Application of Trichoderma aspergillus in reducing fluoride in tea

Abstract

The present invention discloses the application of Trichoderma asperellum in reducing fluoride in tea leaves, and belongs to the field of microbial strains and application technology. The strain is named Trichoderma asperellum M700, which has been deposited in the China Center for Type Culture Collection (CCTC), with a deposit number of CCTCC NO: M 2024579 and a deposit date of March 27, 2024. Beneficial effect: The Trichoderma asperellum strain of the present invention presents white hyphae in the early stage of colony growth and produces green spores in the later stage, resulting in the bacterial body being yellow-green and green, aerobic, and able to survive at a pH of 3.0-10.0 and a temperature of 25-28°C. The strain and the subculture strain retaining this active strain can be applied to tea leaf fluoride reduction treatment, and can be used as a microbial fertilizer to reduce the fluoride 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 microbial strains and applications, and in particular to the application of Trichoderma aspergillus in reducing fluoride in tea leaves. Background Art

[0002] Tea trees are highly fluoride-rich plants, and excessive fluoride content in older, coarse tea leaves is a pressing issue for my country's tea export industry. Consuming tea beverages with excessive fluoride can lead to fluorosis, skeletal fluorosis, and other symptoms of fluorosis. Consequently, scientists in the tea industry are constantly seeking new technological solutions, focusing on screening low-fluoride tea varieties, improving cultivation management, raising harvest grades, refining processing techniques, and optimizing packaging and filtration. Fertilization to reduce fluoride and improving harvest grades are common practices in tea garden management.

[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 CN109006118A discloses a cultivation method for reducing the fluoride content in tea leaves. For low-fluoride spring tea cultivation, root application is used. Before the end of December in the first winter, trenches 15-20 cm deep and 9-10 cm wide are dug along the drip line of the tea row. Selenium fertilizer is then applied to the roots and covered with soil. For low-fluoride summer and autumn tea cultivation, foliar application is used. Before picking the tea leaves in the second summer and autumn, the tea trees are pruned. Before and after germination, foliar selenium fertilizer is evenly sprayed onto the leaves. This method uses biochar and humic acid to alter the fluoride form in the soil, reducing fluoride ion absorption by the tea tree roots. Enzyme bacteria are also used to improve the soil's acidic environment, promoting the presence of selenite in its anionic form, allowing the roots to absorb it better. This achieves the goal of comprehensive treatment to reduce the fluoride content in tea leaves. However, this patent does not utilize microbial strains to reduce the fluoride content in tea plants. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to provide a fluorine-resistant Trichoderma aspergillus strain for use in reducing fluoride in tea.

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

[0007] The first aspect of the present invention provides a strain of Trichoderma acanthosporum Trichoderma asperellum Application of M700 in reducing fluoride in tea ， The deposit number of the strain is CCTCC NO: M 2024579.

[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 Trichoderma aspergillus.

[0009] Preferably, the spore content of Trichoderma aculeatus in the nutrient solution or aqueous solution is 2×10 3 CFU / mL.

[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 root fertilization every 10-12 days, for 5 times or more.

[0012] The advantages of the present invention are:

[0013] Trichoderma acanthoides strain of the present invention Trichoderma asperellum The colony appears as white hyphae in early growth, producing green spores later, resulting in a yellow-green or green color. It is aerobic and can grow in pH ranges of 3.0-10.0 and temperatures of 25-28°C. This strain, as well as subcultures that retain this active strain, can be used in tea leaf fluoride reduction treatments, providing research insights into using it as a microbial fertilizer to reduce fluoride content in tea and improve tea quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The following is a comparison of the growth changes of Trichoderma acanthoides at different pH values, where A is the actual growth graph of the fungus and B is the comparison graph of the fungus amount;

[0015] Figure 2 This is the minimum inhibitory concentration (MIC) test graph of Trichoderma aspergillus against F, where A is the actual growth graph of the fungus and B is the specific test data graph;

