Artificial cultivation method of phyllodium fraternum

Abstract

The application provides an artificial cultivation method of Pholiota nameko, and belongs to the technical field of edible and medicinal mushroom cultivation, and comprises the following steps: providing a primary substrate for preparing Pholiota nameko primary culture; providing a cultivation substrate for cultivating Pholiota nameko cultivation by the primary culture; and cultivating the cultivation by the under-forest soil covering method to obtain Pholiota nameko sclerotia; the cultivation substrate mainly comprises bamboo leaves, and auxiliary materials are soybean meal, wheat bran and corn flour; the under-forest soil covering cultivation adopts the way of ridge cultivation, the upper and lower sides of the cultivation are provided with a main material layer formed by bamboo leaves, and bamboo stems are filled in the gaps of the cultivation. The method has the advantages of fast mycelium growth speed, shortened production period, high substrate biological conversion rate, improved Pholiota nameko yield and quality, and reduced production cost, and the yield and income of the cultivated Pholiota nameko sclerotia are obviously increased, so as to solve the problems that the existing Pholiota nameko mainly relies on wild resources, and the quality control of the wild Pholiota nameko sclerotia is difficult and the consistency is poor.

Description

Technical Field

[0001] The present invention relates to the technical field of edible and medicinal mushroom cultivation, and particularly to an artificial cultivation method of Polyporus mylittae Cooke & Mass. Background Art

[0002] Polyporus mylittae Cooke & Mass. is the dried sclerotium of the fungus Polyporus mylittae Cooke & Mass. belonging to the Basidiomycota, Agaricomycetes, Agaricales, Marasmiaceae, and Gerronema. It is listed among the "Three Polyporus" together with Poria cocos and Polyporus umbellatus, and is one of the commonly used traditional Chinese medicines in China. It is widely distributed in Sichuan, Yunnan, Hunan, Guangdong, Anhui, Gansu, Shaanxi, Hubei, Henan and other places, and mostly grows saprophytically or parasitically on the roots and stumps of bamboos with yellowing and flowering leaves. The mycelium, sclerotium, fruit body and spores of Polyporus mylittae Cooke & Mass. all contain various medicinal components, such as amino acids, polysaccharides, flavonoids, vitamins, sterols, alkaloids and antibiotics and other active substances. The 2015 edition of the Chinese Pharmacopoeia stipulates that the sclerotium is the medicinal part of Polyporus mylittae Cooke & Mass. The sclerotium of Polyporus mylittae Cooke & Mass. is spherical or irregular in shape, with a black-brown or gray-brown surface and slightly raised irregular reticulate fine lines. It is firm in texture and not easy to break. The cross-section is uneven, white or light gray-yellow, often with yellow-white marble-like texture. It has a faint smell, a slightly bitter taste, is cold in nature, has a granular feeling when chewed, is slightly sticky, and has no residue after chewing for a long time. It belongs to the stomach and large intestine meridians. It has the functions of killing insects, promoting digestion and dissipating tumors, and is commonly used for tumors, taeniasis, ancylostomiasis, ascariasis, abdominal pain due to insect accumulation, and infantile malnutrition. Polyporus mylittae protease and Polyporus mylittae polysaccharide are the main active components of Polyporus mylittae Cooke & Mass., which have the effects of inhibiting tumors, anti-inflammatory and hypoglycemic.

[0003] There are more than 140 practical prescriptions containing Polyporus mylittae Cooke & Mass. in Chinese medical prescriptions. Polyporus mylittae Cooke & Mass. is also a hot medicinal material for the research and development of new and efficient anthelmintics, plant virus inhibitors and anticancer drugs at home and abroad, and the market demand has increased sharply. However, the Polyporus mylittae Cooke & Mass. medicinal materials sold in the domestic and foreign markets mainly rely on wild resources. Not only is the output of natural sclerotia limited, the contradiction between supply and demand in the domestic and foreign markets is prominent, but the contents of Polyporus mylittae protease and Polyporus mylittae polysaccharide in different wild sclerotia of Polyporus mylittae Cooke & Mass. vary greatly, making it difficult to control the quality and the consistency is poor, resulting in medication risks. And research shows that there is no obvious difference in the in vitro inhibitory activity of Polyporus mylittae protease obtained by artificial cultivation and natural Polyporus mylittae protease against Taenia solium cysticerci. Therefore, in order to expand the medicinal source, people have successively carried out research on the artificial cultivation of Polyporus mylittae Cooke & Mass. However, there are few reports on the artificial cultivation of Polyporus mylittae Cooke & Mass., resulting in the existing technology of the artificial cultivation of Polyporus mylittae Cooke & Mass. being insufficient to guide production, and there is an urgent need for the update and popularization of artificial cultivation technology to supplement the medicinal source of Polyporus mylittae Cooke & Mass. and improve the quality. Summary of the Invention

[0004] This invention provides an artificial cultivation method for *Lycopodium clavatum* that features rapid mycelial growth, a shortened production cycle, high substrate bioconversion rate, improved yield and quality, and reduced production costs. This method addresses the existing problem that *Lycopodium clavatum* mainly relies on wild resources, and that controlling the quality of wild *Lycopodium clavatum* sclerotia is difficult and inconsistent.

[0005] To achieve the above objectives, the present invention provides a method for the artificial cultivation of *Leiwan* fungus, comprising: providing a primary spawn substrate for preparing *Leiwan* fungus primary spawn; providing a spawn substrate for cultivating *Leiwan* fungus spawn through the primary spawn; and cultivating the spawn using a forest understory soil covering cultivation method to obtain *Leiwan* fungus sclerotia; during primary spawn cultivation, an activated mother spawn that is vigorous in growth, short in age, and whose mycelial layer has not yet shown pigment secretions is inoculated; the spawn substrate uses bamboo leaves as the main material, and soybean meal, wheat bran, and corn flour as auxiliary materials; the forest understory soil covering cultivation method adopts a raised bed planting method, with a main material layer of bamboo leaves set on the upper and lower sides of the spawn, and bamboo stalks filled in the gaps between the spawn.

[0006] The above technical solution utilizes *Lycopodium clavatum* bags as the inoculum source for artificial cultivation. Outdoors, tree materials and natural waste (bamboo stalks and leaves) are used as the main materials. *Lycopodium clavatum* is cultivated by covering it with soil under the forest canopy to obtain sclerotia. This method is beneficial for the induction and formation of sclerotia, improving the yield and quality of artificially cultivated *Lycopodium clavatum*, shortening the overall production time, and eliminating the need for artificial production facilities, thus reducing cultivation costs and promoting the standardized and large-scale artificial cultivation of *Lycopodium clavatum*.

