A white stropharia strain and a cultivation method

Abstract

The invention belongs to the technical field of edible fungus cultivation, and specifically discloses a white Stropharia rugosoannulata strain and a cultivation method. A white Stropharia rugosoannulata strain Stropharia rugosoannulata was obtained for the first time by cobalt 60 irradiation mutagenesis breeding. It was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on April 20, 2023, with a deposit number of CGMCC No. 40581, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The strain has the outstanding feature of a white fruiting body, and its mycelium grows whiter, thicker and more uniformly than that of the original reddish-brown Stropharia rugosoannulata strain. The color of the fruiting body cap is special, which is white. It has strong stress resistance and genetic stability. Compared with the existing known Stropharia rugosoannulata varieties, it has better commerciality and higher quality, and has good promotion value.

Description

Technical Field

[0001] The invention relates to the technical field of edible fungus cultivation, in particular to a white Stropharia rugosa strain and a cultivation method. Background Art

[0002] Stropharia rugosoannulata, belonging to the phylum Basidiomycetes, class Hymenomycetes, order Agaricales, family Strophariaceae, genus Stropharia, also known as wrinkled globe mushroom, wrinkled Stropharia, wine-red Stropharia, and often called "red pine mushroom" in the market, is a nutritious and delicious edible mushroom with great development potential. As one of the edible mushroom cultivation varieties recommended by the Food and Agriculture Organization of the United Nations, Stropharia rugosoannulata is bright in color and delicious in taste. It is rich in 17 kinds of essential amino acids and other amino acids for the human body, and also contains various mineral elements, vitamins, multiple amino acids and other nutrients required by humans. It has antiviral and anti-tumor effects, can also reduce cholesterol content and prevent arteriosclerosis. The fruiting body is rich in dietary fiber, which helps digestion and has a laxative function. Stropharia rugosoannulata has strong resistance to bacteria and stress; it has a wide range of adaptability to temperature; it has strong ability to degrade cellulose and lignin, and has a wide source of cultivation materials, and can use a variety of agricultural and forestry waste resources; it has high yield and has good economic, ecological and social benefits.

[0003] The caps of the Stropharia capiensis varieties commonly sold in the market are reddish brown to wine red, or dark brown. No white Stropharia caps are seen circulating in the market. There are many varieties of Stropharia caps used in production, and the phenomenon of homonymous species and homonymous species is serious. The genetic background is narrow, and new varieties with characteristics are urgently needed to meet market demand. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a white Stropharia officinalis strain and a cultivation method.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] One of the purposes of the present invention is to provide a white giant puffball strain Strophariarugosoannulata, which is Liaobai Puffball No. 1, which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on April 20, 2023, with a deposit number of CGMCC No. 40581, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The second object of the present invention is to provide a method for cultivating white stropharia, comprising the following steps:

[0008] S1. After activating the Stropharia nematodea strain No. 1 with a plate culture medium, a 5 mm punch was used to take a bacterial block and inoculated into a PDA solid culture medium, and cultured at a temperature of 25° C. to obtain a bacterial block;

[0009] S2. Take 30% sawdust, 30% corn cobs, 20% rice husks, 10% wheat bran, 8% soybean meal and 2% lime by weight to prepare the culture raw materials. Mix the culture raw materials and add water to adjust the moisture content to 65-70%. Build a pile for fermentation. The top width of the pile is 0.8m, the bottom width is 2.2m, the height of the pile is 1.2m, and the length is unlimited. After the pile is built, 2 rows of ventilation holes are made in a herringbone shape on both sides of the longitudinal direction, which are connected to the bottom of the pile. The hole spacing is 0.5m and the diameter of the ventilation holes is about 0.1m. When the material temperature reaches 50°C, maintain it for 24h and turn the pile once. When the material temperature rises to 55°C again, maintain it for 24h and turn the pile twice. The total number of turning the pile is 3 to 4 times.

[0010] S3. Take 44% corn stalks, 30% rice husks, 15% sawdust, 8% wheat bran and 3% light calcium carbonate by weight to prepare the cultivation materials. After mixing the cultivation materials, add water to adjust the moisture content to 65-70%. 3-5 days before sowing, irrigate the ground, and after the water penetrates, perform rotary tillage to a depth of 15-20cm, harrow and level the ground at 40g / m 2 Sprinkle quicklime on the surface; spread a layer of 10cm thick cultivation material, sow a layer of mushroom blocks using the V-shaped spot sowing method, cover it with a layer of 10cm thick cultivation material and sow another layer of mushroom blocks, three layers of materials and two layers of mushroom blocks, the dosage of Liaobai Stropharia No. 1 strain is 500-800g / m 2 , the material temperature is maintained at 22-25℃ after sowing, about 7 days after sowing, cover with soil after the mycelium germinates; after covering with soil, spread 2cm thick straw on the bed, spray water appropriately to ensure the soil is moist;

