CN115039638A

CN115039638A - Ganoderma resinatum strain H63 and application thereof

Info

Publication number: CN115039638A
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: 2022-09-13
Anticipated expiration: 2042-04-22
Also published as: CN115039638B

Abstract

The invention relates to a resin ganoderma lucidum strain H63 and application thereof, belonging to the technical field of microorganisms. The ganoderma lucidum strain H63 is already preserved in Guangdong province microbial strain preservation center in China at 12 months and 27 days in 2021, and the preservation number is GDMCC No: 62106. the strain obtained by tissue separation of a specimen collected from a tropical region of Yunnan China is combined with morphological and phylogenetic analysis to determine the classification status, and domestication cultivation finds that the resin lucid ganoderma H63 has strong heat resistance, short growth period of a fruit body, high spore yield and strong stress resistance, the polysaccharide content is 1.26 percent, and the triterpene content is 2.86 percent. The resin ganoderma lucidum H63 strain and the fruiting body of the invention grow well at the high temperature of 28-35 ℃, have high active ingredient content and excellent commodity character, have the potential of cultivation and popularization in low-altitude areas, and can provide important reference for exploring and utilizing the wild ganoderma lucidum resources in Yunnan.

Description

Ganoderma resinatum 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 genus is one of the most studied genera of the Ganoderma family and the Polyporales, and is established by Peter Adolph Karsten, Finnish botanicals, in 1881, and Ganoderma lucidum (W.Cur.Fr.) Karst. which has lacquer-like luster of the cap and stipe of the fruiting body as a model species of this genus.

Modern researches show that some ganoderma lucidum varieties known at present are rich sources of high-bioactivity substances, particularly 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 the main bioactive components in ganoderma.

With the continuous development of medical science, the disease prevention and treatment effects and health care consciousness of people on the lucid ganoderma are gradually improved, and the demand on the lucid ganoderma is continuously increased. Due to the change of ecological environment and the excessive collection of wild ganoderma lucidum resources, the number of wild ganoderma lucidum is gradually reduced, and therefore, ganoderma lucidum products required by the market are artificially cultured as main sources. The resource characteristics and artificial domestication and cultivation of rare ganoderma lucidum strains are developed, thereby being beneficial to perfecting the collection, evaluation and utilization of ganoderma lucidum resources in regions and providing practical guiding significance for the protection and continuous utilization of ganoderma lucidum species.

The resin ganoderma strain is a new strain collected and separated in New Anchou town of Monself city of red river, Yunnan province, and the research report of the strain is not found.

Disclosure of Invention

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

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

a resin glossy ganoderma strain H63, which has been preserved in 27 months at 2021 in the China Guangdong province culture Collection center for microorganisms with the preservation number GDMCC No: 62106.

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

step (1), inoculating resin ganoderma lucidum strain H63 to a PDA culture medium under aseptic condition, sealing and placing in a 28 ℃ incubator for constant-temperature culture until bacterial colonies grow over a culture dish;

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

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

step (3), inoculating the liquid strains cultured in the step (2) into a culture bag, and placing the culture bag in a culture room at 28 ℃ for dark culture; after the hyphae grow over the cultivation bags, carrying out earthing cultivation; earthing and then carrying out ganoderma lucidum output management;

the cultivation bag substrate comprises the following raw materials in parts by weight: broad-leaved tree sawdust 45-60 parts, corncob 25-30 parts, wheat bran 15-20 parts, gypsum 1.0-2.0 parts, lime 0.5-1.0 part, white sugar 1.0-1.5 parts; the pH value is 6-8, and the water content is 55-65%.

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

Further, it is preferable that, in the step (2), the rotation speed of the shaking table is 150-;

the liquid strain culture medium comprises: maltose 20g/L, yeast powder 2g/L, magnesium sulfate 0.5g/L, vitamin B ₁ 100mg, agar 1g, pH 5.5.

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

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

the lucid ganoderma emergence management is 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-.

Further, preferably, in the step (3), the soil-covering cultivation method comprises the following specific steps:

weeding, sterilizing and killing insects on a cultivation field;

mechanically or manually making planting grooves with depth of 0.20-0.30m, width of 0.10-1.50 m and length of no more than 10.0m, wherein the furrow depth in the middle is 25-30 cm;

the cultivation bag full of hypha is transported to the cultivation place at the temperature of 25-28 ℃ and the illumination intensity of 500-; and covering soil on the planting grooves, wherein the thickness of the covered soil is 3-5 cm.