[0007] Further, the preparation steps for the activated mother culture are as follows: The *Tectus spp.* mother culture is inoculated onto an activation mother culture medium and cultured at a constant temperature of 22-26℃ in the dark for 5-10 days. Then, under the same culture conditions, it is transferred once to obtain the activated mother culture of *Tectus spp.* *Tectus spp.* mother cultures are usually preserved at low temperatures during storage, keeping the *Tectus spp.* in a semi-dormant state to ensure that the species characteristics of *Tectus spp.* do not change significantly during storage. Therefore, the *Tectus spp.* mother culture must be activated before culturing the original *Tectus spp.* strain.

[0008] Further, the preparation steps of the original seed substrate are as follows: Select rice grains that are free from mold, rot, and insect infestation, boil them in water until thoroughly cooked, drain and cool them. Based on the weight of the rice grains, add 1.0-10% bamboo leaves, 0.1-1.0% corn flour, 1.5-3.0% gypsum, 0.1-0.3% MgSO4, and 0.2-0.5% KH2PO4, stir and mix well, put them into the original seed bottle, sterilize them at 120-130℃ and 103-110kPa for 1-3 hours, and cool them to room temperature to obtain the final product.

[0009] Bamboo leaves provide nutrients such as bamboo leaf polysaccharides, vitamins, and trace elements. Corn flour serves as both a carbon and nitrogen source. Rice is used as the main raw material to prepare the original seed substrate, which can enhance the absorption and transformation of various nutrients by *Trichoderma repens*, resulting in rapid growth of the original seed and promoting mycelial growth. The mycelia are white and dense. This original seed substrate can solve the problem of slow mycelial growth in the original seed culture process and is not easily contaminated by other bacteria, thus reducing the cost of seed production.

[0010] Further, the inoculation procedure for primary culture is as follows: Inoculation is performed in a clean inoculation room or on a workbench. Using an inoculation needle, take 1-2 cm of the activated mother culture medium. 2 The mycelial blocks of various sizes are transferred into the primary culture bottle; the culture conditions for primary culture are: protection from light, temperature 22-26℃, and culture until the mycelium of *Opuntia ficus-indica* completely covers the primary culture bottle. Preferably, the culture temperature is 24-26℃.

[0011] Further configured, the cultivation substrate is mainly composed of bamboo leaves, and by weight of bamboo leaves, it contains 10-20% wheat bran, 2-12% corn flour, 1-5% soybean meal, 0.5-1% gypsum, 0.2-0.5% KH2PO4, and 0.1-0.5% MgSO4. Preferably, the substrate bag is a polypropylene corner-folding bag.

[0012] The cultivation substrate, made primarily of bamboo leaves and supplemented with soybean meal, wheat bran, and corn flour, and containing mineral elements such as KH2PO4, gypsum, and MgSO4, can accelerate the growth of *Lycopodium clavatum* mycelium, promote vigorous growth of *Lycopodium clavatum*, accelerate the formation of sclerotia, shorten the cultivation cycle of the cultivar, thereby shortening the production cycle of *Lycopodium clavatum*, reducing time costs, and also promoting the accumulation of major active ingredients, resulting in a higher content of major effective active ingredients in the sclerotia of *Lycopodium clavatum* compared to wild *Lycopodium clavatum* sclerotia.

[0013] Further, the inoculation procedure for the cultivation of the spawn is as follows: Inoculate in a clean bench or a sterilized inoculation room. Before inoculation, wipe hands and forceps with 75% disinfectant alcohol, then place the forceps over an alcohol lamp flame to burn. After cooling, take 1-3g of *Gnaphalium affine* spawn from the spawn bottle and transfer it into a material bag filled with the spawn substrate. The cultivation conditions for the spawn are: avoid light, temperature 22-26℃, and cultivate until the *Gnaphalium affine* mycelium has fully grown in the material bag.

[0014] Preferably, the cultivation temperature is 24-26℃. At this temperature, the mycelium of *Lactobacillus thuringiensis* grows rapidly, fully consuming the substrate and improving the utilization rate of the original strain. After the cultivation period, the entire substrate bag is covered with mycelium, resulting in a cultivated strain with white, robust mycelium, a rich aroma, and strong resistance to contamination by other microorganisms. This shortens the production cycle and reduces costs. Furthermore, after being covered with soil, the cultivated strain of *Lactobacillus thuringiensis* can quickly infect the main substrate layer to obtain nutrients, thus playing a positive role in increasing the yield of *Lactobacillus thuringiensis*.

[0015] Further, the understory soil covering cultivation includes the steps of selecting a planting site and preparing materials, as follows: Select a shady forest land at the foot of a hillside with loose and well-drained soil, prepare planting beds 0.5-1m wide and 30-70cm high, and loosen the topsoil with a thickness of 5-10cm; cut the tree material into specifications of 5-10cm in diameter and 50-75cm in length, take half of the tree material and soak it for 36-72 hours, take it out and drain it 12 hours before cultivation, and set it aside.

[0016] Further, the understory soil-covered cultivation also includes the following cultivation steps: a 1-2cm thick layer of sawdust is laid on the surface soil of the cultivation bed, then a layer of main material formed by bamboo leaves is evenly laid on the sawdust layer, tree material is placed on the main material layer in a dry-wet interval, cultivars are evenly placed between the tree material, and bamboo stalks are filled in the gaps between the cultivars, then another layer of main material is evenly laid on top of the cultivars, and finally a 5-6cm thick layer of sandy soil in a tile-back shape is covered to begin cultivation.

[0017] Further settings include using coarse-grained river sand with a moisture content of 40-60% for the sand layer; and cultivation conditions during the cultivation period are: cultivation temperature of 24-26℃ and soil moisture control of 40-60%.

[0018] Further, the total amount of bamboo leaves used in the main material layers on both the upper and lower sides of the cultivated plant is set at 5.0-6.5 kg / m². 2 Furthermore, the amount of bamboo leaves used in the main material layer on each side is the same.

