CN115039638B

CN115039638B - Resin ganoderma lucidum strain H63 and application thereof

Info

Publication number: CN115039638B
Application number: CN202210429574.5A
Authority: CN
Inventors: 李娥贤; 李树红; 何俊; 雷涌涛; 叶洋; 苏开美
Original assignee: Biotechnology and Germplasm Resource Institute of Yunnan Academy of Agricultural Sciences
Current assignee: Biotechnology and Germplasm Resource Institute of Yunnan Academy of Agricultural Sciences
Priority date: 2022-04-22
Filing date: 2022-04-22
Publication date: 2023-12-29
Anticipated expiration: 2042-04-22
Also published as: CN115039638A

Abstract

The invention relates to a resin ganoderma lucidum strain H63 and application thereof, belonging to the technical field of microorganisms. The ganoderma strain H63 is preserved in the microorganism strain collection of Guangdong province in China at 2021, 12 months and 27 days, and the preservation number is GDMCC No:62106. the strain obtained by tissue separation of specimens of tropical areas of Yunnan province in China combines morphology and phylogenetic analysis to determine classification status, and the cultivated and domesticated culture finds that the resin ganoderma lucidum H63 has strong heat resistance, short fruiting body growth period, high spore yield, strong stress resistance, 1.26 percent of polysaccharide content and 2.86 percent of triterpene content. The resin ganoderma lucidum H63 strain and fruiting body of the invention grow well at 28-35 ℃, have high content of active ingredients and good commodity property, have the potential of cultivation and popularization in low-altitude areas, and can provide important reference for the exploitation and utilization of Yunnan wild ganoderma lucidum resources.

Description

Resin ganoderma lucidum strain H63 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a resin ganoderma lucidum strain H63 and application thereof.

Background

Ganoderma is one of the most studied genera of the Ganodermaceae and Polyporaceae, established in 1881 by the finnish plant scholars Peter Adolph Karsten, and has the fruiting body cap and petiole of Ganoderma lucida Ganoderma lucidum (W.Cur. Fr.) Karst.

Modern researches show that some ganoderma varieties known at present are rich sources of high-biological active substances, in particular to various active substances such as saccharides, triterpenes, sterols, trace elements, fatty acids and the like. Among them, polysaccharides and ganoderma triterpenes are widely considered as main bioactive components in ganoderma lucidum.

With the continuous development of medicine science, the effects of people on disease prevention and treatment of ganoderma lucidum and the gradual improvement of health care consciousness are achieved, and the demand for ganoderma lucidum is continuously increased. Because of the change of ecological environment, the excessive collection of wild ganoderma lucidum resources leads to the increasingly reduced quantity of wild ganoderma lucidum, so that ganoderma lucidum products in market demand are mainly artificially cultivated. The development of the resource characteristics and the artificial domestication cultivation of the rare ganoderma lucidum strains is beneficial to perfecting the collection, evaluation and utilization of ganoderma lucidum resources in the region, and is also beneficial to providing practical guiding significance for the protection and continuous utilization of ganoderma lucidum species.

The resin ganoderma lucidum strain is a novel strain collected and separated in New Anzhen of Mongolia of Yunnan province, and is not reported by researches.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides a new strain H63 of resin ganoderma lucidum and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a resin ganoderma lucidum strain H63 was deposited at the microorganism strain collection of Guangdong province in China on the 12 th month 27 th year 2021, accession number GDMCC No:62106.

the invention also provides an artificial culture method of the resin ganoderma lucidum strain H63, which comprises the following steps:

inoculating resin ganoderma lucidum strain H63 to a PDA culture medium under aseptic conditions, sealing and placing the culture medium in an incubator at 28 ℃ for constant temperature culture until colonies grow on a culture dish;

step (2), inoculating the strain cultured in the step (1) into a liquid strain culture medium, and placing the liquid strain culture medium in a shaking table for culturing for 7-10 days at the constant temperature of 25-30 ℃;

the liquid strain culture medium is as follows: 19-21g/L maltose, 1.9-2.1g/L yeast powder, 0.45-0.55g/L magnesium sulfate, vitamin B ₁ 98-102mg, 0.95-1.05g of agar, ph=5.5;

step (3), inoculating the liquid strain cultured in the step (2) into a cultivation bag, and placing the cultivation bag in a 28 ℃ cultivation room for dark cultivation; after the hypha grows up to be full of the cultivation bag, performing earthing cultivation; carrying out fruiting management after earthing;

wherein, the culture bag substrate comprises the following raw materials in parts by weight of dry matter: 45-60 parts of broad-leaved tree wood chips, 25-30 parts of corncobs, 15-20 parts of wheat bran, 1.0-2.0 parts of gypsum, 0.5-1.0 parts of lime and 1.0-1.5 parts of white sugar; pH value is 6-8, and water content is 55-65%.

