A northern cold region morel WZ-23 and a cultivation method of its fruiting body

Abstract

A strain of northern cold-region Morchella WZ-23 and a cultivation method of its fruiting body, the present invention relates to a strain of northern cold-region Morchella WZ-23 and a cultivation method of its fruiting body. The purpose of the present invention is to solve the problem of low yield of existing Morchella under low temperature conditions. The present invention is a strain of northern cold-region Morchella WZ-23, which is Morchella eohespera WZ-23 from three places, preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation date is December 5, 2023, and the preservation number is CCTCC NO: M 20232465. The strain screened by the present invention solves the problem of unstable yield and traits in the northern cold region because the main cultivated varieties are southern varieties. The present invention is applied to the field of edible fungi.

Description

Technical Field

[0001] The invention relates to a northern cold region morel WZ-23 and a cultivation method of its fruiting body. Background Art

[0002] Morchella esculenta (L.) Pers., belonging to the Morchella genus of the Morchaceae family of the Ascomycetes order of fungi, is recognized as a rare edible and medicinal fungus in the world. Morchella prefers low temperature and high humidity environments and is distributed all over the world. The main production areas in my country are Yunnan, Guangdong, Fujian and other places. Morchella is delicious and has a unique taste. It is rich in protein and a variety of essential amino acids for the human body and has a low fat content. It is a high-end food. At the same time, Morchella also has important medicinal value. The ancient Chinese medicine book "Compendium of Materia Medica" records that it "tastes sweet and cold, is neutral in nature, non-toxic, benefits the stomach and intestines, and resolves phlegm and regulates qi." The selenium and various bioactive substances rich in Morchella can inhibit the growth and spread of tumor cells, and have anti-tumor and immune-enhancing effects. Nowadays, as people's understanding of the efficacy of Morchella deepens, Morchella has become a high-end tonic and is widely popular in the market.

[0003] Wild resources of morels are scarce, and they are cultivated in many places across the country. At present, the strains used for cultivation of morels in my country are mostly the six-sister morel and ladder-shaped morel strains domesticated from wild morels in the south. However, these two main varieties cultivated in the south often have unstable yields and traits in the north with lower temperatures, especially in cold regions such as Heilongjiang. Even young mushrooms that grow relatively well often have dead and deformed mushrooms, which seriously restricts the development of the morel industry in the cold regions of the north. Summary of the invention

[0004] The purpose of the invention is to solve the problem of low yield of existing Morchella under low temperature conditions and to provide a northern cold region Morchella WZ-23 and a cultivation method of its fruiting body.

[0005] The present invention discloses a northern cold region morel WZ-23, which is a Sandi Morel (Morchella eohespera) WZ-23, which is preserved in China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation date is December 5, 2023, and the preservation number is CCTCC NO:M 20232465.

[0006] The invention collects the wild strain BF2020 of northern cold-region Morchella in Tongjiang City, Heilongjiang Province at latitude 47.754610 and longitude 132.643900, and obtains the strain WZ-23 after multi-spore separation and mycelium purification.

[0007] Morphological identification of WZ-23: fruiting body in robust stage, 4-10cm high, 3-6cm wide; cap is black, dome-shaped, ovate to elliptical, nearly spherical, with sheep's stomach-like veins on the surface, obvious veins, horizontal and vertical ridges cross to form obvious pits, the bottom of the cap is connected with the stipe, and the junction is obviously concave; the stipe is short, hollow, cylindrical, white, and smooth. The base of the stipe is swollen with irregular grooves. The ascus is an octopus spore ascus. After collecting and arranging relevant information on Morchella species, it was found that the morphological characteristics of the strain WZ-23 of the present invention are consistent with those of the three-place Morchella, and WZ-23 was preliminarily identified as the three-place Morchella.

[0008] DNA of the WZ-23 strain was extracted, and amplified using ITS1 and ITS4 as primers (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'; ITS4: 5'-TCCTCCGCTTATTGATATGC-3'). The results of PCR amplification were detected by agar gel electrophoresis, and the PCR products were recovered using an AxyPrep DNA gel recovery kit and sequenced by Nanjing Paisono Gene Technology Co., Ltd. The DNA sequence information was compared with the data in the NCBI nucleic acid database using the NCBI BLAST tool, and it was found that the ITS sequence of WZ-23 and the ITS sequence of Morchella eohespera were as high as 99.86% similar, indicating that the WZ-23 strain of the present invention is likely to belong to Morchella eohespera in the genus Morchella. In the comparison results, sequences with high similarity were selected for downloading, and a phylogenetic tree was established using MEGA 11.0 software. It was identified as Morchella eohespera in the combination of BLAST and phylogenetic tree results, and finally named Morchella eohespera WZ-23.