[0019] Further settings indicate that the total amount of bamboo stalks used is 3-8 kg / m². 2 The bamboo culms are 5-7 cm in length. The main materials used in the cultivation—tree material, bamboo culms, and bamboo leaves—are all inexpensive and readily available, allowing the cultivar to grow in a simulated wild environment. This promotes the induction and formation of *Echinops latifolius* sclerotia, resulting in a high substrate bioconversion rate and improved material utilization. This reduces cultivation costs and aligns with the wild environment of *Echinops latifolius* and the concept of "turning waste into treasure," thus contributing to ecological environmental protection. This invention employs understory cultivation, enabling the *Echinops latifolius* cultivar to grow rapidly in an outdoor simulated wild environment. This improves the quality of *Echinops latifolius* sclerotia, is suitable for large-scale production, and eliminates the need for artificial production facilities, saving production costs and resulting in higher economic benefits.

[0020] Further settings indicate that the amount of cultivar used is 4-8 packets / m². 2 Preferably, after the spawn is removed from the substrate bag, it is gently broken up and then evenly sprinkled onto the main substrate layer. The spawn carries a large amount of mycelium, which can germinate at multiple points on the main substrate layer, improving the utilization rate of the spawn and increasing the yield of *Sclerotium tumefaciens*, resulting in a significant increase in production and income.

[0021] Further, the present invention is preferably planted in March of each year and can be harvested twice in July and November of the same year.

[0022] The artificial cultivation method for *Leiwan* provided by this invention, compared with the prior art, achieves the following:

[0023] Beneficial effects:

[0024] 1) In this invention, the bag material of *Lycopodium clavatum* is used as the source of spawn for artificial cultivation. The sclerotia of *Lycopodium clavatum* are obtained by outdoor cultivation under forest soil covering. The operation is simple and easy to carry out. The artificial cultivation of *Lycopodium clavatum* yields high output. The production time is shortened by the steps of original spawn preparation and spawn preparation, which reduces the production cost and improves the economic benefits of *Lycopodium clavatum* production. It is conducive to the promotion of standardized and large-scale artificial cultivation of *Lycopodium clavatum*.

[0025] 2) The original seed substrate and cultivation seed substrate of the present invention have a fast mycelial growth rate, which can solve the problem of slow mycelial growth during the cultivation of *Lycoperdon perlatum*, resulting in significant increases in yield and income, and reducing seed production costs. In the outdoor bed planting process, tree material, bamboo stalks and bamboo leaves are used as the main materials, which is conducive to the induction and formation of *Lycoperdon perlatum* sclerotia. Moreover, no artificial production facilities are required, which reduces cultivation costs and is more in line with the wild environment of *Lycoperdon perlatum* and the concept of "turning waste into treasure", which is of certain significance for ecological environmental protection.

[0026] 3) The cultivation method of the present invention has a high substrate bioconversion rate, which is conducive to the induction and formation of Leiwan sclerotia. The yield and quality of Leiwan sclerotia are high, and the accumulation of the main effective active ingredients is also higher than that of wild Leiwan sclerotia. This improves production efficiency and Leiwan quality, reduces production costs, and makes full use of agricultural and forestry waste. It is of great significance for protecting the ecological environment and wild Leiwan resources. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 A schematic diagram showing the effect of different cultivation methods on the content of leiwanin in the sclerotium of *Leymus chinensis*.

[0029] Figure 2 This is a schematic diagram showing the effect of different cultivation methods on the polysaccharide content of *Lycoperdon perlatum* sclerotia. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 also within the scope of protection of the present invention.

[0031] The following examples use conventional instruments and equipment in the art. Unless otherwise specified, the experimental materials and reagents used in the following examples are commercially available and conform to conventional specifications in the art. Any techniques or conditions not specifically described in the following examples can be performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0032] It should be noted that, in this invention and the following embodiments, unless otherwise specified, concentration, ratio, etc. are all weight concentration, weight ratio, etc., "%" all represent weight percentage, and "parts" all represent weight parts. These are common writing habits used by those skilled in the art, and therefore will not be repeated in this invention.

[0033] PDA solid culture medium: 200g potato (peeled), 20g glucose, 5g peptone, 5g potassium dihydrogen phosphate, 3g magnesium sulfate, 15g agar powder, 0.1g vitamin B1, add distilled water to a final volume of 1L, pH 5-5.5. Sterilize at 121℃ and 101kPa for 20min.

[0034] In a specific implementation scheme, the present invention provides a method for the artificial cultivation of *Echinopsis pilosula*, comprising the following steps:

[0035] 1) Activation of microbial strains

[0036] The mother culture of *Lycoperdon terrestris* was inoculated onto an activated mother culture medium (i.e., PDA solid medium) and cultured at a constant temperature of 22-26℃ in the dark for 5-10 days. Then, under the same culture conditions, the culture was transferred once to obtain the activated mother culture of *Lycoperdon terrestris*.

[0037] The mother culture of *Lycoperdon julibrissin* is not limited to self-retained mother cultures, mother cultures preserved in research institutes, or mother cultures from various culture centers. It can also be obtained and preserved by collecting wild *Lycoperdon julibrissin* sclerotia using conventional culture methods. In this embodiment of the invention, the *Lycoperdon julibrissin* mother culture comes from *Lycoperdon julibrissin* (CGMCC No. 5.54) from the China General Microbiological Culture Collection Center.

[0038] 2) Preparation of original seeds

[0039] 2.1) Preparation of original seed substrate

[0040] Select rice grains free from mold, rot, and insect infestation, boil them in water until thoroughly cooked, drain and cool. Based on the weight of the rice grains, add 1.0-10% bamboo leaves, 0.1-1.0% corn flour, 1.5-3.0% gypsum, 0.1-0.3% MgSO4, and 0.2-0.5% KH2PO4. Stir well, put the mixture into a seed bottle, and sterilize at 120-130℃ and 103-110kPa for 1-3 hours. Cool to room temperature to obtain the final product.

[0041] Preferably, the pH of the original seed substrate is 6.0-7.0.

[0042] 2.2) Vaccination

[0043] Perform the inoculation procedure in a clean inoculation room / box or on a workbench. Select a vigorous, short-aged, and activated mother culture that has not yet developed pigment secretions in its mycelial layer for inoculation. Use an inoculation needle to take 1-2 cm of the activated mother culture from the test tube. 2 Transfer the bacterial clumps of different sizes into the original culture bottle. Before inoculation, the inoculation room or workbench should be sterilized with ultraviolet light. First, disinfect your hands and inoculation needle with 75% alcohol. The inoculation needle, test tube mouth, and cotton plug should all be sterilized with an alcohol lamp flame.

