CN114958615A

CN114958615A - Ganoderma leucocontextum strain L4968 and cultivation method thereof

Info

Publication number: CN114958615A
Application number: CN202210435494.0A
Authority: CN
Inventors: 刘丹; 李娥贤; 何俊; 蔡雄; 叶洋; 万涵; 李树红; 李燕
Original assignee: Yunnan Boyan Agricultural Technology Development Co ltd; Biotechnology and Germplasm Resource Institute of Yunnan Academy of Agricultural Sciences
Current assignee: Yunnan Boyan Agricultural Technology Development Co ltd; Biotechnology and Germplasm Resource Institute of Yunnan Academy of Agricultural Sciences
Priority date: 2022-04-24
Filing date: 2022-04-24
Publication date: 2022-08-30
Anticipated expiration: 2042-04-24
Also published as: CN114958615B

Abstract

The invention relates to a ganoderma leucocontextum strain L4968 and a cultivation method thereof, belonging to the technical field of microorganisms. The ganoderma lucidum strain L4968 is preserved in Guangdong province microbial strain preservation center in China at 27 th 12 th 2021, with the preservation number GDMCC No: 62105. collecting and separating wild Ganoderma strain-L4968, determining its classification status by combining morphology and DNA bar code technology, performing strain culture, biological characteristics and artificial domestication, and performing soil covering, domestication and cultivation with bag material to obtain mature fruiting body. The fruiting body of the white-flesh ganoderma lucidum has high nutrient content, good uniformity of the grown ganoderma lucidum, obvious concentric ring lines of pileus, strong lacquer-like luster, thick flesh, good mushroom shape and quality, 33.40g of dry weight per flower and 10.26 percent of biological efficiency, belongs to a medium-low temperature type strain, is suitable for being planted in an altitude area of more than 1800m in Yunnan, and has the potential of cultivation and popularization in the high-altitude area.

Description

Ganoderma leucocontextum strain L4968 and cultivation method thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a ganoderma leucocontextum strain L4968 and a cultivation method thereof.

Background

Ganoderma is distributed in the temperate zone and tropical region of the world, is one of the earliest and most serious edible and medicinal fungi, and has profound influence and important effect in improving human health and establishing auspicious bacteria culture. Ganoderma Ganoderma spp belonging to Ganoderma fungus is a traditional Chinese medicinal material with medicinal history of thousands of years, has high edible and medicinal value, is regarded as a Lingdanmiao medicine capable of treating diseases by Chinese people for thousands of years, and is listed as one of the nine-big Chinese immortals. Ganoderma lucidum is classified as the first-class drug in Shen nong Ben Cao Jing (Shen nong's herbal medicine) in the period of eastern Han, and is classified into 6 types of Ganoderma lucidum, black Ganoderma lucidum, green Ganoderma lucidum, white Ganoderma lucidum, yellow Ganoderma lucidum and purple Ganoderma lucidum according to the shape and color of Ganoderma lucidum. Subsequently, the famous medical scientist Li Shizhen in Ming dynasty made a detailed description of the properties and effects of ganoderma lucidum in Ben Cao gang mu. In recent times, researchers have conducted extensive and intensive research on ganoderma species, so that research on ganoderma becomes a research hotspot in mycology, and potential medicinal value of ganoderma is further exploited. The ganoderma lucidum is gradually and widely cultivated in China, which not only forms an important industry, but also forms ganoderma lucidum culture

In recent years, the DNA barcode technology has been widely applied to species identification, and is one of the essential means for identifying and discovering new species. The wild ganoderma lucidum is influenced by the continuous rising of market demand and price, and the wild ganoderma lucidum resources are excessively collected, so that the wild ganoderma lucidum resources are sharply reduced. The collection and protection of wild ganoderma germplasm resources and the application of variety breeding are imminent.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides a ganoderma leucocontextum strain L4968 and a cultivation method thereof.

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

ganoderma leucocontextum strain L4968, which has been preserved in 27 months 12 and 2021 in the China Guangdong province microbial culture collection center with the preservation number GDMCC No: 62105.

the invention also provides a cultivation method of the ganoderma leucocontextum strain L4968, which is characterized by comprising the following steps:

step (1), mother seed propagation: inoculating Ganoderma Sinense L4968 strain to mother strain PDA culture medium under aseptic condition, sealing, and culturing in 20-26 deg.C incubator until the colony grows over the culture dish;

the mother strain PDA culture medium comprises the following raw materials in parts by weight: 200 parts of potato, 19-21 parts of glucose, 1.9-2.1 parts of peptone, 0.48-0.52 part of ferric trichloride, 16 parts of agar powder and 1000 parts of water; pH 5.5;

step (2), stock preparation: inoculating the mother seeds cultured in the step (1) into an original seed culture bottle, and culturing in dark light, wherein the intensity of the dark light is 20-100Lx, and the temperature is controlled at 22-25 ℃; when the 1/3 bags are full of hypha in the culture bottle, the temperature of the culture chamber is reduced to 20-23 ℃, and the hypha is full of the culture bottle to obtain stock;

the stock culture medium in the stock culture bottle comprises the following raw materials in parts by weight: 60-80 parts of wheat, 10-20 parts of broad-leaved tree sawdust, 10-18 parts of wheat bran, 1.0-2.0 parts of gypsum powder and 1.0-2.0 parts of white sugar;

