CN112662566A - Ganoderma lucidum spore-less variety with high yield of polysaccharide and artificial cultivation method thereof - Google Patents

Abstract

The invention discloses a ganoderma lucidum spore-reduced variety with high yield of polysaccharide and an artificial cultivation method thereof. Ganoderma lucidum (ganoderma lucidum) M624, with accession number: GDMCC No: 60889. the artificial cultivation method comprises the steps of hypha culture of ganoderma lucidum M624, ganoderma lucidum production management and fruiting body collection. The invention screens and obtains a ganoderma lucidum spore-lacking variety with high yield of polysaccharide, namely ganoderma lucidum M624, in substitute cultivation, the content of crude polysaccharide in the sporocarp reaches 2.40 +/-0.09 percent and is far higher than 0.9 percent required by pharmacopeia, and the content of crude polysaccharide in substitute cultivated ganoderma lucidum in industry is generally 0.9-1.5 percent; moreover, the ganoderma lucidum M624 disclosed by the patent has low spore yield, can reduce the influence on the cultivation environment and machines, and meets the requirement of ganoderma lucidum cultivation with sporocarp as a harvesting target. The invention can improve the crude polysaccharide content of the ganoderma lucidum raw material at the source, enrich the ganoderma lucidum varieties in the current market, is also suitable for industrial large-scale production, avoids the influence of a large amount of spore powder generated in the spore spraying period of the ganoderma lucidum on the environment and machines, and meets the requirement of the industrialized development of the ganoderma lucidum.

Description

Ganoderma lucidum spore-less variety with high yield of polysaccharide and artificial cultivation method thereof

The technical field is as follows:

the invention belongs to the technical field of agricultural microorganisms, and particularly relates to a ganoderma lucidum spore-less variety with high yield of polysaccharide and an artificial cultivation method thereof.

Background art:

the efficacy of ganoderma lucidum and related products thereof are well recognized at home and abroad, and are listed in the United states pharmacopoeia as early as 2010, and the annual output value of the cultivation and processing industry (including health-care food, skin care products and the like) of ganoderma lucidum reaches over 1000 million yuan at present. The ganoderma lucidum variety determined in China has 19 such as Shanghanon No.1, wherein 1 is imported abroad, and 3 is obtained by protoplast fusion. However, according to 2016 strain research of researchers at the research and development center of edible fungi of the institute of microbiology, Guangdong province, the main strain produced in China in the main production area of Ganoderma lucidum is: 7 Korean Ganoderma, 2 American Ganoderma, 2 Longzhi, 2 Shannon Ganoderma, 1 Japanese Ganoderma, and 1 undefined variety. Although the Shanghangong and Longzhi series of local breeding are popularized to a certain extent, the number and the popularity of the ganoderma lucidum varieties with independent intellectual property rights in China are far less than those of the introduced varieties, and the varieties such as high-yield polysaccharide or high-yield triterpene are still very lack if the ganoderma lucidum varieties are suitable for industrial cultivation, so the breeding of the ganoderma lucidum strains is a bottleneck problem in the development of the ganoderma lucidum industry at present. With the progress of research on ganoderma, the pharmacological activity of the polysaccharide is proved, and the research shows that the ganoderma polysaccharide has the effects of immunoregulation, tumor resistance, oxidation resistance, anti-inflammation, antibiosis, blood sugar reduction, diabetes and complications thereof, liver protection and the like (Zhao Xiaoping, the research and cultivation status of medicinal fungi ganoderma, edible fungi in China, 2019,38(7): 1-5). Medicinal materials, medicines and health-care products taking ganoderma lucidum polysaccharide as a main component have wide market space and huge commercial value, and related enterprises gradually put the center of gravity into a target strain for high-yield polysaccharide so as to improve the quality of the product. According to literature reports, the polysaccharide content of the ganoderma lucidum variety in the market is generally 0.9-1.5%, the small part of the ganoderma lucidum cultivated by basswood can reach 1.8-2.5%, but the environment is greatly damaged by basswood cultivation, the sustainable development of cultivation production is not facilitated, and the substitute cultivation is a necessary trend of large-scale production of edible fungi. According to the reflection of domestic ganoderma lucidum cultivation enterprises, the ganoderma lucidum variety faces degeneration risk after being used for 1-2 years, which not only shows that the yield is reduced, but also shows that the main components representing the effectiveness of the ganoderma lucidum variety, such as ganoderma lucidum polysaccharide, ganoderma lucidum triterpene and the like are greatly reduced, and the product quality is seriously influenced, so that the breeding work of the ganoderma lucidum variety which is high and stable in yield and is suitable for industrial material-replacing cultivation is very important and urgent.

