CN110938547B - Lentinula edodes strain and cultivation method and application thereof - Google Patents
Lentinula edodes strain and cultivation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H15/00—Fungi; Lichens
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention discloses a wild mushroom strain and a cultivation method and application thereof, wherein the mushroom strain (a)Agaricus subfloccosus) The strain is preserved in the China general microbiological culture Collection center (preservation number CGMCC NO. 18159). Through morphological identification and molecular biological analysis of field collected mushroom strains, the mushroom is identified as the periphyton mushroom, in domestication cultivation of the periphyton mushroom, the mushroom body of the periphyton mushroom is found to have good taste and is an edible mushroom with potential edible development value, the periphyton mushroom has important application value in preparation of food, medicines and health products, and meanwhile, research and development of the periphyton mushroom has important significance for protecting germplasm resources of wild mushrooms and genetic breeding.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to edible value mushroom fungus and application thereof.
Background
Mushroom of quilted margin (Agaricus subfloccosus) Taxonomically belonging to the kingdom Fungi (Fungi), the phylum Basidiomycota (Basidiomycota), the class Agaricales (Agaricamycetes), the order Agaricales (Agaricales), the family Agaricaceae (Agaricaceae), the genus Agaricales (Agaricaceae) (fungus)agaricus). Genus parachuting (A)agaricus）The mushroom is a model genus of the family Agaricus (Agaricaceae), and is mainly characterized in that: the pileus has more scales; the stipe is middle-growing and has a fungus ring; the fungus folds are separated from the raw material, the fungus folds are white in the initial stage and become pink, light brown, reddish brown to dark brown in the later stage; the mushroom meat is mostly composed of non-starch reaction hypha; the spores were light brown to dark brown. The mushrooms are widely distributed all over the world, and more than 400 varieties exist. China's mushrooms range from Liaoning, inner Mongolia, Jilin, Heilongjiang to Yunnan, Sichuan, Gansu, Tibet and Xinjiang, but only about 50 kinds are reported in China.
The national natural protection area of Gansu Qilianshan is located in northern slope of Qilianshan in Gansu province, east, and is across Wuwei, Jinchang, Zhangye 3 City Liangzhou, Tianzhu Tibetan autonomous county, Gulang, Yongchang, Ganzhou, Shandan, folk music, Su nan Gugu county 8 county (area), the geographic location is located between 97 deg. 23 '34' 'to 103 deg. 45' 49 '' of east longitude, 36 deg. 29 '57' 'to 39 deg. 43' 39 '' of northern latitude, total area is 198.72 ten thousand hectares, the functional area is 50.41 ten thousand hects of core area, 38.74 ten thousand hects of buffer area, 109.57 thousand hects of semi-dry area, and belongs to forest grassland climate with high cold area in temperate zone, the average air temperature of drought mountain area is 0.7 deg. C, the annual water reduction amount is 433.6 mm, and the relative humidity is 60% year. During the investigation of large-scale fungi in the natural protection area of Qilian mountain in Gansu province, the authors collected the periphyton mushrooms in the protection area (Agaricus subfloccosus) The mushroom is widely distributed in northern hemisphere, and is edible and delicious (R.W. Kerrigan) et al1999), the present mushroom with the flocculent margin has less literature and more relates to systematic classification, and has fresh reports on biological characteristics and cultivation and domestication. Therefore, the experiment carries out tissue separation and biological characteristic systematic research on the boundary mushrooms and aims to provide a theoretical basis for the protection and utilization of the Qilian mountain mushroom resources.
Disclosure of Invention
The present invention provides a mushroom belonging to the genus Agaricus and having a mushroom-like borderAgaricus subfloccosus) And (3) strain.
The present invention provides a mushroom with a flocculent margin (Agaricus subfloccosus) From wild mushroom Florida: (Agaricus subfloccosus) LQQ20171008005 with the preservation number of CGMCC NO. 18159.