Further, it is preferable that in the step (1), the culture is carried out at a constant temperature in an incubator at 28℃for 5 to 10 days.

Further, it is preferable that in the step (2), the rotation speed of the cradle is 150-160r/min;

the liquid strain culture medium is as follows: maltose 20g/L, yeast powder 2g/L, magnesium sulfate 0.5g/L, vitamin B ₁ 100mg, 1g of agar, ph=5.5.

Further, preferably, in the step (3), the cultivation bag substrate comprises 49% of broadleaf tree wood chips, 29% of corncob, 19% of wheat bran, 1% of gypsum, 1% of lime and 1% of white sugar in percentage by weight of dry matter; pH value is 6-8, and water content is 55-65%.

Further, preferably, in the step (3), the weight of the cultivation bag is 1.0-1.2kg, and each bag is inoculated with 25m L liquid strain;

the fruiting management is specifically to keep the temperature at 25-32 ℃, the relative humidity of air at 85% -95%, the concentration of carbon dioxide at less than 0.2% and the illumination intensity at 300-1000Lx.

Further, preferably, in the step (3), the specific method of soil-covered cultivation is as follows:

weeding, sterilizing and killing insects on the cultivation site;

selecting a planting groove with the depth of 0.20-0.30m, the width of 0.10-1.50 m and the length of not more than 10.0m, wherein the furrow depth in the middle is 25-30cm;

the overgrown mycelium cultivation bags are conveyed to a cultivation place at the temperature of 25-28 ℃ and the illumination intensity of 500-1000Lx for continuous cultivation for 1-2 weeks, then 1/3 fungus bags on the upper parts of the fungus bags are removed and vertically arranged in a planting groove, the open ends are upward, and the distance between fungus sticks is kept at 5-10cm; and (3) covering soil on the planting groove, wherein the thickness of the soil is 3-5 cm.

Further, preferably, the fruiting body is managed until the tender yellow or white color of the edge of the fungus cover completely disappears, the fungus holes start to spray basidiospores outwards, and the spores are released and the color is deepened, so that the fruiting body can be harvested.

The invention also provides application of the mycelium or fruiting body of the resin ganoderma lucidum strain H63 in preparing antitumor drugs or tranquillizing and sleep promoting drugs.

The resin ganoderma lucidum strain H63 contains high content of ganoderan and ganoderma lucidum triterpene compounds. The ganoderma lucidum strain is detected by a detection method for determining ganoderma lucidum polysaccharide and ganoderma lucidum triterpene (calculated by oleanolic acid) in the 2020 edition of Chinese pharmacopoeia, the content of the wild resin ganoderma lucidum strain H63 polysaccharide is 1.05 percent, the content of triterpene is 2.55 percent, the content of the cultivated strain H63 polysaccharide and the content of triterpene are obviously improved, the polysaccharide content is 1.26 percent, the content of triterpene is 2.86 percent, and the ganoderma lucidum polysaccharide has wide pharmacological activity, and can reduce blood sugar, blood fat, resist thrombus, resist oxidation, remove free radicals, resist aging, resist radiation, resist tumors, promote blood circulation, regulate immunity, regulate nucleic acid and protein metabolism, promote DNA synthesis and promote human umbilical blood LAK cell proliferation. The Ganoderma triterpene compound has antiinflammatory, analgesic, tranquilizing, antiaging, tumor cell killing, and anoxia resisting effects.

Compared with the prior art, the invention has the beneficial effects that:

the invention combines the molecular biology and edible fungus breeding techniques to identify, biologically characterize and domesticate the wild ganoderma lucidum strain. H63 is gathered with known resin ganoderma G. Resinaceum in a ganoderma polygenic phylogenetic tree, and the supporting rate of 97% is obtained; the optimal growth temperature of the H63 strain is 30 ℃, and hypha grows slowly or does not grow when the temperature is lower than 24 ℃, and belongs to high-temperature strains.

The growth period of the H63 strain is short, the average mother strain period is 8 days (a culture dish with the thickness of 90 mm), and the maximum growth rate of solid hypha is 14.92mm/d; the average liquid fermentation period was 7 days (200L seed tank); the average cultivation bag growth period is 26 days (bag average wet weight is 1.0-1.2 kg), and compared with other Ganoderma lucidum variety, the cultivation time can be saved by 8-15 days. The cultivated resin ganoderma lucidum H63 has strong heat resistance, which is consistent with the experimental result of the biological characteristics of hypha, and the growth period of fruiting bodies cultivated by the strain is shorter, and only 65-80 days are needed from the cultivation in the lower place to the mature picking of fruiting bodies, which is 20-30 days shorter than the growth period of other ganoderma lucidum. On the other hand, the ganoderma lucidum H63 strain has regular ganoderma lucidum, excellent properties and high spore yield, and each fungus bag entity produces 24.6g of spore powder on average. The mycelium culture stage and the fruiting body growth stage both show stronger disease and pest resistance, and have better strain activity and stress resistance. The H63 strain and fruiting body grow well at the high temperature of 28-35 ℃, have high active ingredient content, have the potential of cultivation and popularization in low-altitude areas, can provide important reference for the development of high-quality wild ganoderma lucidum resources in Yunnan, and are easy to popularize and apply.