[0044] 2.3) Cultivation

[0045] After inoculation, the original culture bottle was transferred to a light-proof culture room and cultured at a temperature of 22-26℃ until the mycelium of *Lycopodium clavatum* completely covered the original culture bottle.

[0046] Preferably, the culture temperature is 24-26℃.

[0047] 3) Preparation of cultivars

[0048] 3.1) Preparation of cultivation substrate

[0049] Weigh bamboo leaves as the main ingredient, and add 10-20% wheat bran, 2-12% corn flour, 1-5% soybean meal, 0.5-1% gypsum, 0.2-0.5% KH2PO4, and 0.1-0.5% MgSO4 by weight of bamboo leaves. Mix all the raw materials evenly, add water until the substrate moisture content reaches 60-80%, mix well, and then pack into bags of 300-500g each, sealing them. Sterilize the bags at 120-130℃ and 103-110kPa for 1-3 hours or at 100-110℃ under normal pressure for 10-12 hours. After sterilization, allow them to cool naturally to room temperature.

[0050] The preferred material bag is a polypropylene corner-fold bag.

[0051] 3.2) Vaccination

[0052] Inoculate in a clean bench or sterilized inoculation room / box. Before inoculation, wipe your hands and tweezers with 75% alcohol, then burn the tweezers over an alcohol lamp flame. After cooling, take 1-3g of *Leptochloa crus-galli* spawn from the spawn bottle and transfer it into a material bag filled with the cultivation substrate.

[0053] 3.3) Cultivation

[0054] After inoculation, transfer the substrate bags to a dark incubation room and incubate them at a temperature of 22-26℃ until the mycelium of *Lycopodium clavatum* completely covers the substrate bags.

[0055] Preferably, the culture temperature is 24-26℃.

[0056] 4) Understory cultivation with soil covering

[0057] 4.1) Site selection and material preparation: Select a shady forest land with loose and well-drained soil at the foot of a hillside. After finding a gap in the forest, level the forest land and remove weeds. Then, prepare planting beds that are 0.5-1m wide and 30-70cm high, and loosen the topsoil that is 5-10cm thick. Cut the tree material into pieces with a diameter of 5-10cm and a length of 50-75cm. Soak half of the tree material for 36-72 hours. Take it out and drain it 12 hours before the breeding and cultivation.

[0058] The aforementioned wood materials are other trees that do not contain oil or have low oil content, excluding pine, fir, camphor, and cypress, such as elm, oak, maple, poplar, and chestnut.

[0059] 4.2) Cultivation: Spread a 1-2cm thick layer of sawdust on the surface soil of the cultivation bed, then evenly spread a layer of main material formed by bamboo leaves on the sawdust layer, then place the tree material on the main material layer in a dry-wet interval, then evenly place the cultivar between the tree material, and fill the gaps between the cultivar with bamboo stalks, then evenly spread another layer of main material on top of the cultivar, and finally cover it with a 5-6cm thick layer of sand in a tile-back shape, and start cultivation. During the cultivation period, the moisture content of the sand layer should be controlled at 40-60%, and the cultivation temperature should be controlled at 24-26℃.

[0060] The aforementioned tree materials are placed parallel to each other along the length of the planting bed, with a spacing of 3-10cm along the length of the planting bed and a spacing of 8-15cm along the width of the planting bed.

[0061] The above-mentioned sandy soil layer uses coarse-grained river sand with a moisture content of 40-60%.

[0062] In the main substrate layers on both sides of the above-mentioned cultivar, the total amount of bamboo leaves used is 5.0-6.5 kg / m². 2 Furthermore, the amount of bamboo leaves used in each main material layer is the same. The amount of bamboo culms used is 3-8 kg / m². 2The bamboo culms are 5-7 cm long. Bamboo leaves and culms can be fresh or dead bamboo branches and leaves. The dosage of the above-mentioned cultivar is 4-8 packs / m². 2 After the spawn is taken out of the bag, it is gently crushed and then evenly sprinkled on the main material layer.

[0063] The artificial cultivation method of the present invention is preferably carried out in March of each year, and the sclerotia of *Lycopodium clavatum* can be harvested twice in July and November of the same year.

[0064] As a further improvement to the aforementioned implementation, 0.5-1.5% barbituric acid and 0.1-1.0% rhamnolipid are added to the cultivation substrate based on the weight of bamboo leaves. The barbituric acid and rhamnolipid work synergistically with other components in the cultivation substrate to stimulate and enhance the growth and metabolic capacity of the original strain of *Lycoperdon perlatum*, promoting faster mycelial growth, increasing the mycelial growth rate, resulting in better mycelial growth, greater mycelial biomass, and shorter cultivation time. This also keeps *Lycoperdon perlatum* in a highly active state, resulting in a more active cultivation strain and more highly active metabolites. Consequently, during the casing cultivation stage, the formation of *Lycoperdon perlatum* sclerotia can be rapidly induced and promoted, and the accumulation of effective active ingredients in the sclerotia can be increased to varying degrees, leading to better quality and increased cultivation yield.

[0065] In a specific example of the present invention, the original seed bottle has a size of 500-1000mL; the polypropylene corner bag has a size of 14cm x 33cm and a thickness of 0.5mm.

[0066] It should be noted that the entire process of cultivating the original strain and cultivar of *Lycoperdon perlatum* in this invention is completed indoors under artificially controlled temperature and humidity conditions. All culture substrates used have been sterilized, and each step of the operation is carried out in an ultra-clean working environment. This avoids contamination problems during inoculation and cultivation, improves production efficiency, achieves high-efficiency production, and greatly increases the yield of *Lycoperdon perlatum* cultivar.

[0067] The present invention will be further described in detail below with reference to embodiments. However, it should be understood that the embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0068] Example 1:

[0069] A method for artificially cultivating *Echinopsis pilosula* includes the following steps:

[0070] 1) Activation of strain: The mother culture of *Lycoperdon terrestris* was inoculated onto PDA solid medium and cultured at a constant temperature of 25°C in the dark for 8 days. Then, under the same culture conditions, it was transferred once to obtain the activated mother culture of *Lycoperdon terrestris*.

[0071] 2) Preparation of original seeds

[0072] 2.1) Preparation of the original seed substrate: Select rice grains that are free from mold, rot, and insect infestation, boil them in water until thoroughly cooked, drain and cool. Based on the weight of the rice grains, add 6.5% bamboo leaves, 0.5% corn flour, 2.0% gypsum, 0.3% MgSO4, and 0.4% KH2PO4, stir and mix well. Adjust the pH of the original seed substrate to 6.0-7.0, then put it into the original seed bottle and sterilize it at 121℃ and 105kPa for 1.5h. Cool to room temperature to obtain the original seed substrate.

