Separation method of achnatherum inebrians Epichulous endophytic fungi
By using potato glucose agar medium containing antibiotics, yeast leaching and water leaching leaf leachate, combined with colony edge purification method, the problems of low separation rate, high contamination rate and slow growth rate of endophytic fungi in the grass were solved, and a significant improvement in the separation rate and accelerated growth rate were achieved.
Patent Information
- Application Number
- CN202510584489.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-07
- Publication Date
- 2025-07-22
AI Technical Summary
In the prior art, the separation rate of endophyte fungi of the wormwood grass is low, the pollution rate is high, and the growth rate is slow.
The potato glucose agar medium containing antibiotics, yeast leaching and water leaching of the genus genus purified by picking the edge of the colony for purifying the endophytic fungi of the genus genus were obtained.
The isolation rate of endophytic fungi in the wormwood grass has been increased from 5% to 18%, and the pollution rate has been reduced from 17% to 6%. The growth rate of endophytic fungi has been increased from 2.6mm/day to 3.1mm/day.
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Figure CN120349900A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biology, and particularly relates to a method for isolating endophytic fungi of Achnatherum inebrians genus. Background Art
[0002] Achnatherum inebrians is a common highly poisonous gramineous weed in the grasslands of China, commonly found in the natural grasslands in the north and west of China. The endophytic fungi ( spp.) infection rate in Achnatherum inebrians plants is nearly 100%, so it is also called the Achnatherum inebrians-endophytic fungi symbiont. At present, two endophytic fungi have been identified, namely Epichloë gansuensis and Epichloë inebrians, and their toxicity is caused by alkaloids produced by endophytic fungi infection.
[0003] Isolating endophytic fungi from Achnatherum inebrians plants for related basic research or endophytic fungi breeding, but currently, whether isolating endophytic fungi from seeds or leaves, the isolation rate is relatively low. Summary of the Invention
[0004] Based on the above technical problems, the purpose of the present invention is to improve the method for isolating endophytic fungi from seeds, increase the isolation rate and growth rate of endophytic fungi of Achnatherum inebrians, and reduce the contamination rate. Specifically, it includes the following contents:
[0005] In the first aspect, the present invention provides a biological culture medium for isolating endophytic fungi of Achnatherum inebrians genus, and the culture medium is a potato dextrose agar medium containing antibiotics and water extract of Achnatherum inebrians leaves.
[0006] Preferably, the culture medium is a potato dextrose agar medium containing antibiotics, yeast extract, and water extract of Achnatherum inebrians leaves.
[0007] Preferably, the antibiotics are potassium penicillin and streptomycin.
[0008] Preferably, the concentration of potassium penicillin in the culture medium is 80 - 100 mg / L; the concentration of streptomycin is 20 - 50 mg / L.
[0009] Preferably, the concentration of potassium penicillin in the culture medium is 100 mg / L; the concentration of streptomycin is 50 mg / L.
[0010] Preferably, the concentration of yeast extract is 1 - 3 g / L.
[0011] Preferably, the concentration of yeast extract is 3 g / L.
[0012] Preferably, the concentration of the aqueous extract of Achnatherum inebrians leaves is 3-5% (v / v).
[0013] Preferably, the concentration of the aqueous extract of Achnatherum inebrians leaves is 5% (v / v).
[0014] Preferably, the method for preparing the aqueous extract of Achnatherum inebrians leaves is as follows: Take Achnatherum inebrians leaves, after subjecting them to low-temperature stress at 4°C for 12 h, add distilled water and then juice, and filter to obtain the filtrate, which is the aqueous extract of Achnatherum inebrians leaves.
[0015] Preferably, the mass-volume ratio of Achnatherum inebrians leaves to distilled water is 1:2.
[0016] Preferably, the method is as follows: Take fresh Achnatherum inebrians leaves, wash and dry them, after subjecting them to low-temperature stress at 4°C in a refrigerator for 12 h, cut them into small pieces about 1 cm in length, weigh 100 g, add 200 mL of distilled water and then juice, filter the obtained liquid through gauze and quantitative filter paper, add distilled water to make up to 200 mL, and store it at 4°C for later use.