Drawings

FIG. 1 is a graph of building a ganoderma phylogenetic tree based on ITS+LSU+TEF1-alpha+RPB2 Maximum Likelihood (ML), with branch node support rates exhibiting bootstrap values above 75%;

FIG. 2 shows growth rate of H63 mycelium of Ganoderma resin in different carbon sources;

FIG. 3 shows growth rate of H63 mycelium of Ganoderma resin in different nitrogen sources;

FIG. 4 shows the growth rate of Ganoderma lucidum H63 mycelia in different inorganic salts;

FIG. 5 shows the growth rate of H63 mycelium of Ganoderma resin at different pH values;

FIG. 6 shows the growth rate of H63 mycelium of Ganoderma resin at different temperatures;

FIG. 7 is a morphology of Ganoderma lucidum H63; wherein, a-b: a fruiting body; c: pore surfaces; d: cutting the fungus cover; e: cortical cells; f: a fungus meat skeleton hypha; g: bacterial tube reproduction hypha; h: the fungus tube is combined with hypha; i-k: a basidion or pseudobasidion; l-o: basidiospore; p-q: pure culture. Scale bar: 30mm (e-h); 10 μm (i-k, o); 5 μm (l-n);

FIG. 8 is a shake flask growth chart of liquid of the mycelium of Ganoderma lucidum H63.

FIG. 9 is a diagram of domestication, cultivation and growth of resin ganoderma lucidum H63; a: primordia and a stipe formation period; b: a fungus cap differentiation period; c-d: maturity of the fruiting body; e-f fruiting body aging period;

the resin ganoderma lucidum strain H63 (Ganoderma resinaceum) of the invention is preserved in the microorganism strain preservation center of Guangdong province in 2021, 12 months and 27 days, and the preservation number is GDMCC No:62106 the preservation address is building 5 of Guangzhou Miao 100 # college of first-vogue 59, and the university of Guangdong province institute of microorganisms.

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.

Example 1

example 2

The artificial culture method of the resin ganoderma lucidum strain H63 comprises the following steps:

step (2), inoculating the strain cultured in the step (1) into a liquid strain culture medium, and placing the liquid strain culture medium in a shaking table at the constant temperature of 28 ℃ for 8 days;

the liquid strain culture medium is as follows: maltose 20g/L, yeast powder 2g/L, magnesium sulfate 0.5g/L, vitamin B ₁ 100mg, 1g of agar, ph=5.5;

wherein, the culture bag substrate comprises the following raw materials in parts by weight of dry matter: 55 parts of broad-leaved tree wood chips, 28 parts of corncobs, 18 parts of wheat bran, 1.5 parts of gypsum, 0.8 part of lime and 1.2 parts of white sugar; pH 7 and water content 60%.

Example 3

step (2), inoculating the strain cultured in the step (1) into a liquid strain culture medium, and placing the liquid strain culture medium in a shaking table at the constant temperature of 25 ℃ for 7 days;

the liquid strain culture medium is as follows: 19g/L maltose, 1.9g/L yeast powder, 0.45g/L magnesium sulfate and vitamin B ₁ 98mg, 0.95g of agar, ph=5.5;

wherein, the culture bag substrate comprises the following raw materials in parts by weight of dry matter: 45 parts of broad-leaved tree wood chips, 25 parts of corncobs, 15 parts of wheat bran, 1.0 part of gypsum, 0.5 part of lime and 1.0 part of white sugar; pH value 6, water content 55%.

In the step (1), the culture was carried out at a constant temperature in an incubator at 28℃for 5 days.

In the step (2), the rotation speed of the cradle is 150r/min;

in the step (3), the weight of the material in the cultivation bags is 1.0kg, and each bag is inoculated with 25mL of liquid strain;

In the step (3), the concrete method of earthing cultivation is as follows:

weeding, sterilizing and killing insects on the cultivation site;

selecting a planting groove with the depth of 0.20m, the width of 1.00 m and the length of not more than 10.0m by machinery or manpower, wherein the depth of a furrow in the middle is 25cm;

the overgrown mycelium cultivation bags are conveyed to a cultivation place at the temperature of 25-28 ℃ and the illumination intensity of 500-1000Lx for continuous cultivation for 1 week, then 1/3 fungus bags on the fungus bags are removed and vertically arranged in a planting groove, the open ends are upward, and the distance between fungus sticks is kept at 5cm; and (5) covering soil on the planting groove, wherein the thickness of the soil is 3 cm.

