoride content of leaves 1-3 of tea seedlings treated with Bacillus was significantly decreased (p<0.05), and the fluoride removal rates reached 21.20%, 25.01% and 32.20%, respectively; as shown in bar graph B, compared with the respective control groups, the water-soluble fluoride content of leaves 4-6 of tea seedlings treated with Bacillus decreased, especially the water-soluble fluoride content of the 0F and 10F treatment groups was significantly decreased (p<0.05), reaching 12.30% and 9.97%, respectively; as shown in Figures C and D, the total fluoride content of leaves 1-3 and 4-6 of tea seedlings inoculated with Bacillus decreased in the different F treatment groups, especially the 10F treatment group, which had a significant effect of reducing total fluoride in leaves 1-3 and 4-6 (p<0.001), reaching 26.62% and 34.46%, respectively, while the total fluoride content of leaves in the 0F and 5F treatment groups decreased but not significantly. The specific fluoride removal effects are shown in Tables 1-3 below.

[0090] Table 1-3 Fluoride removal rate of Bacillus under different fluoride concentrations

[0091]

[0092] Table 1-4 Enrichment coefficients of different parts of Shucha early tea seedlings under fluorine stress

[0093]

[0094] The enrichment coefficient refers to the ratio of the element content in a certain treatment part to the concentration of the exogenously added element. The higher the enrichment coefficient, the stronger the plant's ability to absorb the element. As shown in Tables 1-4, the enrichment coefficients of different parts of Shucha early tea seedlings under different fluorine concentration treatments ranged from 54.19 to 198.97. Among them, the enrichment coefficients of 4-6 leaves of tea trees were generally higher than those of 1-3 leaves, and the enrichment capacity of old leaves was greater than that of young leaves. After inoculation with Bacillus, the enrichment coefficients of the leaves changed. Compared with the CK group, the enrichment coefficients of tea leaves decreased overall, indicating that Bacillus Velezii can interfere with the enrichment of fluorine by tea trees. Combined Figure 4 Based on the results in Tables 1-3, it is speculated that Bacillus velezensis can affect the absorption and utilization of fluoride by tea trees, reduce the fluoride content in leaves, and the fluoride-reducing effect of this strain is more significant under high fluoride stress.

[0095] Field verification trials

[0096] 1. The strain was tested in the Shucha early tea garden in Ningguo County, Xuancheng City, Anhui Province from June to August 2023. The CK group and the Bacillus group were set up, and isolation belts were set up every other row. The test was repeated 5 times, with 10 tea trees in each replicate and 50 tea trees in each group. The roots were irrigated with bacteria once every 10 days (500mL / tree / time, a total of 25L per group, and the bacterial liquid content was 2×10 -4 The 1-3 and 4-6 leaves were harvested and dried at 105°C for 2 hours. The ground samples were then analyzed and their water-soluble and total fluoride contents were calculated.

[0097] 2. The strain was tested in the tea garden of Zhaoliqiao Tea Factory in Chibi City, Hubei Province from October to December 2023. The CK group and the Bacillus group were set up, and isolation belts were set up every other row. The test was repeated 5 times, with each group of 30m 2 , each repetition interval is 1m 2 Root irrigation was performed once every 12 days (1000 mL / m 2 Each time, a total of 30L per group, the bacterial liquid contained 2×10 -4 The 1-3 and 4-6 leaves were harvested and dried at 105°C for 2 hours. The water-soluble and total fluoride contents were then measured and calculated.

[0098] Figure 5 Figures A and B in the figure respectively show the changes in the water-soluble fluoride and total fluoride content in the leaves of Shucha Zaocha tea plantations in Ningguo Tea Garden, Xuancheng. Compared with the CK group, the water-soluble fluoride and total fluoride content in the leaves of tea plants decreased after inoculation with Bacillus. Figure 5 As shown in the A column diagram, the strain significantly reduced the water-soluble content of tea leaves 1-3 and 4-6 (p < 0.01), and its fluoride reduction rate reached 40.40% and 21.83%. Figure 5 The results of the B bar graph show that compared with the CK group, the total fluoride content of the tea tree decreased after inoculation with Bacillus subtilis, especially the total fluoride content of leaves 4-6 decreased significantly (p<0.05), and the fluoride reduction rate reached 21.67%.

[0099] Figure 5 Figures C and D in Figure 3 show the changes in water-soluble and total fluoride content in leaves 1-3 and leaves 4-6, respectively, of tea plants from the Hubei population. As shown in Figure C, compared with the CK group, water-soluble fluoride content in leaves 1-3 decreased slightly after inoculation with Bacillus spores, but this was not statistically significant. However, water-soluble fluoride content in leaves 4-6 decreased significantly (p < 0.0001), representing a fluoride removal rate of approximately 35.22%. As shown in Figure D, compared with the CK group, total fluoride content in leaves 1-3 and leaves 4-6 decreased significantly after inoculation with Bacillus spores (p < 0.001), reaching fluoride reduction rates of 25.24% and 21.88%, respectively.

[0100] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A strain of Bacillus Velez Bacillus velezensis Y500, characterized by The strain has been deposited in the China Center for Type Culture Collection (CCTC) with the accession number CCTCC NO: M 2024562 and the deposit date of March 25, 2024.

2. The Bacillus Velez strain according to claim 1 Bacillus velezensis Y500, characterized by The 16S rDNA sequence of the strain is shown in SEQ ID NO.

1.

3. The Bacillus Velez strain according to claim 1 Bacillus velezensis Y500, characterized by The optimum growth temperature of the strain is 15-37°C, and the optimum growth pH range is 5.0-9.

0.

4. The Bacillus Velez strain according to claim 1 Bacillus velezensis Y500, characterized by The strain was cultured in LB medium, and the inoculum size in the expanded culture was 1%.

5. A microbial agent comprising the Bacillus Velezii strain according to claim 1 Bacillus velezensis Y500.

6. The microbial agent according to claim 5, characterized in that Also includes excipients.

7. The microbial agent according to claim 5, characterized in that The bacterial agent is in the form of powder, granule or suspension.

8. The microbial agent according to claim 7, characterized in that The bacterial agent is in the form of powder.

9. The microbial agent according to claim 7, characterized in that The bacterial agent is a granule.

10. The microbial agent according to claim 7, characterized in that The bacterial agent is a suspension agent.

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