[0017] In a second aspect, the present invention provides the use of the culture medium described in the first aspect above for isolating endophytic fungi of Achnatherum inebrians genus.
[0018] In a third aspect, the present invention provides a method for isolating endophytic fungi of Achnatherum inebrians genus, the method comprising: disinfecting Achnatherum inebrians seeds and then culturing them in the culture medium described in the first aspect above, picking and purifying and identifying the hyphae at the edge of the colony without contamination to obtain endophytic fungi of Achnatherum inebrians genus.
[0019] Preferably, the method comprises:
[0020] (1) Seed disinfection: Soak Achnatherum inebrians seeds in 50% sulfuric acid and then disinfect them successively with 75% ethanol and 10% sodium hypochlorite;
[0021] (2) Seed culture: Place the Achnatherum inebrians seeds disinfected in step (1) in the culture medium according to any one of claims 1-5 and culture them in the dark at 22-25°C;
[0022] (3) Sub-culturing: After culturing for 10-14 days, use a sterilized inoculation needle to pick the vigorously growing and uncontaminated hyphae at the edge of the colony and transfer them to a fresh potato dextrose agar medium without antibiotics until a single and uniform colony is formed, thus obtaining endophytic fungi of Achnatherum inebrians genus.
[0023] Preferably, step (1) is:
[0024] Soak the fresh seeds of Achnatherum inebrians in 50% sulfuric acid for 30 minutes, and then wash them 5 times with sterile water. After that, disinfect them with 75% ethanol for 3 minutes first, then with 10% sodium hypochlorite for 3 minutes, and finally rinse them 5 times with sterile water and then air-dry them naturally or blot them dry with filter paper under sterile conditions.
[0025] Preferably, step (2) is as follows: Place the seeds of Achnatherum inebrians disinfected in step (1) in the culture medium according to any one of claims 1-5, so that the seeds are embedded 1-2 mm into the culture medium, and are evenly spaced and distributed in an X shape. After sealing with a breathable sealing film, culture them in the dark at 22°C. During the culture process, timely remove the contaminated seeds, and gently wipe the edges with 1% sodium hypochlorite to prevent the contamination from spreading to other areas. If the contamination has spread, timely transfer the uncontaminated seeds to a new culture medium for culture.
[0026] Preferably, after the seeds of Achnatherum inebrians are collected, air-dry them in time to remove the surface moisture, and then store them in a paper bag or envelope, avoiding using plastic bags.
[0027] Preferably, the seeds of Achnatherum inebrians should be separated from endophytic fungi within 48 hours after collection. If they cannot be separated in time, refrigerate them in a 4°C refrigerator.
[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0029] (1) The present invention first provides a biological culture medium for separating endophytic fungi of Achnatherum inebrians genus.
[0030] (2) Secondly, the present invention provides a method for separating endophytic fungi of Achnatherum inebrians genus. The method is as follows: After disinfecting the seeds of Achnatherum inebrians and culturing them in the above culture medium for 10-14 days, take the mycelia at the edge of the colony without contamination and purify them to obtain endophytic fungi of Achnatherum inebrians genus.
[0031] (3) Compared with the traditional method, the method of the present invention increases the separation rate of endophytic fungi of Achnatherum inebrians from 5% to 18%, reduces the contamination rate from 17% to 6%, and increases the growth rate of endophytic fungi from 2.6 mm / day to 3.1 mm / day. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.
[0033] Figure 1 Pictures of Achnatherum inebrians seed culture. Specific implementation manners
[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part rather than all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.
[0035] In the following embodiments, the reagents, materials, etc. used are commercially available or prepared according to conventional methods unless otherwise specified.
[0036] Potassium penicillin was purchased from Harbin Heping Veterinary Drug Co., Ltd.
[0037] Streptomycin sulfate was purchased from Jiangxi Baoling Animal Health Products Co., Ltd.