And (3) the fruiting body is managed until the tender yellow or white color of the edge of the fungus cover completely disappears, the fungus holes start to spray basidiospores outwards, and the spores are released and the color is deepened, so that the fruiting body can be harvested.

Example 4

step (2), inoculating the strain cultured in the step (1) into a liquid strain culture medium, and placing the liquid strain culture medium in a shaking table at a constant temperature of 30 ℃ for 10 days;

the liquid strain culture medium is as follows: 21g/L maltose, 2.1g/L yeast powder, 0.55g/L magnesium sulfate and vitamin B ₁ 102mg, agar 1.05g, ph=5.5;

wherein, the culture bag substrate comprises the following raw materials in parts by weight of dry matter: 60 parts of broad-leaved tree wood chips, 30 parts of corncobs, 20 parts of wheat bran, 2.0 parts of gypsum, 1.0 part of lime and 1.5 parts of white sugar; pH 8 and water content 65%.

In the step (1), the culture was carried out at a constant temperature in an incubator at 28℃for 10 days.

In the step (2), the rotation speed of the cradle is 160r/min;

in the step (3), the weight of the material in the cultivation bags is 1.2kg, and each bag is inoculated with 25mL of liquid strain;

In the step (3), the concrete method of earthing cultivation is as follows:

weeding, sterilizing and killing insects on the cultivation site;

selecting a planting groove with the depth of 0.30m, the width of 1.50 m and the length of not more than 10.0m by machinery or manpower, wherein the depth of a furrow in the middle is 30cm;

the overgrown mycelium cultivation bags are conveyed to a cultivation place at the temperature of 25-28 ℃ and the illumination intensity of 500-1000Lx for continuous cultivation for 2 weeks, then 1/3 fungus bags on the upper parts of the fungus bags are removed and vertically arranged in a planting groove, the open ends are upward, and the distance between fungus sticks is kept at 10cm; and (5) covering soil on the planting groove, wherein the thickness of the covering soil is 5 cm.

Example 5

wherein, the culture bag substrate comprises the following raw materials in parts by weight of dry matter: 49% of broad-leaved tree wood chips, 29% of corncob, 19% of wheat bran, 1% of gypsum, 1% of lime and 1% of white sugar; pH 7 and water content 60%.

In the step (1), the culture was carried out at a constant temperature in an incubator at 28℃for 8 days.

In the step (2), the rotation speed of the cradle is 155r/min;

In the step (3), the culture bag substrate comprises the following components in percentage by weight of dry matter,

in the step (3), the weight of the material in the cultivation bags is 1.1g, and each bag is inoculated with 25mL of liquid strain;

In the step (3), the concrete method of earthing cultivation is as follows:

weeding, sterilizing and killing insects on the cultivation site;

selecting a planting groove with the depth of 0.25m, the width of 1.20 m and the length of not more than 10.0m by machinery or manpower, wherein the depth of a furrow in the middle is 28cm;

the overgrown mycelium cultivation bags are conveyed to a cultivation place at the temperature of 25-28 ℃ and the illumination intensity of 500-1000Lx for continuous cultivation for 1.5 weeks, then 1/3 fungus bags on the fungus bags are removed and vertically arranged in a planting groove, the open ends are upward, and the distance between fungus sticks is kept at 8cm; soil is covered on the planting groove, and the thickness of the covered soil is 4 cm.

Application instance

1 materials and methods

1.1 test strains

The resin ganoderma Ganoderma resinaceum (strain number: H63) is obtained from New Anshizhen of Mongolia, yunnan province, and is separated and purified by a tissue separation method. The specific form is shown in fig. 7.

1.2 test Medium

PDA medium raw material: 200g (peeling) of potato, 20g of glucose, 18g of agar and 1000 ml of distilled water. The preparation and sterilization method of the culture medium comprises the steps of chopping 200g of peeled potatoes, adding water to 1L of the chopped potatoes, thoroughly boiling, filtering potato residues by using gauze to obtain 1L of potato starch solution, sequentially adding 18g of agar and 20g of glucose into a conical flask with the concentration of 2 ml, stirring and boiling, pouring the mixture into the conical flask with the concentration of 2 ml, covering with special asbestos paper, fastening the mixture by using rubber strings, and preventing the culture medium from being blown out at high temperature. Sterilizing at 121deg.C under high temperature and high pressure for 25min, taking out when air pressure drop is 0, pouring into a flat plate with diameter of 5cm at the side of alcohol lamp on an ultra-clean workbench, pouring amount of 1/3 of the height of the lower layer of the flat plate, and cooling for use.