Further, it is preferable that the fungus is discharged until a ring of bright yellow or white color disappears at the cap edge, basidiospores start to be sprayed outwards, and spores are released and deepened to collect.

The invention also provides application of the mycelium or the fruiting body of the resin ganoderma lucidum strain H63 in preparation of anti-tumor drugs or drugs for soothing nerves and promoting sleep.

The resin ganoderma lucidum strain H63 contains higher content of ganoderma lucidum polysaccharide and ganoderma lucidum triterpenoids. The detection method is adopted for detection by ganoderma lucidum polysaccharide determination and ganoderma triterpene determination (based on oleanolic acid) of the first edition of Chinese pharmacopoeia 2020, page 196, the content of polysaccharide of wild resin ganoderma lucidum strain H63 is 1.05%, the content of triterpene is 2.55%, the content of polysaccharide and triterpene of cultivated strain H63 is obviously improved, the content of polysaccharide is 1.26%, the content of triterpene is 2.86%, and the ganoderma lucidum polysaccharide has wide pharmacological activity, can reduce blood sugar and blood fat, resist thrombus and oxidation, remove free radicals, resist aging, radiation and tumors, promote blood circulation, regulate immunity, regulate nucleic acid and protein metabolism, promote DNA synthesis and promote proliferation of LAK cells in cord blood of a human body. Ganoderma triterpene compounds have antiinflammatory, analgesic, tranquilizing, antiaging, tumor cell poisoning, and anoxia resisting effects.

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

the invention combines the technologies of molecular biology, edible fungus breeding and the like to carry out the identification, biological characteristics and domestication cultivation of the strains of the wild ganoderma lucidum. H63 is gathered with known resin ganoderma G.resinaceum in the multigenic phylogenetic tree of ganoderma, and 97% of support rate is obtained; the optimum growth temperature of the H63 strain is 30 ℃, hypha grows slowly or does not grow when the temperature is lower than 24 ℃, and the H63 strain belongs to a high-temperature type strain.

The H63 strain has short growth cycle, the average mother seed cycle is 8 days (90mm culture dish), and the maximum growth rate of solid hypha is 14.92 mm/d; average liquid fermentation period was 7 days (200L seed tank); the average growth period of the cultivation bags is 26 days (the average humidity of the bags is 1.0-1.2kg), and the cultivation time can be saved by 8-15 days compared with other ganoderma lucidum varieties. The heat resistance of the cultivated resin ganoderma lucidum H63 is strong, which is consistent with the experiment result of hypha biological characteristics, the growth period of the cultivated fruiting body of the strain is short, only 65-80 days are needed from underground cultivation to mature picking of the fruiting body, and the growth period is 20-30 days shorter than that of other ganoderma lucidum. On the other hand, the resin ganoderma H63 strain produces uniform ganoderma, excellent properties and high spore yield, and each bag fruiting body produces 24.6g of spore powder on average. The hypha cultivation stage and the fruiting body growth stage show strong disease and pest resistance, and have good strain activity and stress resistance. The H63 strain and the fruiting body grow better under the high temperature condition of 28-35 ℃, the active ingredient content is high, and the method has the potential of cultivation and popularization in low-altitude areas.

Drawings

FIG. 1 is a diagram of the construction of a Ganoderma phylogenetic tree based on the ITS + LSU + TEF1- α + RPB2 Maximum Likelihood (ML) with branch node support rates showing lead values above 75%;

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

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

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

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

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

FIG. 7 is a morphological diagram of Ganoderma lucidum H63; wherein, a-b: fruiting bodies; c: a pore surface; d: cutting pileus into sections; e: cortical cells; f: hypha of mushroom meat skeleton; g: spawn tube reproductive hyphae; h: the fungus tube combines the hypha; i-k: a basilar or pseudo-basilar; l-o: basidiospores; p-q: and (5) pure culture. Scale bar: 30mm (e-h); 10 μm (i-k, o); 5 μm (l-n);

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

FIG. 9 is a diagram of domesticated and cultivated growth of Ganoderma lucidum H63; a: primordium and stipe formation phase; b: the stage of differentiation of the pileus; c-d: mature period of sporophore; e-f fruiting body aging period;

the resin Ganoderma lucidum strain H63(Ganoderma resinaceum) is preserved in the China Guangdong province microorganism strain preservation center at 12 months and 27 days in 2021, and the preservation number is GDMCC No: 62106, the preservation address is No. 59 building No. 5 building of Mirabhi 100 college of Mirabhi, Guangzhou province academy of sciences of Guangdong province.