[0009] The cultivation method of the fruiting body of the northern cold region Morchella WZ-23 of the present invention is carried out according to the following steps:

[0010] 1. Preparation of mother culture: put the mother culture medium into a test tube, sterilize it, and then condense it into a slant for later use; inoculate the Morchella WZ-23 bacterial block into the slant test tube under a sterile environment, and the culture temperature is 16-18°C to obtain the WZ-23 mother culture;

[0011] 2. Stock seed production: Inoculate the WZ-23 mother seed onto the stock seed culture medium under sterile conditions and culture at a temperature of 18-20°C, relative humidity ≤40%, and keep away from light to obtain the WZ-23 stock seed;

[0012] 3. Cultivated seed production: Inoculate the WZ-23 stock into the cultivated seed culture medium under sterile conditions and culture at a temperature of 18-20°C, relative humidity ≤40%, and keep away from light to obtain WZ-23 cultivated seeds;

[0013] 4. Land preparation and ridge making: before sowing, weeds on the greenhouse ground should be removed. The ridge should be 100-120cm wide, 15-20cm high, 25-30cm apart, and the surface should be flat.

[0014] 5. Sowing and covering: Before sowing, adjust the soil moisture to 25-30%, sow the cultivated seeds, spray water after sowing, and cover with film;

[0015] 6. Management of fungus growth: Control the temperature in the greenhouse at 10-18°C, relative humidity at 65-70%, and cultivate under dark light;

[0016] 7. Placement of nutrient bags: When white mycelium appears on the surface of the bed, place the nutrient bags at a density of 5 to 6 bags / m 2 , coating;

[0017] 8. Management of mushroom stimulants: For greenhouse cultivation, spray water 45-60 days after sowing. After the winter, remove the mulch film on the bed, remove the nutrient bags, and spray water.

[0018] IX. Management of mushroom production: The temperature in the greenhouse is controlled at 10-18℃, the ground temperature is controlled at 8-16℃; the relative humidity of the air is 85-95%, the soil humidity of the bed is 25-30%; the light duration is 4-6 hours per day;

[0019] 10. Harvesting.

[0020] Beneficial effects of the present invention:

[0021] 1. The WZ-23 of the present invention has good low temperature adaptability and is suitable for cold-region cultivation. The suitable culture temperature of WZ-23 mycelium is 16-18°C, which is lower than the suitable culture temperature of 20-25°C of general morel mycelium. At the cold-region cultivation temperature of 12°C, the growth of WZ-23 is significantly better than the currently commonly cultivated six-sister variety, with a faster growth rate, higher mycelium density, and more neat and obvious colony edges. Therefore, compared with the currently commonly cultivated morel varieties, WZ-23 has better low temperature adaptability and is suitable for planting in the northern cold regions. It can solve the problem of unstable yield and properties of morels in the northern cold regions due to differences between regions and acclimatization caused by the main cultivated varieties being southern varieties. According to measurements, the yield of WZ-23 per mu reaches 175-200kg, and the yield per mu benefit can reach 50,000 to 100,000 yuan.

[0022] 2. The WZ-23 of the present invention can be widely promoted and cultivated in the northern cold regions. It solves the problem of unstable yield and traits in the northern cold regions because the main cultivated varieties are southern varieties, and will further promote the industrialization and large-scale development of Morchella cultivation in the northern cold regions. At the same time, it can make up for the vacancy in the fresh Morchella market caused by the inability to produce mushrooms due to the temperature rise in the south from May to June, bring higher income to mushroom farmers, and help rural revitalization.