Basal medium: is used for screening and optimizing a carbon source (20 g/L), a nitrogen source (2 g/L), inorganic salt (0.5 g/L), pH and temperature, and after the ingredients are finished, the materials are put into a high-pressure steam pot to be sterilized at the high temperature of 120-125 ℃ for 30 minutes, and then are taken out to be cooled for standby.

Liquid strain medium: maltose 20g, yeast extract 2g, magnesium sulfate 0.5g, vitamin B ₁ 100mg, 1g of agar and 1L of purified water. Sterilizing in a high-pressure steam kettle at 120-125deg.C for 30 min, taking out, and cooling.

The culture bag substrate formula comprises 49% of broadleaf tree sawdust, 29% of corncob, 19% of wheat bran, 1% of gypsum, 1% of lime and 1% of white sugar according to the weight percentage of dry matters. pH value is 6-8, and water content is 55-65%. The wood chips are soaked in water for 24 hours in advance, so that the wood chips are soaked thoroughly, incomplete sterilization is prevented, the cultivation materials are weighed according to the cultivation seed formula, mixed, filled in 35cm multiplied by 17cm multiplied by 0.005cm polypropylene angle folding plastic bags by a bagging nest opening integrated machine, placed in a 121 ℃ high-pressure steam pot, sterilized for 2 hours, taken out and cooled for standby.

1.3 molecular biological Studies

The method comprises the steps of taking a strain separated from a wild ganoderma lucidum fruiting body specimen as an extraction material, extracting DNA (deoxyribonucleic acid) by using a plant genome kit of Beijing qingke new industry biological technology Co., ltd.) for carrying out ITS, nrLSU, tef-alpha and rpb gene sequence amplification, and carrying out homology analysis and phylogenetic analysis of BLAST comparison data. The PCR reaction system (25. Mu.l) included: 2.5. Mu.l of PCR buffer, 2.5. Mu.l of 0.2% BSA, 2. Mu.l of dNTPs (2.5 mmol), 100 pmol/. Mu.l of each of the upstream and downstream primers, 1. Mu.l of DNA solution and 16. Mu.l of sterile ddH ₂ O。

Four gene segments selected: ITS, LSU, TEF-1a and RBP2, the base composition of the specific primers is as follows:

ITS primer pair:

ITS1-F：5'-cttggtcatttagaggaagtaa-3'；(SEQ ID NO.1)

ITS4：5'-tcctccgcttattgatatgc-3'；(SEQ ID NO.2)

nLSU primer pair:

LR0R：5'-acccgctgaacttaagc-3'；(SEQ ID NO.3)

LR5：5'-tcctgagggaaacttcg-3'；(SEQ ID NO.4)

TEF-1. Alpha. Primer pair:

983F：5'-gcyccygghcaycgtgayttyat-3'；(SEQ ID NO.5)

1567R：5'-achgtrccrataccaccratctt-3'；(SEQ ID NO.6)

RBP2 primer pair:

fRrbp2-6F：5'-tggggyatggtntgyccygc-3'；(SEQ ID NO.7)

fRrbp2-7cR：5'-cccatrgcttgyttrcccat-3'。(SEQ ID NO.8)；

note that: bases include a, t, c, g, with other letters appearing in the primer representing degenerate bases.

1.4 study of biological Properties

PDA culture medium is used as the culture medium of the activated strain, and after the transfer, the colony grows to a culture dish for carrying out biological characteristic research. Inoculating the fungus blocks into liquid strain culture mediums with different conditions by using a puncher with the diameter of 7mm for culturing. All experiments except the temperature experiment were placed in a constant temperature incubator at 28℃for cultivation. Each treatment was set up for 8 replicates. The colony size is measured every 24 hours by a cross streaking method, until one of the culture medium colonies is full of growth, the measurement is stopped, the mycelium morphology and growth vigor are observed and recorded, and the data result is subjected to statistical analysis by Excel2019 and PSS 20.0.

1.4.1 carbon source experiments: taking a basic culture medium as a control, respectively setting 5 carbon sources: glucose, sucrose, maltose, lactose and soluble starch were used as treatment groups at a concentration of 20g/L.

1.4.2 nitrogen source experiments: setting 5 nitrogen sources by taking a basal medium as a control: peptone, ammonium chloride, ammonium sulfate, urea, yeast powder were used as treatment groups at a concentration of 2g/L.

1.4.3 inorganic salt experiments: setting 5 inorganic salts by taking a basal medium as a control: magnesium sulfate, ferric trichloride, calcium carbonate, ferrous sulfate and sodium chloride are used as treatment groups, and the concentration is 0.5g/L.

