Fluor-tolerant myceliopthora and a microbial agent prepared therefrom

Abstract

The application discloses a kind of fluorine-resistant trichoderma asperellum and the fungicide prepared by it, belong to microbial strain and application technical field.The strain is named Trichoderma asperellum M700, has been preserved in China typical culture preservation center (CCTC), and its preservation number is CCTCC NO: M 2024579, and the preservation date is March 27, 2024.The beneficial effect: the trichoderma asperellum strain of the application Trichoderma asperellum. Colony growth early stage presents white mycelium, later generation green spore, causes the body to present yellow green, green, aerobic, can survive at ph 3.0-10.0, temperature is 25-28 DEG C.The strain has very good resistance, and can grow when the fluorine ion concentration is below 800 mg / L.Can very good dispel fluorine element in water body.

Description

Technical Field

[0001] This invention relates to the field of microbial strains and their application technology, specifically to a fluoride-resistant Trichoderma and its prepared inoculum. Background Technology

[0002] *Trichoderma* is a fast-growing soil fungus that produces a large number of spores (Monfifile et al., 2014). *Trichoderma* reproduces in two ways: sexually (named *Hypocrea*) and asexually (named *Trichoderma*). Sexual reproduction is a unique form of reproduction that has promoted the evolution of the *Trichoderma* genus and increased its genetic diversity. Some *Trichoderma* species have lost their sexual reproduction and evolved into independent species. *Trichoderma* can adapt to a variety of ecosystems and plays an important role in ecosystem health.

[0003] Trichoderma can promote plant growth by enhancing nutrient absorption, such as through nitrogen fixation and phosphorus solubilization. Studies have shown that after Trichoderma colonizes plant roots, its extra-root hyphae can absorb different forms of nitrogen and transport the assimilated nitrogen to the host plant roots. Furthermore, Trichoderma can promote phosphorus absorption from the soil through various pathways. The phosphorus-solubilizing Trichoderma tang-10-11 can promote the conversion of insoluble phosphorus in the rhizosphere soil of pepper plants into readily available phosphorus, thereby promoting plant growth and increasing yield and quality. In addition, Trichoderma can promote the absorption of micronutrients such as iron. While soils are generally rich in iron, under neutral or alkaline conditions, iron often exists in the form of iron oxides and cannot be directly absorbed and utilized by plants. Trichoderma can promote or inhibit iron absorption by plants through various pathways.

[0004] Fluoride pollution in environmental water bodies is currently severe, and there is a lack of efficient and green methods for its treatment. The concept of using microorganisms for environmental remediation is well-known, but so far no fluoride-resistant Trichoderma strains have been discovered and applied to the treatment of fluoride pollution in environmental water bodies.

[0005] Chinese patent application CN111777437A discloses a Trichoderma echinococcosis fertilizer and its preparation method. The fertilizer is produced using molasses alcohol concentrate and soybean paste as raw materials, utilizing only factory waste to achieve resource utilization. The preparation method is simple, and the resulting Trichoderma echinococcosis fertilizer promotes plant growth. However, this patent does not disclose the fluoride-resistant Trichoderma echinococcosis of this invention or its application in the removal of fluoride from water. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to provide a fluoride-resistant Trichoderma sp. strain.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] The first aspect of the present invention provides a Trichoderma asperellum strain named Trichoderma asperellum M700, which has been deposited at the China Center for Type Culture Collection (CCTC) with accession number CCTCC NO: M2024579 and deposit date of March 27, 2024.

[0009] Preferably, the ITS sequence of this strain is shown in SEQ ID NO.1.

[0010] (SEQ ID NO.1)

[0011] Preferably, the optimal growth temperature for this strain is 25-28℃.

[0012] Preferably, the suitable pH range for the growth of this strain is 3.0-10.0.

[0013] Preferably, the culture medium for this strain is potato dextrose medium, and the inoculum size in the expansion culture is 1%.

[0014] Preferably, the minimum inhibitory concentration (MIC) of this strain is 800 mg / L.

[0015] A second aspect of the present invention provides a microbial agent comprising the above-mentioned Trichoderma echinosporum strain.

[0016] Preferably, it also includes auxiliary materials.

[0017] Preferably, the microbial agent is a powder, granules, or suspension.

[0018] A third aspect of the present invention proposes the application of the above-mentioned Trichoderma echinosporum strain or the above-mentioned microbial agent in the defluorination of water.