[0011] S4. The temperature in the material bed is 22-25℃. The air in the spawning environment is kept fresh. Ventilate 2-3 times a day for 20-30 minutes each time to ensure that the soil is moist. The mycelium can grow into the covering soil in 30-35 days, and then enter the fruiting management stage;

[0012] S5. Management of mushroom production: Scatter light, spray large amounts of water for 1 hour to allow moisture to penetrate the covering soil and seep into the surface of the culture medium. The relative humidity of the air should be 80% to 85%. Increase ventilation to promote mycelium entanglement. Maintain the temperature of the material bed at 20 to 25°C. The first wave of mushroom buds will be produced in 5 to 7 days.

[0013] Compared with the prior art, the present invention uses a systematic breeding method to obtain for the first time a white giant stropharia strain Stropharia rugosoannulata, which was preserved in the General Microbiology Center of the China Microbiological Culture Collection Administration on April 20, 2023, with a preservation number of CGMCC No. 40581, and a preservation address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The outstanding feature of this strain is that the fruiting body is white, and the mycelium growth is denser than that of the original reddish-brown giant stropharia strain. The color of the fruiting body cap is more special, which is white, and it has strong stress resistance and genetic stability. Compared with the existing known giant stropharia varieties, it has better commerciality and higher quality, and has better promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of antagonism test of different strains.

[0015] Figure 2 The ISSR molecular marker identification results of different strains.

[0016] Figure 3 The effect of different carbon sources on the mycelium growth rate and growth potential of Stropharia nematodes 1 (XQ).

[0017] Figure 4 The effect of different nitrogen sources on the mycelium growth rate and growth potential of Stropharia nematodes 1 (XQ).

[0018] Figure 5 This paper shows the effects of different inorganic salts on the mycelium growth rate and growth potential of Stropharia nematodes 1 (XQ).

[0019] Figure 6 This is the growth status of Liaobai Stropharia No. 1 (XQ) at different temperatures.

[0020] Figure 7 This is a photo of the fruiting body of Liaobai Stropharia No. 1. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0022] Example 1: Screening and identification of Stropharia nematodes strain No. 1

[0023] 1) Parent strain: FQ-1, isolated from fruiting body tissue collected in Yixian County, Jinzhou City, Liaoning Province. The strain is preserved in the Liaoning Edible Fungi Germplasm Resource Bank.

[0024] 2) Spore acquisition and processing: After the FQ-1 strain has produced mushrooms, select 10 fresh large ball cap bodies with strong fruiting bodies, typical characteristics, and about to open the umbrella, cut off the stems, put a sterilized kraft paper at the bottom of the wide-mouthed tissue culture bottle, place the fruiting bodies with the gills facing down in a sterile tissue culture bottle, cover the bottle mouth with a double layer of sterile newspaper, and put it tightly with a leather cover. Leave it at room temperature for 24 hours to obtain 10 spore prints. Cut 1cm of each spore print 2 Place in a 250 ml Erlenmeyer flask containing 100 ml of sterile water and shake well.

[0025] 3) Mutagenesis treatment: The flask containing spores was placed under a 60Co-γ-ray radiation source. The radiation conditions were 0.8, 1.2, and 1.6 KGy, and the dose rate was 10 Gy / min.

[0026] 4) Screening of mutant strains: After 60Co-γ-ray irradiation, on the clean bench, mix the spore liquid of two different fruiting bodies and take 100 microliters with a pipette, and spread it on the plate containing PDA culture medium, a total of 200 plates. Incubate inverted in the dark in an incubator at 25°C. After 10 days, pick out the regenerated spore colonies in the culture dish, and pick out the discontinuous hyphae after they are full and inoculate them in a new dish, marked as the first generation. This cycle of activation is repeated for 5 generations to find strains with relatively stable traits. Use a microscope to observe whether the induced strain has a lock-like union, and eliminate strains without a lock-like union.

[0027] 5) Mutant strain fruiting test: The mutants obtained after screening were expanded and cultured to prepare mother strains and cultivars. Then, they were managed for field cultivation in a solar greenhouse according to conventional cultivation methods, and the mycelium germination time, primordium formation time, and fruiting time were recorded; the agronomic traits of the fruiting bodies were investigated, including the long diameter of the cap, the short diameter of the cap, the length and diameter of the stipe, the cap thickness, the cap color, and the yield.