1.4.4ph experiments: the basic culture medium formula is prepared by adjusting acid with 1.0mol/L HCl and alkali with 1.0mol/L NaO H solution, and 5 initial pH gradients are respectively adjusted: 5.0, 5.5, 6.0, 6.5, 7.0.

1.4.5 temperature experiments: after inoculation with basal medium, the dishes were placed separately: culturing at 20deg.C, 22deg.C, 24deg.C, 26deg.C, and 28deg.C in a constant temperature incubator.

1.4.6 orthogonal experiments: combining 5 single-factor experiment bases, selecting 4 factors with great influence on the growth speed of H63 hyphae, and selecting 3 better horizontal factors from each factor. Select L ₉ (3 ⁴ ) The orthogonality table is used for carrying out orthogonality test on carbon source, nitrogen source, inorganic salt and temperature which have obvious influence on hypha growth.

1.5 domestication cultivation

Taking out the separated and stored Ganoderma strain from the refrigerator, standing at room temperature for 4-6 hr for activation, collecting purified mycelium blocks, inoculating to new PDA culture medium, and culturing in a constant temperature incubator at 28deg.C until mycelium grows. 2-3 ganoderma lucidum mother seeds with the size of broad bean grains are taken and inoculated into 250ml of liquid strain culture solution, and liquid shaking table 10d is carried out under the condition of 28 ℃ and 160 r/min. Then selecting 35cm x 17cm polypropylene plastic bags, weighing 1.0-1.2kg of each bag, sterilizing at 121deg.C for 2 hr, cooling, inoculating liquid strain after shake culture into culture bags, inoculating 25ml liquid strain per bag, and placing into 28 deg.C culture room for dark culture. And (5) after 25-30 days, the hypha grows up to be full of the cultivation bag, and the soil-covered cultivation is carried out.

Before cultivation, weeds in the field are removed, lime is spread for ploughing and insolating, and a biological agent or a physical trapping and killing method is adopted for sterilizing and killing insects in the cultivation environment. After the fungus growing of the cultivation fungus bag is completed, the cultivation fungus bag is conveyed to a cultivation place at the temperature of 25-28 ℃ and the illumination intensity of 500-1000Lx for continuous cultivation for 1-2 weeks, and after-ripening cultivation is completed. Selecting a planting groove with the depth of 0.20-0.30m, the width of 1.00-1.50 m and the length of not more than 10.0m, wherein the furrow depth in the middle is 25-30cm. A blade is adopted to open small holes at the top end of the fungus bag and then vertically arranged in a planting groove or a planting hole, the open ends face upwards, and the distance between fungus sticks is kept at 5cm to 10cm; and (3) covering soil on the planting grooves or planting holes, wherein the number of bags is 4000-4200 bags per mu, and the thickness of the covering soil is 3-5 cm.

And (3) carrying out ganoderma lucidum production management after earthing, maintaining the temperature at 25-32 ℃, atomizing and spraying water to the greenhouse space at regular time, maintaining the surface soil humidity and the space relative humidity at 85-95%, and carrying out ventilation frequently to maintain the carbon dioxide concentration below 0.2%, and carrying out shading treatment to ensure that the illumination intensity is 300-1000Lx. When the ganoderma lucidum grows to a mature period, the fungus cover is not increased and thickened, the light yellow edge of the fungus cover disappears, the fungus holes start to spray basidiomycetes outwards, the spores are released, and the color of the fungus cover is deepened, so that the ganoderma lucidum can be harvested. The stem pedicel is reserved for 1cm by cutting off along the base of the fungus stem with a blade or a scissors during collection.

2 results and analysis

2.1 phylogenetic analysis

Relevant reference sequences were downloaded from genbank and Tomophagus colossus as an outer cluster. H63 is aggregated with known resin ganoderma G.resinaceum in ganoderma polygenic phylogenetic tree and obtains 97% support rate, as shown in figure 1.

Characterization sequence of H63 strain: the ITS, nrLSU, tef 1-alpha and rpb2 gene sequences are shown in SEQ ID NO. 9-SEQ ID NO. 12.

2.2 biological Property study results

2.2.1 Single factor experiment results

As can be seen from FIG. 2, H63 had the fastest growth rate of hyphae under the condition of maltose as a carbon source. As can be seen from FIG. 3, H63 grows at the fastest rate under the condition that yeast powder is used as the nitrogen source. As can be seen from fig. 4, H63 grows at the fastest rate under the condition that magnesium sulfate is an inorganic salt. As can be seen from FIG. 5, H63 has an optimum pH of 5.5. As can be seen from FIG. 6, the optimum temperature of H63 is 30 ℃.