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 invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.

Example 1

example 2

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

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

the liquid strain culture medium comprises: maltose 20g/L, yeast powder 2g/L, magnesium sulfate 0.5g/L, vitamin B ₁ 100mg, agar 1g, pH 5.5;

step (3), inoculating the liquid strains cultured in the step (2) into a culture bag, and placing the culture bag in a culture room at 28 ℃ for dark culture; after hyphae grow over the cultivation bags, carrying out soil covering cultivation; performing ganoderma lucidum production management after earthing;

the cultivation bag substrate comprises the following raw materials in parts by weight: 55 parts of broad-leaved tree sawdust, 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 of 60%.

Example 3

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

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

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

step (3), inoculating the liquid strains cultured in the step (2) into a culture bag, and placing the culture bag in a 28 ℃ culture room for dark culture; after the hyphae grow over the cultivation bags, carrying out earthing cultivation; performing ganoderma lucidum production management after earthing;

the cultivation bag substrate comprises the following raw materials in parts by weight: 45 parts of broad-leaved tree sawdust, 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 6, water content 55%.

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

In the step (2), the rotating speed of the shaking table is 150 r/min;

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

In the step (3), the soil-covering cultivation method comprises the following specific steps:

weeding, sterilizing and killing insects on a cultivation field;

mechanically or manually opening a planting groove with the depth of 0.20m, the width of 1.00 m and the length of not more than 10.0m, wherein the furrow depth in the middle is 25 cm;

the cultivation bag full of hypha is transported to the cultivation area for continuous cultivation for 1 week at the temperature of 25-28 ℃ and the illumination intensity of 500-1000Lx, then the upper 1/3 fungus bags of the fungus bags are removed and vertically arranged in the cultivation groove, the opening end is upward, and the distance between the fungus sticks is kept at 5 cm; and covering soil on the planting grooves, wherein the thickness of the covered soil is 3 cm.

And (4) managing the ganoderma to be grown until a circle of bright yellow or white on the edge of a pileus disappears, spraying basidiospores outwards from the fungus pores, releasing the spores, and deepening the color to harvest the ganoderma.

Example 4

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

the liquid strain culture medium comprises: maltose 21gL, 2.1g/L of yeast powder, 0.55g/L of magnesium sulfate and vitamin B ₁ 102mg, agar 1.05g, pH 5.5;

step (3), inoculating the liquid strains cultured in the step (2) into a culture bag, and placing the culture bag in a culture room at 28 ℃ for dark culture; after the hyphae grow over the cultivation bags, carrying out earthing cultivation; performing ganoderma lucidum production management after earthing;

the cultivation bag substrate comprises the following raw materials in parts by weight: 60 parts of broad-leaved tree sawdust, 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; the pH value is 8, and the water content is 65 percent.

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

In the step (2), the rotating speed of the shaking table is 160 r/min;

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

weeding, sterilizing and killing insects on a cultivation field;

mechanically or manually opening a planting groove with the depth of 0.30m, the width of 1.50 m and the length of not more than 10.0m, wherein the depth of a furrow in the middle is 30 cm;

the cultivation bag full of hypha is transported to a cultivation place for continuous cultivation for 2 weeks at the temperature of 25-28 ℃ and the illumination intensity of 500-1000Lx, then 1/3 fungus bags on the upper part of the fungus bags are taken off and vertically arranged in a cultivation groove, the opening end is upward, and the distance between fungus sticks is kept at 10 cm; and covering soil on the planting grooves, wherein the thickness of the covered soil is 5 cm.

Example 5

the cultivation bag substrate comprises the following raw materials in parts by weight: 49% of broad-leaf tree wood chips, 29% of corncobs, 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 in an incubator at 28 ℃ for 8 days.

In the step (2), the rotating speed of the shaking table is 155 r/min;

In the step (3), the culture bag substrate is measured according to the weight percentage of dry matters,

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

In the step (3), the specific method of soil-covering cultivation is as follows:

weeding, sterilizing and killing insects on a cultivation field;

mechanically or manually opening a planting groove with the depth of 0.25m, the width of 1.20 m and the length of not more than 10.0m, wherein the depth of a furrow in the middle is 28 cm;

the cultivation bag full of hypha is transported to the cultivation place at the temperature of 25-28 ℃ and the illumination intensity of 500-; and covering soil on the planting grooves, wherein the thickness of the covered soil is 4 cm.