The invention also provides a method for cultivating the mushroom (A)Agaricus subfloccosus) LQQ20171008005 CGMCC No.18159, which comprises the steps of mixing the mushroom with the soybean milk (mushroom)Agaricus subfloccosus) LQQ20171008005 CGMCC No.18159 is used for the culture step in the culture medium for culturing mushrooms, and the steps are as follows:
firstly, hypha of the margin mushrooms is propagated to obtain a mother seed; performing stock culture on the mother strain to obtain a stock; culturing the stock seeds on the culture material to obtain culture seeds, and covering soil for fruiting to realize the cultivation of the margin-flocculated mushrooms;
the culture medium in the method is as follows:
hypha culture medium: 200g of potato, 20g of glucose, 18g of agar, 1g of peptone, 2g of monopotassium phosphate, 2g of magnesium sulfate and 1000ml of water;
the formula of the stock culture medium is as follows: 500 g/bottle, 97% of wheat grain, 1% of gypsum powder and 1.5% of calcium carbonate, and autoclaving at 121 ℃ for 2 hours;
the formula of the cultivation material is as follows: 500 g/bag, 79% of wood chips, 5% of sorghum seeds, 5% of corn flour and 1% of gypsum, and carrying out autoclaving at 121 ℃ for 2 hours;
hypha culture conditions: culturing at 16-18 deg.C in dark for 30 days;
stock culture conditions: culturing at 16-18 deg.C in dark for 60 days;
culture conditions of the cultivation material are as follows: culturing at 16-18 deg.C in dark for 90 days;
the fruiting culture comprises the following steps: covering 3-5cm of soil on the cultivation material, sealing the cultivation bag, continuously culturing at 14-18 deg.C, opening the bag for fruiting 1 week after primordium appears, and culturing at 15-20 deg.C;
the said mushroom isAgaricus subfloccosus) LQQ20171008005 CGMCC No.18159 is used for preparing food;
the said mushroom isAgaricus subfloccosus) LQQ20171008005 CGMCC No.18159 is used for preparing medicines;
the said mushroom isAgaricus subfloccosus) LQQ20171008005 CGMCC No.18159 is used for preparing health products;
the said mushroom isAgaricus subfloccosus) LQQ20171008005 CGMCC No.18159 is applied to cultivation of edible mushroom with flocculent margin;
mushroom of quilted margin (Agaricus subfloccosus) In 2019, 09 and 2 months, the strain is preserved in the China general microbiological culture Collection center (CGMCC for short, address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, and the preservation number of the strain is CGMCC NO.18159, and the strain is classified and named as the mushroom: (A)Agaricus subfloccosus)。
The invention has the beneficial effects that:
the invention carries out morphological and molecular biological analysis based on the ITS sequence of the universal barcode gene of the fungus on the cultivated mushroom and the strain which are obtained by collecting the specimen in the field and domesticating artificially, and identifies the mushroom as the periophyte mushroom. In domestication and cultivation of the Binfo mushrooms, the growth speed of hyphae of the Binfo mushrooms is high, the mushroom bodies have good taste, the Binfo mushrooms are edible mushrooms with developable value, and research and development of the Binfo mushrooms are of great significance for protecting wild germplasm resources and genetic breeding of the edible mushrooms in China.
Drawings
FIG. 1 shows the wild mushroom (Flock edge mushroom)Agaricus subfloccosus) Macroscopic morphological characteristics.
FIG. 2 shows artificially cultivated Binchuan mushroomsAgaricus subfloccosus) Macroscopic morphological characteristics.
FIG. 3 shows the mushroom of different growing periods under artificial cultivation conditions (Agaricus subfloccosus) (the preservation number of the strain is CGMCC NO. 18159). Forming primordium of mushroom body; B-D cultivating the fruiting body artificially.
FIG. 4 shows the mushroom with the catkin (Agaricus subfloccosus) The colony and the microscopic morphological characteristic A of the culture medium are cultured; b spore, C-F basidiophore, G lateral blastocyst and H pith.
FIG. 5 is a phylogenetic tree of Agaricus based on the Maximum Likelihood (ML) analysis of ITS1-5.8S-ITS2 sequences.
Wherein the sequence MN503475 is obtained from a wild specimen LQQ 20171008005; the sequence MN510483 is from a strain CGMCC NO.18159 separated from a wild specimen; sequence MN510484 is from mushroom body after artificial domestication.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1.
Cultivation method of mushroom with flocculent margin
First, specimen separation and identification
1. Separation of field specimens
The wild specimen was collected from the natural protection area (height above sea level 2680 m) of the Kelianshan country in Nancounty, Zhangye, Gansu province in 10 months in 2017, and the specimen collection number was LQQ 20171008005.