[0038] Yeast extract was purchased from Biosharp.
[0039] The preparation method of the water extract of Achnatherum inebrians leaves is as follows: Take fresh Achnatherum inebrians leaves, wash and dry them, subject them to low-temperature stress at 4°C in the refrigerator for 12 h, cut them into small pieces about 1 cm in length, weigh 100 g, add 200 mL of distilled water and then juice. The obtained liquid is filtered through gauze and quantitative filter paper, and distilled water is added to make up to 200 mL and then stored refrigerated at 4°C for later use.
[0040] Example 1 Achnatherum inebrians Isolation method of endophytic fungi
[0041] 1. Preparation of culture medium
[0042] Culture medium 1: The culture medium is a potato dextrose agar medium (PDA) containing antibiotics, yeast extract, and the water extract of Achnatherum inebrians leaves. Specifically: Weigh about 200 g of potatoes, wash, peel and cut them into pieces, add 1000 mL of water and boil for 15 min, filter through gauze and then add water to make up to 1000 mL, add 20 g of glucose and 18 g of agar powder, boil until the agar powder completely melts and then dispense. The dispensed culture medium is sterilized by high-pressure steam moist heat. At a steam pressure of 0.1 MPa, the temperature reaches 121°C and is maintained for 20 min. When the PDA culture medium cools to about 50°C, potassium penicillin, streptomycin, yeast extract, and the leaf extract of Achnatherum inebrians are added to the PDA culture medium. The concentration of potassium penicillin is 100 mg / L, the concentration of streptomycin is 50 mg / L, the concentration of yeast extract is 3 g / L, and the concentration of the leaf extract of Achnatherum inebrians is 5% (v / v).
[0043] Medium 2: The medium is a potato dextrose agar medium (PDA) containing antibiotics and an extract of Achnatherum inebrians leaves. Specifically, potassium penicillin, streptomycin, yeast extract, and an extract of Achnatherum inebrians leaves are added to the PDA medium, where the concentration of potassium penicillin is 100 mg / L, the concentration of streptomycin is 50 mg / L, and the concentration of the Achnatherum inebrians leaf extract is 5% (v / v). The rest is the same as the preparation of Medium 1.
[0044] Medium 3: The medium is a potato dextrose agar medium (PDA) containing only antibiotics. Specifically, potassium penicillin and streptomycin are added to the PDA medium, where the concentration of potassium penicillin is 100 mg / L and the concentration of streptomycin is 50 mg / L. The rest is the same as the preparation of Medium 1.
[0045] Medium 4: The medium is a potato dextrose agar medium (PDA) containing antibiotics and yeast extract. Specifically, potassium penicillin, streptomycin, yeast extract, and an extract of Achnatherum inebrians leaves are added to the PDA medium, where the concentration of potassium penicillin is 100 mg / L, the concentration of streptomycin is 50 mg / L, and the concentration of yeast extract is 3 g / L. The rest is the same as the preparation of Medium 1.
[0046] 2. Achnatherum inebrians Isolation of endophytic fungi
[0047] (1) Treatment of Achnatherum inebrians seeds: After collecting Achnatherum inebrians seeds, they are promptly air-dried to remove surface moisture, and then stored in a paper bag or envelope, avoiding the use of plastic bags. The Achnatherum inebrians seeds should be subjected to the isolation of endophytic fungi within 48 h after collection. If isolation cannot be carried out in a timely manner, they are refrigerated in a 4°C refrigerator.
[0048] (2) Seed disinfection: First, soak fresh Achnatherum inebrians seeds in 50% sulfuric acid for 30 min and then wash them 5 times with sterile water. Subsequently, disinfection treatment is carried out in a laminar flow hood. First, disinfect with 75% ethanol for 3 min, then disinfect with 10% sodium hypochlorite for 3 min, and then rinse with sterile water 5 times. Subsequently, use sterile and dry filter paper to completely absorb the moisture on the seed surface. In addition, the seeds can be allowed to air-dry naturally in a sterile workbench to ensure that the seeds are completely dry.