[0023] 3. The present invention breeds cold-resistant Morchella that is suitable for planting in northern cold regions, thus enriching the germplasm resources of Morchella. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a photo of the fruiting body of WZ-23;

[0025] Figure 2 The results of agar gel electrophoresis after PCR amplification of the ITS sequence of WZ-23;

[0026] Figure 3 This is the phylogenetic tree diagram of WZ-23;

[0027] Figure 4 This is a graph of the mycelium growth of WZ-23;

[0028] Figure 5 This is a comparison of the growth of WZ-23 and Morchella Liumei under 12℃ culture conditions. DETAILED DESCRIPTION

[0029] Specific implementation method 1: In this implementation method, a strain of northern cold-region morel WZ-23 is Morchella eohespera WZ-23, which is preserved in the China Type Culture Collection Center, the preservation address is Wuhan University, Wuhan, China, the preservation date is December 5, 2023, and the preservation number is CCTCC NO:M 20232465.

[0030] In this embodiment, the wild strain BF2020 of northern cold-region Morchella was collected in Tongjiang City, Heilongjiang Province at latitude: 47.754610, longitude: 132.643900, and the WZ-23 strain was obtained after multi-spore separation and mycelium purification.

[0031] Specific implementation method 2: Classification and identification of strain WZ-23:

[0032] 1. Morphological identification of WZ-23

[0033] In the robust stage of fruiting body, the height is 4-10cm and the width is 3-6cm; Figure 1 As shown, the cap is black, dome-shaped, ovate to elliptical, nearly spherical, with sheep's stomach-like veins on the surface, the veins are obvious, and the horizontal and vertical ridges cross to form obvious pits. The bottom of the cap is connected to the stipe, and the junction is obviously sunken; the stipe is short, hollow, cylindrical, white, and smooth. The base of the stipe is swollen and has irregular grooves. The ascus is an octosporic ascus. It was found that the morphological characteristics of the strain WZ-23 of the present invention are consistent with those of the Sandi Morel, and WZ-23 was preliminarily identified as Sandi Morel.

[0034] 2. Molecular biological identification of WZ-23

[0035] The ITS sequencing process is as follows:

[0036] WZ-23 DNA was extracted and amplified using ITS1 and ITS4 as primers. The sequences of ITS1 and ITS4 are as follows:

[0037] ITS1

[0038] 5′-TCCGTAGGTGAACCTGCGG-3′;

[0039] ITS4

[0040] 5′-TCCTCCGCTTATTGATATGC-3′.

[0041] PCR reaction system: total volume 50.0uL: genomic DNA (20ng / uL) 1.0uL, 10× Buffer (containing 2.5mMMg 2+ )5.0uL, Taq polymerase (5u / uL) 1.0uL, dNTP (10mM) 1.0uL, ITS1 primer (10uM) 1.5uL, ITS4 primer (10uM) 1.5uL, ddH2O 39.0uL.

[0042] PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 1 min; final extension at 72°C for 7 min; number of cycles: 35.

[0043] The results of PCR amplification were detected by agarose gel electrophoresis. Figure 2 As shown. The PCR product was recovered using an AxyPrep DNA gel recovery kit and sequenced by Nanjing Paisonno Gene Technology Co., Ltd. to obtain the nucleotide sequence (ITS sequence) of the DNA fragment. The sequence information is shown in SEQ ID No. 1, and the sequence is a 712bp DNA fragment. The obtained DNA sequence information was compared with the data in the NCBI nucleic acid database using the NCBI BLAST tool, and it was found that the ITS sequence of WZ-23 and the ITS sequence of Morchella sandii were as similar as 99.86%, indicating that the WZ-23 strain of the present invention is likely to belong to the Morchella sandii genus in terms of classification. Sequences with high similarity were selected from the comparison results for downloading. A phylogenetic tree was established using MEGA11.0 software ( Figure 3), it was found that the strain WZ-23 of the present invention and Morchella eohespera can form a stable branch with a credibility of 98%, and the BLAST comparison results showed that the strain WZ-23 was different from any known Morchella eohespera strain. Therefore, the strain WZ-23 of the present invention was identified as a new Morchella eohespera strain through the BLAST comparison results combined with the phylogenetic tree, and was finally named Morchella eohespera WZ-23.

