Application of Bacillus Velez in reducing fluoride in tea

Abstract

The invention discloses application of Bacillus velezensis in reducing fluoride in tea leaves, and belongs to the technical field of application of microbial strains. The strain belongs to Bacillus velezensis, is named Bacillus velezensis Y500, and has a preservation number of CCTCC NO:M 2024562. The invention has the following beneficial effects: the Bacillus velezensis strain Bacillus velezensis Y500 proposed by the invention has good tolerance to fluorine element, can adsorb and accumulate fluorine element, the strain and the subculture strain retaining the active strain can be applied to tea leaf fluoride reduction treatment, and can be used as a microbial fertilizer to reduce the fluorine content in tea leaves, thereby providing a research idea for improving the quality of tea leaves.

Description

Technical Field

[0001] The invention relates to the technical field of application of microbial strains, and in particular to application of Bacillus velez in reducing fluoride in tea leaves. Background Art

[0002] Tea trees are super-fluorine-rich plants, and the excessive fluoride content in rough and old tea leaves is an urgent problem to be solved by my country's tea export industry. Drinking tea drinks with excessive fluoride content can lead to fluorosis, fluorosis and other fluorosis. Therefore, scientists in the tea-related field are constantly looking for new technical solutions, starting with measures such as screening low-fluoride tea varieties, improving cultivation management, improving picking grades, improving processing technology, and optimizing packaging and filtration. Among them, fertilization to reduce fluoride and improving picking grades in cultivation management are more commonly used measures in tea garden management.

[0003] The cultivation and management of tea trees is an important part of the quality of tea gardens. Fertilization in tea gardens is also an important means to reduce the fluoride content of tea leaves, which mainly includes 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 performance, broad prospects, and are more environmentally friendly and sustainable. Although the market for microbial fertilizers is becoming more mature, with a wide range of choices and diverse combinations, there are few reports on endophytic fertilizers for tea trees to reduce fluoride. The research on endophytes of tea trees is becoming more and more extensive and in-depth. Using the characteristics of the tea tree's own strains, fluoride-resistant bacteria are screened, and tea tree fluoride reduction experiments are conducted, which has good economic and ecological value for the development of the tea industry.

[0004] The Chinese patent application document with publication number CN109006118A discloses a planting method for reducing the fluorine content in tea leaves. When cultivating low-fluorine spring tea, the root application method is adopted. Before the end of December in the first winter, the tea row drip line is used to dig a trench with a depth of 15-20cm and a width of 9-10cm, and the root selenium fertilizer is applied into the trench and covered with soil; when cultivating low-fluorine summer and autumn tea, the foliar spraying method is adopted. Before picking the summer and autumn tea in the second year, the tea trees are pruned first, and the foliar selenium fertilizer is evenly sprayed on the leaves of the tea trees before and after the germination of the tea trees. It changes the occurrence form of fluorine in the soil through biochar and humic acid, reduces the absorption of fluoride ions by the roots of tea trees, and uses enzyme bacteria to jointly improve the acidic environment of the soil, promotes the existence of selenite in anionic state, and makes the roots of tea trees absorb selenite better, so as to achieve the purpose of comprehensive management and reduce the fluorine content of tea leaves. However, the patent does not use microbial strains to reduce the fluorine content of tea leaves. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to provide an application of Velezius fluoride-resistant Bacillus in reducing the fluoride content of tea.

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

[0007] The first aspect of the present invention provides the use of Bacillus velezensis Y500 in reducing fluoride in tea leaves. The deposit number of the strain is CCTCC NO: M 2024562.

[0008] The second aspect of the present invention provides a cultivation method for reducing the fluorine content in tea tree leaves, comprising the following steps: watering the roots of the tea trees with a nutrient solution or aqueous solution containing the above-mentioned Bacillus Velezii.

[0009] Preferably, the bacterial content of the nutrient solution or aqueous solution is 2×10 -4 CFU.

[0010] Preferably, the root irrigation volume is 500 mL / tree / time or 1000 mL / m 2 / Second-rate.

[0011] Preferably, the frequency of irrigation is once every 10-12 days, for 5 times or more.