step (3), preparing cultivars: inoculating the stock culture medium and the hyphae in the step (2) into a culture material bag, placing the culture material bag in a culture room at the temperature of 20-26 ℃ for dark culture, keeping the air humidity at 60-65%, and obtaining a culture bag when the hyphae grow over the culture bag;

the culture medium in the culture bag comprises the following raw materials in parts by weight: broad-leaved tree sawdust 45-60 parts, cottonseed hull 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%;

step (4), earthing cultivation and lucid ganoderma output management of bag materials: removing 1/3 fungus bags on the upper part of the furrow according to the depth of the furrow of 20-30cm and the width of 0.8-1.0m, placing the cultivation bags vertically, arranging the fungus bags at the distance of 5-10cm, and making the furrow between the two furrows with the width of 48-52cm and the depth of 28-32 cm; then covering soil 2-5cm higher than the surface of the fungus bag; and (5) covering soil, and performing fruiting management on the ganoderma lucidum.

Further, it is preferable that, in the step (1), the culture is carried out in an incubator at 24 ℃ for 10 to 12 days at a constant temperature.

Further, preferably, in the step (1), the mother seed PDA medium comprises the following raw materials in parts by weight: 200 parts of potato, 20 parts of glucose, 2 parts of peptone, 0.5 part of ferric trichloride, 16 parts of agar powder and 1000 parts of water; pH 5.5; the preparation method comprises the following steps: chopping potatoes, adding water until the potatoes are completely boiled, filtering the potato residues by using gauze to obtain a potato starch solution, sequentially adding agar, glucose, peptone and ferric chloride, stirring and boiling until the agar, the glucose, the peptone and the ferric chloride are boiled, and adjusting the pH value; sterilizing at 121 deg.C for 25min, pouring into flat plate, and cooling.

Further, in the step (2), it is preferable that the stock seed of the step (1) is selected from the group consisting of 1: carrying out propagation with a propagation coefficient of 10; the distance between the original strain culture bottles is 5-10 cm; the diameter of the original seed bottle is 8cm multiplied by 11 cm; each flask of stock culture medium was 250 g.

Further, preferably, in the step (2), the wheat, the broad-leaved tree wood chips and the wheat bran in the stock culture medium are respectively soaked in lime water for 24 hours, taken out, added with gypsum powder and white sugar, uniformly mixed, sterilized at 121 ℃ under 0.103MPa for 2 hours, and then bottled.

Further, it is preferable that, in the step (3), the stock seed obtained in the step (2) is mixed in a ratio of 1: performing propagation with a propagation coefficient of 20; each bag of cultivation material weighs 1.0-1.2 kg.

Further, preferably, in the step (3), the cultivation bag is a polyethylene bag with the length of 17cm, the height of 17cm and the thickness of 35cm, and the thickness of 0.05 cm; sterilizing at 110 deg.C for 8-10h after filling with cultivation material.

The ganoderma leucocontextum strain L4968 contains higher content of ganoderma lucidum polysaccharide and ganoderma lucidum triterpenoids. The detection method of ganoderma lucidum polysaccharide determination and ganoderma lucidum triterpene determination (based on oleanolic acid) is adopted for detection in the first part of China pharmacopoeia 2020 edition, page 196, the content of polysaccharide is 1.47 percent, the content of triterpene is 3.25 percent, which are all higher than the medicinal ganoderma lucidum standard of the pharmacopoeia of the people's republic of China, and the ganoderma lucidum polysaccharide has wide pharmacological activity, can reduce blood sugar, blood fat, thrombus and oxidation, can eliminate free radicals, has the functions of aging resistance, radiation resistance and tumor resistance, can promote blood circulation, regulate immunity, regulate nucleic acid and protein metabolism, can promote DNA synthesis and promote proliferation of LAK cells in human umbilical blood. Ganoderma triterpene compounds have antiinflammatory, analgesic, tranquilizing, antiaging, tumor cell poisoning, and anoxia resisting effects.

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

the ganoderma leucocontextum strain L4968 is a new strain collected and separated from yerba mate of Guishan Zhenjiang of Shilin county of Kunming, Yunnan province, and relevant research reports of the strain are not found through research.

The invention combines DNA bar code identification and strain culture utilization technology to develop biological characteristics of wild ganoderma lucidum, liquid fermentation culture and domestication culture experiments, L4968 forms a single branch with reported white ganoderma lucidum in ganoderma phylogenetic tree, and 94% support rate is obtained; the optimal growth temperature of the L4968 strain is 24 ℃, and the growth speed of hyphae is sharply reduced when the temperature exceeds 26 ℃, so that the strain belongs to a medium-low temperature type strain. Domestication cultivation results such as separation, purification, screening, fruiting test, area and production test and the like show that the ganoderma leucocontextum strain L4968 has excellent agronomic characters, high and stable yield, the per-bag yield is 80 g/bag, and the per-mu yield is 480 kg. And the content of active substances is higher than that of the ganoderma lucidum which is widely cultivated, the fruiting uniformity and the stress resistance are good, and the hereditary character is stable.