[0028] 6) Obtaining white spherical cap strain: Two white mutant fruiting bodies were obtained in the fruiting experiment. The mutant strain was retained and two more fruiting experiments were conducted. It was found that the yellow cap trait can be stably inherited, and the mycelium growth rate is faster than that of the original strain. It also has bright colors, high yield and excellent quality.

[0029] 7) ITS Identification

[0030] The genomic DNA of 'Liaobai Stropharia No. 1' was extracted according to the kit operation steps and detected by 1% agarose gel electrophoresis. The universal primer ITS1 of the fungal ribosomal gene transcribed spacer region was used.

[0031] (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO:1) and ITS4

[0032] (5'-TCCTCCGCTTA-TTGATATGC-3', SEQ ID NO:2), the primer was synthesized by Shanghai Sangon Biotechnology Co., Ltd. The extracted white giant puffball genomic DNA was used as a template, ITS1 and ITS4 (diluted to 10uM with ddH2O) were used as forward and reverse primers for PCR amplification, and the total PCR reaction amplification system was 25uL: Mix: 21uL, 1uL of forward and reverse primers ITS1 and ITS4, and 2uL of genomic DNA. After fully mixing, the amplification reaction was carried out in a PCR instrument. The PCR amplification program was: 96℃-5min, 96℃-30s, 56℃-30s, 72℃-1min, 35 cycles, 72℃-5min, 4℃-∞. The PCR amplification product was detected by 1% agarose gel electrophoresis, and the remaining product was sent to Shanghai Sangon Biotechnology Co., Ltd. for bidirectional sequencing to obtain a 650bp sequence:

[0033] 。

[0034] Enter NCBI, use BLAST to search the sequence for homology, and perform similarity analysis with the sequences of each strain in the database. The coverage rate with the sequences of each strain of Stropharia rugosoannulata in the database reaches 99%-100%, so the strain is identified as Stropharia rugosoannulata. It was deposited in the General Microbiology Center of China Microbiological Culture Collection on April 20, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 40581.

[0035] Example 2: Genetic Identification of Stropharia nematodes No. 1

[0036] The strains of 'Liaobai Stropharia No. 1 (XQ)', the original 'FQ-1' and the local main cultivated variety 'FQ-2' were inoculated and cultured in a culture dish filled with PDA comprehensive culture medium. The inoculation points were 2 cm apart and cultured at a constant temperature of 25℃ for 15 days.

[0037] Results: The culture results were as follows Figure 1 As shown, the left one is 'Liaobai Stropharia No. 1', and the right ones are 'FQ-1' and 'FQ-2'; at the junction of the confronting culture colonies, the antagonistic lines of 'Liaobai Stropharia No. 1' and the original 'FQ-1' and 'FQ-2' are shallow grooves, indicating that the screened 'Liaobai Stropharia No. 1' has differences with the original 'FQ-1' and 'FQ-2', and can be preliminarily judged as a new strain different from the original strain and the local main cultivated varieties.

[0038] ISSR molecular marker identification

[0039] Using 100 ISSR random primers published by Columbia University, after three rounds of screening, two primers (ISSR3 GAGAGAGAGAGAGAGAC; ISSR4GAGAGAGAGAGAGAGAAC) were found out from the 100 primers that could pair with 'Liaobai Stropharia No. 1' and the original starting strain 'FQ-1' and amplify polymorphic bands. Amplification was performed to observe the genetic diversity.

[0040] The results are as follows Figure 2 As shown: ISSR molecular markers were used to identify the obtained Liaobai Stropharia No. 1 and the original FQ-1 to test the specificity of the germplasm. It was found that 43 bands were amplified by using 5 primers to amplify the two strains, of which 23 were polymorphic bands, accounting for 54%. Among them, primer ISSR3 amplified a 250bp marker band that was different from the original strain FQ in the 'Liaobai Stropharia No. 1' strain; primer ISSR4 amplified two marker bands that were different from the original strain FQ at a position close to 500bp in the 'Liaobai Stropharia No. 1' strain.

[0041] Example 3, biological properties study

[0042] 1. Carbon source test

[0043] Replace the glucose in the basal medium with 20g of fructose, maltose, soluble starch, and sucrose, respectively. Use a 5mm diameter puncher to take an activated mycelium block for inoculation, place it in a constant temperature incubator at 25℃ and incubate it in the dark, and repeat it 3 times. 4 days after inoculation, use the "cross" streak method to draw a line along the growth edge of the colony and measure its diameter. Then measure it every 2 days, record the data, and calculate the mycelium growth rate.