[0073] 2.2) Inoculation: Inoculation should be performed in a clean inoculation room / box or on a workbench. Select a vigorous, short-aged, and pre-existing activated mother culture with no pigment secretions in the mycelial layer for inoculation. Use an inoculation needle to take 1-2 cm of the activated mother culture from the test tube. 2 Transfer the bacterial clumps of different sizes into the original culture bottle. Before inoculation, the inoculation room or workbench should be sterilized with ultraviolet light. First, disinfect your hands and inoculation needle with 75% alcohol. The inoculation needle, test tube mouth, and cotton plug should all be sterilized with an alcohol lamp flame.

[0074] 2.3) Cultivation: Transfer the inoculated primary culture bottle to a light-proof cultivation room and cultivate at 25℃ until the mycelium of *Lycopodium clavatum* completely covers the primary culture bottle.

[0075] 3) Preparation of cultivars

[0076] 3.1) Preparation of Cultivation Substrate: Weigh bamboo leaves as the main material, and add 16% wheat bran, 10% corn flour, 3% soybean meal, 1% gypsum, 0.5% KH2PO4, and 0.3% MgSO4 by weight of bamboo leaves. Mix all cultivation materials evenly, add water until the cultivation substrate reaches 70% water content, mix well, and then pack into bags of 500g each, sealing the bags. Sterilize the bags at 122℃ and 105kPa for 2 hours, and then allow them to cool naturally to room temperature. The preferred material bags are polypropylene folded corner bags.

[0077] 3.2) Inoculation: Inoculate in a clean bench or in a sterilized inoculation room / box. Before inoculation, wipe hands and forceps with 75% disinfectant alcohol, then place the forceps over an alcohol lamp flame to burn. After cooling, take 3g of *Leptochloa crus-galli* spawn from the spawn bottle and transfer it into a material bag filled with the cultivation substrate.

[0078] 3.3) Cultivation: Transfer the inoculated bags to a light-proof cultivation room and cultivate at 25℃ until the mycelium of *Lycopodium clavatum* fully colonizes the bags.

[0079] 4) Understory cultivation with soil covering:

[0080] 4.1) Site Selection and Material Preparation: Select a shady woodland at the foot of a hillside with loose and well-drained soil. After finding suitable gaps in the forest, level and weed the woodland, then prepare planting beds 1m wide and 70cm high, and loosen the top 10cm layer of soil. Cut the tree material into pieces 5-10cm in diameter and 65-75cm in length. Soak half of the tree material for 72 hours, then drain it 12 hours before the breeding and cultivation period. The tree material should be branches of elm, poplar, etc.

[0081] 4.2) Cultivation: Spread a 1-2cm thick layer of sawdust on the surface soil of the cultivation bed, then evenly spread a layer of main material formed by bamboo leaves on the sawdust layer, then place the tree material on the main material layer in a dry-wet interval, then evenly place the cultivar between the tree material, and fill the gaps between the cultivar with bamboo stalks, then evenly spread another layer of main material on top of the cultivar, and finally cover it with a 5-6cm thick layer of sand in a tile-back shape, and start cultivation. During the cultivation period, the moisture content of the sand layer should be controlled at 40-60%, and the cultivation temperature should be controlled at 24-26℃.

[0082] The aforementioned tree material is laid parallel to the length of the planting bed, with a spacing of 5 cm along the length and 10 cm along the width. The sand layer uses coarse-grained river sand with a moisture content of 55%. The total amount of bamboo leaves used in the main material layers on both sides of the planting bed is 6 kg / m². 2 Furthermore, the amount of bamboo leaves used in each main material layer is the same. The amount of bamboo culm used is 5 kg / m². 2 The bamboo culms are 5-7 cm long. Bamboo leaves and culms can be fresh or dead bamboo branches and leaves. The recommended usage for the above-mentioned cultivar is 6 packs / m². 2 After the spawn is taken out of the bag, it is gently crushed and then evenly sprinkled on the main material layer.

[0083] Example 2:

[0084] A method for artificially cultivating *Echinopsis pilosula* includes the following steps:

[0085] 1) Same as step 1) of Example 1;

[0086] 2) Same as step 2) in Example 1;

[0087] 3) The only difference from step 3) of Example 1 is:

[0088] 3.1) Preparation of cultivation substrate: Weigh bamboo leaves as the main material, and add 16% wheat bran, 10% corn flour, 3% soybean meal, 1% gypsum, 0.5% KH2PO4, 0.3% MgSO4, 1.0% barbituric acid and 0.5% rhamnolipid based on the weight of bamboo leaves.

[0089] 4) Same as step 4) in Example 1.

[0090] Comparative Example 1:

[0091] A method for artificially cultivating *Echinopsis pilosula* includes the following steps:

[0092] 1) Same as step 1) of Example 1;

[0093] 2) The only difference from step 2) of Example 1 is:

[0094] 2.1) Preparation of the original seed substrate: Based on the weight of rice grains, add 0.5% corn flour, 2.0% gypsum, 0.3% MgSO4, and 0.4% KH2PO4. That is, no bamboo leaves were added to the original seed substrate.

[0095] 3) Same as step 3) in Example 1;

[0096] 4) Same as step 4) in Example 1.

[0097] Comparative Example 2:

[0098] A method for artificially cultivating *Echinopsis pilosula* includes the following steps:

[0099] 1) Same as step 1) of Example 1;

[0100] 2) Same as step 2) in Example 1;

[0101] 3) The only difference from step 3) of Example 1 is:

[0102] 3.1) Preparation of cultivation substrate: Weigh sawdust as the main material, and add 16% wheat bran, 10% corn flour, 3% soybean meal, 1% gypsum, 0.5% KH2PO4 and 0.3% MgSO4 by weight of sawdust.

[0103] 4) Same as step 4) in Example 1.

[0104] Comparative Example 3:

[0105] A method for artificially cultivating *Echinopsis pilosula* includes the following steps:

[0106] 1) Same as step 1) of Example 1;

[0107] 2) Same as step 2) in Example 1;

[0108] 3) Same as step 3) in Example 1;

[0109] 4) The only difference from step 4) of Example 1 is:

[0110] 4.2) Cultivation: A 1-2cm thick layer of sawdust is laid on the surface soil of the cultivation bed. Then, a layer of bamboo leaves is evenly spread on top of the sawdust. Tree materials are then arranged in a dry-wet alternating pattern on the main material layer. Cultivation seeds are then evenly placed between the tree materials. Another layer of main material is then evenly spread on top of the cultivation seeds. Finally, a 5-6cm thick layer of sand in a tile-back shape is placed on top. Cultivation begins, and the moisture content of the sand layer is controlled at 40-60%, and the cultivation temperature is controlled at 24-26℃. That is, bamboo stalks are not placed between the cultivation seeds.