[0012] The beneficial effects of the present invention are:

[0013] The Bacillus velezensis Y500 proposed in the present invention has good tolerance to fluorine element and can adsorb and accumulate fluorine element. The strain and the subculture strain retaining the active strain can be used in tea fluoride reduction treatment, and can be used as a microbial fertilizer to reduce the fluorine content in tea and improve the quality of tea, providing research ideas. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The graphs of Bacillus activity and pH value changes of LB medium under different pH value treatments;

[0015] Figure 2 The graphs of Bacillus activity and pH value of LB medium under different F treatment concentrations;

[0016] Figure 3 The graphs show the changes of Bacillus activity, fluoride removal rate, adsorption capacity, pH value and intracellular and extracellular fluoride content under different F treatment concentrations;

[0017] Figure 4 This is the effect of Bacillus on the fluorine content in the leaves of Shucha early tea seedlings under different F treatments;

[0018] Figure 5 This is a graph showing the effect of Bacillus on the fluoride content in 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

[0019] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are 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 creative work are within the scope of protection of the present invention.

[0020] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0021] 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 art or according to the product instructions.

[0022] Embodiment 1:

[0023] Screening of fluoride-resistant endophytes in tea plants:

[0024] Samples were collected from a tea garden in Chibi, 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 washed with Cl - Soak in 3% sodium hypochlorite for 3 minutes, and finally wash with sterile water 3 times. Take sterile filter paper to absorb excess water, cut the leaves with sterile scissors, put them in a sterile mortar and add a small amount of sterile quartz sand to grind. Then add 10 times the volume of phosphate buffer (pH = 7) for homogenization, let it stand for 5 minutes, and take the suspension for 10-fold serial dilution, a total of 6 concentrations. Take 100μL of each concentration and apply it on a solid culture medium containing 20ml LB, the F concentration is 50mg / L, and each gradient is repeated 3 times. Seal with sealing film and invert it in a 37℃ constant temperature incubator for dark culture. To verify the effectiveness of the plant's in vitro sterilization process, take 100μL of the last washed sterile water and evenly apply it on the LB solid culture medium as a control, and culture it at 37℃ for one week. The control without any colony growth is used as an effective surface sterilization. Endophytic bacteria isolated from the 50 mg / L fluoride treatment group with different dilution gradients were inoculated onto LB solid medium containing 100 mg / L F and inverted in a constant temperature incubator at 37°C. The F concentration was then gradually increased by 50 mg / L and isolated and purified at the same time, and the obtained strains were identified and preserved.

[0025] The result of 16SrDNA bacterial species identification was Bacillus velezensis of the Bacilales family, Firmicutes, and it was deposited in the China Center for Type Culture Collection (CCTC) on March 25, 2024, with the preservation number CCTCC NO:M 2024562, and it was also named Bacillus velezensis Y500.

[0026] Testing the growth of Bacillus Velez at different pH:

[0027] Adjust the pH of the LB medium, use 4.1mol / L NaOH and 10% nitric acid to adjust the pH to 3, 4, 5, 6, 7, 8, 9, 10, set up 3 replicates, inoculate 1% of Velez's bacillus in a flask containing 100mL LB liquid, and place it in a constant temperature shaking incubator at 37°C and 150r / min. After 5 days, measure the changes in pH and activity. The results are as follows Figure 1 As shown in B, the growth of Bacillus Velezii is more active between pH values ​​of 5, 6, 7, 8, and 9, with the most suitable pH value for growth being 6.

[0028] The bacterial solution was centrifuged at 6000r / 10min in a centrifuge and the pH value of the supernatant was measured. Figure 1 As shown in A in the figure, compared with the set results, the pH value of the supernatant when the initial pH value of the culture medium is 5, 6 and 7 increased significantly, while the pH value of the supernatant when the initial pH value of the culture medium is 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.

[0029] Testing the minimum inhibitory concentration (MIC) and pH changes of Bacillus velez:

[0030] 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, 1% of Bacillus Velezii was inoculated and placed in a constant temperature shaking incubator at 37°C and 150 r / min for 24 h in the dark. Starting from the 8th hour after inoculation, samples of the culture fluid were taken every 2 hours to measure the OD 600 The results are as follows Figure 2 As shown. Figure 2From A, we can see the activity change of the strain in the fluoride-containing medium for 24 hours. The activity trends of Bacillus Velezii under different F concentrations and in different time periods are different. Except for the strain containing 700 mg / L F concentration, 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 concentrations, especially at 22 hours, the growth of 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.

[0031] The Bacillus Velez bacteria solutions 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. Figure 2 As can be seen in B, in the CK group (sterile) treatment group, with the increase of F concentration, the pH value of LB medium changed, but all treatment groups were in weak acidic conditions. After the treatment with Bacillus Velezii, except for the strain containing F concentration of 700 mg / L, the pH of other LB medium changed significantly, and all treatment groups were in an alkaline environment. Figure 1 It can be seen that the growth of Bacillus Velezii can change the pH value of LB medium, making the pH value of the solution around 9, showing alkalinity; after the 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 the 500F and 600F groups were less than 8, with a smaller increase.