Examples of the applications

1 materials and methods

1.1 test strains

Ganoderma resinatum Ganoderma resinaceum (strain number: H63) is collected from the town of New Anhui city of Mongolia, Red river, Yunnan province, and separated and purified by tissue separation method. The specific form is shown in fig. 7.

1.2 test Medium

Raw materials of PDA culture medium: 200g of potato (peeled), 20g of glucose, 18g of agar and 1000 ml of distilled water. A preparation method of a culture medium and a sterilization method comprise the steps of chopping 200g peeled potatoes, adding water to 1L, boiling thoroughly, filtering potato residues with gauze to obtain 1L of potato starch solution, sequentially putting 18g of agar and 20g of glucose into a conical flask with 500ml, stirring and boiling, pouring into the conical flask, covering with special asbestos mesh paper, and tightly binding with rubber bands to prevent the culture medium from being popped out at high temperature. Sterilizing at 121 deg.C for 25min under high temperature and high pressure, taking out when the air pressure drops to 0, pouring into a 5 cm-diameter plate beside an alcohol lamp on a superclean bench, pouring 1/3 with the height of the lower layer of the plate, and cooling.

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

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

The cultivation bag substrate formula comprises 49% of broad-leaved tree sawdust, 29% of corncobs, 19% of wheat bran, 1% of gypsum, 1% of lime and 1% of white sugar in percentage by weight of dry substances. The pH value is 6-8, and the water content is 55-65%. Soaking sawdust in water for 24h in advance to thoroughly soak the sawdust and prevent incomplete sterilization, weighing the culture materials according to the formula of the cultivated species, stirring the materials, filling the culture materials into polypropylene folded angle plastic bags of 35cm multiplied by 17cm multiplied by 0.005cm by a bagging and opening folding all-in-one machine, sterilizing the plastic bags in a high-pressure steam kettle at the temperature of 121 ℃ for 2 hours, taking out the plastic bags and cooling the plastic bags for later use.

1.3 molecular biology Studies

Strains separated from a wild ganoderma lucidum fruiting body specimen are used as extraction materials, liquid nitrogen is used for grinding, DNA is extracted by using a plant genome kit of Beijing Optimalaceae New Biotechnology Limited company to carry out ITS, nrLSU, tef 1-alpha and rpb2 gene sequence amplification, and homology analysis and phylogenetic analysis of BLAST comparison data are carried out. The PCR reaction system (25. mu.l) included: 2.5. mu.l of PCR reaction buffer, 2.5. mu.l of 0.2% BSA, 2. mu.l of dNTP (2.5mmol), 0.5. mu.l of each of the upstream and downstream primers at a concentration of 100 pmol/. mu.l, 1. mu.l of DNA solution and 16. mu.l of sterile ddH ₂ O。

Four gene fragments were selected: ITS, LSU, TEF-1a and RBP2, and the base composition of 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 set:

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

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

primer pair RBP 2:

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

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

note: the bases include a, t, c and g, and other letters appearing in the primer represent degenerate bases.

1.4 biological Property Studies

And (3) taking the PDA culture medium as an activated strain culture medium, and after the transfer, growing the bacterial colony to a culture dish for carrying out biological characteristic research. Inoculating the bacterium blocks into liquid strain culture media with different conditions by using a puncher with the diameter of 7mm for culture. All the experiments except the temperature experiment were cultured in a constant temperature incubator at 28 ℃. Each treatment was set to 8 replicates. And measuring the sizes of the colonies every 24h by adopting a cross-hatch method until one culture medium colony grows full, observing and recording the hypha form and the growth potential, and statistically analyzing the data result by adopting Excel2019 and PSS 20.0.

1.4.1 carbon source experiments: with a basic culture medium as a control, 5 carbon sources are respectively set: glucose, sucrose, maltose, lactose, soluble starch as treatment groups, all at a concentration of 20 g/L.

1.4.2 Nitrogen Source experiments: with a basic culture medium as a control, 5 nitrogen sources were set: peptone, ammonium chloride, ammonium sulfate, urea and yeast powder are taken as treatment groups, and the concentration is 2 g/L.