[0111] Comparative Example 4:

[0112] A method for artificially cultivating *Echinopsis pilosula* includes the following steps:

[0113] 1) Same as step 1) of Example 1;

[0114] 2) Same as step 2) in Example 1;

[0115] 3) Same as step 3) in Example 1;

[0116] 4) The only difference from step 4) of Example 1 is:

[0117] 4.2) Cultivation: A 1-2cm thick layer of sawdust is laid on the surface soil of the cultivation bed. Tree material is then placed on the sawdust layer in a dry-wet alternating pattern. Cultivation seeds are then evenly placed between the tree material. Finally, a 5-6cm thick layer of sand in a tile-back shape is evenly covered over the cultivation seeds. Cultivation begins, and the moisture content of the sand layer is controlled at 40-60%, and the cultivation temperature is controlled at 24-26℃. Specifically, no main material layer of bamboo leaves is placed above or below the cultivation seeds, and no bamboo culms are filled between the cultivation seeds.

[0118] Comparative Example 5:

[0119] A method for artificially cultivating *Echinopsis pilosula* includes the following steps:

[0120] 1) Same as step 1) of Example 1;

[0121] 2) Same as step 2) in Example 1;

[0122] 3) The only difference from step 3) of Example 1 is:

[0123] 3.1) Preparation of cultivation substrate: Weigh bamboo leaves as the main material, and add 16% wheat bran, 10% corn flour, 3% soybean meal, 1% gypsum, 0.5% KH2PO4, 0.3% MgSO4 and 1.5% barbituric acid according to the weight of bamboo leaves.

[0124] 4) Same as step 4) in Example 1.

[0125] Comparative Example 6:

[0126] A method for artificially cultivating *Echinopsis pilosula* includes the following steps:

[0127] 1) Same as step 1) of Example 1;

[0128] 2) Same as step 2) in Example 1;

[0129] 3) The only difference from step 3) of Example 1 is:

[0130] 3.1) Preparation of cultivation substrate: Weigh bamboo leaves as the main material, and add 16% wheat bran, 10% corn flour, 3% soybean meal, 1% gypsum, 0.5% KH2PO4, 0.3% MgSO4 and 1.5% rhamnolipid based on the weight of bamboo leaves.

[0131] 4) Same as step 4) in Example 1.

[0132] Comparative Example 7:

[0133] A method for artificially cultivating *Echinopsis pilosula* includes the following steps:

[0134] 1) Same as step 1) of Example 1;

[0135] 2) Same as step 2) in Example 1;

[0136] 3) The only difference from step 3) of Example 1 is:

[0137] 3.1) Preparation of cultivation substrate: Weigh bamboo leaves as the main material, and add 16% wheat bran, 10% corn flour, 3% soybean meal, 1% gypsum, 0.5% KH2PO4, 0.3% MgSO4, 3.0% barbituric acid and 0.5% rhamnolipid based on the weight of bamboo leaves.

[0138] 4) Same as step 4) in Example 1.

[0139] Comparative Example 8:

[0140] A method for artificially cultivating *Echinopsis pilosula* includes the following steps:

[0141] 1) Same as step 1) of Example 1;

[0142] 2) Same as step 2) in Example 1;

[0143] 3) The only difference from step 3) of Example 1 is:

[0144] 3.1) Preparation of cultivation substrate: Weigh bamboo leaves as the main material, and add 16% wheat bran, 10% corn flour, 3% soybean meal, 1% gypsum, 0.5% KH2PO4, 0.3% MgSO4, 1.0% barbituric acid and 2.0% rhamnolipid based on the weight of bamboo leaves.

[0145] 4) Same as step 4) in Example 1.

[0146] Experimental Example 1:

[0147] Effects of different substrates on the growth of *Gnaphalium affine* mycelium

[0148] Measurement method: The original and cultivated strains of *Trichoderma repens* were cultured according to the original and cultivated strain culture steps in Examples 1, 2, 1, 2, 5-8, respectively. During the culture period, the mycelial growth of *Trichoderma repens* was observed, and the daily mycelial growth (cm²) was recorded as the measurement. 2 / d) Statistical analysis of the data was performed. All original culture bottles were standardized to 650 mL, and each experiment had three replicates, with the average value taken. The results are shown in Table 1.

[0149] Table 1

[0150]

[0151]

[0152] During cultivation, the mycelia of both the original strain and the cultivated strain are white and dense. After the original strain is inoculated into the cultivation strain substrate bag, new mycelia can be seen growing in about 2-3 days and can quickly branch into multiple levels until the substrate bag is completely covered. Comparing Comparative Example 1 with Example 1, it can be seen that the mycelia of the original strain in Comparative Example 1, which does not contain bamboo leaves, grows more slowly and takes the longest to fill the bottle. Furthermore, the poor growth of the original strain will continue to affect the growth of the cultivated strain, resulting in slower mycelia growth and a longer time to fill the bag for the cultivated strain in Comparative Example 1.

[0153] Comparing Comparative Example 2 with Example 1, it can be seen that when the growth difference of the original species is not obvious, the mycelial growth of the cultivated species in Comparative Example 2 is lower and the time to fill the bag is also longer than that in Example 1. This indicates that bamboo leaves as the main material of the cultivated species substrate has a better effect on promoting growth than sawdust.

[0154] A comparison of Examples 1 and 2 with Comparative Examples 1, 2, 5-8 shows that, under conditions where the differences in the growth of the original strain are not significant, Example 2 exhibits the highest mycelial growth rate and the shortest time to fill the bag. Comparative Examples 5 and 6 show only slight improvements over Example 1, and while Comparative Examples 7 and 8 show greater improvements than Comparative Examples 5 and 6, they are still inferior to Example 2. Considering the added cost, Example 2 is more advantageous. In summary, the addition of a specific ratio of barbituric acid and rhamnolipids to the cultivation substrate in Example 2 of this invention is more conducive to synergistically promoting faster mycelial growth, increasing the mycelial growth rate, shortening the cultivation time, and reducing time costs.