The fruiting body of the white ganoderma lucidum has high nutrient content, 1.47 percent of polysaccharide, 3.25 percent of triterpene, good uniformity of the produced ganoderma lucidum, obvious concentric ring lines of pileus, strong lacquer-like luster, thick meat, good mushroom shape and quality, 33.40g of dry weight per flower, 10.26 percent of biological efficiency, proper cultivation temperature of 20-26 ℃, belongs to a medium-low temperature type strain, is suitable for being planted in an altitude area of more than 1800m in Yunnan area, has the potential of cultivation and popularization in the medium-high altitude area, and provides an important way for the utilization of new rare wild white ganoderma lucidum resources in the southwest area.

Drawings

FIG. 1 is a phylogenetic tree of Ganodermataceae constructed based on the Maximum Likelihood (ML) sequence of ITS + LSU + TEF1- α + RPB 2; note: ML boottrap only shows support values greater than 75% branching;

FIG. 2 shows the growth rate of L4968 mycelia in different carbon sources;

FIG. 3 shows the growth rate of L4968 hyphae in different nitrogen sources;

FIG. 4 shows the growth rate of L4968 mycelia in different inorganic salts;

FIG. 5 shows the growth rate of L4968 hyphae at different pH;

FIG. 6 shows the growth rate of L4968 hyphae at different temperatures;

FIG. 7 shows the fruiting body of ganoderma lucidum cultivated in soil; wherein, a: the primary stage; b: in the bud period; c-e: differentiation period of fruiting body; f-g: semi-mature period of fruiting body; h: mature period of sporophore; i-j: aging period of fruiting body;

FIG. 8 is a morphogram of Ganoderma lucidum karst; wherein, a-c: a basil fruit; d: a pore surface; e: cutting pileus into sections; f: a pericarp cell; g: hypha of mushroom meat skeleton; h, i: the fungus tube combines the hypha; j: germ cells of the fungal tube; k: basidiospores; scale bar: 20 μm (f, g), 10 μm (h-j), 5 μm (k).

The Ganoderma leucocontextum strain (Ganoderma leucocontextum) L4968 is preserved in the China Guangdong province microbial strain preservation center in 12-27 months in 2022, 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 cultivation method of the ganoderma leucocontextum strain L4968 comprises the following steps:

step (1), mother seed propagation: inoculating the ganoderma leucocontextum strain L4968 to a mother strain PDA culture medium under aseptic conditions, sealing and placing in a 24 ℃ incubator for constant-temperature culture until colonies grow to fill a culture dish;

the mother strain PDA culture medium comprises the following raw materials in parts by weight: 200 parts of potato, 20 parts of glucose, 2 parts of peptone, 0.5 part of ferric chloride, 16 parts of agar powder and 1000 parts of water; pH 5.5;

step (2), stock preparation: inoculating the mother seeds cultured in the step (1) into an original seed culture bottle, and culturing in dark light, wherein the intensity of the dark light is 20-100Lx, and the temperature is controlled at 24 ℃; when the 1/3 bags are full of hypha in the culture bottle, the temperature of the culture chamber is reduced to 22 ℃, and the hypha is full of the culture bottle to obtain stock;

the stock culture medium in the stock culture bottle comprises the following raw materials in parts by weight: 70 parts of wheat, 15 parts of broad-leaved tree sawdust, 15 parts of wheat bran, 1.5 parts of gypsum powder and 1.6 parts of white sugar;

step (3), preparing cultivars: inoculating the stock culture medium and the hyphae in the step (2) into a culture material bag, placing the culture material bag in a culture room at 23 ℃ for dark culture, keeping the air humidity at 60-65%, and obtaining a culture bag when the hyphae grow over the culture bag;

the culture medium in the culture bag comprises the following raw materials in parts by weight: 55 parts of broad-leaved tree sawdust, 28 parts of cottonseed hulls, 18 parts of wheat bran, 1.5 parts of gypsum, 0.8 part of lime and 1.3 parts of white sugar; the pH value is 6-8, and the water content is 55-65%;

step (4), earthing cultivation and lucid ganoderma output management of bag materials: removing 1/3 fungus bags on the upper part of the furrow according to the depth of 25cm and the width of 0.9m, standing the cultivation bags, arranging the fungus bags at the interval of 7cm, and making a furrow between two furrows with the width of 50cm and the depth of 30 cm; then covering soil, wherein the soil is 4cm higher than the surface of the fungus bag; and (5) covering soil, and performing fruiting management on the ganoderma lucidum.

Example 3

step (1), mother seed propagation: inoculating Ganoderma Sinense L4968 strain to mother strain PDA culture medium under aseptic condition, sealing, and culturing in 20 deg.C incubator until the colony grows over the culture dish;

the mother strain PDA culture medium comprises the following raw materials in parts by weight: 200 parts of potato, 19 parts of glucose, 1.9 parts of peptone, 0.48 part of ferric trichloride, 16 parts of agar powder and 1000 parts of water; pH 5.5;

step (2), stock preparation: inoculating the mother seeds cultured in the step (1) into an original seed culture bottle, and culturing in dark light, wherein the intensity of the dark light is 20-100Lx, and the temperature is controlled at 22 ℃; when the 1/3 bags are full of hypha in the culture bottle, the temperature of the culture chamber is reduced to 20 ℃, and the hypha is full of the culture bottle to obtain stock;