1.4.3 inorganic salt experiments: with a basic culture medium as a control, 5 inorganic salts were set: magnesium sulfate, ferric trichloride, calcium carbonate, ferrous sulfate and sodium chloride are taken as a treatment group, and the concentration is 0.5 g/L.

1.4.4pH assay: the basic culture medium formula is adjusted to be acid by using 1.0mol/L HCl and adjusted to be alkali by using 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 experiment: after inoculation with basal medium, the petri dishes were placed in: culturing in a constant temperature incubator at 20 deg.C, 22 deg.C, 24 deg.C, 26 deg.C, and 28 deg.C.

1.4.6 orthogonal experiments: and (3) selecting 4 factors which have large influence on the growth speed of H63 hyphae by combining 5 single-factor experimental bases, and selecting 3 optimal horizontal factors in each factor. Selecting L ₉ (3 ⁴ ) And the orthogonal table is used for carrying out orthogonal test by taking carbon sources, nitrogen sources, inorganic salts and temperature which have obvious influence on hypha growth as direct factors.

1.5 domestication and cultivation

Taking the Ganoderma strain out of refrigerator, standing at room temperature for 4-6 hr for activation, selecting purified mycelium block, inoculating on new PDA culture medium, and culturing in constant temperature incubator at 28 deg.C until mycelium grows. 2-3 pieces of broad bean-sized ganoderma lucidum mother seeds are taken and inoculated into 250ml of liquid strain culture solution, and the liquid shaking table is carried out for 10d under the conditions of 28 ℃ and 160 r/min. Selecting polypropylene plastic bags of 35cm × 17cm in size, each bag weighing 1.0-1.2kg, sterilizing at 121 deg.C under high pressure for 2 hr, cooling, inoculating liquid strain cultured by shaking table into cultivation bags, inoculating 25ml liquid strain in each bag, and dark culturing in 28 deg.C cultivation room. And (5) after 25-30 days, the cultivation bags are full of mycelia, and soil covering cultivation is carried out.

Before cultivation, weeds in the field are removed, lime is spread on the field for ploughing and insolation, and a biological agent or a physical trapping and killing method is adopted to carry out sterilization and insecticidal treatment on the cultivation environment. After the cultivation of the cultivation fungus bag is finished, the cultivation fungus bag is transported to a cultivation place for further cultivation for 1-2 weeks at the temperature of 25-28 ℃ and the illumination intensity of 500-. Mechanically or manually making planting grooves with depth of 0.20-0.30m, width of 1.00-1.50 m and length of no more than 10.0m, and making furrow depth in the middle of 25-30 cm. A blade is adopted to make a small hole at the top end of the fungus bag, and then the fungus bag is vertically arranged in a planting groove or a planting hole, the hole opening end is upward, and the distance between fungus sticks is kept to be 5-10 cm; covering soil on the planting grooves or the planting holes, wherein the number of the placing bags is 4000 and 4200 bags per mu, and the thickness of the covered soil is 3-5 cm.

And (3) performing ganoderma lucidum output management after covering soil, keeping the temperature at 25-32 ℃, spraying water into the greenhouse space in a atomizing manner at regular time, keeping the ground surface soil humidity at 85-95% of the relative space humidity, keeping the carbon dioxide concentration below 0.2% by ventilation frequently, and shading treatment to ensure that the illumination intensity is 300-1000 Lx. When the ganoderma lucidum grows to the mature period, the pileus is not increased and thickened any more, the faint yellow edge of the pileus disappears, basidiospores begin to be sprayed outwards, spores are released, and the color of the pileus is deepened, so that the ganoderma lucidum can be harvested. When in collection, a blade or a scissors is used for shearing along the basal part of the stipe, and 1cm of the stipe base is reserved.

2 results and analysis

2.1 phylogenetic analysis

Relevant reference sequences were downloaded from Gen Bank and Tomophagus colossus was used as the exo-group. H63 was clustered with the known resin Ganoderma G.resinaceum in the polygenic phylogenetic tree of Ganoderma genus and achieved 97% support as shown in FIG. 1.

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

2.2 results of the biological Properties

2.2.1 Single factor test results

As is clear from FIG. 2, H63 showed the highest growth rate of mycelia under the condition of maltose as a carbon source. As can be seen from fig. 3, H63 grew most rapidly under the condition that yeast powder was used as a nitrogen source. As can be seen from fig. 4, H63 grew most rapidly under the condition of magnesium sulfate as an inorganic salt. As can be seen from FIG. 5, H63 had an optimum pH of 5.5. As can be seen from FIG. 6, the optimum temperature of H63 was 30 ℃.