The invention content is as follows:

the first purpose of the invention is to provide a new ganoderma lucidum (ganodermalucidum) M624 with high polysaccharide yield and low spore yield.

Ganoderma lucidum (ganoderma lucidum) M624 of the present invention, which was deposited at Guangdong province microbial culture Collection (GDMCC) on 11/7 in 2019, address: the preservation number of the Guangzhou city Pieli Zhongluo No. 100 large yard No. 59 building 5 is as follows: GDMCCNo: 60889.

the second purpose of the invention is to provide an artificial cultivation method of the ganoderma lucidum M624, which comprises the steps of hypha cultivation of the ganoderma lucidum M624, ganoderma lucidum growth management and fruiting body harvesting.

Specifically, it is preferable that:

a. hypha culture: inoculating Ganoderma M624 block on mother culture medium until mycelia overgrow the slant, transferring mother culture mycelia to production culture medium, performing expanded culture, culturing until mycelia overgrow the bag, inoculating production strain mycelia on the culture medium, and culturing for post-maturation period;

b. and (4) lucid ganoderma management: and (4) performing bud promotion after the post-maturation period of the mycelia is over, changing fresh air when the pileus begins to differentiate, and harvesting the ganoderma lucidum sporocarp when the sporocarp pileus stops growing flatly.

The hypha culture is preferably as follows:

inoculating Ganoderma M624 to mother culture medium under aseptic condition, culturing at 25 deg.C in dark at constant temperature with relative humidity of 50-60% for 7-10 days until mycelia overgrow the slant, transferring mother mycelia to production culture medium, culturing at 25 deg.C in dark at constant temperature until mycelia overgrow the slant, inoculating production strain mycelia to cultivation material, culturing at 25 deg.C in dark at constant temperature with relative humidity of 50-60%, ventilating at regular time to keep carbon dioxide concentration below 4000ppm, culturing for 20-25 days until mycelia overgrow the band, and culturing under the same condition for 23-25 days for post-maturation;

the mother culture medium is as follows: comprises 20 percent of potato, 2 percent of glucose, 1 percent of peptone, 2 percent of agar, 0.3 percent of monopotassium phosphate, 0.15 percent of magnesium sulfate, 10.001 percent of vitamin B and the balance of water according to mass fraction;

the production seed culture medium comprises: comprises 98-99% of sorghum and 1-2% of calcium carbonate by mass fraction, and has natural pH;

the cultivation material is as follows: the composite material comprises 50% of sawdust, 38% of cottonseed hulls, 10% of wheat bran and 2% of calcium carbonate by mass, the water content is 60-65%, and the pH is natural.

The fruiting body management and harvesting method comprises the following steps:

and (3) after the hypha is mature, bud promotion is carried out, the cover of the cultivation bag is opened, the cultivation bag is placed in a vertical row, the temperature is 25-28 ℃, the relative humidity of air is kept at 75% -90%, scattered light is used for illuminating for 10h/d, primordium begins to form at the opening position of the cultivation material after the glossy ganoderma is grown for 8-10 days, the humidity is adjusted to be 80-90%, when the pileus begins to differentiate, fresh air is changed for 2 times every day, 1h is carried out every time, and when the sporocarp pileus stops flat growth, sporocarp is collected.

The method comprehensively evaluates the agronomic characters and the polysaccharide content of a plurality of wild ganoderma lucidum strains, screens and obtains a ganoderma lucidum spore variety-ganoderma lucidum M624 with high polysaccharide yield, wherein in substitute cultivation, the content of crude polysaccharide in a sporocarp reaches 2.40 +/-0.09 percent and is far higher than 0.9 percent required by pharmacopeia, and the content of crude polysaccharide in substitute cultivated ganoderma lucidum in the industry is generally 0.9-1.5 percent; moreover, the ganoderma lucidum M624 disclosed by the patent has low spore yield, can reduce the influence on the cultivation environment and machines, and meets the requirement of ganoderma lucidum cultivation with sporocarp as a harvesting target. The invention can improve the crude polysaccharide content of the ganoderma lucidum raw material at the source, enrich the ganoderma lucidum varieties in the current market, is also suitable for industrial large-scale production, avoids the influence of a large amount of spore powder generated in the spore spraying period of the ganoderma lucidum on the environment and machines, and meets the requirement of the industrialized development of the ganoderma lucidum. The invention improves the quality of the ganoderma lucidum product at the aspect of strains, and is beneficial to the processing and application of ganoderma lucidum polysaccharide.