[0044] Results: By Figure 3 As shown in Table 1, Stropharia nephritis 1 can grow normally on the five carbon source media tested, but different carbon sources have significant effects on the mycelial growth rate of 'Stropharia nephritis 1', and the growth is also different. Stropharia nephritis 1 strain grows fastest on sucrose medium, but the growth is the weakest, and the mycelium is transparent. On maltose medium, the growth rate is slower than sucrose, but faster than other carbon sources, and the growth is the best. Therefore, the most suitable carbon source for Stropharia nephritis 1 is maltose.

[0045] Table 1 Effects of different carbon sources on the mycelial growth rate and growth potential of Stropharia nematodes No. 1

[0046]

[0047] 2. Nitrogen source screening

[0048] The peptone in the basal medium was replaced with 3g urea, beef extract, yeast extract, and soy peptone, respectively. The rest of the operations were the same as those in the carbon source test.

[0049] Results: The mycelial growth of Stropharia nematodes 1 on 5 different nitrogen source media is shown in Table 2 and Figure 4 The mycelium growth rate on the medium with soybean peptone as nitrogen source was the fastest and the strongest, followed by beef extract and yeast extract, and the slowest growth was in urea medium. Therefore, the best carbon source for Stropharia nematode strain 1 was soybean peptone.

[0050] Table 2 Effects of different nitrogen sources on the mycelial growth rate and growth potential of Stropharia nematodes No. 1

[0051]

[0052] 3. Inorganic salt screening test

[0053] 3g of potassium dihydrogen phosphate, magnesium sulfate, dipotassium hydrogen phosphate, and disodium hydrogen phosphate were added to the basal medium to replace the peptone in the basal medium. The rest of the operation was the same as the carbon source test.

[0054] Results: The mycelial growth of Stropharia nematodes 1 on 5 different inorganic salt media is shown in Tables 3 and Figure 5 The growth performance of the Liaobai Stropharia No. 1 strain on different inorganic salt culture media was different. The Liaobai Stropharia No. 1 strain grew fastest and best on potassium dihydrogen phosphate culture medium, and grew slowest on magnesium sulfate culture medium. Therefore, the most suitable inorganic salt for the growth of Liaobai Stropharia No. 1 mycelium is potassium dihydrogen phosphate.

[0055] Table 3 Effects of different inorganic salts on the mycelial growth rate and growth potential of Stropharia nematodes No. 1

[0056]

[0057] 4. Temperature test

[0058] After the mother culture plate culture medium is activated, a 5mm puncher is used to take the bacterial block and inoculate it into the PDA culture medium. Six culture temperatures are set, and the culture is carried out at 5, 10, 15, 20, 25, and 30°C, respectively. Each temperature condition is set for 5 repetitions. The number of days of mycelial growth and mycelial growth potential are recorded, and the mycelial growth rate at different temperatures is calculated.

[0059] Results: As shown in Table 4 Figure 6 As shown in the results, the experiment found that Liaobai Stropharia No. 1 (XQ) can grow in the range of 5-30℃, but the growth rate is significantly different. As the temperature rises, the mycelium growth rate accelerates. At 25℃, the mycelium growth rate reaches the fastest, and then decreases. Therefore, the most suitable temperature for the mycelium growth of Liaobai Stropharia No. 1 is 25℃.

[0060] Table 4 Mycelial growth rate of Stropharia nematodes No. 1 under different temperature conditions

[0061]

[0062] Embodiment 4, cultivation and fruiting of mushrooms

[0063] S1. After activating the Stropharia nematodea strain No. 1 with a plate culture medium, a 5 mm punch was used to take a bacterial block and inoculated into a PDA solid culture medium, and cultured at a temperature of 25° C. to obtain a bacterial block;

[0064] S2. Take 30% sawdust, 30% corn cobs, 20% rice husks, 10% wheat bran, 8% soybean meal and 2% lime by weight to prepare the culture raw materials. Mix the culture raw materials and add water to adjust the moisture content to 65%. Build a pile for fermentation. The top width of the pile is 0.8m, the bottom width of the pile is 2.2m, the height of the pile is 1.2m, and the length is unlimited. After the pile is built, 2 rows of ventilation holes are made in a herringbone shape on both sides of the longitudinal direction, which are connected to the bottom of the pile. The hole spacing is 0.5m and the diameter of the ventilation holes is about 0.1m. When the material temperature reaches 50°C, maintain it for 24h and turn the pile once. When the material temperature rises to 55°C again, maintain it for 24h and turn the pile twice. The total number of turning the pile is 3 to 4 times.