[0044] Specific implementation method 3: The cultivation method of Morchella eohespera WZ-23 fruiting body is carried out according to the following steps:

[0045] 1. Preparation of mother stock

[0046] The formula of the mother culture medium is: 200g peeled potato, 20g glucose (sucrose), 15-20g agar, 1000mL distilled water. The preparation method is: cut the peeled potato into small pieces, heat it in water for 15-20min, filter it with gauze after boiling, and obtain potato extract. Then add 20g glucose and 15-20g agar, and make the volume to 1000mL to obtain the mother culture medium. The culture medium is divided into test tubes, and the amount of culture medium accounts for about 3 / 1 of the test tube. Tie the test tube tightly with kraft paper, put it into a high-pressure steam sterilizer at 121℃ for 30min, and immediately place it in the lower layer of the test tube rack after sterilization, and condense it into a slope for use. Then, in a sterile environment, use a sterilized inoculation shovel to inoculate the WZ-23 bacterial block into the inclined test tube. The culture temperature is 16-18℃, and the test tube can be full in 7-10 days to obtain the WZ-23 mother culture.

[0047] 2. Original seed production

[0048] The formula of the original culture medium is: 73% wheat, 20% sawdust, 5% rice husk, 1% lime, 1% gypsum or 40% wheat, 38% sawdust, 15% wheat bran, 5% humus, 1% lime, 1% gypsum. Pre-wet the rice husk, corn cob, and humus according to the proportion of the original culture medium formula; the wheat can be soaked in water for 24 to 48 hours, or the wheat grains can be steamed with water until there is no white core, then the water is removed and the water is controlled, mixed with sawdust, and other raw materials in the formula are added, stirred evenly, and the water content is 60 to 65%, and the pH is 7.0 to 7.5. For bagging, use a 550 to 1000mL strain bottle or a polyethylene bag or polypropylene bag with a size of (12 to 14cm) × (24 to 28cm) × (0.004 to 0.006cm). The mixed materials on the same day should be packed on the same day. The packed bottles or bags should be tight on the top and loose on the bottom, the material surface should be flat, and the inoculation holes should be punched. The bottle mouth can be sealed with a sealing film, and the bag mouth can be sealed with a cotton-free cover. Autoclave, the temperature reaches 121-125℃, maintain for 2-3 hours, and then simmer for 0.5-1.5 hours after stopping the fire. When the temperature drops below 28℃, dig out the mother seed obtained in step 1 under sterile conditions and inoculate it on the original seed culture medium for cultivation. The culture room must be clean and light-proof, the temperature should be controlled at 18-20℃, the relative humidity should be controlled below 40%, and light-proof management should be carried out throughout the process to keep the indoor air fresh to obtain the WZ-23 original seed.

[0049] 3. Cultivated species production

[0050] The formula of the culture medium for cultivation is: 73% wheat, 20% sawdust, 5% rice husk, 1% lime, 1% gypsum or 40% wheat, 38% sawdust, 15% wheat bran, 5% humus, 1% lime, 1% gypsum. Pre-wet the rice husk, corn cob and humus according to the proportion of the culture medium formula; the wheat can be soaked in water for 24 to 48 hours, or the wheat grains can be steamed with water until there is no white core, then the wheat grains are removed and the water is controlled, mixed with sawdust, and other raw materials in the formula are added, stirred evenly, and the water content is 60 to 65%, and the pH is 7.0 to 7.5. For bagging, use polyethylene bags or polypropylene bags of 16cm×(35 to 37cm)×(0.004 to 0.006cm). The mixed materials should be packed on the same day, and the packed bottles or bags should be tight on the top and loose on the bottom, with the material surface flat and inoculated holes punched. The bottle mouth can be sealed with a sealing film, and the bag mouth can be sealed with a cotton-free cover. High pressure sterilization, the temperature reaches 121 ~ 125 ℃, maintain for 2 ~ 3 hours, and then simmer for 0.5 ~ 1.5 hours after stopping the fire. When the temperature drops below 28 ℃, the WZ-23 original seed is inoculated into the culture medium for cultivation. The culture temperature is controlled at 18 ~ 20 ℃, the relative humidity is controlled below 40%, and the whole process is kept away from light to keep the indoor air fresh to obtain the WZ-23 cultivar.

[0051] 4. Land preparation and ridge making

[0052] Before sowing, weeds should be removed from the greenhouse ground every 667m 2Evenly spread 75 - 100 kg of quicklime on the land, rototill the soil to a depth of 25 - 30 cm, expose it to the sun for at least 7 days, water the soil 1 - 3 times until thoroughly wet, and make ridges in a timely manner. The width of the ridge bed is 100 - 120 cm, the height is 15 - 20 cm, the spacing is 25 - 30 cm, and the length depends on the length of the greenhouse, with the surface being flat.

