Cultivation method of mycorrhizal edible fungus symbiotic seedlings

Abstract

The invention discloses a cultivation method of mycorrhizal edible fungus symbiotic seedlings, and relates to the technical field of edible fungus cultivation, and the cultivation method specifically comprises the following steps: S1, culturing symbiotic plant seedlings; s2, mycelium culture of the mycorrhizal edible fungi: S21, mycelium culture; s22, performing purification culture: selecting the hyphae in the step S21, inoculating the hyphae into a new solid culture medium, and performing standing culture at 24-26 DEG C until the hyphae are overgrown; s23, fermentation culture: inoculating the hypha in the step S22 on a liquid fermentation culture medium, and culturing on a constant-temperature shaking table at 25 DEG C to obtain a liquid strain; s3, infection: injecting the liquid strain in the step S23 into the symbiotic seedling composite culture medium in the step S13, placing the symbiotic seedling composite culture medium in an illumination incubator for culture, and regularly supplementing a nutrient solution during culture; and culturing for 60-90 days to form the mycorrhizal edible fungus symbiotic seedling. The method is high in mycorrhiza infection rate, high in symbiotic seedling survival rate and suitable for popularization.

Description

Technical Field

[0001] This invention relates to the field of edible fungi cultivation technology, specifically to a method for cultivating mycorrhizal edible fungi symbiotic seedlings. Background Technology

[0002] Some edible fungi live in symbiosis with plant roots, with their mycelium twining around the root surface or penetrating deep into the root. The symbiotic plant provides the fungi with carbohydrates synthesized through photosynthesis, while the mycelium absorbs water, inorganic salts, and secretes vitamins and growth hormones from the soil to supply the plant. This forms a close, mutually beneficial relationship, and this type of edible fungi is called mycorrhizal edible fungi.

[0003] The formation of mycorrhizae in edible fungi is an ecological relationship that has developed over a long period in the natural environment. This relationship is easily disrupted or altered, negatively impacting the lives of both the plants and the fungi, and may even prevent them from living normally. Therefore, the mycorrhizal infection rate and survival rate of artificially cultivated symbiotic plant-mycorrhizal edible fungi seedlings are currently low, posing significant challenges to the development of artificial cultivation.

[0004] Therefore, developing a cultivation method for mycorrhizal edible fungi symbiotic seedlings with high mycorrhizal infection rate and high survival rate is a technical problem that urgently needs to be solved by people in this field. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for cultivating mycorrhizal edible fungi symbiotic seedlings, which solves the problems of low mycorrhizal infection rate and low survival rate of artificially cultivated symbiotic plant-mycorrhizal edible fungi symbiotic seedlings in the prior art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a method for cultivating symbiotic seedlings of mycorrhizal edible fungi, specifically comprising the following steps:

[0008] S1: Cultivation of symbiotic plant seedlings:

[0009] S11: Disinfection and sterilization: Select healthy, plump, and pest-free symbiotic plant seeds, soak them in a 1% potassium permanganate solution for 1-2 hours for surface disinfection and sterilization, and then rinse them with sterile water.

[0010] S12: Germination: Soak the symbiotic plant seeds from step S11 in the germination solution for 2-3 hours;

[0011] S13: Cultivation: Sow the symbiotic plant seeds from step S12 into the symbiotic seedling composite culture medium, and add nutrient solution to the culture medium regularly until 2-3 roots grow.

[0012] S2: Culture of mycorrhizal edible fungi mycelium:

[0013] S21: Mycelial culture: Collect healthy and disease-free wild mycorrhizal edible fungi, extract the flesh tissue, inoculate it on a solid culture medium, and culture it at 24-26℃ until it is covered with mycelium;

[0014] S22: Purification culture: Pick the mycelia from step S21, inoculate them into a new solid culture medium, and incubate them statically at 24-26℃ until they are fully colonized with mycelia;

[0015] S23: Fermentation culture: The mycelium from step S22 is inoculated onto the liquid fermentation medium and cultured on a constant temperature shaker at 25℃ to obtain the liquid culture.

[0016] S3: Infection: Inject the liquid inoculum from step S23 into the symbiotic seedling composite culture medium in step S13, place it in a light incubator for cultivation, and replenish the nutrient solution regularly during the cultivation period; after 60-90 days of cultivation, mycorrhizal edible fungi symbiotic seedlings can be formed.

[0017] Further, in step S12, the raw material composition and weight parts of the germination solution are: 0.05g of 1% calcium chloride, 0.1g of 2% copper sulfate, 0.2g of gibberellin, 2g of chitosan, and 1L of water, sterilized at 121℃ for 3h.