[0019] The advantages of this invention are:

[0020] The *Trichoderma asperellum* strain of this invention exhibits white mycelium in the early stages of colony growth, later producing green spores, resulting in a yellowish-green or green colony. It is aerobic and can survive at pH 3.0-10.0 and temperatures of 25-28°C. This strain has excellent fluoride tolerance, growing even at fluoride ion concentrations below 800 mg / L, and can effectively remove fluoride from water. Attached Figure Description

[0021] Figure 1 This is a comparison chart of the growth changes of Trichoderma hygroscopicum under different pH values, where A is the actual growth of the fungus and B is the comparison chart of the amount of fungus.

[0022] Figure 2 The graph shows the minimum inhibitory concentration (MIC) test results of Trichoderma hygroscopicum against F, where A is the actual growth graph of the fungus and B is the specific test data graph.

[0023] Figure 3 The fluoride removal rate, adsorption and accumulation capacity of F by Trichoderma hygroscopicum;

[0024] Figure 4 The effect of different F concentrations of Trichoderma acicularis on the fluoride content of leaves of early-maturing tea seedlings of Shucha;

[0025] Figure 5 The effect of Trichoderma hygroscopicum on the fluoride content of leaves of tea trees in the Shucha early and Hubei population varieties is shown in the figure. "*" represents p<0.05, "**" represents p<0.01, "***" represents p<0.001, and "****" represents p<0.0001. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of 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 some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0028] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0029] Example 1:

[0030] Samples were taken from the stems of the early-maturing tea plant. The tea stem segments, washed with pure water, were placed in 70% ethanol in a clean bench for 2 minutes, ensuring complete submersion. They were then washed four times with sterile water, followed by chlorine... - The stems were soaked in 3% sodium hypochlorite solution for 3 minutes, then washed three times with sterile water. Excess water was absorbed using sterile filter paper, and the stem segments were longitudinally cut with sterile scissors. Four sections were placed on 20 ml of PDA medium containing 50 mg / L of ferrous sulfate (F). The experiment was repeated three times. The sections were sealed with sealing film and incubated upside down in a 28°C incubator in the dark. To verify the effectiveness of the in vitro sterilization process, 100 μL of the final rinse sterile water was evenly spread on PDA medium as a control and incubated at 28°C for one week. Treatment with no bacterial growth was considered effective surface sterilization. The isolated endophytic bacteria were inoculated onto PDA medium containing 100 mg / L of F and incubated upside down in a 28°C incubator. Subsequently, the F concentration was gradually increased in 50 mg / L increments while simultaneously isolating and purifying the bacteria. The resulting bacterial strains were identified and preserved.

[0031] The strain was identified by ITS as *Trichoderma asperellum* and deposited at the China Center for Type Culture Collection (CCTC) on March 27, 2024, with accession number CCTCC NO: M2024579, and also named *Trichoderma asperellum* M700.

[0032] Testing the growth of Trichoderma echinosporum at different pH levels:

[0033] PDB medium was prepared, and its pH was adjusted to 3, 4, 5, 6, 7, 8, 9, and 10 using 4.1 mol / L NaOH and 10% nitric acid, respectively, with three replicates. After aseptic treatment, one loopful of *Trichoderma echinosporum* was inoculated into 100 mL of PDB medium and placed in a constant temperature shaking incubator at 28°C and 150 rpm for 5 days in the dark. The growth status was observed, and the bacterial culture was centrifuged at 8000 rpm for 10 minutes, and the pH change of the supernatant was measured. The precipitate was washed three times with 0.85% NaCl, thoroughly dried at 70°C, and the dry weight of the bacterial cells was measured. The growth status and dry weight changes are shown below. Figure 1 As shown in A and B, this strain can grow in PDA medium with pH 3-10. The highest cell count was observed in mediums with pH 3 and 4, while the cell count was lower in medium with pH 7. The cell counts in the other treatment groups were relatively similar.

[0034] Minimum inhibitory concentration (MIC) for testing Trichoderma echinosporum:

[0035] Different concentrations of sodium fluoride (F) were prepared in PDA medium (0, 5, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800 mg / L, 900 mg / L) and placed in 250 mL Erlenmeyer flasks. The pH of each medium was measured. Then, under aseptic conditions, one loopful of *Trichoderma echinococcus* was inoculated into the center of a plate and incubated in a constant temperature incubator at 28°C for 77 h. Starting from the 8th hour after inoculation, the radius of the inoculum was measured every 2 hours. The growth status and growth rate of the fungus are shown below. Figure 2 As shown in Figures A and B, the activity changes of *Trichoderma echinococcus* varied under different F concentrations and at different time points. Except for the treatment group with an F concentration of 900 mg / L, which showed no growth from beginning to end, the activity trends of the strains under other F concentrations became clear after 42 hours. The growth rates of the 300F (where 300F represents 300 mg / L, and the same applies to the others) treatment group and the 0F control group were relatively close. The growth rates of the 0F, 5F, 20F, 50F, 100F, and 200F treatment groups were higher than that of the 0F control group, while the growth rates of the 400F, 500F, 600F, 700F, and 800F treatment groups were significantly lower than that of the 0F control group. Therefore, F concentrations of 200 mg / L and below can promote the growth of *Trichoderma echinococcus*, while F concentrations of 400 mg / L and above will inhibit the growth rate of this strain.

[0036] Testing the adsorption and accumulation capacity of Trichoderma echinosporum for fluoride:

[0037] The F concentrations in 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. After ultrasonic dissolution, the pH was adjusted to 5.0 ± 0.2, and each treatment was repeated three times. After aseptic treatment, the actual F content was measured again. One loop of strain M700 was picked from a clean bench and inoculated into 100 mL of sterile PDB culture medium. The medium was then placed in a constant temperature incubator at 28°C and shaken at 150 rpm for 5 days. The bacterial culture was centrifuged at 8000 rpm for 10 min 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 used to calculate the fluoride removal rate using formula (1-1). The precipitated bacterial cells were washed three times with 0.85% NaCl, dried thoroughly in an oven at 70°C, and the dry weight was measured. The adsorption amount was then calculated by substituting the dry weight into formula (1-2).

[0038]

[0039]

[0040] In the formula: R is the adsorption rate (%), C0 and C t The values ​​are the initial actual F concentration and the F concentration after adsorption in the supernatant (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).

[0041] The results are as follows Figure 3 As shown in Figure A, the addition of strain M700 reduced the water-soluble fluoride content in the culture medium in all different F treatment groups, with varying fluoride removal rates and adsorption capacities. The 0F group, due to the presence of fluoride in its culture medium and the fact that strain M700 was extracted from fluoride-containing tea plant organs, achieved a fluoride removal rate of approximately 9%. The 600F group followed with a removal rate of 7.84%, while the 50F and 700F groups had removal rates of approximately 3%. The remaining F treatment groups exhibited removal rates ranging from 3.89% to 6.38%. In different F concentration treatment groups, 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, the F adsorption capacity began to decrease in the 700F treatment group.

[0042] Weigh 0.1500 g (accurate to 0.0001 g) of the dried bacterial cells and place them in a 30 mL nickel crucible. Add 1.00 g of solid NaOH and mix well. Cover the crucible and place it in a muffle furnace at 300 °C for 30 min, then at 600 °C for 1 h for alkali digestion. After digestion, turn off the muffle furnace and allow it to cool to room temperature. Remove the crucible and wash the inner wall with 5.0 mL of 10% (v / v) dilute nitric acid solution. Adjust the pH to 8.0-9.0. Transfer the sample to a 50 mL volumetric flask and make up to volume. Mix well and filter through filter paper. Take the filtrate and mix it with TISAB buffer at a volume ratio of 1:1. Measure the fluoride content using a fluoride ion selective electrode and substitute the fluoride content into formula (1-4). The results are as follows: Figure 3 As shown in Figure B, with the increase of F concentration in the treatment groups, the intracellular F accumulation of the bacteria gradually increased, reaching its highest level in the 600F treatment group, and then began to decrease in the 700F treatment group. Figure 3 The adsorption trend of A was consistent, with the overall fluoride content in different fluoride treatment groups ranging from 231.53 to 4544.80 mg / kg. This may be related to the strain's own tolerance range to fluoride. Figure 1 , 2 The results of 3 indicate that the strain itself contains fluorine, and fluorine may be involved in the growth and development of the strain.

[0043] Preparation of strain suspension of Trichoderma hygroscopicum

[0044] *Trichoderma echinococcus* was placed in PDB medium and cultured at 28℃ / 100 rpm for 5 days. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded. The sample was washed with sterile water and centrifuged again at 8000 rpm for 10 min, discarding the supernatant. The spore suspension was then filtered through four layers of sterile lens paper using sterile water vortexing. The spore count was determined using a counting chamber to ensure that the spore content of *Trichoderma echinococcus* in the nutrient solution or aqueous solution was 2 × 10⁻⁶. -3 CFU.

[0045] Preparation and use of tea tree nutrient solution

[0046] The stock solution of the nutrient solution is prepared as shown in Table 1-1. When using it, dilute it to the required concentration as shown in Table 1-2, and adjust its pH value with 16.8 mol / L NaOH and 20% nitric acid, as shown in the table.