[0032] Combination 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 own resistance to fluoride in the environment.

[0033] Testing the adsorption and accumulation ability of Bacillus Velez on F:

[0034] The F concentration of LB medium was set to 0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 50 mg / L, respectively. After ultrasonic dissolution, the pH was adjusted to 5.0 ± 0.02, and each treatment was repeated 3 times. Then the actual fluoride content of different F treatment groups was determined again by sterile treatment. Under sterile conditions, 1 ring of strain Y500 was accurately picked and inoculated into 100 mL of sterile LB medium, and placed in a constant temperature incubator at 37 ° C and 150 r / min for dark culture for 120 hours. First take 200 μL to measure its OD 600The remaining bacterial solution was centrifuged at 6000r / min for 10min in a 50mL sterile centrifuge tube, and 2mL of 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, and the residual F concentration was measured with a fluoride ion selective electrode and then entered into formula (1-1) to calculate the fluoride removal rate. The precipitated bacteria were washed three times with 0.85% NaCl, dried thoroughly at 70℃, and the dry weight of the bacteria was measured and then entered into formula (1-2) to calculate the amount of F adsorbed by the bacteria. The results are as follows Figure 3 As shown in A, B and C, compared with the control group containing F at 0 mg / L, the activity of the strains in the treatment groups containing F at 5, 10, 20 and 50 mg / L increased, indicating that F may be involved in the growth and development of the strain; the amount of F adsorbed by the bacteria increased with the increase of F concentration, while the fluoride removal rate of the supernatant decreased with the increase of F concentration, and the overall fluoride removal rate was between 1-4%. The pH value of the supernatant increased significantly as a whole, and the culture medium changed from weakly acidic to alkaline.

[0035]

[0036] 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).

[0037] 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 min, centrifuged at 6000 r / min for 10 min, and 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.

[0038] 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 melting 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 solution in a volume ratio of 1:1. The fluoride content was determined by a fluoride ion selective electrode and then entered into formula (1-4) to calculate the intracellular fluoride content. The results are as follows: Figure 3As 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 amount of F inside and outside the cell, under different F concentration treatments, the intracellular accumulation and extracellular attachment ability of Bacillus Velezii to F were positively proportional to the fluoride concentration in the growth environment, and the change trends of the two were consistent.

[0039] Preparation of Bacillus Velez strain suspension:

[0040] Place Bacillus Velez 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.

[0041] Tea tree nutrient solution preparation and use:

[0042] 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 using 16.8 mol / L NaOH and 20% nitric acid within the range shown in the table.

[0043] Table 1-1 Preparation of nutrient solution mother solution

[0044]

[0045]

[0046] Table 1-2 Nutrient solution dilution concentration

[0047]

[0048] Preparation of TISAB buffer solution: Take 58g of NaCl and 68g of Na3C6H5O7·2H2O and put them in a beaker, add 700mL of ultrapure water, mix and dissolve by ultrasonic, add 57mL of glacial acetic acid in a fume hood, mix and let stand for 10min, adjust the pH value to between 5.2-5.3 with 16.8mol / L NaOH, transfer to a volumetric flask and make up to 1L, cool to room temperature before use.

[0049] Determination of water-soluble fluoride content: weigh 0.1500g of tea sample (accurate to 0.0001g) and put it into a 50ml centrifuge tube, add 20ml of ultrapure water, put it into a 100℃ water bath for 30min, take it out and cool it to room temperature, filter it with filter paper, add tea sample and TISAB buffer solution in a 1:1 volume ratio, mix it 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.

[0050]

[0051] 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).

[0052] Determination of total fluorine content: weigh about 0.2500g (accurate to 0.0001g) of powder of 1-3 leaves and 4-6 leaves respectively, put the tea sample into a 30ml nickel crucible, add 2.5g solid NaOH and mix well, cover and put into a muffle furnace and gradually heat up, 300℃ / 30min, 600℃ / 1h, after alkaline melting digestion, turn off the muffle furnace, wait until it cools to room temperature, take out the crucible, add 5.0mL 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 50mL volumetric flask and make up to volume with ultrapure water, mix well and 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.

[0053]

[0054] 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).