[0065] S3. Take 44% corn stalks, 30% rice husks, 15% sawdust, 8% wheat bran and 3% light calcium carbonate by weight to prepare the cultivation materials. After mixing the cultivation materials evenly, add water to adjust the moisture content to 65%. 3 to 5 days before sowing, irrigate the ground, and after the water penetrates, perform rotary tillage to a depth of 15 to 20 cm, harrow and level the ground at 40 g / m 2 Sprinkle quicklime on the surface; spread a layer of 10cm thick cultivation material, sow a layer of mushroom blocks using the V-shaped spot sowing method, cover it with a layer of 10cm thick cultivation material and sow another layer of mushroom blocks, three layers of materials and two layers of mushroom blocks, the dosage of Liaobai Stropharia No. 1 strain is 500-800g / m 2 , the material temperature is maintained at 25℃ after sowing, about 7 days after sowing, cover with soil after the mycelium germinates; after covering with soil, spread 2cm thick straw on the bed, spray water appropriately to ensure the soil is moist;

[0066] S4. The temperature in the material bed is 25℃. The air in the spawning environment is kept fresh. Ventilate 2-3 times a day for 20-30 minutes each time to ensure that the soil is moist. The mycelium can grow into the covering soil in 30-35 days, and then enter the fruiting management stage;

[0067] S5. Management of mushroom production: Scatter light, spray large amounts of water for 1 hour to allow moisture to penetrate the covering soil and seep into the surface of the culture medium. The relative humidity of the air is 80% to 85%. Increase ventilation to promote mycelium entanglement. Maintain the temperature of the material bed at 25°C. The first wave of mushroom buds will be produced in 5 to 7 days.

[0068] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A white Stropharia officinalis strain ( Stropharia rugosoannulata , characterized in that, The strain is Liaobai Stropharia No. 1, which was deposited in the General Microbiology Center of China Culture Collection Administration on May 15, 2023. The deposit number is CGMCC No. 40581, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. A method for cultivating white stropharia as claimed in claim 1, characterized in that: The following steps are involved: S1. After activating the Stropharia nematodea strain No. 1 with a plate culture medium, a 5 mm borer was used to take a bacterial block and inoculated it into a PDA solid culture medium, and the block was cultured at a temperature of 25°C to obtain a bacterial block; S2. Take 30% sawdust, 30% corn cobs, 20% rice husks, 10% wheat bran, 8% soybean meal and 2% lime by weight to prepare the culture materials. Mix the culture materials well and add water to adjust the moisture content to 65-70%. Build a pile for fermentation. The top width of the pile is 0.8m, the bottom width is 2.2m, the height is 1.2m, and the length is unlimited. After the pile is built, 2 rows of ventilation holes are made in a triangular shape on both sides of the longitudinal direction, which are connected to the bottom of the pile. The hole spacing is 0.5m and the diameter of the ventilation holes is 0.1m. When the material temperature reaches 50℃, maintain it for 24 hours and turn the pile once. When the material temperature rises to 55℃ again, maintain it for 24 hours and turn the pile twice. The total number of turnings is 3-4 times. S3. Take 44% corn stalks, 30% rice husks, 15% sawdust, 8% wheat bran and 3% light calcium carbonate by weight to prepare the cultivation materials. Mix the cultivation materials evenly and add water to adjust the moisture content to 65-70%. 3-5 days before sowing, irrigate the ground, and after the water penetrates, perform rotary tillage to a depth of 15-20 cm, harrow and level the ground at 40 g / m 2 Sprinkle quicklime on the surface; spread a layer of cultivation material with a thickness of 10cm, sow a layer of mushroom blocks using the V-shaped spot sowing method, cover it with a layer of cultivation material with a thickness of 10cm and sow another layer of mushroom blocks, three layers of materials and two layers of mushroom blocks, the dosage of Liaobai Stropharia No. 1 strain is 500-800 g / m 2 After sowing, the material temperature is maintained at 22-25℃. After sowing for 7 days, cover the soil after the mycelium germinates. After covering the soil, spread 2 cm thick straw on the bed and spray water appropriately to ensure that the soil is moist. S4. The temperature in the material bed is 22-25℃. The air in the spawning environment is kept fresh. Ventilate 2-3 times a day for 20-30 minutes each time. Ensure that the soil is moist. The mycelium can grow into the covering soil in 30-35 days, and then enter the fruiting management stage. S5. Management of mushroom production: Scatter light and spray water for 1 hour to allow moisture to penetrate the covering soil and seep into the surface of the culture medium. The relative humidity of the air is 80%~85%. Increase ventilation to promote mycelium twisting. Maintain the temperature of the material bed at 20~25℃. The first wave of mushroom buds will be produced in 5~7 days.

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