[0053] 5. Sowing and Covering Soil

[0054] Before sowing, adjust the soil moisture to 25 - 30%. Sow the WZ - 23 cultivated species by broadcast sowing and drill sowing. For every 667 m 2 The amount of spawn used is 180 - 200 kg, and the thickness of the covered soil is 2 - 3 cm. After sowing, spray water in a fine mist, water thoroughly, cover with black plastic film, and press the film down with soil.

[0055] 6. Spawn Run Management

[0056] Regulate the temperature in the greenhouse to 10 - 18°C, the relative humidity in the air to 65 - 70%, cultivate in low light, and keep the air in the greenhouse fresh.

[0057] 7. Placement of Nutrient Bags

[0058] When white mycelium (mycelial frost) appears on the surface of the ridge bed, nutrient bags can be placed. Make 2 longitudinal slits on one side of the nutrient bag, with a length of 10 cm and an interval of 2.5 - 3.0 cm between the slits. Gently press the side with the slits down to make full contact with the surface of the ridge bed, and arrange them in a "pin" shape. The placement density is 5 - 6 bags / m 2 , cover with black plastic film, and press it down with soil.

[0059] 8. Mushroom Primordium Induction Management

[0060] For greenhouse cultivation, 45 - 60 days after sowing, when the mycelial frost on the surface of the ridge bed begins to fade and water droplets appear on the mycelium, spray a thorough watering. Keep scattered light in the greenhouse. After the winter ends, when the thawing depth of the soil layer in the greenhouse reaches 15 - 20 cm and the temperature at 10 cm below the soil layer reaches about 8°C, remove the black plastic film on the ridge bed, remove the nutrient bags, and spray a thorough watering.

[0061] 9. Fruitbody Formation Management

[0062] Control the temperature in the greenhouse at 10 - 18°C and the ground temperature between 8 - 16°C; the relative humidity in the air is 85 - 95%, and the soil moisture of the ridge bed is 25 - 30%; the daily light duration is 4 - 6 h, with scattered light, and maintain good ventilation and fresh air.

[0063] 10. Harvesting

[0064] When the top of the fruitbody begins to change color and the honeycomb - shaped ascoma part has unfolded, harvest in a timely manner.

[0065] The fruitbodies cultivated in this embodiment are applied in foods, health products, beverages, and pharmaceuticals.

[0066] Specific embodiment 4: This embodiment is different from specific embodiment 3 in that the formula of the mother culture medium is: 200 g peeled potatoes, 20 g glucose, 15-20 g agar, and 1000 mL distilled water. The rest is the same as specific embodiment 3.

[0067] Specific embodiment 5: This embodiment is different from specific embodiment 3 or 4 in that the original seed culture medium formula is: 73% wheat, 20% sawdust, 5% rice husk, 1% lime, 1% gypsum by mass fraction. The rest is the same as specific embodiment 3 or 4.

[0068] Specific embodiment 6: This embodiment is different from specific embodiments 3 to 5 in that the original seed culture medium formula is: 40% wheat, 38% sawdust, 15% wheat bran, 5% humus, 1% lime, 1% gypsum by mass fraction. The rest is the same as specific embodiments 3 to 5.

[0069] Specific embodiment 7: This embodiment is different from any one of specific embodiments 3 to 6 in that the culture medium formula is: 73% wheat, 20% sawdust, 5% rice husk, 1% lime, 1% gypsum. The rest is the same as any one of specific embodiments 3 to 6.

[0070] Specific embodiment 8: This embodiment is different from specific embodiments 3 to 7 in that the culture medium formula is: 40% wheat, 38% sawdust, 15% wheat bran, 5% humus, 1% lime, and 1% gypsum. The rest is the same as specific embodiments 3 to 7.

[0071] Specific implementation method 9: This implementation method is different from any one of the specific implementation methods 3 to 8 in that: in step 5, every 667m 2 The amount of soil used for cultivation is 180-200 kg, and the thickness of soil covering is 2-3 cm. The rest is the same as that of the third to eighth embodiments.