[0055] Hydroponic experiment

[0056] Under different F treatments, inoculation with Bacillus could affect the changes in fluorine content in tea seedling leaves. Figure 4 As shown in the bar graph A, 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 1-3 leaves 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 4-6 leaves of tea seedlings treated with Bacillus was decreased, especially the water-soluble fluoride content of 0F and 10F treatment groups was significantly decreased (p<0.05), reaching 12.30% and 9.97%, respectively; as shown in the results in Figures C and D, in different F treatment groups, the total fluoride content of 1-3 leaves and 4-6 leaves of tea seedlings inoculated with Bacillus was decreased, especially in the 10F treatment group, the total fluoride reduction effect of 1-3 leaves and 4-6 leaves was significant (p<0.001), reaching 26.62% and 34.46%, respectively, while the total fluoride content of leaves of 0F and 5F treatment groups was decreased but not significantly. The specific defluorination effect is shown in Table 1-3 below.

[0057] Table 1-3 Fluoride removal rate of Bacillus under different fluoride concentrations

[0058]

[0059] Table 1-4 Enrichment coefficients of different parts of Shucha early tea seedlings under fluorine stress

[0060]

[0061] 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 the tea tree 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 coefficient of the leaves changed. Compared with the CK group, the enrichment coefficient of the tea leaves decreased overall, indicating that Bacillus Velezii can interfere with the enrichment of fluorine by tea trees. Combined with Figure 4 According to the results in Tables 1-3, it is speculated that Bacillus Velezii can affect the absorption and utilization of fluoride by tea trees, reduce the enrichment of fluoride in leaves, and the fluoride reduction effect of this strain is more significant under high fluoride stress.

[0062] Field validation trials

[0063] 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 in alternate rows. The test was repeated 5 times, with 10 tea trees in each repeat and 50 tea trees in each group. The roots were irrigated with bacteria once every 10 days (500mL / tree / time, 25L per group, and the bacterial liquid content was 2×10 -4The harvested leaves 1-3 and 4-6 were dried at 105°C for 2 hours and ground to determine their water-soluble fluoride and total fluoride content.

[0064] 2. The strain was tested in the group tea garden of Zhaoliqiao Tea Factory in Chibi City, Hubei Province from October to December 2023. CK group and Bacillus group were set up, and isolation belts were set up every other row. The test was repeated 5 times, with 30m per group. 2 , each repetition interval is 1m 2 Root irrigation was performed once every 12 days (1000 mL / m 2 / time, each group totaled 30L, the bacterial liquid contained 2×10 -4 The harvested leaves 1-3 and 4-6 were dried at 105°C for 2 hours, and the water-soluble fluoride and total fluoride contents were determined by grinding.

[0065] 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 tree in Ningguo Tea Garden, Xuancheng. Compared with the CK group, the water-soluble fluoride and total fluoride content of tea leaves decreased after inoculation with Bacillus. Figure 5 As shown in the A column chart, the strain significantly reduced the water-soluble content of leaves 1-3 and 4-6 of the tea tree (p<0.01), and the fluoride reduction rate reached 40.40% and 21.83%. Figure 5 From the results of the B bar graph, we can see that compared with the CK group, the inoculation of Bacillus reduced the total fluoride content of the tea tree, especially the total fluoride content of leaves 4-6 decreased significantly, and the fluoride reduction rate reached 21.67%.

[0066] Figure 5 Figures C and D in the figure respectively show the changes in the water-soluble fluoride and total fluoride content of leaves 1-3 and leaves 4-6 of tea trees in the Hubei population. As shown in Figure C, compared with the CK group, the water-soluble fluoride content of leaves 1-3 of tea trees decreased after inoculation with Bacillus, but there was no statistical significance; while the water-soluble fluoride content of leaves 4-6 decreased significantly, and the fluoride removal rate was about 35.22%. As shown in Figure D, compared with the CK group, after inoculation with Bacillus, the total fluoride content of leaves 1-3 and leaves 4-6 of tea trees decreased significantly, and the fluoride reduction rates reached 25.24% and 21.88%, respectively.

[0067] 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 the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. 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 embodiments of the present invention.

Claims

1. Bacillus Velezii strains Bacillus velezensis The application of Y500 in reducing fluoride in tea leaves is characterized by: The deposit number of the strain is CCTCC NO: M 2024562.

2. A method for reducing the fluorine content in tea leaves, characterized in that: The following steps are involved: The nutrient solution or aqueous solution containing the Bacillus Velez subtilis according to claim 1 is watered at the roots of tea trees.

3. The method for reducing the fluorine content in tea leaves according to claim 2, characterized in that: The bacterial content of the nutrient solution or aqueous solution is 2×10 -4 CFU.

4. The method for reducing the fluorine content in tea leaves according to claim 2, characterized in that: The root irrigation volume is 500mL / tree / time or 1000mL / m 2 / Second-rate.

5. The method for reducing the fluorine content in tea leaves according to claim 2, characterized in that: The frequency of watering is once every 10-12 days, and it should be continued for at least 5 times.

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