[0047] Table 1-1 Preparation of Nutrient Solution Stock Solution

[0048]

[0049]

[0050] Table 1-2 Nutrient solution dilution concentration

[0051]

[0052] Preparation of TISAB buffer solution: Place 58g of NaCl and 68g of Na3C6H5O7·2H2O in a beaker, add 700mL of ultrapure water, mix well and sonicate to dissolve. Add 57mL of glacial acetic acid in a fume hood, mix well and let stand for 10min. Adjust the pH to between 5.2 and 5.3 with 16.8mol / L NaOH. Transfer to a volumetric flask and make up to 1L. Cool to room temperature before use.

[0053] Determination of water-soluble fluoride content: Weigh 0.1500g of sample (accurate to 0.0001g) and place it in a 50ml centrifuge tube. Add 20ml of ultrapure water and place the tube in a 100℃ water bath for 30min. Remove the tube and cool it to room temperature. Filter the solution with filter paper. Add tea sample and TISAB buffer solution in a 1:1 volume ratio and mix them in the centrifuge tube. Measure the fluoride content using a fluoride ion electrode and then use the result to calculate the final water-soluble fluoride content using formula (1-3).

[0054]

[0055] In the formula, X is the final F content of the sample (mg / kg), A is the fluorine concentration of the sample (mg / L), A0 is the fluorine concentration of the blank solution (mg / L), V is the total volume of the sample (mL), and m is the sample mass (g).

[0056] Total fluoride content determination: Weigh approximately 0.2500 g (accurate to 0.0001 g) of tea samples (1-3 leaves and 4-6 leaves) into a 30 ml nickel crucible, add 2.5 g of solid NaOH and mix well. Cover and place in a muffle furnace and gradually heat to 300℃ / 30 min, 600℃ / 1 h. After alkali melting and digestion, turn off the muffle furnace and allow it to cool to room temperature. Remove the crucible and add 5.0 mL of 10% (v / v) 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 at a volume ratio of 1:1. Measure the fluoride content using a fluoride ion selective electrode and then use formula (1-4) to calculate the final total fluoride content.

[0057]

[0058] In the formula, ω is the final F content of the sample (mg / kg), A is the fluorine concentration of the sample (mg / L), A0 is the 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).

[0059] hydroponic experiment

[0060] Depend on Figure 4The results show that inoculation of tea seedlings with *Trichoderma hygroscopica* under different fluoride ion treatments can affect the fluoride content in tea leaves. Figure 4 Bar charts A and B show that in different fluoride treatment groups (0, 5, 10 mg·L⁻¹), -1 In the study, compared with their respective control groups, the water-soluble fluoride content in the 1-3 leaves and 4-6 leaves of tea seedlings treated with *Trichoderma hygroscopicum* was reduced, and the water-soluble fluoride content in the 10F treatment group was significantly reduced (p<0.05), with a fluoride reduction rate of 24.67% in the 1-3 leaves and 11.68% in the 4-6 leaves; Figure 4 In the C-bar chart, compared with their respective control groups, the total fluoride content of tea seedlings in the 10F treatment group was significantly reduced in the first 1-3 leaves after Trichoderma echinosporum treatment (p<0.0001), with a fluoride reduction rate of 36.67%; Figure 4 In the D-bar chart, the total fluoride content in the 4-6 leaves of tea seedlings was significantly reduced under the 0F and 10F treatments (p<0.05), reaching 23.05% and 10.15%, respectively. The specific fluoride removal effects are shown in Tables 1-3 below.

[0061] Table 1-3 Fluoride removal rate of Trichoderma hygroscopicum under different fluoride concentration treatments

[0062]

[0063] Table 1-4 Enrichment coefficients of different parts of early-maturing tea seedlings under fluoride stress

[0064]

[0065] The enrichment coefficient refers to the ratio of the element content in a certain treatment part to the concentration of that element added exogenously. The higher the enrichment coefficient, the stronger the plant's ability to absorb that element. As shown in Table 1-4, the enrichment coefficients of different parts of the Shucha early tea seedlings under different concentrations of fluoride treatment ranged from 46.17 to 217.71. Among them, the enrichment coefficient of leaves 4-6 was generally higher than that of leaves 1-3, that is, the enrichment capacity of older leaves was greater than that of younger leaves. After inoculation with Trichoderma acicularis, the enrichment coefficient of the leaves changed. Compared with the CK group, the enrichment coefficient of the tea seedling leaves decreased overall, indicating that Trichoderma acicularis can interfere with the enrichment of fluoride in tea plants. Combining Table 1-3 and... Figure 4 The results suggest that Trichoderma hygroscopicum can affect the absorption and utilization of fluoride by tea seedling leaves, reduce the fluoride content in the leaves, and the fluoride-reducing effect of this strain is more significant under high fluoride stress.