[0072] The low temperature adaptability of Morchella eohespera WZ-23 of this embodiment was verified:

[0073] Test 1, WZ-23 growth rate determination experiment:

[0074] The WZ-23 bacterial block was inoculated in the center of the PDA plate under a sterile environment, 6 replicates were set up, and cultured at 17°C. Figure 4 The graph of WZ-23 mycelium growth is shown in Table 1. The mycelium growth was measured and recorded every 24 hours using the cross method. Each plate was measured three times until the mycelium covered the entire plate. The growth rate of WZ-23 was then determined. The experimental results are shown in Table 1:

[0075] Table 1 WZ-23 growth rate measurement data

[0076]

[0077] Experiment 2: Comparative experiment on low temperature adaptability of WZ-23 and Morchella liumei at 12℃

[0078] In a sterile environment, WZ-23 and Liumei bacteria were inoculated in the center of the PDA plate and cultured at 12°C. Six replicates were set for each sample. The mycelial growth was measured and recorded using the cross method every 24 hours. Each plate was measured three times until the mycelium covered the entire plate. The mycelial density and the neatness of the colony edges were observed and recorded. Figure 5 As shown in Table 2:

[0079] Table 2 Differences in low temperature adaptability between WZ-23 and Morchella liumei at 12℃

[0080]

[0081] From the data in Table 2, it can be seen that under the low temperature culture condition of 12℃, the growth rate of WZ-23 is faster than that of Morchella liumei, the mycelium is denser, and the colony edge is more neat. It can be concluded that WZ-23 grows better than Morchella liumei under the low temperature cultivation environment of 12℃ and has stronger low temperature adaptability.

Claims

1. A strain of northern cold region Morchella WZ-23, characterized in that It is Morchella eohespera WZ-23, which is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is December 5, 2023, and the deposit number is CCTCC NO:M 20232465.

2. The method for cultivating the fruiting body of the northern cold region morel WZ-23 as claimed in claim 1, characterized in that The cultivation method is carried out according to the following steps:

1. Preparation of mother culture: the mother culture medium is divided into test tubes, then sterilized, and then condensed into a slant for standby use; inoculate the Morchella WZ-23 bacterial block into the slant test tube under a sterile environment, and the culture temperature is 16-18°C to prepare the WZ-23 mother culture; the formula of the mother culture medium is: 200g peeled potato, 20g glucose, 15-20g agar, and 1000mL distilled water; 2. Stock seed production: Inoculate WZ-23 mother seed onto stock seed culture medium under sterile conditions, culture at a temperature of 18-20°C, relative humidity ≤40%, and keep away from light to obtain WZ-23 stock seed; wherein the stock seed culture medium formula is as follows by mass fraction: 73% wheat, 20% sawdust, 5% rice husk, 1% lime, 1% gypsum; or as follows by mass fraction: 40% wheat, 38% sawdust, 15% wheat bran, 5% humus, 1% lime, 1% gypsum; III. Cultivated seed production: inoculate WZ-23 stock onto cultivar culture medium under sterile conditions, culture at a temperature of 18-20°C, relative humidity ≤40%, and keep away from light to obtain WZ-23 cultivars; wherein the cultivar culture medium formula is: 73% wheat, 20% sawdust, 5% rice husk, 1% lime, 1% gypsum; or the formula is: 40% wheat, 38% sawdust, 15% wheat bran, 5% humus, 1% lime, 1% gypsum; 4. Land preparation and ridge making: before sowing, weeds on the greenhouse ground should be removed. The ridge should be 100-120cm wide, 15-20cm high, 25-30cm apart, and the surface should be flat.

5. Sowing and covering: Before sowing, adjust the soil moisture to 25-30%, sow the cultivated seeds, spray water after sowing, and cover with film; Every 667m 2 The amount of soil used for cultivation is 180-200 kg, and the thickness of soil covering is 2-3 cm; 6. Management of fungus growth: Control the temperature in the greenhouse to 10-18℃, relative humidity to 65-70%, and cultivate in dark light; 7. Placement of nutrient bags: When white mycelium appears on the surface of the bed, place the nutrient bags at a density of 5 to 6 bags / m 2 , coating; 8. Management of mushroom stimulants: For greenhouse cultivation, spray water 45-60 days after sowing. After the winter, remove the mulch film on the bed, remove the nutrient bags, and spray water. IX. Management of mushroom production: The temperature in the greenhouse is controlled at 10-18℃, the ground temperature is controlled at 8-16℃; the relative humidity of the air is 85-95%, the soil humidity of the bed is 25-30%; the light duration is 4-6 hours per day; 10. Harvesting.

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