the stock culture medium in the stock culture bottle comprises the following raw materials in parts by weight: 60 parts of wheat, 10 parts of broad-leaved tree sawdust, 10 parts of wheat bran, 1.0 part of gypsum powder and 1.0 part of white sugar;

step (3), preparing cultivars: inoculating the stock culture medium and the hyphae in the step (2) into a culture material bag, placing the culture material bag in a culture room at 20 ℃ for dark culture, keeping the air humidity at 60-65%, and obtaining a culture bag when the hyphae grow over the culture bag;

the culture medium in the culture bag comprises the following raw materials in parts by weight: 45 parts of broad-leaved tree sawdust, 25 parts of cottonseed hulls, 15 parts of wheat bran, 1.0 part of gypsum, 0.5 part of lime and 1.0 part of white sugar; the pH value is 6-8, and the water content is 55-65%;

step (4), earthing cultivation and lucid ganoderma output management of the bag materials: removing 1/3 fungus bags on the upper part of the furrow according to the depth of the furrow of 20cm and the width of 0.8m, placing the cultivation bags vertically, arranging the fungus bags at the interval of 5cm, and making a furrow between two furrows with the width of 48cm and the depth of 28 cm; then covering soil, wherein the soil is 2cm higher than the surface of the fungus bag; and (5) covering soil, and performing fruiting management on the ganoderma lucidum.

In the step (2), the mother seeds in the step (1) are mixed according to the proportion of 1: carrying out propagation with a propagation coefficient of 10; the distance between the original strain culture bottles is 5 cm; the diameter of the original seed bottle is 8cm multiplied by 11 cm; each flask of stock culture medium was 250 g.

In the step (2), the wheat, broad-leaved tree sawdust and wheat bran in the stock culture medium are respectively soaked in lime water for 24h, then taken out, added with gypsum powder and white sugar, uniformly mixed, sterilized at 121 ℃ under 0.103MPa for 2h, and then bottled.

In the step (3), the stock seeds obtained in the step (2) are mixed according to the ratio of 1: performing propagation with a propagation coefficient of 20; each bag of cultivation material weighs 1.0 kg.

In the step (3), the cultivation bags are bagged by adopting polyethylene bags with the length multiplied by the height multiplied by the thickness of 17cm multiplied by 35cm multiplied by 0.05 cm; sterilizing at 110 deg.C for 8 hr after filling with culture material.

Example 4

step (1), mother seed propagation: inoculating the white-flesh ganoderma lucidum strain L4968 to a mother strain PDA culture medium under aseptic conditions, sealing and placing in an incubator at 26 ℃ for constant-temperature culture until colonies grow to fill the culture dish;

the mother strain PDA culture medium comprises the following raw materials in parts by weight: 200 parts of potato, 21 parts of glucose, 2.1 parts of peptone, 0.52 part of ferric trichloride, 16 parts of agar powder and 1000 parts of water; pH 5.5;

step (2), stock preparation: inoculating the mother seeds cultured in the step (1) into an original seed culture bottle, and culturing in dark light, wherein the intensity of the dark light is 20-100Lx, and the temperature is controlled at 25 ℃; when the hypha in the culture bottle overgrows 1/3 bags, the temperature of the culture room is reduced to 23 ℃, and the hypha overgrows the culture bottle to obtain stock seeds;

the stock culture medium in the stock culture bottle comprises the following raw materials in parts by weight: 80 parts of wheat, 20 parts of broad-leaved tree sawdust, 18 parts of wheat bran, 2.0 parts of gypsum powder and 2.0 parts of white sugar;

step (3), preparing cultivars: inoculating the stock culture medium and the hyphae in the step (2) into a cultivation material bag, placing the cultivation material bag in a 26 ℃ cultivation room for dark cultivation, keeping the air humidity at 60-65%, and obtaining a cultivation bag when the hyphae grow over the cultivation material bag;

the culture medium in the culture bag comprises the following raw materials in parts by weight: 60 parts of broad-leaved tree sawdust, 30 parts of cottonseed hulls, 120 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 6-8, and the water content is 55-65%;

step (4), earthing cultivation and lucid ganoderma output management of the bag materials: removing 1/3 fungus bags on the upper part of the furrow according to the furrow depth of 30cm and the width of 1.0m, placing the cultivation bags vertically, arranging the fungus bags at the interval of 10cm, and making furrows with the width of 52cm and the depth of 32cm between two furrows; then covering soil, wherein the soil is 5cm higher than the surface of the fungus bag; and (5) covering soil, and performing fruiting management on the ganoderma lucidum.

In the step (2), the mother seeds in the step (1) are mixed according to the proportion of 1: carrying out propagation with a propagation coefficient of 10; the distance between the original strain culture bottles is 10 cm; the diameter of the original seed bottle is 8cm multiplied by 11 cm; each flask of stock culture medium was 250 g.

In the step (3), the stock seeds obtained in the step (2) are mixed according to the ratio of 1: performing propagation with a propagation coefficient of 20; each bag of cultivation material weighs 1.2 kg.

In the step (3), the cultivation bags are bagged by polyethylene bags with the length multiplied by the height multiplied by the thickness of 17cm multiplied by 35cm multiplied by 0.05 cm; sterilizing at 110 deg.C for 10 hr after filling with culture material.