[0016] Figure 3 is the fluoride removal rate, F adsorption and accumulation capacity of Trichoderma acanthosporum;

[0017] Figure 4 The effect of Trichoderma aspergillus on the fluoride content in the leaves of Shucha early tea seedlings under different fluoride ion F concentrations;

[0018] Figure 5 The effect of Trichoderma aspergillus on the fluoride content in the leaves of Shuchazao and Hubei tea plantations; "*" in the figure representsp <0.05, “**” represents p <0.01, “***” represents p <0.001, “****” represents p <0.0001. DETAILED DESCRIPTION

[0019] 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.

[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 field or according to the product instructions.

[0022] Example 1:

[0023] Samples were taken from the stems of the tea tree. The tea stems were washed with pure water and placed in 70% ethanol in a clean bench for 2 minutes, and the samples were completely submerged. The samples were washed with sterile water for 4 times and then washed with Cl - After soaking in 3% sodium hypochlorite for 3 minutes, the stems were then washed three times with sterile water. Excess moisture was then removed using sterile filter paper. The stem segments were then cut longitudinally with sterile scissors, and four sections were placed on 20 ml of PDA medium containing 50 mg / L fluoride. This experiment was repeated three times. The sections were sealed with parafilm and incubated inverted in the dark at 28°C in a constant temperature incubator. To verify the effectiveness of the in vitro sterilization process, 100 μL of the sterile water from the final wash was evenly spread on the PDA medium as a control. The cells were incubated at 28°C for one week, and the absence of any colony growth was considered effective surface sterilization. The isolated endophytes were inoculated onto PDA medium containing 100 mg / L fluoride ions and incubated inverted in a constant temperature incubator at 28°C. The fluoride concentration was then gradually increased by 50 mg / L, and the resulting strains were isolated and purified. The resulting strains were identified and stored.

[0024] The results of TIS identification showed that the fungus was Trichoderma acanthosporum ( Trichoderma asperellum. ) and deposited in China Center for Type Culture Collection (CCTC) on March 27, 2024, with the accession number CCTCC NO: M 2024579, and named Trichoderma asperellum M700.

[0025] Testing the growth of Trichoderma aculeatus at different pH values:

[0026] Prepare PDB culture medium, use 4.1mol / L NaOH and 10% nitric acid to adjust its pH value to 3, 4, 5, 6, 7, 8, 9, 10, and repeat 3 times. After sterilization, pick 1 ring of Trichoderma spinulosa and inoculate it into 100mL of PDAB culture medium, and place it in a constant temperature shaking incubator at 28℃, 150r / min. After dark culture for 5 days, observe its growth status, centrifuge the bacterial solution at 8000r / 10min in a centrifuge, and measure the change in the pH value of the supernatant. Wash the precipitate with 0.85% NaCl 3 times, dry it thoroughly at 70℃, and measure the dry weight of the bacteria. The growth status of the bacteria and the change in dry weight are as shown below. Figure 1 As shown in A and B, the strain can grow in PDA culture medium with a pH of 3-10, among which the bacterial amount is the highest in the culture medium with a pH of 3 and 4, the bacterial amount is the least in the culture medium with a pH of 7, and the bacterial amount of the other treatment groups is relatively similar.

[0027] Testing the minimum inhibitory concentration (MIC) of Trichoderma aculeatus:

[0028] PDB culture medium with different fluoride ion F concentrations (0, 5, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800 mg / L, 900 mg / L) was prepared with sodium fluoride and placed in a 250 mL Erlenmeyer flask. The pH of each culture medium was measured. Then, one ring of Trichoderma spinulosa was inoculated in the center of the plate under aseptic conditions. The plate was placed in a constant temperature incubator at 28°C for 77 hours in the dark. Starting from the 8th hour after inoculation, the change in its radius was measured every 2 hours. The growth state and growth rate of the bacteria are as follows. Figure 2 As shown in Figures A and B, the activity trends of Trichoderma aspergillus varied under different fluoride ion (F) concentrations and over different time periods. With the exception of the 900 mg / L fluoride concentration group, where the strain ceased growth from the beginning, the activity trends under the other F concentrations varied, becoming clearly stable after 42 hours. The growth rates of the 300 F (300 F here represents 300 mg / L, and the same applies to the others) treatment group and the 0 F control group were similar. The growth rates of the 0 F, 5 F, 20 F, 50 F, 100 F, and 200 F treatment groups were higher than that of the 0 F control group, while the growth rates of the 400 F, 500 F, 600 F, 700 F, and 800 F treatment groups were much lower than those of the 0 F control group. This indicates that treatments with 200 mg / LF or lower F concentrations promoted the growth of Trichoderma aspergillus, while treatments with 400 mg / LF or higher F concentrations inhibited the growth of the strain.

[0029] Testing the adsorption and accumulation capacity of Trichoderma acanthoides:

[0030] The F concentrations in the liquid PDB culture medium were set at 0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, and 700 mg / L, respectively. After sonication, the pH was adjusted to 5.0 ± 0.2, and each treatment was repeated three times. After sterilization, the actual F content was measured again. Within the clean bench, one loopful of strain M700 was inoculated into 100 mL of sterile PDB culture medium and incubated in a constant temperature shaking incubator at 28°C and 150 rpm for 5 days. The culture solution was centrifuged at 8000 rpm for 10 minutes in a centrifuge tube. 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 applied to the solution to calculate the fluoride removal rate using formula (1-1). The precipitated bacteria were washed three times with 0.85% NaCl and dried thoroughly in an oven at 70°C. The dry weight was measured and then used in formula (1-2) to calculate the adsorption capacity.

[0031] (1-1)

[0032] (1-2)

[0033] 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).

[0034] The results are as follows Figure 3 As shown in Figure A, in the different fluoride (F) treatment groups, the addition of strain M700 reduced the water-soluble fluoride content in the culture medium, resulting in different fluoride removal rates and adsorption capacities. Compared with their respective control groups, the 0F group, due to the fluoride content in its culture medium and the fact that strain M700 was extracted from fluoride-containing tea plant organs, had a fluoride removal rate of approximately 9%. This was followed by the 600F treatment group with a fluoride removal rate of 7.84%, while the 50F and 700F treatment groups had fluoride removal rates of approximately 3%. The remaining F treatment groups had fluoride removal rates ranging from 3.89% to 6.38%. In the treatment groups with different F concentrations, the strain's F adsorption capacity ranged from 4 to 14230.77 mg / g. When the fluoride content was between 0 and 600 mg / kg, the adsorption capacity increased with increasing F concentration, reaching 14230.77 mg / g at 600F. However, F adsorption began to decrease in the 700F treatment group.

[0035] Weigh 0.1500g (accurate to 0.0001g) of the dried bacteria above, place it in a 30mL nickel crucible, add 1.00g of solid NaOH and mix well, cover and place in a muffle furnace at 300℃ / 30min, 600℃ / 1h, after alkaline dissolution, turn off the muffle furnace, wait for it to cool to room temperature, take out the crucible, add 5.0mL of 10% volume fraction dilute nitric acid solution to wash the inner wall of the crucible, adjust the pH to 8.0-9.0, transfer the sample to a 50mL volumetric flask and make up the volume, mix well and filter with filter paper, take the filtrate and mix it with TISAB buffer at a volume ratio of 1:1, measure it with a fluoride ion selective electrode and substitute it into formula (1-2) to calculate its fluoride content. The results are as follows Figure 3 As shown in Figure B, with the increase of F concentration in the treatment group, the strain's ability to accumulate F in the cell gradually increased. The fluoride content of the strain was the highest in the 600F treatment group and began to decline in the 700F treatment group. Figure 3 The adsorption trend of A was consistent, and the overall fluoride content of the different fluoride treatment groups was between 231.53-4544.80 mg / kg, which was similar to the fluoride content of the bacteria at 200F, which may be related to the strain's own tolerance to fluoride. Figure 1 、 2 The results of 3 show that the strain itself contains fluoride, and fluoride may be involved in the growth and development of the strain.