Ganoderma lucidum M624, deposited at the guangdong collection of microorganisms and cell cultures (GDMCC) on 7/11/2019, address: the preservation number of the Guangzhou city Pieli Zhongluo No. 100 large yard No. 59 building 5 is as follows: GDMCCNo: 60889.

description of the drawings:

FIG. 1 is a diagram showing the fruit body growth of Ganoderma lucidum M624;

FIG. 2 is an ML method for constructing a phylogenetic tree;

FIG. 3 is an NJ method for constructing a phylogenetic tree;

FIG. 4 shows the domesticated cultivation yield of Ganoderma lucidum, wherein M624 is wild Ganoderma lucidum strain; MC-GL-0046 and MC-GL-0054 are market seeds;

FIG. 5 is a standard curve of glucose standards at different concentrations;

FIG. 6 shows the content of crude polysaccharides in artificially cultured fruiting bodies of wild Ganoderma strains, wherein A532, Z381, M311, Z112, W201, and M624 are wild Ganoderma strains; MC-GL-0046 and MC-GL-0054 are market seeds; the difference significance analysis is that P is more than 0.01 and P is more than 0.05;

FIG. 7 shows the artificial cultivation of Ganoderma M624, MC-GL-0046 and MC-GL-0054.

Detailed Description

The invention will be further explained with reference to specific embodiments with reference to the drawings. The examples themselves are not intended to limit the invention in any way.

Example 1: obtaining method of Ganoderma lucidum (Ganoderma lucidum) M624 as a Ganoderma lucidum spore variety with high yield of polysaccharide

According to the situation that researchers at the research and development center of edible fungi of Guangdong institute of microbiology, Guangdong province research and research for a long time investigate wild large-scale fungi resources, the lucid ganoderma is mainly distributed in Guangdong, Hunan, Anhui, Henan and the like. Researchers of edible fungus research and development center of Guangdong institute of microbiology, from Liuan City, Anhui province, latitude 31 degrees 33 ', longitude 116 degrees 09', altitude 70M, scattered growth, forest facies being broad-leaved forest, humus soil matrix collecting wild strain HMGIM-M624 fruiting body (namely Ganoderma (Ganoderma lucidum) M624), which has lateral growing stalk, and canopy flat-open type, diameter of 2.8cm and length of 6.5 cm; mostly yellow brown pileus, pale yellow and blunt edges, white surface of the orifice, light brown pileus (fig. 1).

1. Tissue isolation

Wiping the surface of the strain HMGIM-M624 fruiting body collected in the field with 75% alcohol, tearing and clamping the inner flesh tissue of 0.2-0.5mm multiplied by 0.2-0.5mm in a sterile environment, and inoculating into enriched comprehensive PDA culture medium. Placing in an incubator at 25 deg.C, dark culturing at constant temperature, and taking tip mycelium for transfer purification culture after the mycelium grows over the inclined plane.

2. Molecular identification

ITS identification method

Collecting strain HMGIM-M624 mycelium, drying at low temperature, grinding at normal temperature, extracting DNA genome with Ezup column type fungus genome DNA extraction kit (Biotechnology engineering (Shanghai) Co., Ltd.), and refrigerating the obtained DNA solution (DNA template) at-20 deg.C for use. ITS-PCR experiments were performed with fungal ribosomal intergenic region universal primers ITS1/ITS4(ITS1: TCCGTAGGTGAACCTGCGG, ITS4: TCCTCCGCTTATTGAT ATGC, synthesized by Biotechnology, Inc. (Shanghai) and amplification was performed on a Biometra PCR instrument, with PCR reaction solutions (50. mu.l total) as in Table 1 and with reaction procedures as in Table 2:

TABLE 1 fungal ITS PCR System

TABLE 2 fungal ITS PCR amplification procedure

The PCR product is directly checked for bidirectional sequencing and completed by Huada gene.