2.2.2 orthogonal experiment results

The results of the orthogonal experiments are shown in Table 1.

TABLE 1

Note that: data are mean of 8 replicates; kn represents the average growth rate of hypha of the resin ganoderma lucidum strain on the level of n, and R is very bad; "+", "++", "+++". Indicating hyphae growth vigor is enhanced in turn

As can be seen from the orthogonal visual analysis of the 4 factor 3 level, the strain carbon source was the greatest, r=3.42, followed by temperature, nitrogen source and inorganic salts. The maximum growth rate of X3 in the carbon source is 14.92mm/d, the maximum growth rate of X2 in the nitrogen source is 13.94mm/d, the maximum growth rate of X3 in the inorganic salt is 12.50mm/d, and the maximum growth rate of X3 in the temperature is 14.73mm/d. In combination with analysis of variance, the significant differences of 4 factors are, in order from large to small, carbon source > temperature > nitrogen source > inorganic salt. Analysis of comprehensive data results, and optimal growth conditions of strains: the carbon source is maltose, the nitrogen source is yeast powder, the inorganic salt is magnesium sulfate, and the temperature is 30 ℃.

Through the pH test of the resin ganoderma lucidum mycelia, the strain mycelia are mainly suitable for the growth under the meta-acid condition, and when the pH exceeds 7.0, the mycelia can germinate, but the growth is very slow. The most suitable pH of hypha growth is 5.5, and the hypha growth is rapid and thicker.

2.3 domestication cultivation results

The liquid shake flask growth chart of the resin ganoderma lucidum H63 mycelium is shown in fig. 8, and the domestication cultivation growth chart is shown in fig. 9. The H63 ganoderma lucidum fruiting body is successfully domesticated by bag material earthing cultivation, and the growth cycle of the resin ganoderma lucidum H63 is short, the average mother seed cycle is 8 days (a culture dish with the diameter of 90 mm), and the maximum growth rate of solid hypha is 14.92mm/d from the aspects of producing seeds and cultivating ganoderma lucidum; the average liquid fermentation period was 7 days (200L seed tank); the average cultivation bag growth period is 26 days (bag average wet weight 1.0-1.2 kg); the total production of the cultivation bags 300 is 3 bags polluted and the pollution rate is 1 percent. The yield of the cultivated glossy ganoderma is 98%, the bud period is 15 days, the differentiation period is 29 days, the harvesting period is 77 days, the average fresh weight of the single fruiting body is 45.20g, the average dry weight of the single fruiting body is 17.80g, and the biological efficiency is 9.04%. The invention adopts DNA bar code molecular sequence phylogenetic analysis, H63 and reported resin ganoderma lucidum are in one phylogenetic branch, and 97% supporting rate is obtained, as shown in figure 1.

The experimental strain can grow under different carbon source conditions, the conditions of glucose and maltose grow faster, and lactose grows slowest. Under different nitrogen source conditions, the H63 strain grows at the fastest speed under the condition of yeast powder and the slowest speed under the condition of urea. The growth difference of hyphae of the H63 strain is obvious under different inorganic salt conditions, the growth speed of the hyphae is fastest under magnesium sulfate conditions, the optimal growth temperature of the H63 strain is 30 ℃, and the growth speed of the hyphae is reduced or does not grow when the temperature is lower than 24 ℃, so that the strain belongs to high-temperature strains.

The ganoderma lucidum strain H63 bag material of the invention is covered with soil for domestication, and the result shows that the ganoderma lucidum strain H63 resin has regular fruiting, good properties and high spore yield, and each fungus bag entity produces 24.6g of spore powder on average. The mycelium culture stage and the fruiting body growth stage both show stronger disease and pest resistance, and have better strain activity and stress resistance. Belongs to high-temperature strain and has the potential of cultivation and popularization in low-altitude areas.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> institute of biotechnology and germplasm research of the academy of agricultural sciences of Yunnan province

<120> a resin ganoderma lucidum strain H63 and application thereof

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<170> SIPOSequenceListing 1.0

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achgtrccra taccaccrat ctt 23

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gtctcacaaa cgagacggtt agaagctcgc caaaacgctt cacggtcgcg gcgtagacat 180

tatcacaccg agagccgatc cgcaaggaat caagctaata catttaagag gagccgaccg 240

aaacacggcc gacaagcctc caagtccaag cctacaaacc cgcaaagatc tgtaagttga 300

agatttcatg acactcaaac aggcatgctc ctcggaatac caaggagcgc aaggtgcgtt 360

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tcttcatcga tgcgagagcc aagagatccg ttgctgaaag ttgtattata gatgcgttac 480