[0036] Preparation of suspension of Trichoderma aculeatus strain

[0037] The Trichoderma acanthoides was placed in PDB medium, cultured at 28°C / 100r for 5 days, centrifuged at 8000r / 10min, the supernatant was discarded, washed with sterile water, centrifuged again at 8000r / 10min, the supernatant was discarded, vortexed with sterile water, and the spore liquid was filtered out with 4 layers of sterile lens paper. The number of spores was determined by counting the plate, so that the spore content of the Trichoderma acanthoides in the nutrient solution or aqueous solution was 2×10 3 CFU / mL.

[0038] Preparation and use of tea tree nutrient solution

[0039] 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.

[0040] Table 1-1 Preparation of nutrient solution mother solution

[0041]

[0042] Table 1-2 Nutrient solution dilution concentration

[0043]

[0044] Preparation of TISAB buffer solution: Take 58g of NaCl and 68g of Na3C6H5O7•2H2O and place them in a beaker, add 700mL of ultrapure water, mix well and dissolve by ultrasonication, add 57mL of glacial acetic acid in a fume hood, mix well and let stand for 10min, use 16.8mol / L NaOH to adjust the pH value to between 5.2-5.3, transfer to a volumetric flask and make up to 1L, and cool to room temperature before use.

[0045] Determination of water-soluble fluoride content: Weigh 0.1500 g of 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 tea sample and TISAB buffer solution in a 1:1 volume ratio and mix well in a centrifuge tube. Use a fluoride ion electrode to determine the fluoride content and then enter it into formula (1-3) to calculate the final water-soluble fluoride content.

[0046] (1-3)

[0047] Where X is the final F content 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).

[0048] Determination of total fluorine content: Weigh approximately 0.2500 g (accurate to 0.0001 g) of tea samples (1-3 leaves and 4-6 leaves) respectively and place them 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 and 600°C / 1 h. After alkaline dissolution, turn off the muffle furnace and wait until it cools to room temperature. Remove the crucible and add 5.0 mL of 10% by volume dilute nitric acid solution to wash the inner wall of the crucible. Transfer the washing solution to a beaker and 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. Mix well and filter with filter paper. Take the filtrate and mix it with TISAB buffer in a volume ratio of 1:1. Determine the fluorine content with a fluoride ion selective electrode and then enter it into formula (1-4) to calculate the final total fluorine content.

[0049] (1-4)

[0050] Where ω is the final F content 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).

[0051] Hydroponic experiment

[0052] Depend on Figure 4The results show that under different fluoride ion treatments, tea seedlings inoculated with Trichoderma acanthoides can affect the changes in fluoride content in tea leaves. Figure 4 As can be seen from the A and B bar graphs, 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 in the 1-3 leaves and 4-6 leaves of the tea seedlings treated with Trichoderma aspergillus was reduced, and the water-soluble fluoride content in the 10F treatment group was significantly reduced ( p <0.05), the fluoride reduction rate of 1-3 leaves reached 24.67%, and the fluoride reduction rate of 4-6 leaves reached 11.68%; Figure 4 In the C column, compared with the respective control groups, the total fluoride content of the 1-3 leaves of the tea seedlings in the 10F treatment group decreased significantly after the treatment with Trichoderma aspergillus ( p <0.0001), the fluoride reduction rate reached 36.67%; Figure 4 In the D bar graph, the total fluoride in the 4-6 leaves of tea seedlings under 0F and 10F treatments was significantly reduced ( p <0.05), reaching 23.05% and 10.15% respectively. The specific fluoride removal effects are shown in Tables 1-3 below.