2.1 taxonomic status determination

The sequencing result (the nucleotide sequence is shown as SEQ ID NO. 1) is subjected to sequence comparison in NCBI GenBank, and the ITS identification result shows that the strain is a Ganoderma lucidum (Ganoderma lingzhi) strain.

In order to further confirm the variety, a maximum likelihood Method (ML) and an adjacency method (NJ) are used for constructing a phylogenetic tree, and the Yunzhi (Trametes versicolor) is used as an outer group to respectively construct the phylogenetic tree (see the figure 2-3). In a phylogenetic tree built by two algorithms, HMGIM-M624 and Ganoderma lingzhi are in the same branch and are grouped into a class, the genetic relationship is close, and the HMGIM-M624 can be determined to be a Ganoderma lucidum (Ganoderma lingzhi) strain named as Ganoderma lucidum (Ganoderma lingzhi) M624. Ganoderma lucidum recorded in pharmacopoeia of China is dry fruiting body of Ganoderma lucidum G.lucidum or Ganoderma sinensis G.sinense, which is a fungus of Polyporaceae, but with the progress of phylogenetic research, the research of professor Dayucheng Daochan of taxonomic experts in China in 2013 shows that the Ganoderma lucidum widely distributed and cultivated in China is different from G.lucidum produced in Europe, and Wasser et al (2006) and Wasser (2011) also indicate that the name of G.lucidum is wrongly used in medicinal Ganoderma lucidum. In fact, Chinese ganoderma (commonly known as ganoderma lucidum) is a new species: ganoderma lingzhi S.H.Wu, Y.Cao & Y.C.Dai (Cao et al.2012), therefore the Chinese pharmacopoeia should describe the Ganoderma lucidum latin name as G.lingzhi, but not G.lucidum, the patent protects the strain as the Ganoderma lucidum G.lingzhi wild strain native to China, but because the current pharmacopoeia is still using G.lucidum as the Ganoderma lucidum latin name, the patent protects the strain deposit name as Ganoderma lucidum (Ganoderma lucidum) M624 temporarily, and the pharmacopoeia is corrected after modification. The strain HMGIM-M624 was therefore named Ganoderma lucidum (Ganoderma lucidum) M624, which was deposited at 11/7 of 2019 with the collection of microorganisms and cell cultures (GDMCC) of guangdong province, address: the preservation number of the Guangzhou city Pieli Zhongluo No. 100 large yard No. 59 building 5 is as follows: GDMCCNo: 60889.

example 2: domestication cultivation of ganoderma lucidum M624 and evaluation of fruiting body crude polysaccharide content

Study on domestication and cultivation of wild ganoderma lucidum strains

Formula and configuration of culture medium

1. Mother culture medium (enriched integrated PDA): comprises 20 percent of potato, 2 percent of glucose, 1 percent of peptone, 2 percent of agar, 0.3 percent of monopotassium phosphate, 0.15 percent of magnesium sulfate, 10.001 percent of vitamin B and the balance of water by mass fraction, and the preparation method comprises the following steps: firstly, cleaning and peeling potatoes, weighing 200g of potatoes, cutting the potatoes into small pieces, adding water, boiling the small pieces thoroughly (boiling for 20-30 minutes and being capable of being punctured by a glass rod), filtering the small pieces with eight layers of gauze, adding 20g of glucose, 10g of peptone, 20g of agar, 3g of monopotassium phosphate, 1.5g of magnesium sulfate and 10.01g of vitamin B into filtrate, uniformly stirring the filtrate, slightly cooling the filtrate, then supplementing water to 1000 milliliters, carrying out high-temperature high-pressure moist heat sterilization at the temperature of 0.11MPa and 121 ℃ for 30 minutes, and placing the filtrate on an inclined plane for cooling and shaping.

2. Production of seed culture medium: comprises 99 percent of sorghum and 1 percent of calcium carbonate by mass fraction, and has natural pH. Weighing sorghum according to the required proportion, soaking overnight, cooking till the sorghum is thoroughly penetrated, taking out, filtering, removing redundant water, adding calcium carbonate according to the proportion, uniformly stirring, filling into high-temperature-resistant transparent polypropylene bags of 13cm multiplied by 25cm, and converting each bag into 200 grams of dry materials and 250 grams of dry materials. Sterilizing at high temperature and high pressure of 121 deg.C under 0.11MPa for 30 min.