Example 5

step (1), mother seed propagation: inoculating Ganoderma leucocontextum strain L4968 to mother strain PDA culture medium under aseptic condition, sealing, and placing in 24 deg.C incubator for constant temperature culture for 10 days until the colony grows over the culture dish;

the stock culture medium in the stock culture bottle comprises the following raw materials in parts by weight: 70 parts of wheat, 15 parts of broad-leaved tree sawdust, 15 parts of wheat bran, 1.5 parts of gypsum powder and 1.5 parts of white sugar;

step (3), preparing cultivars: inoculating the stock culture medium and the hyphae in the step (2) into a culture material bag, placing the culture material bag in a culture room at 24 ℃ for dark culture, keeping the air humidity at 60-65%, and obtaining a culture bag when the hyphae grow over the culture bag;

the culture medium in the culture bag comprises the following raw materials in parts by weight: 50 parts of broad-leaved tree sawdust, 28 parts of cottonseed hulls, 18 parts of wheat bran, 1.5 parts of gypsum, 0.8 part of lime and 1.2 parts of white sugar; the pH value is 6-8, and the water content is 55-65%;

step (4), earthing cultivation and lucid ganoderma output management of the bag materials: removing 1/3 fungus bags on the upper part of the furrow according to the depth of 25cm and the width of 0.9m, standing the cultivation bags, arranging the fungus bags at the interval of 8cm, and making furrows with the width of 50cm and the depth of 30cm between two furrows; then covering soil, wherein the soil is 4cm higher than the surface of the fungus bag; and (5) covering soil, and performing fruiting management on the ganoderma lucidum.

In the step (1), the mother strain PDA culture medium comprises the following raw materials in parts by weight: 200 parts of potato, 20 parts of glucose, 2 parts of peptone, 0.5 part of ferric chloride, 16 parts of agar powder and 1000 parts of water; pH 5.5; the preparation method comprises the following steps: chopping potatoes, adding water until the potatoes are completely boiled, filtering the potato residues by using gauze to obtain a potato starch solution, sequentially adding agar, glucose, peptone and ferric chloride, stirring and boiling until the agar, the glucose, the peptone and the ferric chloride are boiled, and adjusting the pH value; sterilizing at 121 deg.C for 25min, pouring into flat plate, and cooling.

In the step (2), the mother seeds in the step (1) are mixed according to the proportion of 1: carrying out propagation with a propagation coefficient of 10; the distance between the original strain culture bottles is 8 cm; the diameter of the stock seed bottle is 8cm multiplied by 11 cm; each bottle of stock culture medium is 250 g.

In the step (3), the stock seeds obtained in the step (2) are mixed according to the ratio of 1: performing propagation with a propagation coefficient of 20; each bag of cultivation material weighs 1.1 kg.

In the step (3), the cultivation bags are bagged by polyethylene bags with the length multiplied by the height multiplied by the thickness of 17cm multiplied by 35cm multiplied by 0.05 cm; sterilizing at 110 deg.C for 9 hr.

Examples of the applications

1 materials and methods

1.1 test strains

Ganoderma leucocontextum G.leucocontextuum (strain number: L4968) is obtained from Guiyashan Zhenyan of Shilin county of Kunming, Yunnan province, and is separated and purified by tissue separation method.

1.2 test Medium and compost formula

Raw materials of mother strain PDA culture medium: 200g of potato, 20g of glucose, 2g of peptone, 0.5g of ferric chloride, 16g of agar powder and 1000mL of water; pH 5.5; soy peptone is preferably used;

a preparation method of culture medium and sterilization method comprises 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 agar, glucose, peptone and ferric chloride into a conical flask with 2 volumes of 500ml, stirring and boiling, pouring into the conical flask, covering with special asbestos mesh paper, and tightening with rubber band to prevent the culture medium from being splashed 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.

The formula of the stock culture medium comprises: 60-80 parts of wheat, 10-20 parts of broad-leaved tree sawdust, 10-18 parts of wheat bran, 1.0-2.0 parts of gypsum powder and 1.0-2.0 parts of white sugar; soaking wheat, broad-leaved tree sawdust and wheat bran in the stock culture medium with lime water for 24h, taking out, adding Gypsum Fibrosum powder and white sugar, mixing, sterilizing at 121 deg.C under 0.103MPa for 2h, and bottling.

The formula of the cultivated species matrix comprises: 45-60 parts of broad-leaved tree sawdust, 25-30 parts of cottonseed hulls, 15-20 parts of wheat bran, 1.0-2.0 parts of gypsum, 0.5-1.0 part of lime and 1.0-1.5 parts of white sugar. The pH value is 6-8, and the water content is 55-65%. Pre-wetting broad-leaf tree sawdust, cottonseed hull and wheat bran for more than 24h in advance, adding gypsum, lime and white sugar while adding water, and stirring until the water content is 55-65%; sterilizing with normal pressure steam at 110 deg.C for 8-10h, naturally cooling, and transferring the cultivation bag to a cooling chamber for natural cooling when the temperature in the sterilizer is reduced to below 60 deg.C.

1.3 molecular biology Studies

Taking strains separated from a wild ganoderma lucidum fruiting body specimen as an extraction material, grinding by using liquid nitrogen, extracting DNA by using a plant genome kit of Beijing Optimalaceae New Biotechnology Limited company, carrying out ITS, nrLSU, tef 1-alpha and rpb2 sequence amplification, and carrying out phylogenetic analysis after analysis and treatment of a detection result.