Separating strains by adopting a tissue separation method: collecting the tissues at the junction of pileus and stipe of fruiting body of Pleurotus Abutili in field, placing on the slant of test tube culture medium containing PDA culture medium under aseptic environment, culturing at 16 deg.C in dark to obtain Pleurotus Abutili mycelium, and purifying to obtain the strain.
PDA culture medium: cutting 200g of fresh potato into blocks of 1cm in size, adding 1L of distilled water, boiling for 30min, filtering, diluting to 1L, adding 20g of glucose and 18g of agar.
2. Specimen identification
1) Morphological identification the fruiting body is first observed for its constituent parts and morphological characteristics
Longitudinally cutting the fruiting body of fresh mushroom (LQQ 20171008005) with scalpel, and observing the cap composition, color, texture, fold shape, growth condition, stipe composition, stipe texture, and middle or hollow characteristics of the fruiting body;
microscopic morphology observation: placing a field collected mushroom fold in the center of a clean slide, adding a drop of 10% Congo red coloring agent, draining the coloring agent with absorbent paper after 1min, adding 5% potassium hydroxide aqueous solution to prepare a microscopic examination material, covering a glass with tweezers, taking care to avoid gas generation, and observing the microscopic characteristics under an optical microscope.
The results are shown in FIGS. 1, 2, 3 and 4: wild basidiomycetes fruit is single-grown (figure 1), single-grown or double-grown (figures 2 and 3) under artificial cultivation conditions, fresh mushroom has mushroom fragrance, fruiting body is medium and large, pileus is 4-7cm and is primarily hemispherical, pileus is gradually flattened after the fruiting body is mature, the center is slightly sunken, white color is slightly brownish and unsmooth, flaky scale is formed, the edge is inward curled, gear-shaped scale is formed, the diameter is 4-7cm (figures 2-C, D), pileus flesh is white, and damage is light pink; the fungal folds are 1-3mm, are far away, are not equal in length, are dense, are pink when young, and are brown when mature; the stipe is nearly cylindrical, (4.2-8.5) × (0.5-1.5) cm, white, and the stipe meat is tough; the medium-upper part of the stipe is the sclerotium of the sclerotium in the middle of the sclerotium, white and membranous; microscopic characteristics, spore oval, (5-7.5) μm × (3.75-5.5) μm, smooth, colorless (FIG. 4-B); basidion rod-shaped (17.5-27.5) μm × (5.5-8.5) μm, rod-shaped, small stalk 4, mostly 4 basidiospores (FIG. 4-C-F); the fruiting body has a shape of capsule with lateral growth, and has a shape of rod (8.5-14.8 × 4.3-6.8) μm, and is colorless (fig. 4-G); the diameter of the pith (22.5-52.5X 52.5-85) μm and the edge (22.5-42.5X 40-55) μm (FIG. 4-H).
2) Molecular systematics analysis
Extraction of laboratory DNA: in the experiment, a specimen LQQ20171008005 is collected in the field, a strain (with the preservation number of CGMCC NO.18159) is obtained in the field, DNA is extracted from an artificially cultured fruiting body by adopting a CTAB method, and the ITS1 (5'-TCCGTAGGTGAACCTGCGC-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') universal primers are used for PCR amplification.
PCR reaction (25) μ L: 1 μ LDNA template, 2 μ LdNTP, 1 μ LITS1, 1 μ L ITS4, 2 XTQ PCR Master Mix (200 Md NTP, 4.0 mM MgCl)22.5U TaqDNA polymerase), dd H2And (4) supplementing and finishing. The amplification procedure was pre-denaturation at 95 ℃ for 5 min, followed by denaturation at 95 ℃ for 60s, annealing at 50 ℃ for 60s, extension at 72 ℃ for 60s, 35 cycles, and final extension at 72 ℃ for 10 minThe PCR amplification product was sequenced by Beijing Okagaku sequencing Biotech.
The ITS sequences from the wild specimen, the hypha CGMCC NO.18159 and the artificially domesticated mushroom body are all consistent (shown as a sequence 1). According to the constructed phylogenetic map of the Agaricus (figure 5), the Agaricus and the reported fruiting mushroom are in one phylogenetic branch, and the posterior probability support rate of 0.95 is obtained, and the research result supports that the Agaricus and the fruiting mushroom are the fruiting mushrooms of the same species.