3. Cultivation material: comprises 50 percent of mixed wood dust, 38 percent of cottonseed hull, 10 percent of wheat bran and 2 percent of calcium carbonate by mass percent, and has natural pH. The formula is fully mixed, water is added until the water content is 60-65%, and the mixture is filled into a 17 cm-35 cm high-temperature-resistant transparent polypropylene strain bag, wherein the amount of the dry materials in each bag is 400-420 g. After the materials are filled, a hole is punched in the bag materials by a punching rod, the hole is deep to the bottom of the bag, then a plastic ring is sleeved on the bag opening, and a matched cover is buckled, so that the prepared original seed bag materials are obtained. Performing wet heat sterilization at 0.147MPa and 128 deg.C for 90 min.

(II) cultivation technical parameters

1. Hypha culture: inoculating 0.5cm × 0.5cm Ganoderma M624 preserved strain block in mother culture medium (enriched comprehensive PDA) under aseptic condition, culturing at 25 deg.C in dark for 7-10 days with relative humidity of 50% -60%, and allowing mycelia to grow on the slant. Transferring the mother seed mycelium to a production seed culture medium for enlarged culture, culturing at a constant temperature of 25 ℃ in the dark at a relative humidity of 50-60% until the mycelium is full of bags, and obtaining the production seed. Inoculating mycelium of Ganoderma M624 production strain into the cultivation material, culturing at 25 deg.C in dark at constant temperature with relative humidity of 50% -60%, periodically ventilating to keep carbon dioxide concentration below 4000ppm, culturing for 20-25 days until the mycelium is full of band, and culturing under the same conditions for 23-25 days for post maturation period.

2. And (4) lucid ganoderma management: bud forcing is carried out after the post-maturation period of hyphae is over, the cover of the cultivation bag is opened, the cultivation bags are placed in a vertical row (the distance between the bags is 15cm), the temperature is 25-28 ℃, the relative humidity of air is kept at 75% -90%, scattered light illumination is carried out for 10h/d, primordium begins to be formed at the opening position of the cultivation material after the ganoderma lucidum is produced and managed for 8-10 days, the relative humidity is adjusted to be 80-95%, and fresh air is changed for 2 times every day when the mushroom cover begins to differentiate, wherein the time for each time is 1 h.

3. Harvesting: and (4) after the primordium is formed, the fruiting body of the lucid ganoderma is mature 35 to 40 days, and the first tide of mushroom is collected.

Domesticating 6 wild Ganoderma strains (HMGIM-A532, HMGIM-Z381, HMGIM-M311, HMGIM-Z112, HMGIM-W201 and HMGIM-M624, abbreviated as A532, Z381, M311, Z112, W201 and M624 in the diagram) according to the same method, and selecting two Ganoderma varieties mainly cultivated in the market as control, wherein MC-GL-0046 is introduced Korean Ganoderma II foreign, and has small spore yield; MC-GL-0054 is a locally screened high-quality Ganoderma lucidum Hunong I, and has strong stress resistance and large spore yield; the two control strains are popularized and cultivated in Anhui, Zhejiang, Fujian, Shanghai and other places for many years, and are ganoderma lucidum strains which are stable and mature in technology at present.

The average bag yield of artificially cultivated ganoderma lucidum M624 is 34.91 +/-1.15 g (figure 4), the diameter of pileus is 10.73 +/-0.34 cm, the yield is equivalent to the yield of control strains MC-GL-0054 and MC-GL-0046, the yield has no obvious advantage under the cultivation method, and the yield of ganoderma lucidum M624 can be improved by subsequently formulating a proper cultivation formula. As shown in FIG. 7, the yield of spores of the substitute cultivated Ganoderma lucidum M624 is low, and no spore data can be obtained because data analysis cannot be performed. The spore ejection capacity of the ganoderma lucidum is weak because a layer of colloid substance is covered on the reticular structure of the fungus tube in the fungus tube, only a small amount of spores are ejected in the mature process, but the synthesis of secondary metabolites such as polysaccharide and the like cannot be influenced due to the loss of a spore forming mechanism. Compared with the spore-producing ganoderma, the fruiting body of the ganoderma lucidum with few spores is thick and solid, can accumulate more active ingredients in the growth and development process, has stronger biological activity, and meets the product requirement of the ganoderma lucidum market taking the fruiting body as the raw material.