2.2.2 results of orthogonal experiments

The results of the orthogonality experiment are shown in table 1.

TABLE 1

Note: data are the average of 8 replicates; kn represents the average growth rate of hyphae of the resin ganoderma strain on the level of n, and R is the range; "+", "+ + + + +" and "+ + + +" indicate that the hyphal growth is enhanced sequentially

It can be seen by an orthogonal visual analysis at the 4-factor 3 level that the strain carbon source is the worst, R3.42, followed by temperature, nitrogen source and inorganic salts. The growth rate of X3 in the carbon source is 14.92mm/d at most, the growth rate of X2 in the nitrogen source is 13.94mm/d at most, the growth rate of X3 in the inorganic salt is 12.50mm/d at most, and the growth rate of X3 in the temperature is 14.73mm/d at most. By combining with anova, the significant difference of the 4 factors is carbon source > temperature > nitrogen source > inorganic salt from large to small. And (4) analyzing the result of the comprehensive data, wherein the optimal growth conditions of the strain are as follows: the carbon source is maltose, the nitrogen source is yeast powder, the inorganic salt is magnesium sulfate, and the temperature is 30 ℃.

Through a pH test of ganoderma lucidum mycelia, the strain mycelia are mainly suitable for growing under a slightly acidic condition, and when the pH exceeds 7.0, the mycelia can germinate but grow very slowly. The pH value of the hyphae is most suitable for 5.5, and the hyphae grow quickly and are dense.

2.3 acclimatization of cultivation results

The shake flask growth pattern of the Ganoderma lucidum H63 mycelium liquid is shown in FIG. 8, and the acclimatized cultivation growth pattern is shown in FIG. 9. The H63 lucid ganoderma fruiting body is successfully domesticated through earthing cultivation of a bag material, the growth cycle of the resin lucid ganoderma H63 is short from the aspects of seed production and lucid ganoderma cultivation, the average mother seed cycle is 8 days (a 90mm culture dish), and the maximum growth rate of solid hypha is 14.92 mm/d; the average liquid fermentation period was 7 days (200L seedtank); the average growth period of the cultivation bags is 26 days (the average humidity of the bags is 1.0-1.2 kg); the cultivation bags are 300 bags in total, the pollution is 3 bags, and the pollution rate is 1%. The culture rate of the cultivated ganoderma is 98%, the budding period is 15 days, the differentiation period is 29 days, the harvesting period is 77 days, the average fresh weight of single fruiting bodies is 45.20g, the average dry weight of the single fruiting bodies 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% support rate is obtained, as shown in figure 1.

The experimental strain can grow under different carbon source conditions, the growth vigor of glucose and maltose is higher, and the growth vigor of lactose is slowest. Under different nitrogen source conditions, the H63 strain grows at the fastest speed under the yeast powder condition, and grows at the slowest speed under the urea condition. The strain H63 has remarkable hypha growth difference under different inorganic salt conditions, the hypha grows at the fastest speed under the magnesium sulfate condition, the optimal growth temperature of H63 is 30 ℃, and the hypha grows at a slower speed or does not grow when the temperature is lower than 24 ℃, and belongs to a high-temperature strain.

The bag material of the ganoderma lucidum strain H63 is covered with soil and acclimatized, the result shows that the ganoderma lucidum strain H63 has neat lucid ganoderma, excellent properties and high spore yield, and the sporocarp of each bag produces 24.6g of spore powder on average. The hypha cultivation stage and the sporocarp growth stage show stronger disease and insect resistance, and have better strain activity and stress resistance. Belongs to high-temperature strains and has the potential of cultivation and popularization in low-altitude areas.