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 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 22 ℃ incubator. 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 is full, observing and recording the morphology and the growth potential of the hyphae, and statistically analyzing the data result by adopting Excel2019 and PSS 20.0.

1.4.1 carbon source experiments: sucrose, maltose, lactose and soluble starch are used to replace glucose in the mother PDA culture medium, and the concentration is 20 g/L.

1.4.2 Nitrogen Source experiments: ammonium chloride, ammonium sulfate, urea, yeast extract and diammonium phosphate are used to replace soybean peptone in the PDA culture medium of the mother seeds, and the concentration is 2 g/L.

1.4.3 inorganic salt experiments: ferrous sulfate, sodium chloride, calcium carbonate and zinc sulfate heptahydrate are used to replace ferric trichloride in the basic culture medium, and the concentration is 0.5 g/L.

1.4.4pH assay: the initial pH was adjusted with 1.0mol/L hydrochloric acid and 1.0mol/L sodium hydroxide solution, and 5 initial pH gradients were set: 5.0, 5.5, 6.0, 6.5, 7.0.

1.4.5 temperature experiment: the culture dishes are respectively placed in a constant temperature incubator with the temperature of 18 ℃, 20 ℃, 22 ℃, 24 ℃, 26 ℃ and 28 ℃ for culture.

1.5 domestication and cultivation

Taking the separated and stored L4968 strain out of a refrigerator, placing the strain at room temperature for 2-3 hours for activation, selecting a purified hypha block, inoculating the hypha block on a new mother strain PDA culture medium, and culturing in a constant temperature incubator at 22 ℃ for later use. Taking the mother seeds, mixing the mother seeds with 1: and (2) expanding propagation coefficient of 10, inoculating mycelium into stock culture bottles (specification: 8cm multiplied by 11cm), placing the stock bottles on a spawn running rack in order, culturing for 25-35 days at constant temperature of 22-25 ℃, selecting polypropylene plastic bags with the size of 17cm multiplied by 35cm, weighing 1-1.2kg of materials in each bag, sterilizing under high pressure at 121 ℃ for 2 hours, cooling, and mixing the cultured stock bottles in a proportion of 1: inoculating into culture bag with propagation coefficient of 20, culturing in dark at 20-26 deg.C in culture room with air humidity of 60-65%, and culturing in soil-covered culture bag until mycelia grow over the bag.

The furrow making and arranging are carried out according to the furrow depth of 20-30cm, the width of 0.8-1.0m, the furrow length determined according to the length of the greenhouse, the width of 50cm between two furrows and the depth of 30 cm. Placing the fungus bags vertically, placing the fungus bags at the interval of 5-10cm, and then covering loose soil with the thickness of 2-5 cm. And after covering soil, performing fruiting management on the lucid ganoderma.

The ganoderma fruiting management comprises the following steps: bud forcing and bud outlet management, humidity management, temperature management and illumination and ventilation management

And (3) bud promotion and bud emergence management: the temperature is kept at 22-26 ℃, and the relative humidity of air is 60% -90%; the white primordia can be formed by ventilating for 2h and 7-10d in the morning and afternoon every day. At the early stage of bud differentiation, part of inferior Ganoderma buds can be removed according to commodity requirements.

Humidity management: when the buds are formed and the mushroom pieces are cut, atomizing and spraying water to the greenhouse space at regular time, keeping the soil humidity on the ground surface, keeping the relative humidity of the space between 80 and 95 percent, basically cutting the sporocarp enough, keeping the air humidity between 75 and 85 percent when the edges of the mushroom caps are slightly yellow, leading the sporocarp to be mature, and keeping the relative air humidity above 60 percent.

Temperature management: the optimum temperature for primordium differentiation and fruiting body development is 22-26 deg.C, and the temperature in hypha growth stage is controlled at 20 deg.C to promote the formation of bud, and the optimum temperature is 24-26 deg.C when fruiting body is mature.

Illumination and ventilation management: after the sporocarp is completely sliced, the air is ventilated for more than 3 hours every day in a convection way to reduce the concentration of carbon dioxide. The illumination generally adopts the principle of 'three fens positive and seven minutes negative', and the uniform scattered light is kept, and the light intensity is 1000-5000 Lx.

2 results and analysis

2.1 phylogenetic analysis

Relevant reference sequences were downloaded from GenBank and Tomophagus colossus was used as the exo-group. L4968 forms a single branch with the reported ganoderma leucocontextum in phylogenetic trees of the genus Ganoderma, and achieves 94% support (see FIG. 1). The strain is determined to be ganoderma leucocontextum G.leucococcus textum by combining macroscopic and microscopic morphological characteristics (as shown in figure 8).

The characteristic sequence of the L4968 strain is shown as SEQ ID NO.9, wherein 1-632 bases are: an ITS; 633-1516 bases are: nrLSU; 1517-2092 bases are: tef1 α; 2093 2822 bases are: rpb2 are provided.

2.2 results of the study of biological Properties

As is clear from FIG. 2, L4968 showed the highest growth rate of mycelia under the condition of glucose as a carbon source. As can be seen from FIG. 3, L4968 grows most rapidly on soybean peptone as a nitrogen source. As can be seen from FIG. 4, L4968 grows at the fastest growth rate under the condition of ferric trichloride as an inorganic salt, and as can be seen from FIG. 5, the optimum pH of L4968 is 5.5. As can be seen from FIG. 6, the optimum temperature for L4968 is 26 ℃.