[0049] (3) Seed culture: Use forceps to pick up the seeds of Achnatherum inebrians sterilized in step (2) and place them separately into the culture medium prepared in (1) above. When adding the seeds, gently press the seeds with forceps so that the seeds are embedded in the culture medium by 1-2 mm. Add 5 seeds to each petri dish, and make them evenly spaced and distributed in an X shape. After the petri dish is sealed with a breathable sealing film, culture it in the dark at 22°C. When contaminated seeds are found during the culture process, remove them in time and gently wipe the edges with 1% sodium hypochlorite to prevent the contamination from spreading to other areas. If the contamination has spread, transfer the uncontaminated seeds to a new culture medium in time. The seed culture of Achnatherum inebrians is as Figure 1 shown.
[0050] (4) Subculture: After culturing for 10 days, use a sterilized inoculation needle to pick up the vigorously growing and uncontaminated mycelia at the edge of the colony and transfer them to a fresh potato dextrose agar medium without antibiotics. Wait until its growth diameter is about 2 cm;
[0051] (5) Repeat step (4) until a single and uniform colony is formed.
[0052] 3. Identification of endophytic fungi
[0053] After the mycelia have grown on the new culture medium for one week, extract the DNA of the strain for sequencing. Through the combined identification of morphology and molecules, confirm that the strain is genus endophytic fungi.
[0054] 4. Determination of isolation rate
[0055] (1) Determination method
[0056] After the colonies grown on the seeds are purified and their growth diameter is about 2 cm, conduct molecular identification on them. After determining that they are endophytic fungi of Achnatherum inebrians, count them. The isolation rate refers to the proportion of successfully isolating the target endophytic fungi from a certain number of seeds.
[0057] Among them, the isolation rate (%) = (the number of seeds with growing colonies / the total number of inoculated seeds) × 100%.
[0058] (2) Results
[0059] When using culture medium 3 to isolate endophytic fungi of Achnatherum inebrians, the isolation rate of genus endophytic fungi is 5%; when using culture medium 4 to isolate endophytic fungi of Achnatherum inebrians, the isolation rate of genus endophytic fungi is 6%; when using culture medium 2 to isolate endophytic fungi of Achnatherum inebrians, the isolation rate of genus endophytic fungi is 16%; when using culture medium 1 to isolate endophytic fungi of Achnatherum inebrians, The isolation rate of the genus endophyte is 18%. Compared with culture media 3 and 4, the culture media described in the present application significantly increase the isolation rate of the endophyte in Achnatherum inebrians.
[0060] 5. Determination of contamination rate
[0061] (1) Determination method
[0062] During the cultivation of seeds, when contamination of the seeds in the culture medium is detected, the contaminated samples are observed and counted. Subsequently, the contaminants are removed and the edges are gently wiped with 1% sodium hypochlorite to prevent the spread of contamination to other areas.
[0063] Among them, the contamination rate (%) = (number of contaminated seeds counted / total number of inoculated seeds) × 100%.
[0064] (2) Results
[0065] When isolating the endophyte of Achnatherum inebrians using culture medium 3, the contamination rate of the genus endophyte is 17%; when isolating the endophyte of Achnatherum inebrians using culture medium 4, the contamination rate of the genus endophyte is 13%; when isolating the endophyte of Achnatherum inebrians using culture medium 2, the contamination rate of the genus endophyte is 6%; when isolating the endophyte of Achnatherum inebrians using culture medium 1, the contamination rate of the genus endophyte is 6%. Compared with culture media 3 and 4, using the culture media described in the present application significantly reduces the contamination rate of the endophyte in Achnatherum inebrians.
[0066] 6. Determination of the growth rate of endophytes
[0067] (1) Determination method
[0068] The cross method is used to measure the colony diameter: draw two perpendicular lines on the back of the petri dish to form a cross as the measurement reference line. During measurement, pass through the center of the colony in two directions and record the distance between the colony edges. Repeat 2 times, and take the arithmetic mean of the finally measured diameters as the final diameter of the colony. Among them, the growth rate = colony diameter / number of culture days.