Through the identification, the strain (i.e. mycelium) from the wild mushroom LQQ20171008005 is preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of Xilu No.1 Beijing area of the Korean district in Beijing) in 29 th of 2019 and 29 th of 2019, the preservation number of the strain is CGMCC NO.18159, and the strain is classified and named as mushroom (A) AAgaricus subfloccosus）。
Example 2.
Biological characteristics of mushroom
(1) Effect of different carbon sources on hyphal growth carbon-free medium was used as a blank and 6 carbon-source media (sucrose, maltose, mannitol, glycerol, soluble starch and glucose) were used as treatment groups, each treatment being 5 replicates. Punching the same radius of the strain culture medium by using a puncher, taking a strain block, inoculating the strain block on a 6-carbon source culture medium, culturing at constant temperature, measuring the growth length of hyphae in the same time period by using a cross method, stopping culturing in the whole experiment when one treatment hyphae is full, and repeating 5 treatments each time.
(2) The influence of different nitrogen sources on the growth of hyphae was controlled by using a carbon-free medium as a blank and 6 nitrogen source media (ammonium sulfate, urea, potassium nitrate, yeast powder, ammonium nitrate and peptone) as treatment groups. Other operations were performed in the same manner as in the carbon source influence experiment.
(3) The influence of different inorganic salts on the growth of hyphae was controlled by using a non-inorganic salt culture medium as a blank and 6 different inorganic salts (ferric sulfate, zinc sulfate, copper sulfate, manganese sulfate, magnesium sulfate and calcium sulfate) as treatment groups. Other operations were performed in the same manner as in the carbon source influence experiment.
(4) The effect of different pH on hypha growth was set to 6 pH gradients (5, 6, 7, 8, 9, 10), and other operations were performed in the same carbon source effect experiment.
(5) 6 temperature gradients (16 ℃, 18 ℃, 22 ℃, 24 ℃, 26 ℃ and 28 ℃) are set for the influence of different temperatures on the growth of hyphae, and other operations are carried out with the carbon source influence experiment.
(6) The influence of different vitamins on hypha growth was controlled by using a non-vitamin culture medium as a blank and 6 different Vitamins (VB)1、VB2、VB3、VB6、VB12、VBCompounding) To treat the groups, other manipulations were performed in parallel with the carbon source impact experiments.
(7) Orthogonal experiment: on the basis of a single-factor experiment, 3 factors which have great influence on the growth of the hyphae of the mushroom at the edge of the flocculent are selected. Selecting L9(34) And (3) performing three-factor three-level orthogonal experiments by taking the optimal carbon source, inorganic salt and pH which are obvious in hypha growth as direct factors, culturing at constant temperature of 16 ℃, repeating each treatment for 9 times, and performing other operations and recording data which are the same as those of carbon source screening experiments.
TABLE 1 Effect of different monogenic conditions on the growth of hyphae of Florida mushrooms
Note: "+ ++" indicates that the hyphae grow fast, exuberantly, densely and white, "+ ++" indicates that the hyphae turn yellow and grow well, "+ + +" indicates that the hyphae grow slowly and sparsely, and "+" indicates that the hyphae do not grow. Lower case letters in the same column indicate variability at the 0.05 level and upper case letters indicate variability at the 0.01 level.
From table 1, it is known that the hyphae of the marginal mushroom can grow on 6 different carbon sources, the hyphae grow fastest in a sucrose culture medium and slowest in a glycerol culture medium, the growth vigor and the growth speed of the hyphae are integrated, and the most suitable carbon source is sucrose; the mushroom hyphae can grow in different nitrogen source culture mediums, the hyphae grow fastest in potassium nitrate and peptone culture mediums, the hyphae grow slowest in urea culture mediums, and the growth vigor of the hyphae is combinedThe most suitable nitrogen source is potassium nitrate; the mushroom hypha can grow in other inorganic salt culture mediums except manganese sulfate, the hypha is thick and white, the hypha grows fastest in a magnesium sulfate culture medium, the growth vigor is good, and the most suitable inorganic salt is magnesium sulfate; when the pH value is more than 8, the growth of hyphae is inhibited, when the pH value is =6, the hyphae are thick and white, grow fast and grow best, and the pH value which is considered to be the most suitable for the growth of the hyphae is = 6; the hypha of the mushroom grows on different vitamin culture media and at vitamin VB12The growth speed of the culture medium is fastest, and the vitamin VB is3The growth speed of the culture medium is slowest, and the optimal vitamin is VB by integrating12(ii) a The hyphae are thick white and grow fastest (0.153 cm/d) at the temperature of 16 ℃.