[0066] Field validation trial

[0067] 1. This strain was tested in the Shucha early-maturing tea garden in Ningguo County, Xuancheng City, Anhui Province from June to August 2023. A control group (CK) and a Trichoderma echinosporum group were set up, with isolation strips placed every other row. The experiment was repeated 5 times, with 10 tea trees per replicate and 50 tea trees per group. The roots were drenched with the strain every 10 days (500 mL / tree / drench, totaling 25 L per group), with a bacterial concentration of 2 × 10⁻⁶. -3 The fluoride content (CFU) was measured and calculated in six batches over a period of two months. The harvested leaves (1-3 and 4-6) were dried at 105°C for two hours, then ground to determine and calculate their water-soluble fluoride and total fluoride content.

[0068] 2. This strain was subjected to a field validation trial in the tea plantation of Zhao Liqiao Tea Factory in Chibi City, Hubei Province, from October to December 2023. A control group (CK) and a Trichoderma echinococcosis group were set up, with isolation strips placed every other row. The trial was repeated 5 times, with each group measuring 30m. 2 Each repetition interval is 1m 2 Drench the roots with a fungicide every 12 days (1000 mL / m²). 2 Each batch consists of 30L, with a bacterial count of 2×10⁶. -3 The fluoride content (CFU) was measured five times over a period of two months. The harvested leaves (1-3 and 4-6) were dried at 105°C for two hours, then ground to determine and calculate their water-soluble fluoride and total fluoride content.

[0069] Figure 5 Figures A and B in the figure represent the changes in water-soluble fluoride and total fluoride content in the leaves of early-maturing tea trees from the Ningguo Tea Garden in Xuancheng, respectively. Compared with the control group, inoculation with Trichoderma hygroscopicum resulted in a decrease in both water-soluble fluoride and total fluoride content in the tea leaves. The water-soluble fluoride content in leaves 1-3 of the tea tree decreased significantly (p<0.01), with a fluoride reduction rate of 51.52%. Compared with the control group, inoculation with Trichoderma hygroscopicum resulted in a significant decrease in the total fluoride content in leaves 1-3 and 4-6 of the tea tree (p<0.05), with fluoride reduction rates of 29.19% and 19.33%, respectively.

[0070] Figure 5 Figures C and D in the diagram represent the changes in water-soluble fluoride and total fluoride content in leaves 1-3 and 4-6 of the tea plantation population in Hubei Province, respectively. Compared with the control group, after inoculation with Trichoderma hygroscopicum, the water-soluble fluoride and total fluoride content in leaves 1-3 of the tea plantation decreased significantly (p<0.05), with a fluoride reduction rate of 22.8%; while the fluoride reduction rates in leaves 4-6 reached 27.61% and 24.03%, respectively, with a significant decrease in fluoride content (p<0.01).

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of *Trichoderma echinosporum*, characterized in that, The strain, named Trichoderma asperellum M700, has been deposited at the China Center for Type Culture Collection (CCTC) with accession number CCTCC NO: M 2024579 and deposit date of March 27, 2024.

2. The *Trichoderma hygroscopica* strain according to claim 1, characterized in that, The ITS sequence of this strain is shown in SEQ ID NO.

1.

3. The *Trichoderma hygroscopica* strain according to claim 1, characterized in that, The optimal growth temperature for this strain is 25-28℃.

4. The *Trichoderma hygroscopica* strain according to claim 1, characterized in that, The optimal pH range for this strain to grow is 3.0-10.

0.

5. The *Trichoderma echinosporum* strain according to claim 1, characterized in that, The strain was cultured on potato dextrose medium, and the inoculum size for scaling-up was 1%.

6. The *Trichoderma echinosporum* strain according to claim 1, characterized in that, The minimum inhibitory concentration (MIC) of this strain is 800 mg / L.

7. A microbial inoculant, characterized in that, It contains the Trichoderma hyacinth strain as described in claim 1.

8. The microbial agent according to claim 7, characterized in that, It also includes auxiliary materials.

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

10. The application of the Trichoderma echinosporum strain according to claim 1 and the microbial agent according to any one of claims 7-9 in the defluorination of water.

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