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gatccacaag ctcccataag gagcccgctt ccacaacgtc tagaacccac agtaagtgca 600

caggtgtaga gtggatgagc agggcgtgca catgcctcgg aaggccagct acaacccagt 660

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cgggtcccaa catacatgct ctaccgcgga tccgtcagag aacgtcaggt ccgggcgtcg 180

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gcaaaagcaa ggctgagttc cggacaacgc gactgacttc aagcgtttcc ctttcagcaa 540

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gcggaaagca cttctccaga ctacaactcg gacggccaaa gaccgccaga ttttaaattt 840

gagcttttcc cgcttcactc gcagttacta ggggaatcct tgttagtttc ttttcctccg 900

cttattgata tg 912

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<212> DNA

<213> 11 (Artificial sequence)

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tggagtctct agaagagaat gctcatgctt caacrccttg cacgaaggta tttgtgaatg 180

gcgtttggat gggtgtccac cgagatcctg tgaagctcgt cagcacactc cggaaactcc 240

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aagaggagac agtcatgatc tgtatgacac cggaggattt ggagaactct aggctccagg 540

ctgccggtat cgacccaaac gcagacgacg agaatgacga cccctcggct cgattgaagg 600

cggcgacctc tgcacacaca tggacgcatt gcgagattca tccaagtatg attttgggcg 660

tctgtgccag tattattccc ttccccgacc acaaccaggt aagctcgggc taggaggtca 720

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tggg 784

Claims

1. An artificial culturing method of a resin ganoderma lucidum strain H63, which is characterized in that the resin ganoderma lucidum strain H63 is preserved in the microorganism strain preservation center of Guangdong province in 2021, 12 months and 27 days, and the preservation number is GDMCC No:62106;

the artificial culture method comprises the following steps:

step (2), inoculating the strain cultured in the step (1) into a liquid strain culture medium, and placing the liquid strain culture medium in a shaking table at the constant temperature of 25-30 ℃ for 7-10 days;

the liquid strain culture medium is as follows: 19-21g/L maltose, 1.9-2.1g/L yeast powder, 0.45-0.55g/L magnesium sulfate, vitamin B ₁ 98-102mg, agar 0.95-1.05g，pH=5.5；

wherein, the culture bag substrate comprises the following raw materials in parts by weight of dry matter: 45-60 parts of broad-leaved tree wood chips, 25-30 parts of corncobs, 15-20 parts of wheat bran, 1.0-2.0 parts of gypsum, 0.5-1.0 parts of lime and 1.0-1.5 parts of white sugar; pH value is 6-8, water content is 55-65%;

in the step (1), the culture is carried out for 5 to 10 days at a constant temperature in an incubator at 28 ℃.

2. The artificial culture method of the resin ganoderma lucidum strain H63 according to claim 1, wherein in the step (2), the rotation speed of the shaking table is 150-160r/min;

3. The artificial culture method of the resin ganoderma lucidum strain H63 according to claim 1, wherein in the step (3), the culture bag substrate comprises, by weight percentage of dry matter, 49% of broad-leaved tree wood chips, 29% of corncob, 19% of wheat bran, 1% of gypsum, 1% of lime and 1% of white sugar; pH value is 6-8, and water content is 55-65%.

4. The artificial culture method of the resin ganoderma lucidum strain H63 according to claim 1, wherein in the step (3), the weight of the cultivation bag is 1.0-1.2kg, and each bag is inoculated with 25mL of liquid strain;

5. The artificial culture method of the resin ganoderma lucidum strain H63 according to claim 1, wherein in the step (3), the specific method of the earthing cultivation is as follows:

weeding, sterilizing and killing insects on the cultivation site;

selecting a planting groove with the depth of 0.20-0.30m, the width of 1.00-1.50 m and the length of not more than 10.0m, wherein the furrow depth in the middle is 25-30cm;

continuously culturing the mycelium cultivation bag to a cultivation place at 25-28 ℃ and 500-1000Lx illumination intensity for 1-2 weeks, removing 1/3 fungus bag at the upper part of the fungus bag, vertically arranging the fungus bag in a planting groove, opening the bag end upwards, and keeping the distance between fungus sticks at 5-10cm; and (3) covering soil on the planting groove, wherein the thickness of the soil is 3-5 cm.

6. The artificial culture method of the resin ganoderma lucidum strain H63 according to claim 1, wherein the ganoderma lucidum is cultivated until the tender yellow color or white color of the edge of the fungus cover is completely disappeared, the fungus holes start to spray basidiospores outwards, and the spores are released and the color is deepened, so that the ganoderma lucidum can be harvested.

7. Use of mycelium or fruiting body of Ganoderma resina strain H63 according to any one of claims 1 to 6 in the preparation of antitumor or tranquillizing and sleep promoting medicines.