However, subsequent studies show that the optimal liquid culture medium conditions of the L4968 strain are as follows: the carbon source is glucose, the nitrogen source is soybean peptone, the inorganic salt is ferric trichloride, the temperature is 24 ℃, and the growth speed of hyphae is optimal under the condition.

2.3 acclimatization of cultivation results

The ganoderma lucidum fruiting bodies are successfully domesticated by covering soil for cultivation of substitute materials, as shown in figures 7 and 8. Combining production seed production and soil covering cultivation, carrying out symbiotic production on 400 bags, wherein the average period from inoculation to full growth of the cultivation bags is 28d, a small amount of primordia begin to appear after the cultivation bags are placed in a fruiting field for 15d, the primordia begin to differentiate after 30 days, and the sporophores begin to mature and pick after 17 days. Through statistical analysis, the fruiting rate is more than 97%, the dry weight is 33.40 g/flower, the fresh weight of the bagged yield is 80g, the biological efficiency is 10.26%, and the yield per mu is 480 kg.

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 L4968 strain is slowest in growth speed under urea conditions. The difference of hypha growth of the L4968 strain is obvious under different inorganic salt conditions, and the hypha grows fast under the condition of ferric trichloride, which is probably due to the fact that the strain acts on Fe ³⁺ Has better adaptability. The optimal temperature of L4968 is 24-26 deg.C, and when the temperature exceeds 28 deg.C, the growth rate of hyphae decreases rapidly, and the strain belongs to middle and low temperature type strain.

The ganoderma lucidum strain L4968 is domesticated by covering soil in a bag material, and the result shows that the L4968 is not resistant to high temperature environment, the high temperature can cause no fruiting or deformed mushrooms, the suitable cultivation temperature is 20-26 ℃, which is consistent with the experimental result of hypha biological characteristics, and the domesticated cultivation of the strain has the advantages of excellent fruiting agronomic characters, high yield and stability, higher active substance content compared with that of the widely cultivated ganoderma lucidum, good fruiting uniformity and stress resistance, and stable genetic characters. Therefore, the research suggests that the strain has the potential of cultivation and popularization in high and high altitude areas

The foregoing shows and describes the general 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, 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> Yunan Bo derived agricultural science and technology development Co., Ltd

Institute of biotechnology and germplasm resources, Yunnan Academy of Agricultural Sciences

<120> Ganoderma leucocontextum strain L4968 and cultivation method thereof

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<170> SIPOSequenceListing 1.0

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cttggtcatt tagaggaagt aa 22

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<212> DNA

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tcctccgctt attgatatgc 20

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<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

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<212> DNA

<213> 5 (Artificial sequence)

<400> 5

gcyccygghc aycgtgaytt yat 23

<210> 6

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<212> DNA

<213> 6 (Artificial sequence)

<400> 6

achgtrccra taccaccrat ctt 23

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<211> 20

<212> DNA

<213> 7 (Artificial sequence)

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tggggyatgg tntgyccygc 20

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<211> 20

<212> DNA

<213> 8 (Artificial sequence)

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cccatrgctt gyttrcccat 20

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acctgcggaa ggatcattat cgagttctga ctgggttgta gctggccttc cgaggcacgt 60