From visual analysis of the 3-factor 3 level of the orthorhombic analyzer (table 2) it can be seen that the carbon source is the most badly bad, R =0.50, followed by inorganic salts and pH. In combination, the optimal formulation was determined, carbon source a3, inorganic salt B2, growth factor C1, i.e. sucrose 25 g, magnesium sulfate 2g, pH = 5. The results of the cross experiments were analyzed for variance (table 3), with the carbon source having the largest F value followed by inorganic salts and pH. Therefore, the significant difference of the 3 factors is carbon source > inorganic salt > pH from large to small, which is consistent with the visual analysis result.
TABLE 2 visual analysis of orthogonal experimental results for hypha growth of Fleckedflesh mushrooms
Note: data are mean and standard deviation of 6 runs; kn represents the average growth rate of hyphae on the level of n, and R is the range. "+", "+ + + + + + + + + +", "+ + + + + + + +", and "+ + + + + + + + +" indicate that the hyphal growth is enhanced accordingly.
TABLE 3 analysis of variance of orthogonal experimental results for hypha growth of Florida mushrooms
R square =.998 (adjusting R square =.990)
Example 3
Artificial cultivation of cultivating mushroom
First, preparation of culture medium
Hypha culture medium: 200g of potato, 20g of glucose, 18g of agar, 1g of peptone, 2g of monopotassium phosphate, 2g of magnesium sulfate and 1000mL of water;
the formula of the stock culture medium is as follows: 500 g/bottle, 97% of wheat grains, 1% of gypsum powder and 1.5% of calcium carbonate, and autoclaving at 121 ℃ for 2 hours;
the formula of the cultivation material is as follows: 500 g/bag, 79% of wood chips, 5% of sorghum seeds, 5% of corn flour and 1% of gypsum, and carrying out autoclaving at 121 ℃ for 2 hours;
second, cultivation method
1. And (3) mother seed propagation: a small amount of hyphae of Agaricus campestris (CGMCC NO.18159) were picked from the slant of the centrifuge tube of example 1, and placed in a 18X 180 tube containing PDA medium, and cultured in dark at 16 ℃ for 30 days to obtain the hyphae after propagation.
2. Inoculation cultivation
Cutting the mycelia into small pieces after the test tubes are full of mycelia, inoculating the small pieces into an original seed culture medium, culturing at 16 ℃ in a dark place for 60 days, inoculating the original seeds full of mycelia with a cultivation material, culturing at 16 ℃ in the dark place for 90 days, covering the bags with 3-5cm of soil, culturing in an artificial intelligent climate culture room at the temperature of 14 ℃ and the air humidity of 60%, opening the bags after the original medium appears for 1 week, and culturing at the fruiting temperature of 14 ℃ and the humidity of 85%.
The results show that the strain of the margined mushroom obtained in example 1 can grow normally on the cultivation material which is bagged by polypropylene and takes sawdust and cottonseed hulls as main materials and takes corn flour, gypsum and monopotassium phosphate as auxiliary materials, the bottle is full of the original seeds of wheat for 60 days, the bag is full of the cultivated seeds for about 90 days, the primordium appears after the full cultivation by covering soil for 20-30 days, fresh mushrooms begin to be picked 2 weeks after the primordium is differentiated, and the fruiting bodies of the artificially cultivated margined mushroom are obtained after the original seeds are inoculated and the fruiting bodies are picked for 135 days.
Example 4
Edibility of mushroom
The artificially cultivated mushroom with the two mushrooms has faint scent, is stewed and fried uniformly and has delicious taste,has good taste, good toughness and good mouthfeel, and can be fried for eating. Therefore, the present invention provides a mushroom with a margin of flocculation (Agaricus subfloccosus) LQQ20171008005 and CGMCC NO.18159 have edibility and can be developed into artificially cultivated edible fungi.