[0053] Table 1-3 Fluoride removal rate of Trichoderma spinulosa under different fluoride concentrations

[0054]

[0055] Table 1-4 Enrichment coefficients of different parts of Shucha early tea seedlings under fluorine stress

[0056]

[0057] 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 Table 1-4, the enrichment coefficients of different parts of Shucha early tea seedlings under different fluorine concentration treatments ranged from 46.17 to 217.71. Among them, the enrichment coefficients of 4-6 leaves of the tea tree were generally higher than those of 1-3 leaves, that is, the enrichment capacity of old leaves was greater than that of young leaves. After inoculation with Trichoderma aspergillus, the enrichment coefficients of the leaves changed. Compared with the CK group, the enrichment coefficients of the tea seedling leaves decreased overall, indicating that Trichoderma aspergillus can interfere with the enrichment of fluorine by tea trees. Combined with Table 1-3 and Figure 4 Based on the results, it is speculated that Trichoderma acanthosporum can affect the absorption and utilization of fluoride by tea seedling leaves, reduce the fluoride content in the leaves, and the fluoride reduction effect of this strain is more significant under high fluoride stress.

[0058] Field verification trials

[0059] 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 Trichoderma acanthophorum 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 the fungus once every 10 days (500 mL / tree / time, a total of 25 L per group, and the bacterial liquid content was 2×10 3 CFU / mL) for a total of six times over a two-month period. Harvested leaves 1-3 and 4-6 were dried at 105°C for 2 hours, ground, and the water-soluble and total fluoride contents were determined and calculated.

[0060] 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 Trichoderma acanthophorum group were set up, and isolation belts were set up every other row. The test was repeated 5 times, with each group having an area 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 3 CFU / mL) for a total of five times over a two-month period. Leaves 1-3 and 4-6 were harvested and dried at 105°C for 2 hours. Samples were then ground and analyzed for water-soluble and total fluoride content.

[0061] Figure 5 Figures A and B in the figure show the changes in water-soluble and total fluoride content in the leaves of Shucha Zaocha tea trees from the Ningguo Tea Garden in Xuancheng. Compared with the CK group, the water-soluble and total fluoride content of tea leaves decreased after inoculation with Trichoderma aspergillus, with the water-soluble content in leaves 1-3 significantly decreasing ( p <0.01), and the fluoride reduction rate reached 51.52%; compared with the CK group, the total fluoride content of leaves 1-3 and 4-6 of tea trees inoculated with Trichoderma acanthoides was significantly reduced ( p <0.05), and the fluoride reduction rates reached 29.19% and 19.33%, respectively.

[0062] Figure 5 Figures C and D in Figure 3 show the changes in water-soluble fluoride and total fluoride content in leaves 1-3 and leaves 4-6 of tea trees from the Hubei population, respectively. Compared with the CK group, the water-soluble fluoride and total fluoride content in leaves 1-3 of tea trees after inoculation with Trichoderma aspergillus were significantly reduced ( p <0.05), the fluoride reduction rate reached 22.8%; while the fluoride reduction rates of water-soluble fluoride and total fluoride in 4-6 leaves reached 27.61% and 24.03% respectively, and the fluoride content was significantly reduced ( p <0.01).

[0063] 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 Trichoderma acanthosporum ( Trichoderma asperellum The application of M700 in reducing fluoride in tea leaves is characterized by: The deposit number of the strain is CCTCC NO: M 2024579.

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 Trichoderma as claimed in claim 1 is watered at the roots of the tea trees.

3. The method for reducing the fluorine content in tea leaves according to claim 2, characterized in that: The spore content of Trichoderma acanthoides in the nutrient solution or aqueous solution is 2×10 3 CFU / mL.

4. The method for reducing the fluorine content in tea leaves according to claim 3, 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 4, characterized in that: The frequency of irrigation is once every 10-12 days, and continued for 5 times or more.

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