4. Method for measuring polysaccharide content of ganoderma lucidum cultivated sporocarp

The polysaccharide content of the 8 ganoderma lucidum sporocarp is measured according to a sulfuric acid-anthrone colorimetric method in ganoderma lucidum from Chinese pharmacopoeia, and the method comprises the following steps:

a) preparing an anthrone sulfate solution: 0.1g of anthrone is precisely weighed, 100ml of sulfuric acid is added for dissolution, and the mixture is shaken up for standby. Preparing a reference solution: precisely weighing 3mg of glucose standard substance, and fixing the volume to 25ml, wherein the concentration is 0.12 mg/ml.

b) Preparing a standard curve: precisely weighing reference substance solutions 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2ml, respectively placing in 10ml test tubes with plugs, respectively adding water to 2.0ml, rapidly and precisely adding anthrone sulfate solution 6ml, shaking up immediately, standing for 15 min, immediately placing in ice bath for cooling for 15 min, taking out, measuring absorbance at 625nm wavelength with corresponding reagent as blank, and drawing standard curve with absorbance as ordinate and concentration as abscissa (FIG. 5).

c) And (3) sample determination: crushing a ganoderma lucidum fruiting body sample, precisely weighing 2.00g of the sample, adding 60ml of pure water, heating and refluxing for 4h, filtering while the sample is hot, washing a filter and filter residues with a small amount of hot water, placing the filter residues and filter paper into a flask, adding 60ml of water, heating and refluxing for 3h, filtering while the sample is hot, combining the filtrate, carrying out rotary evaporation to remove water, dissolving the residues with 5ml of water, slowly dropwise adding 75ml of ethanol while stirring, shaking uniformly, placing at 4 ℃ for 12h, centrifuging, discarding the supernatant, dissolving the precipitate with hot water and transferring to a 50ml volumetric flask. Taking a proper amount of the solution, centrifuging at 8000rpm, precisely measuring 3ml of supernate, and fixing the volume to 25 ml. Precisely measuring 2ml of a sample solution, placing the sample solution into a 10ml test tube with a plug, quickly and precisely adding 6ml of anthrone sulfate solution, immediately shaking up, standing for 15 minutes, immediately placing the test tube into an ice bath for cooling for 15 minutes, taking out, taking a corresponding reagent as a blank, measuring absorbance at a wavelength of 625nm by an ultraviolet-visible spectrophotometry, taking 3 repeated samples, and calculating the content of polysaccharide according to a standard curve.

The experimental result of the evaluation of the polysaccharide content of the artificially cultivated fruiting body of the wild ganoderma lucidum strain is shown in figure 6, and the content of crude polysaccharide is ranked in high and low order: ganoderma lucidum M624, HMGIM-M311, MC-GL-0054, HMGIM-W201, HMGIM-A532, MC-GL-0046, HMGIM-Z381, and HMGIM-Z112; wherein, the content of the polysaccharide of the ganoderma lucidum M624 is 2.40 +/-0.09%, which is remarkably higher than that of other wild strains, and is more than 1.5 times of that of two markets of a control group MC-GL-0046 and a control group MC-GL-0054. And is far higher than 0.9 percent of the requirement of pharmacopeia, and the content of the substitute cultivated ganoderma lucidum crude polysaccharide in the industry is generally 0.9 to 1.5 percent. According to the agronomic characters and the polysaccharide content of a plurality of wild ganoderma lucidum strains, the ganoderma lucidum M624 can be determined to be a high-quality ganoderma lucidum strain with high polysaccharide content and low spore yield.

In conclusion, the research team establishes a wild ganoderma germplasm library through series of works such as field collection, purification preservation, molecular identification and the like, 6 strains with strong activity are screened from the wild ganoderma germplasm library for domestication cultivation and polysaccharide content evaluation, and finally a ganoderma lucidum spore-less variety with high polysaccharide yield, namely ganoderma lucidum M624, is obtained.