Sequence listing
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<120> separation and artificial domestication cultivation method of new wild edible fungi
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<170> PatentIn version 3 .5
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<212>Mushroom of quilted margin (Agaricus subfloccosus)
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TTGGCATAGGATATGTTTTCTAGATGGGTTGTAGCTGGCTCTTCGGAGCATGTGCACACCTGTTTGGACTTCATTTTCATCCACCTGTGCACCTTTTGTAGTCTTTCAGGTATTGAAGGAAGTGGTCAGCCTATCAGCTCTTTGCTGGATG TAAGGACTTGCAGTGTGAAAACAGTGCTGTCCTTTACCTTGACCATGGACTCTTTTTCCTGTTAGAGTCTATGTTATTCATTATACTCTTAGAATGTCATTGAATGTCTTTACATGGGCTATGCCTATGAAAATTATTATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCATCTTGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATTATATTCTCAACTCCCCAATACCTTGTTGTAAAGGAGAGCTTGGATTGTGGAGGTTTGCTGGCCCCTGCTTGGGGTCAGCTCCTCTGAAATGCATTAGCGGAACCGTCTGCGATCTGCCACAAGTGTGATAACTTATCTACACTGGCGAGGGGATTGCTTTCTGTGATGTTCAGCTTCTAATCGTCTAAGGACAAATTCTTGAATGCTTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
Sequence listing
<110> riverside academy
<120> separation and artificial domestication cultivation method of new wild edible fungi
<160> 1
<170> PatentIn version 3 .5
<210> 1633
<211> DNA
<212> Flock margin mushroom (Agaricus subfloccosus)
<400> 1
TTGGCATAGGATATGTTTTCTAGATGGGTTGTAGCTGGCTCTTCGGAGCATGTGCACACCTGTTTGGACTTCATTTTCATCCACCTGTGCACCTTTTGTAGTCTTTCAGGTATTGAAGGAAGTGGTCAGCCTATCAGCTCTTTGCTGGATGTAAGGACTTGCAGTGTGAAAACAGTGCTGTCCTTTACCTTGACCATGGACTCTTTTTCCTGTTAGAGTCTATGTTATTCATTATACTCTTAGAATGTCATTGAATGTCTTTACATGGGCTATGCCTATGAAAATTATTATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCATCTTGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATTATATTCTCAACTCCCCAATACCTTGTTGTAAAGGAGAGCTTGGATTGTGGAGGTTTGCTGGCCCCTGCTTGGGGTCAGCTCCTCTGAAATGCATTAGCGGAACCGTCTGCGATCTGCCACAAGTGTGATAACTTATCTACACTGGCGAGGGGATTGCTTTCTGTGATGTTCAGCTTCTAATCGTCTAAGGACAAATTCTTGAATGCTTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
Claims (1)
1. The cultivation method of the margin-flocculent mushrooms is characterized by comprising the following steps: firstly, hypha of the margin mushrooms is propagated to obtain a mother seed; performing stock culture on the mother strain to obtain a stock; culturing the stock seeds on the culture material to obtain culture seeds, and covering soil for fruiting to realize the cultivation of the margin-flocculated mushrooms; the strain of the Pleurotus catbrosus is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 18159;
the culture medium in the method is as follows:
hypha culture medium: 200g of potato, 20g of glucose, 18g of agar, 1g of peptone, 2g of monopotassium phosphate, 2g of magnesium sulfate and 1000ml of water;
the formula of the stock culture medium is as follows: 500 g/bottle, 97% of wheat grain, 1% of gypsum powder, 1.5% of calcium carbonate and autoclaving at 121 ℃ for 2 hours;
the formula of the cultivation material is as follows: 500 g/bag, 79% of wood chips, 5% of sorghum seeds, 5% of corn flour and 1% of gypsum, and carrying out autoclaving at 121 ℃ for 2 hours;
hypha culture conditions: culturing at 16 deg.C in dark for 30 days;
stock culture conditions: culturing at 16-18 deg.C in dark for 60 days;
culture conditions of the cultivation material are as follows: culturing at 16-18 deg.C in dark for 90 days;
the fruiting culture comprises the following steps: covering 3-5cm of soil on the cultivation material, sealing the cultivation bag, continuously culturing at 14-18 deg.C, opening the bag after primordium appears, and fruiting at 15-20 deg.C and humidity of 80-90%.
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