[0069] (2) Results
[0070] When isolating the endophyte of Achnatherum inebrians using culture medium 3, the growth rate of the genus endophyte is 2.6 mm / day; when isolating the endophyte of Achnatherum inebrians using culture medium 4, the growth rate of the genus endophyte is 3.1 mm / day; when isolating the endophyte of Achnatherum inebrians using culture medium 2, The growth rate of the endophytic fungus belonging to the endophytic fungus is 2.7 mm / day; when isolating the endophytic fungus of Achnatherum inebrians using Medium 1, The growth rate of the endophytic fungus belonging to the endophytic fungus is 3.1 mm / day. Compared with Medium 3, the medium described in this application significantly promotes the growth rate of the endophytic fungus of Achnatherum inebrians.
[0071] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A biological medium for isolating endophytic fungi of the genus Achnatherum inebrians , characterized in that The culture medium is a potato dextrose agar medium containing antibiotics and an aqueous extract of Achnatherum inebrians leaves.
2. The culture medium according to claim 1, wherein The culture medium is a potato dextrose agar medium containing antibiotics, yeast extract, and an aqueous extract of Achnatherum inebrians leaves.
3. The culture medium according to claim 1 or 2, characterized in that, The antibiotics are potassium penicillin and streptomycin.
4. The culture medium according to claim 3, characterized in that, In the culture medium, the concentration of potassium penicillin is 80 - 100 mg / L; the concentration of streptomycin is 20 - 50 mg / L.
5. The culture medium according to claim 2, wherein The concentration of the yeast extract is 1 - 3 g / L.
6. The culture medium according to claim 1 or 2, characterized in that, The concentration of the aqueous extract of Achnatherum inebrians leaves is 3 - 5% (v / v).
7. Use of the culture medium according to any one of claims 1-6 for isolating endophytic fungi from Achnatherum inebrians 8. An Achnatherum inebrians A method for isolating endophytic fungi, characterized in that The method includes: disinfecting the seeds of Achnatherum inebrians and culturing them in the culture medium according to any one of claims 1-6, picking the mycelia at the edge of the colony without contamination for purification and identification to obtain the endophytic fungus of Achnatherum inebrians endophyte 9. The method according to claim 8, wherein The method includes: (1) Seed disinfection: Soak the Achnatherum inebrians seeds in 50% sulfuric acid and then disinfect them successively with 75% ethanol and 10% sodium hypochlorite. (2) Seed cultivation: Place the Achnatherum inebrians seeds disinfected in step (1) in the culture medium described in any one of claims 1 - 5 and cultivate them in the dark at 22 - 25°C. (3) Subculture: After culturing for 10 - 14 days, use a sterilized inoculation needle to pick the vigorously growing and uncontaminated hyphae at the edge of the colony, and transfer them to a fresh potato dextrose agar medium without antibiotics until a single and uniform colony is formed, thus obtaining the endophytic fungus of Achnatherum inebrians. It belongs to the endophytic fungus.
10. The method according to claim 9, wherein Step (1) is as follows: Soak the fresh Achnatherum inebrians seeds in 50% sulfuric acid for 30 min and then wash them 5 times with sterile water; then disinfect them with 75% ethanol for 3 min, then disinfect them with 10% sodium hypochlorite for 3 min, and finally rinse them 5 times with sterile water and then air-dry them naturally or blot them dry with filter paper under sterile conditions. Step (2) is as follows: Place the Achnatherum inebrians seeds disinfected in step (1) in the culture medium described in any one of claims 1 - 5, embed the seeds 1 - 2 mm into the culture medium, and distribute them evenly and in an X shape; after sealing with a breathable sealing film, cultivate them in the dark at 22 - 25°C; during the cultivation process, promptly remove the contaminated seeds and gently wipe the edges with 1% sodium hypochlorite to prevent the contamination from spreading to other areas; if the contamination has spread, promptly transfer the uncontaminated seeds to a new culture medium for cultivation.