[0018] Further, in step S13, the raw material composition and weight parts of the nutrient solution are as follows: urea 2g, potassium dihydrogen phosphate 1g, glucose 0.5g, seaweed polysaccharide 0.5g, ferric citrate 0.01g, magnesium sulfate 0.2g, zinc sulfate 0.01g, ferric chloride 0.02g, copper sulfate 0.01g, and water 1L.

[0019] Further, in step S13, the formulation of the symbiotic seedling composite culture medium is as follows: 1g vermiculite, 1.2g maifanite, 2g humus, 1.5g corn flour, 5g Chinese medicinal herb residue, 2g feather meal, 5g peptone, 2g yeast extract powder, 2g glucose, 2g chitosan, 1g K2HPO4, 0.05g MgSO4·7H2O, 0.01g CaCl2, 0.01g NaCl, 100mL distilled water, pH=5-6, sterilized at 121℃ for 3h.

[0020] Further, in step S21, the solid culture medium is formulated as follows: 5g peptone, 2g yeast extract, 20g glucose, 10g chitosan, 1g K2HPO4, 0.5g MgSO4·7H2O, 0.05g CaCl2, 0.1g NaCl, 20g agar, 1L distilled water, pH=5-6, sterilized at 121℃ for 3h.

[0021] Further, in step S23, the liquid fermentation culture medium is formulated as follows: 5.0g peptone, 2.0g yeast extract, 20.0g glucose, 10g chitosan, 1.0g K2HPO4, 0.5g MgSO4·7H2O, 0.05g CaCl2, 0.1g NaCl, 100mL traditional Chinese medicine extract, 900mL distilled water, pH=5-6, sterilized at 121℃ for 3h.

[0022] Furthermore, the raw material components and weight proportions of the medicinal residue are as follows: 10 parts of Gynostemma pentaphyllum, 6 parts of prepared licorice root, 8 parts of Ligusticum chuanxiong, 4 parts of pumpkin seeds, and 4 parts of jujube. The preparation method of the medicinal residue is as follows: take Gynostemma pentaphyllum, prepared licorice root, Ligusticum chuanxiong, pumpkin seeds, and jujube according to the weight proportions, add 6 times the amount of purified water, soak for 2 hours, boil for 2 hours, filter, and obtain the extract; repeat the extraction 3 times, combine the extracts obtained from the 3 extractions, and obtain the medicinal residue and medicinal extract respectively, sterilize at 121℃ for 3 hours, and set aside for later use.

[0023] In this invention, the effects and functions of traditional Chinese medicinal materials are as follows: Gynostemma pentaphyllum, prepared licorice root, and Ligusticum chuanxiong can significantly improve the antioxidant capacity of symbiotic seedlings, while pumpkin seeds and jujubes are not only rich in nutrients, but can also improve the stress resistance of symbiotic seedlings.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] In this invention, by germinating and culturing symbiotic plant seeds in a symbiotic seedling composite culture medium, the germination rate and speed of the symbiotic plant seeds are improved, enhancing the antioxidant capacity and stress resistance of the symbiotic plants. Through three stages of cultivation of mycorrhizal edible fungi mycelium in solid and liquid fermentation media, robust mycelial growth is promoted, enhancing mycelial stress resistance and thus increasing the mycorrhizal infection rate, thereby improving the survival rate of the symbiotic seedlings. Based on this, the method of this invention has a high mycorrhizal infection rate and a high survival rate of symbiotic seedlings, making it suitable for widespread application. Detailed Implementation

[0026] To make the objectives and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0027] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0028] In this embodiment, the formulations of each culture medium are as follows:

[0029] The raw materials and weight proportions of the germination solution are: 0.05g of 1% calcium chloride, 0.1g of 2% copper sulfate, 0.2g of gibberellin, 2g of chitosan, and 1L of water. The solution is sterilized at 121℃ for 3 hours.

[0030] The raw materials and weight proportions of the nutrient solution are as follows: urea 2g, potassium dihydrogen phosphate 1g, glucose 0.5g, seaweed polysaccharide 0.5g, ferric citrate 0.01g, magnesium sulfate 0.2g, zinc sulfate 0.01g, ferric chloride 0.02g, copper sulfate 0.01g, and water 1L.