[0155] Experimental Example 2:

[0156] Effects of different cultivation methods on the content of the main active ingredients in *Sclerotium truncatum*

[0157] Sample testing: *Gerronema* was cultivated according to the methods of Examples 1 and 2, and Comparative Examples 2, 5-8. Cultivation began in April, and sclerotia of each test case were harvested on the same day in November of the same year. Each *Gerronema* sclerotia sample was dried at low temperature (40℃), pulverized, and passed through an 80-mesh sieve to obtain the test samples for later use. Wild *Gerronema* sclerotia served as the control group. These wild *Gerronema* sclerotia were identified as belonging to the genus *Gerronema* by the Teaching and Experimental Center for Biological Science and Engineering of Shaanxi University of Technology.

[0158] (1) Determination of lewanin content: Following the method specified in the 2015 edition of the Chinese Pharmacopoeia: 0.3 g of sample was taken, and extracted with water by ultrasonication (400 W power, 40 kHz frequency) for 30 min. After centrifugation, 1 mL of the supernatant was taken, treated with Folin-phenol reagent, and the absorbance of the standard and sample solutions treated in the same way was determined at 650 nm using ultraviolet-visible spectrophotometry. A standard curve was plotted, and the lewanin content in each sample was calculated. Figure 1 As shown.

[0159] (2) Determination of polysaccharide content of Leiwan: Accurately weigh 10g of each sample, add 30mL of 95% ethanol, shake in a shaker (100r / min) for 20min, soak at room temperature for 6h, filter, evaporate the ethanol, take the filter residue, add 300mL of pure water and 0.24g of cellulase, place in a 50℃ water bath for 90min, then boil in a water bath for 10min; then extract by ultrasonication (power 500W, frequency 40kHz, 20min, 70℃) once, centrifuge and take the supernatant; combine the supernatant obtained by filtration and centrifugation, concentrate in a rotary evaporator to 50mL; add an appropriate amount of 95% ethanol, let stand for 12h, collect the precipitate; take the above precipitate, add an appropriate amount of pure water to dissolve, freeze dry, and obtain crude polysaccharide of Leiwan. The polysaccharide content was determined according to the method described in the literature [Xu Mingfeng et al. Extraction, separation and antioxidant activity of polysaccharides from Leiwan [J]. Chinese Journal of Food Science, 2011, 11(6):42.]. Samples and glucose reference solutions were prepared, treated with the phenol-sulfuric acid method, and the absorbance of the reference and sample solutions was measured at 490 nm. A standard curve was plotted, and the polysaccharide content was calculated. Figure 2 As shown.

[0160] Figure 1 This diagram illustrates the effect of different cultivation methods on the content of lepidotin in the sclerotium of *Lepidium apetalum*. Figure 2 This diagram illustrates the effect of different cultivation methods on the content of *Lycopodium clavatum* polysaccharide in *Lycopodium clavatum* sclerotia. As shown in the figure, the content of *Lycopodium clavatum* extract in all samples was higher than the standard in the Chinese Pharmacopoeia (0.6%). Furthermore, the content of *Lycopodium clavatum* extract in artificially cultured sclerotia was significantly higher than that in wild sclerotia, and the content of *Lycopodium clavatum* polysaccharide in artificially cultured sclerotia was also significantly higher than that in wild sclerotia. Among all experimental examples, Example 2 had the highest contents of *Lycopodium clavatum* extract (3.47%) and *Lycopodium clavatum* polysaccharide (4.21%), significantly higher than Example 1 (1.63 times higher for *Lycopodium clavatum* extract and 1.33 times higher for *Lycopodium clavatum* polysaccharide). Example 1 was also significantly higher than the control group (1.15 times higher for *Lycopodium clavatum* extract and 2.02 times higher for *Lycopodium clavatum* polysaccharide). The quality of *Lycopodium clavatum* sclerotia cultivated in this invention is significantly superior to that of wild *Lycopodium clavatum* sclerotia.

[0161] Comprehensive comparison revealed that using bamboo leaves as the main material in the cultivation substrate is more effective than using sawdust in promoting the quality improvement of the cultivation spores. This is beneficial for increasing the content of the main effective active ingredients in the sclerotium of *Lycopodium clavatum*. Furthermore, in Example 2 of this invention, adding a special proportion of barbituric acid and rhamnolipid to the cultivation substrate is more conducive to synergistically obtaining more active cultivation spores and more highly active metabolites. As a result, the accumulation of effective active ingredients in the sclerotium of *Lycopodium clavatum* is increased to varying degrees, leading to better quality of *Lycopodium clavatum*.

[0162] Experimental Example 3:

[0163] Determination of Sclerotium sclerotium yield in *Leiwan*

[0164] Test method: *Gynostemma pentaphyllum* was cultivated according to the methods of Examples 1, 2, and Comparative Examples 1-8, starting in April. The amount of spawn used was 6 packs / m³. 2 In November of the same year, sclerotia of each experimental case were harvested on the same day. The fresh weight of the sclerotia was weighed, and the bioconversion rate was calculated: Bioconversion rate (%) = Fresh weight of sclerotia / Dry weight of the main substrate layer × 100%. The results are shown in Table 2.

[0165] Table 2. Yield and bioconversion rate of *Sclerotium rayeri* under different cultivation methods.

[0166] <![CDATA[Output g / m 2 > <![CDATA[Dry weight of main ingredient kg / m 2 > Bioconversion rate % Example 1 1457 6.0 24.28 Example 2 1646 6.0 27.43 Comparative Example 1 1402 6.0 23.37 Comparative Example 2 1363 6.0 22.72 Comparative Example 3 1376 6.0 22.93 Comparative Example 4 1348 6.0 22.47 Comparative Example 5 1495 6.0 24.92 Comparative Example 6 1523 6.0 25.38 Comparative Example 7 1576 6.0 26.27 Comparative Example 8 1603 6.0 26.72

[0167] The characteristics of the harvested *Lycopodium clavatum* sclerotia are as follows: oval to ovoid or irregular clumps, 0.5-4.0 cm in diameter; the outer skin is grayish-brown to brownish-brown, with slightly raised fine reticulate wrinkles; after drying, the texture is firm, the cross-section is white to light yellowish-white, with irregular reticulate textures of tightly interwoven translucent and opaque parts; it has a bitter taste.