Sequence listing

<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)

<120> ganoderma lucidum spore-lacking variety with high polysaccharide yield and artificial cultivation method thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 606

<212> DNA

<213> Ganoderma lucidum M624(Ganoderma lucidum)

<400> 1

cggaaggatc attatcgagt tttgaccggg ttgtagctgg ccttccgagg catgtgcacg 60

ccctgctcat ccactctaca cctgtgcact tactgtgggc ttcagattgt gaggcacgct 120

ctttaccggg cttgcggagc atatctgtgc ctgcgtttat cacaaactct ataaagtaac 180

agaatgtgta ttgcgatgta acacatctat atacaacttt cagcaacgga tctcttggct 240

ctcgcatcga tgaagaacgc agcgaaatgc gataagtaat gtgaattgca gaattcagtg 300

aatcatcgaa tctttgaacg caccttgcgc tccttggtat tccgaggagc atgcctgttt 360

gagtgtcatg aaatcctcaa cctacaagct tttgtggttt gtaggcttgg acttggaggc 420

ttgtcggccg ttatcggtcg gctcctctta aatgcattag cttggttcct tgcggatcgg 480

ctctcggtgt gataatgtct acgccgcgac cgtgaagcgt ttggcgagct tctaaccgtc 540

ttataagaca gctttatgac ctctgacctc aaatcaggta ggactacccg ctgaacttaa 600

gcatat 606

Claims (5)

1. Ganoderma lucidum (ganoderma lucidum) M624, with accession number: GDMCC No: 60889.

2. the artificial cultivation method of ganoderma lucidum M624 as claimed in claim 1, comprising mycelium culture of ganoderma lucidum M624, management of ganoderma lucidum emergence and fruit body harvesting.

3. The artificial cultivation method as claimed in claim 2, which is specifically:

a. hypha culture: inoculating Ganoderma M624 block on mother culture medium until mycelia overgrow the slant, transferring mother culture mycelia to production culture medium, performing expanded culture, culturing until mycelia overgrow the bag, inoculating production strain mycelia on the culture medium, and culturing for post-maturation period;

b. and (4) lucid ganoderma management: and (4) performing bud promotion after the post-maturation period of the mycelia is over, changing fresh air when the pileus begins to differentiate, and harvesting the ganoderma lucidum sporocarp when the sporocarp pileus stops growing flatly.

4. The artificial cultivation method according to claim 3, wherein the hypha culture is specifically:

inoculating Ganoderma M624 to mother culture medium under aseptic condition, culturing at 25 deg.C in dark at constant temperature with relative humidity of 50-60% for 7-10 days until mycelia overgrow the slant, transferring mother mycelia to production culture medium, culturing at 25 deg.C in dark at constant temperature until mycelia overgrow the slant, inoculating production strain mycelia to cultivation material, culturing at 25 deg.C in dark at constant temperature with relative humidity of 50-60%, ventilating at regular time to keep carbon dioxide concentration below 4000ppm, culturing for 20-25 days until mycelia overgrow the band, and culturing under the same condition for 23-25 days for post-maturation;

the mother culture medium is as follows: comprises 20 percent of potato, 2 percent of glucose, 1 percent of peptone, 2 percent of agar, 0.3 percent of monopotassium phosphate, 0.15 percent of magnesium sulfate, 10.001 percent of vitamin B and the balance of water according to mass fraction;

the production seed culture medium comprises: comprises 98-99% of sorghum and 1-2% of calcium carbonate by mass fraction, and has natural pH;

the cultivation material is as follows: the composite material comprises 50% of sawdust, 38% of cottonseed hulls, 10% of wheat bran and 2% of calcium carbonate by mass, the water content is 60-65%, and the pH is natural.

5. The artificial cultivation method as claimed in claim 3, wherein the management of fruiting is as follows:

and (3) after the hypha is mature, bud promotion is carried out, the cover of the cultivation bag is opened, the cultivation bag is placed in a vertical row, the temperature is 25-28 ℃, the relative humidity of air is kept at 75% -90%, scattered light is used for illuminating for 10h/d, primordium begins to form at the opening position of the cultivation material after the glossy ganoderma is grown for 8-10 days, the humidity is adjusted to be 80-90%, when the pileus begins to differentiate, fresh air is changed for 2 times every day, 1h is carried out every time, and when the sporocarp pileus stops flat growth, sporocarp is collected.

Images