[0031] The formulation of the symbiotic seedling composite culture medium is as follows: 1g vermiculite, 1.2g maifanite, 2g humus, 1.5g corn flour, 5g Chinese medicinal herb residue, 2g feather meal, 5g peptone, 2g yeast extract, 2g glucose, 2g chitosan, 1g K₂HPO₄, 0.05g MgSO₄·7H₂O, 0.01g CaCl₂, 0.01g NaCl, 100mL distilled water, pH=5-6, sterilized at 121℃ for 3h. (The vermiculite has a particle size of 0.3-1mm, the maifanite has a particle size of 40-60 mesh, and the humus is uniformly sized humus from wild forests after screening and high-temperature sterilization.)

[0032] The solid culture medium is formulated as follows: 5g peptone, 2g yeast extract, 20g glucose, 10g chitosan, 1g K2HPO4, 0.5g MgSO4·7H2O, 0.05g CaCl2, 0.1g NaCl, 20g agar, 1L distilled water, pH=5-6, sterilized at 121℃ for 3h.

[0033] The liquid fermentation medium consists of: 5.0g peptone, 2.0g yeast extract, 20.0g glucose, 10g chitosan, 1.0g K2HPO4, 0.5g MgSO4·7H2O, 0.05g CaCl2, 0.1g NaCl, 100mL traditional Chinese medicine extract, and 900mL distilled water. The pH is 5-6, and the medium is sterilized at 121℃ for 3 hours.

[0034] Preparation Example 1

[0035] The preparation method of Chinese herbal medicine residue and Chinese herbal medicine extract is as follows: Take 10 parts of Gynostemma pentaphyllum, 6 parts of prepared licorice root, 8 parts of Ligusticum chuanxiong, 4 parts of pumpkin seeds, and 4 parts of jujube, add 6 times the amount of purified water, soak for 2 hours, boil for 2 hours, filter, and obtain the extract; repeat the extraction 3 times, combine the extracts obtained from the 3 extractions, and you can obtain Chinese herbal medicine residue and Chinese herbal medicine extract respectively. Sterilize at 121℃ for 3 hours and set aside.

[0036] Example 1

[0037] This embodiment provides a method for cultivating mycorrhizal edible fungi symbiotic seedlings, specifically including the following steps:

[0038] S1: Cultivation of seeds from the symbiotic plant Pinus sylvestris:

[0039] S11: Disinfection and sterilization: Select healthy, plump, disease-free Pinus sylvestris seeds, soak them in a 1% potassium permanganate solution for 1 hour for surface disinfection and sterilization, and then rinse them with sterile water.

[0040] S12: Germination: Soak the seeds of Pinus sylvestris in step S11 in the germination solution for 2 hours;

[0041] S13: Cultivation: Sow the seeds of Pinus sylvestris in step S12 into the symbiotic seedling composite culture medium, and add nutrient solution to the culture medium regularly until two roots grow.

[0042] S2: Culture of mycorrhizal edible fungi mycelium:

[0043] S21: Mycelial culture: Collect healthy and disease-free blood red rivet mushrooms, take the flesh tissue, inoculate it on a solid culture medium, and culture it at 24℃ until it is covered with mycelium;

[0044] S22: Purification culture: Pick the mycelia from step S21, inoculate them into a new solid culture medium, and incubate them statically at 24℃ until they are fully grown.

[0045] S23: Fermentation culture: The mycelium from step S22 is inoculated onto the liquid fermentation medium and cultured on a constant temperature shaker at 25℃ to obtain the liquid culture.

[0046] S3: Infection: Inject the liquid inoculum from step S23 into the symbiotic seedling composite culture medium in step S13, and place it in a light incubator (set temperature to 25℃, humidity to 55%, intensity to 3500 Lux, 12h light, 12h darkness) for cultivation. During the cultivation period, replenish the nutrient solution regularly. After 60 days of cultivation, mycorrhizal edible fungi symbiotic seedlings can be formed.

[0047] Example 2

[0048] This embodiment provides a method for cultivating mycorrhizal edible fungi symbiotic seedlings, specifically including the following steps:

[0049] S1: Cultivation of symbiotic plant spruce seeds:

[0050] S11: Disinfection and sterilization: Select healthy, plump spruce seeds free from pests and diseases, soak them in a 1% potassium permanganate solution for 1.5 hours for surface disinfection and sterilization, and then rinse them with sterile water.

[0051] S12: Germination: Soak the spruce seeds from step S11 in the germination solution for 2.5 hours;

[0052] S13: Cultivation: Sow the spruce seeds from step S12 into the symbiotic seedling composite culture medium, and add nutrient solution to the culture medium regularly until 3 roots grow.