gcacgccctg ctcatccact ctacacctgt gcacttactg tgggtttcag atctgcgaag 120

cgtgctcctt gcggggcttc gtgaagcgcg tctgtgcctg cgtttatcac aaactctata 180

aagtattaga atgtgtattg cgatgtaacg catctatata caactttcag caacggatct 240

cttggctctc gcatcgatga agaacgcagc gaaatgcgat aagtaatgtg aattgcagaa 300

ttcagtgaat catcgaatct ttgaacgcac cttgcgctcc ttggtattcc gaggagcatg 360

cctgtttgag tgtcatgaaa tcttcaacct acaagctttt gcggtttgta ggcttggact 420

tggaggcttg tcggccctct gtcggtcggc tcctcttaaa tgcattagct tgattccttg 480

cggatcggct ctcggtgtga taatgtctac gccgcgaccg tgaagcgttt ggcgagcttc 540

taaccgtctt cgcttgaaga cagctttatg acctctgacc tcaaatcagg taggacwacc 600

cgctgaactt aagcatatca aaagcccgga gggttcgatt agtctttcgc ccctataccc 660

aaatttgacg atcgatttgc acgtcagaat cgctacgagc ctccaccaga gtttcctctg 720

gcttcaccct attcaggcat agttcaccat ctttcgggtc ccaacataca tgctctaccg 780

cggatccttc agagaacgtc aggtccgggc gtcgatgccc tccacgacag aggtctcaac 840

tttcactttc attacgcgct cgggttttcc acccaaacac tcgcaggtat gttagactcc 900

ttggtccgtg tttcaagacg ggtcgtttaa agccattatg ccagcatcct aagcgcgaaa 960

gtgggcgaac ccctgccttg cggcgcgctg cgttcctcga tcccaaccgc cgtatgcgac 1020

tagagtctat aacacacccg gaggtgccac attactccag cccttttccg acggtcaaaa 1080

tcgatgctga cccgtcatcc ggaaagtgca ccaagcgaaa gcaaggctga gttccggacg 1140

acgcgactga cttcaagcgt ttccctttca gcaatttcac gtactgttta actctctttc 1200

caaagtgctt ttcatctttc cctcacggta cttgttcgct atcggtctct cgccaatatt 1260

tagctttaga tggaattcac cacccatttt gagctgcatt cccaaacaac tcgactcttt 1320

gagagcgcat cacaaagcac tggtagtccg tgtcaaagac gggattctca ccctctatga 1380

cgctctgttc caagagactt atacacggtc cagcgcggaa agcacttctc cagactacaa 1440

ctcggacggc cgaagaccgc cagattttaa atttgagctt ttcccgcttc actcgcagtt 1500

actaggggaa tccttgcatc aagaacatga tcaccggtac ctcacaggct gactgcgcta 1560

tcctcatcat cgccgctggt accggtgagt tcgaggctgg tatctccaag gatggccaga 1620

cccgcgagca cgcccttctt gccttcaccc tcggtgtcag gcagctcatc gtcgccgtca 1680

acaagatgga cacaaccaag gttcgtcgtc gcgtgaatca acatacgcgc ttgctctgac 1740

tcgagttcgc agtggtccga agaccgtttc aacgaaatca tcaaggagac gtccaccttc 1800

atcaagaagg tcggttacaa cccgaaggcg gttgcgttcg tccccatctc tggctggcac 1860

ggcgacaaca tgttggagga gtccagcaag tgagtgtatg cgctatactg tctgatgact 1920

cgtcaccctg acccttatat tttagcatga cctggtacaa gggttggacg aaggagacca 1980

aggctggtgt tgtcaagggg aagacccttt tggacgctat tgatgctatt gagccccccg 2040

tccgtccctc cgacaagccc ctccgtctcc ctctccagga tgtctacaag at 2092

Claims

1. Ganoderma leucocontextum strain L4968, which has been deposited at 27.12.2021 in the China Guangdong province culture Collection center for microorganisms with the deposit number GDMCC No: 62105.

2. the method for cultivating ganoderma leucocontextum strain L4968 as claimed in claim 1, comprising the steps of:

the mother strain PDA culture medium comprises the following raw materials in parts by weight: 200 parts of potato, 19-21 parts of glucose, 1.9-2.1 parts of peptone, 0.48-0.52 part of ferric trichloride, 16 parts of agar powder and 1000 parts of water; pH = 5.5;

step (4), earthing cultivation and lucid ganoderma output management of the bag materials: removing 1/3 fungus bags on the upper part of the furrow according to the depth of the furrow of 20-30cm and the width of 0.8-1.0m, placing the cultivation bags vertically, arranging the fungus bags at the distance of 5-10cm, and making the furrow between the two furrows with the width of 48-52cm and the depth of 28-32 cm; then covering soil 2-5cm higher than the surface of the fungus bag; and (5) covering soil, and performing fruiting management on the ganoderma lucidum.

3. The method for cultivating ganoderma leucocontextum strain L4968 as claimed in claim 2, wherein in step (1), the strain is cultivated in an incubator at 24 ℃ for 10-12 days.

4. The cultivation method of ganoderma leucocontextum strain L4968 as claimed in claim 2, wherein in step (1), said mother strain PDA culture medium comprises the following raw materials in parts by weight: 200 parts of potato, 20 parts of glucose, 2 parts of peptone, 0.5 part of ferric chloride, 16 parts of agar powder and 1000 parts of water; pH = 5.5; the preparation method comprises the following steps: chopping potatoes, adding water until the potatoes are completely boiled, filtering the potato residues by using gauze to obtain a potato starch solution, sequentially adding agar, glucose, peptone and ferric chloride, stirring and boiling until the agar, the glucose, the peptone and the ferric chloride are boiled, and adjusting the pH value; sterilizing at 121 deg.C for 25min, pouring into flat plate, and cooling.

5. The method for cultivating ganoderma leucocontextum strain L4968 according to claim 2, wherein in step (2), the stock in step (1) is cultivated in a ratio of 1: carrying out propagation with a propagation coefficient of 10; the distance between the original strain culture bottles is 5-10 cm; the diameter of the original seed bottle is 8cm multiplied by 11 cm; each bottle of stock culture medium is 250 g.

6. The method for cultivating ganoderma leucocontextum strain L4968 as claimed in claim 2, wherein in step (2), the wheat, broad-leaved tree wood chips and wheat bran in the stock culture medium are respectively soaked in lime water for 24h, then taken out, added with gypsum powder and white sugar, mixed uniformly, sterilized at 121 ℃ and 0.103MPa for 2h, and finally bottled.

7. The method for cultivating ganoderma leucocontextum strain L4968 as claimed in claim 2, wherein in step (3), the stock seed obtained in step (2) is cultured in a ratio of 1: performing propagation with a propagation coefficient of 20; each bag of cultivation material weighs 1.0-1.2 kg.

8. The method for cultivating ganoderma leucocontextum strain L4968 as claimed in claim 2, wherein in step (3), the cultivation bags are bagged with polyethylene bags 17cm x 35cm x 0.05cm long x high x thick; sterilizing at 110 deg.C for 8-10h after filling with cultivation material.