[0168] The results of Comparative Examples 1 and 2 showed that adding bamboo leaves to the original spore substrate had a positive effect on increasing the yield of *Sclerotium truncatum*. The cultivar substrate with bamboo leaves as the main material was more effective than sawdust in promoting the yield of *Sclerotium truncatum*. This may be because bamboo leaves can provide a growth environment similar to the wild environment, which promotes the growth of both the original spore and the cultivar, thereby increasing the yield of *Sclerotium truncatum*.

[0169] Comparing the yields and bioconversion rates of Examples 1, 3, and 4, it was found that using bamboo leaves as the main substrate and supplementing it with bamboo stalks to fill the gaps between the cultivars in understory soil covering cultivation resulted in higher sclerotium yields and bioconversion rates. This improved the economic benefits of *Gnaphalium affine* production and facilitated the standardized and large-scale artificial cultivation of *Gnaphalium affine*. Furthermore, it made full use of agricultural and forestry waste, which is of great significance for protecting the ecological environment. Comparing the results of Examples 1, 2, and 5-8, it was found that adding a specific ratio of barbituric acid and rhamnolipids to the cultivar substrate in Example 2 of this invention was more conducive to synergistically obtaining more active cultivars and more highly active metabolites. This allowed for rapid induction and promotion of *Gnaphalium affine* sclerotium formation during the soil covering cultivation stage, resulting in increased cultivation yields and bioconversion rates.

[0170] It should be noted that some detailed steps of the operation are not described in this invention, but are existing technologies known to those skilled in the art, and therefore will not be repeated here. Furthermore, in this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible secondary ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0171] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. In this invention, not all possible combinations of the various technical features in each embodiment or implementation are described. As long as the combinations of these technical features do not contradict each other, the various technical features in each embodiment or implementation can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification. 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for the artificial cultivation of *Echinopsis pilosula*, characterized in that, include: Provide the original culture substrate for preparing the original culture of *Ophiocortis radiata*; Provide a culture medium for culturing *Ophiocortis tinctoria* culture spawn using the original strain; as well as, The cultivar was cultivated using a forest understory soil covering method to obtain *Oryza sativa* sclerotia. During the original culture, an activated mother culture that is growing vigorously, has a short age of mycelium, and whose mycelial layer has not yet shown pigment secretions is inoculated. The preparation steps of the original seed substrate are as follows: Select rice grains that are free from mold, rot, and insect infestation, boil them in water until thoroughly cooked, drain and cool them. Based on the weight of the rice grains, add 1.0-10% bamboo leaves, 0.1-1.0% corn flour, 1.5-3.0% gypsum, 0.1-0.3% MgSO4, and 0.2-0.5% KH2PO4, stir and mix well, put them into the original seed bottle, sterilize them at 120-130℃ and 103-110kPa for 1-3 hours, and cool them to room temperature to obtain the product. The cultivation substrate is mainly composed of bamboo leaves, and by weight of bamboo leaves, it contains 10-20% wheat bran, 2-12% corn flour, 1-5% soybean meal, 0.5-1% gypsum, 0.2-0.5% KH2PO4, 0.1-0.5% MgSO4, 0.5-1.5% barbituric acid and 0.1-1.0% rhamnolipid. The understory soil-covered cultivation adopts the method of planting in raised beds. A main material layer of bamboo leaves is set on both the upper and lower sides of the cultivated plants, and bamboo stalks are filled in the gaps between the cultivated plants.

2. The method for artificial cultivation of *Leiwan* according to claim 1, characterized in that, The inoculation procedure for the original culture is as follows: Inoculation is performed in a clean inoculation room or on a workbench. Using an inoculation needle, take 1-2 cm of the activated mother culture medium. 2 The mycelial blocks of various sizes are transferred into the original culture bottle; the culture conditions for the original culture are: in the dark, at a temperature of 22-26℃, and cultured until the mycelium of *Ophiopogon japonicus* completely fills the original culture bottle.

3. The method for artificial cultivation of *Leiwan* according to claim 1, characterized in that, The inoculation procedure for the cultivation of the cultivar is as follows: Inoculate in a clean bench or a sterilized inoculation room. Before inoculation, wipe hands and forceps with 75% disinfectant alcohol, then place the forceps over an alcohol lamp flame to burn. After cooling, take 1-3g of *Lycoperdon perlatum* cultivar from the original culture bottle and transfer it into a material bag filled with the cultivation substrate. The cultivation conditions for the cultivar are: avoid light, temperature 22-26℃, and cultivate until the *Lycoperdon perlatum* mycelium has fully grown in the material bag.

4. The method for artificial cultivation of *Lycopodium clavatum* according to claim 1, characterized in that, The understory soil covering cultivation includes the steps of selecting a planting site and preparing materials, as follows: Select a shady forest land at the foot of a hillside with loose and well-drained soil, prepare planting beds 0.5-1m wide and 30-70cm high, and loosen the topsoil with a thickness of 5-10cm; cut the tree material into specifications of 5-10cm in diameter and 50-75cm in length, take half of the tree material and soak it for 36-72 hours, take it out and drain it 12 hours before cultivation, and set it aside.

5. The method for artificial cultivation of *Leiwan* according to claim 4, characterized in that, The understory soil-covered cultivation also includes the following cultivation steps: a 1-2cm thick layer of sawdust is laid on the surface soil of the cultivation bed, then a layer of main material formed by bamboo leaves is evenly laid on the sawdust layer, tree material is placed on the main material layer in a dry-wet interval, cultivars are evenly placed between the tree material, and bamboo stalks are filled in the gaps between the cultivars, then another layer of main material is evenly laid on top of the cultivars, and finally a 5-6cm thick layer of sandy soil in a tile-back shape is covered to begin cultivation.

6. The method for artificial cultivation of *Lycopodium clavatum* according to claim 5, characterized in that, The sandy soil layer uses coarse-grained river sand with a moisture content of 40-60%; the cultivation conditions during the cultivation period are: cultivation temperature 24-26℃, and soil moisture control at 40-60%.

7. The method for artificial cultivation of *Lycopodium clavatum* according to claim 1 or 5, characterized in that, The total amount of bamboo leaves used in the main material layers on both sides of the cultivated variety is 5.0-6.5 kg / m². 2 Furthermore, the amount of bamboo leaves used in the main material layer on each side is the same.

8. The method for artificially cultivating *Lycopodium clavatum* according to claim 1 or 5, characterized in that, The amount of the cultivar used is 4-8 packs / m³. 2 The amount of bamboo stalks used is 3-8 kg / m². 2 The length of the bamboo stalk is 5-7cm.

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