The foregoing shows and describes the general principles, essential 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, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> institute of biotechnology and germplasm resources of academy of agricultural sciences in Yunnan province

<120> Ganoderma resinatum strain H63 and application thereof

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213> 1 (Artificial sequence)

<400> 1

cttggtcatt tagaggaagt aa 22

<210> 2

<211> 20

<212> DNA

<213> 2 (Artificial sequence)

<400> 2

tcctccgctt attgatatgc 20

<210> 3

<211> 17

<212> DNA

<213> 3 (Artificial sequence)

<400> 3

acccgctgaa cttaagc 17

<210> 4

<211> 17

<212> DNA

<213> 4 (Artificial sequence)

<400> 4

tcctgaggga aacttcg 17

<210> 5

<211> 23

<212> DNA

<213> 5 (Artificial sequence)

<400> 5

gcyccygghc aycgtgaytt yat 23

<210> 6

<211> 23

<212> DNA

<213> 6 (Artificial sequence)

<400> 6

achgtrccra taccaccrat ctt 23

<210> 7

<211> 20

<212> DNA

<213> 7 (Artificial sequence)

<400> 7

tggggyatgg tntgyccygc 20

<210> 8

<211> 20

<212> DNA

<213> 8 (Artificial sequence)

<400> 8

cccatrgctt gyttrcccat 20

<210> 9

<211> 880

<212> DNA

<213> 9 (Artificial sequence)

<400> 9

atctgaactc aggatcactc gtgttacact gcaatcgcgt gtcgcccttt cctccgctta 60

ttgatatgct taagttcagc gggtagtcct acctgatttg aggtcagagg tcataaagct 120

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

caaagattcg atgattcact gaattctgca attcacatta cttatcgcat ttcgctgcgt 420

tcttcatcga tgcgagagcc aagagatccg ttgctgaaag ttgtattata gatgcgttac 480

atcgcaatac acattctaat actttataga gtttgtgata aacgcaggca caggcacgcc 540

gatccacaag ctcccataag gagcccgctt ccacaacgtc tagaacccac agtaagtgca 600

caggtgtaga gtggatgagc agggcgtgca catgcctcgg aaggccagct acaacccagt 660

caaaactcga taatgatcct tccgcaggtt cacctacgga aagggcgaca cgcgattgca 720

gtcttgagtc cacctgaagg atgtcaaact tggtcatagc tgtttcctgt gtgaaattgt 780

tatccgcttc catagacaca acatacgagc cggaacagaa agtcaaaagc ctccgaccgg 840

aggcttttga cttgatcggc acgtaagagg ttccaacttt 880

<210> 10

<211> 912

<212> DNA

<213> 10 (Artificial sequence)

<400> 10

tcgattagtc tttcgcccct atacccaaat ttgacgatcg atttgcacgt cagaatcgct 60

acgagcctcc accagagttt cctctggctt caccctattc aggcatagtt caccatcttt 120

cgggtcccaa catacatgct ctaccgcgga tccgtcagag aacgtcaggt ccgggcgtcg 180

atgctcccca cgacagggat ctcaactttc actttcatta cgcgctcggg ttttccaccc 240

aaacactcgc aggtatgtta gactccttgg tccgtgtttc aagacgggtc gtttaaagcc 300

attatgccag catcctaagc gcgaaagtgg gcgaacccct gccttgcggc gcgctgcgtt 360

cctcgatccc aaccgccgta tgcgaccrga gtctataaca cacccggagg tgccacatta 420

ctccagccct tttccgacgg tcaaaatcga tgctgacccg tcatccggaa agtgcaccaa 480

gcaaaagcaa ggctgagttc cggacaacgc gactgacttc aagcgtttcc ctttcagcaa 540

tttcacgtac tgtttaactc tctttccaaa gtgcttttca tctttccctc acggtacttg 600

ttcgctatcg gtctctcgcc aatatttagc tttagatgga attcaccacc cattttgagc 660

tgcattccca aacaactcga ctctttgaga gcgcatcaca aagcactggt agtccgtgtc 720

aaagacggga ttctcaccct ctatgacgct ctgttccaag agacttatac acggtccagc 780

gcggaaagca cttctccaga ctacaactcg gacggccaaa gaccgccaga ttttaaattt 840

gagcttttcc cgcttcactc gcagttacta ggggaatcct tgttagtttc ttttcctccg 900

cttattgata tg 912

<210> 11

<211> 576

<212> DNA

<213> 11 (Artificial sequence)