[0053] S2: Culture of mycorrhizal edible fungi mycelium:

[0054] S21: Mycelial culture: Collect healthy, disease-free wild chanterelles, extract the mycelial tissue, inoculate it on a solid culture medium, and culture it at 25℃ until it is covered with mycelium;

[0055] S22: Purification culture: Pick the mycelium from step S21, inoculate it into a new solid culture medium, and incubate it at 25°C until the mycelium is fully grown.

[0056] S23: Fermentation culture: The mycelium from step S22 is inoculated onto the liquid fermentation medium and cultured on a constant temperature shaker at 25℃ to obtain the liquid culture.

[0057] S3: Infection: Inject the liquid inoculum from step S23 into the symbiotic seedling composite culture medium in step S13, and place it in a light incubator (set temperature to 25℃, humidity to 55%, intensity to 4500 Lux, 12h light, 12h darkness) for cultivation. During the cultivation period, replenish the nutrient solution regularly. After 75 days of cultivation, mycorrhizal edible fungi symbiotic seedlings can be formed.

[0058] Example 3

[0059] This embodiment provides a method for cultivating mycorrhizal edible fungi symbiotic seedlings, specifically including the following steps:

[0060] S1: Cultivation of seeds from the symbiotic plant poplar:

[0061] S11: Disinfection and sterilization: Select healthy, plump poplar seeds free from pests and diseases, soak them in a 1% potassium permanganate solution for 1-2 hours for surface disinfection and sterilization, and then rinse them with sterile water.

[0062] S12: Germination: Soak the poplar seeds from step S11 in the germination solution for 3 hours;

[0063] S13: Cultivation: Sow the poplar seeds from step S12 into the symbiotic seedling composite culture medium, and add nutrient solution to the culture medium regularly until 3 roots grow.

[0064] S2: Culture of mycorrhizal edible fungi mycelium:

[0065] S21: Mycelial culture: Collect healthy, disease-free wild Boletus edulis, extract the flesh tissue, inoculate it on a solid culture medium, and culture it at 26℃ until it is covered with mycelium;

[0066] S22: Purification culture: Pick the mycelia from step S21, inoculate them into a new solid culture medium, and incubate them statically at 26°C until they are fully grown.

[0067] S23: Fermentation culture: The mycelium from step S22 is inoculated onto the liquid fermentation medium and cultured on a constant temperature shaker at 25℃ to obtain the liquid culture.

[0068] S3: Infection: Inject the liquid inoculum from step S23 into the symbiotic seedling composite culture medium in step S13, and place it in a light incubator (set temperature to 25℃, humidity to 55%, intensity to 5000 Lux, 12h light, 12h darkness) for cultivation. During the cultivation period, replenish the nutrient solution regularly. After 90 days of cultivation, mycorrhizal edible fungi symbiotic seedlings can be formed.

[0069] The mycorrhizal infection rate and symbiotic seedling survival rate of the methods in Examples 1-3 above were statistically analyzed (parallel measurements were performed 3 times), and the results are shown in Table 1 below.

[0070] Table 1 Statistical Results (Average)

[0071] Mycorrhizal infection rate / % Survival rate of symbiotic seedlings / % Example 1 87.6％ 94.4％ Example 2 88.1％ 96.2％ Example 3 85.3％ 95.7％

[0072] As can be seen from the results in Table 1, the mycorrhizal infection rate of the cultivation methods in Examples 1-3 is all above 85%, and the survival rate of symbiotic seedlings can reach 94.4%-96.2%. It is evident that the methods in this example have high mycorrhizal infection rates and high survival rates of symbiotic seedlings.

[0073] In this embodiment, by germinating the seeds of the symbiotic plant and culturing them in a symbiotic seedling composite culture medium, the germination rate and speed of the symbiotic plant seeds were improved, enhancing the antioxidant capacity and stress resistance of the symbiotic plant. Furthermore, by culturing the mycorrhizal edible fungus hyphae in three stages—solid culture medium and liquid fermentation culture medium—the mycorrhizal growth was promoted to be robust, enhancing the mycorrhizal resistance and thus increasing the mycorrhizal infection rate, thereby improving the survival rate of the symbiotic seedlings. Based on this, the method of the present invention has a high mycorrhizal infection rate and a high survival rate of symbiotic seedlings, making it suitable for widespread application.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for cultivating symbiotic seedlings of mycorrhizal edible fungi, characterized in that: Specifically, it includes the following steps: S1: Cultivation of symbiotic plant seedlings: S11: Disinfection and sterilization: Select healthy, plump, and pest-free symbiotic plant seeds, soak them in a 1% potassium permanganate solution for 1-2 hours for surface disinfection and sterilization, and then rinse them with sterile water. S12: Germination: Soak the symbiotic plant seeds from step S11 in the germination solution for 2-3 hours; S13： Cultivation: Sow the symbiotic plant seeds from step S12 into the symbiotic seedling composite culture medium, and add nutrient solution to the culture medium regularly until 2-3 roots grow. S2: Culture of mycorrhizal edible fungi mycelium: S21: Mycelial culture: Collect healthy and disease-free wild mycorrhizal edible fungi, extract the flesh tissue, inoculate it on a solid culture medium, and culture it at 24-26℃ until it is covered with mycelium; S22: Purification culture: Pick the mycelia from step S21, inoculate them into a new solid culture medium, and incubate them statically at 24-26℃ until they are fully colonized with mycelia; S23: Fermentation culture: The mycelium from step S22 is inoculated onto the liquid fermentation medium and cultured on a constant temperature shaker at 25℃ to obtain the liquid culture. S3: Infection: Inject the liquid inoculum from step S23 into the symbiotic seedling composite culture medium in step S13, place it in a light incubator for cultivation, and replenish the nutrient solution regularly during the cultivation period; after 60-90 days of cultivation, mycorrhizal edible fungi symbiotic seedlings can be formed.