<400> 11

atcaagaaca tgatcactgg tacctcgcag gctgactgcg ctatcctcat catcgccgct 60

ggtaccggtg agttcgaggc tggtatctcc aaagatggcc agacccgcga gcacgccctc 120

cttgccttca cccttggtgt caggcagctc atcgttgccg tcaacaagat ggacaccacg 180

aaggtttgtt gtcacgtgcg tcctcatgcg ctgatgtcct gactcggagc tcgcagtggt 240

ccgaggaccg tttcaacgaa atcatcaagg agacctccac cttcatcaag aaggttggtt 300

acaaccccaa ggccgttgcg ttcgtcccca tctctggctg gcacggcgac aacatgttgg 360

aggagtccac caagtcagtc tatccgcacc cttgtttggt gattcgttgc cctgaccttg 420

ttgcttcagc atgccctggt acaagggctg gaccaaggag actaaggccg gtgttgttaa 480

gggcaagacc cttttggacg ctatcgatgc tatcgagccc cccgtccgtc cctccgacaa 540

gcccctccgt ctcccccttc aggatgtcta caagat 576

<210> 12

<211> 784

<212> DNA

<213> 12 (Artificial sequence)

<400> 12

gtcctgccga aaccccagaa ggacaagctt gcggtctcgt caagaacttg tcgctcatgt 60

cttgcatctc cgtcggtacc ctctctgcac ccgtcatcga gttcttggag gaatggggcc 120

tggagtctct agaagagaat gctcatgctt caacrccttg cacgaaggta tttgtgaatg 180

gcgtttggat gggtgtccac cgagatcctg tgaagctcgt cagcacactc cggaaactcc 240

gccgcaaaga cgacatcaac tgcgaagtat ccgtcgtccg tgacatccga gagcgagagc 300

tccgccttta cacggatgct ggacgtgttt gccggccgct tttcatcgtc gagaaccagc 360

agctcctcat ccagaagcga cacatcgaga gcatagtacg tgccaaggac gacccgacgt 420

tatcatacaa ctgggacagc cttctcaagg atggtgtcat tgagttgcta gatgcggagg 480

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

tcttcagtcg agttactgac atgcatcaaa gtcgcctcgt aakacgtaac caatctgcca 780

tggg 784

Claims

1. A resin ganoderma strain H63, which has been preserved in the China Guangdong province microbial culture collection center at 27 th 12 th 2021 with the preservation number GDMCC No: 62106.

2. the artificial culture method of ganoderma resinatum strain H63 of claim 1, comprising the steps of:

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

3. The artificial culture method of ganoderma resinatum strain H63, according to claim 2, wherein in step (1), the ganoderma resinatum strain H63 is cultured in an incubator at 28 ℃ for 5-10 days.

4. The artificial culture method of Ganoderma resinate strain H63, as claimed in claim 2, wherein, in step (2), the rotation speed of the shaking table is 150-160 r/min;

the liquid strain culture medium comprises: maltose 20g/L, yeast powder 2g/L, magnesium sulfate 0.5g/L, vitamin B ₁ 100mg, agar 1g, pH = 5.5.

5. The artificial culture method of Ganoderma resinate strain H63, according to claim 2, wherein in step (3), the cultivation bag substrate comprises, by weight percentage of dry substance, 49% of broad-leaved tree sawdust, 29% of corncob, 19% of wheat bran, 1% of gypsum, 1% of lime, 1% of white sugar; the pH value is 6-8, and the water content is 55-65%.

6. The artificial culture method of Ganoderma resinate strain H63 according to claim 2, wherein in step (3), the weight of the cultivation bag is 1.0-1.2kg, and each bag is inoculated with 25mL of liquid spawn;

7. The artificial culture method of ganoderma resinatum strain H63 as claimed in claim 2, wherein in step (3), the specific method of earthing cultivation is as follows:

weeding, sterilizing and killing insects on a cultivation field;

mechanically or manually making planting grooves with depth of 0.20-0.30m, width of 1.00-1.50 m and length of no more than 10.0m, wherein the furrow depth in the middle is 25-30 cm;

continuously culturing the mycelium overgrown cultivation bags at the temperature of 25-28 ℃ and the illumination intensity of 500-1000Lx for 1-2 weeks, then removing 1/3 fungus bags on the upper parts of the fungus bags, vertically arranging the fungus bags in the cultivation tank, enabling the bag opening ends to face upwards, and keeping the distance between fungus sticks to be 5-10 cm; and covering soil on the planting grooves, wherein the thickness of the covered soil is 3-5 cm.

8. The method for artificially culturing Ganoderma resinatum strain H63 according to claim 2, wherein Ganoderma lucidum is cultured until the bright yellow or white color of pileus disappears completely, spore is sprayed from the fungus hole, and the spore is released and deepened.

9. The use of mycelium or fruiting body of the resin ganoderma strain H63 according to claims 1-8 in the preparation of anti-tumour or sedative sleep-promoting medicaments.