2. The method for cultivating mycorrhizal edible fungi symbiotic seedlings according to claim 1, characterized in that: In step S12, the raw material composition and weight percentage of the germination solution are: 0.05g of 1% calcium chloride, 0.1g of 2% copper sulfate, 0.2g of gibberellin, 2g of chitosan, and 1L of water, sterilized at 121℃ for 3h.

3. The method for cultivating mycorrhizal edible fungi symbiotic seedlings according to claim 1, characterized in that: In step S13, the raw material composition and weight proportions of the nutrient solution are as follows: urea 2g, potassium dihydrogen phosphate 1g, glucose 0.5g, seaweed polysaccharide 0.5g, ferric citrate 0.01g, magnesium sulfate 0.2g, zinc sulfate 0.01g, ferric chloride 0.02g, copper sulfate 0.01g, and water 1L.

4. The method for cultivating mycorrhizal edible fungi symbiotic seedlings according to claim 1, characterized in that: In step S13, the formulation of the symbiotic seedling composite culture medium is as follows: 1g vermiculite, 1.2g maifanite, 2g humus, 1.5g corn flour, 5g Chinese herbal medicine residue, 2g feather meal, 5g peptone, 2g yeast extract powder, 2g glucose, 2g chitosan, 1g K2HPO4, 0.05g MgSO4·7H2O, 0.01g CaCl2, 0.01g NaCl, 100mL distilled water, pH=5-6, sterilized at 121℃ for 3h.

5. The method for cultivating mycorrhizal edible fungi symbiotic seedlings according to claim 1, characterized in that: In step S21, the solid culture medium is formulated as follows: 5g peptone, 2g yeast extract, 20g glucose, 10g chitosan, 1g K2HPO4, 0.5g MgSO4·7H2O, 0.05g CaCl2, 0.1g NaCl, 20g agar, 1L distilled water, pH=5-6, sterilized at 121℃ for 3h.

6. The method for cultivating mycorrhizal edible fungi symbiotic seedlings according to claim 1, characterized in that: In step S23, the liquid fermentation culture medium is formulated as follows: 5.0g peptone, 2.0g yeast extract, 20.0g glucose, 10g chitosan, 1.0g K2HPO4, 0.5g MgSO4·7H2O, 0.05g CaCl2, 0.1g NaCl, 100mL traditional Chinese medicine extract, 900mL distilled water, pH=5-6, sterilized at 121℃ for 3h.

7. The method for cultivating mycorrhizal edible fungi symbiotic seedlings according to claim 4, characterized in that: The raw material components and weight parts of the medicinal residue are as follows: 10 parts of Gynostemma pentaphyllum, 6 parts of prepared licorice root, 8 parts of Ligusticum chuanxiong, 4 parts of pumpkin seeds, and 4 parts of jujube. The preparation method of the medicinal residue is as follows: take Gynostemma pentaphyllum, prepared licorice root, Ligusticum chuanxiong, pumpkin seeds, and jujube according to the weight parts, add 6 times the amount of purified water, soak for 2 hours, boil for 2 hours, filter, and obtain the extract; repeat the extraction 3 times, combine the extracts obtained from the 3 extractions, and obtain the medicinal residue and medicinal extract respectively. Sterilize at 121℃ for 3 hours and set aside for later use.

8. The method for cultivating mycorrhizal edible fungi symbiotic seedlings according to claim 6, characterized in that: The herbal extract is the herbal extract described in claim 7.