CN103891523B - A kind of Inonotus obliquus artificial cultivation method - Google Patents

Abstract

The invention discloses a method for artificially cultivating Inonotus obliquus. 1) separation and purification of Inonotus obliquus strains; Use sucrose as the carbon source medium, soybean flour as the nitrogen source, the carbon-to-nitrogen ratio is 20:1, the pH is 7, and the temperature is 30°C; 3) Inonotus obliquus is artificially domesticated and cultivated. The invention can form the fruiting body of Inonotus obliquus.

Description

A kind of artificial cultivation method of Inonotus obliquus

technical field

The invention belongs to the field of biotechnology, and relates to an artificial cultivation method of Inonotus obliquus.

Background technique

Inonotus obliquus has high medicinal value. (1) Anti-tumor and anti-cancer effects: the extract of Inonotus obliquus has therapeutic effects on liver cancer, lung cancer, gastric cancer, cervical cancer, etc. Its polysaccharides and amino acids can protect and promote the immune system of the body, improve The immunity of the human body maintains the vitality and metabolism of normal cells in the human body. (2) Prevention and treatment of diabetes: its polysaccharide products (β-glucan, heteropolysaccharide and protein complex) can enhance the function of islet cells, balance the glucose metabolism process in the human body, thereby lowering blood sugar, and can maintain 3-48 Hour. (3) Prevention and treatment of AIDS and anti-virus effects: β, 3-D-glucan in the extract components has a significant inhibitory effect on SARS-like infectious viruses, while the lignin derivatives with high relative molecular weight in the water-soluble components Drugs can resist the activity of HIV-I protease and then be used for the treatment of AIDS. (4) Anti-oxidation and anti-aging: Catechol in Inonotus obliquus has a strong anti-oxidation effect and has a protective effect on genes. (5) Other functions: Finland, Canada and other European and American countries use high-standard extracts of Inonotus obliquus to treat influenza with the effect of enhancing body immunity. Inonotus obliquus is a blood cleanser. It has obvious therapeutic effects on food poisoning, gastrointestinal disorders, hepatitis, nephritis, and high blood pressure. It can also increase appetite, relieve pain, and improve allergic constitution. There are always birch The tea and medicinal wine specially made by Inonotus obliquus, and it is also made into aerosol to treat various diseases.

With the in-depth research on active substances in large-scale medicinal and edible fungi, the medicinal value, nutritional value and economic value of Inonotus obliquus have attracted widespread attention from the people and become a hot development field in various industries. Multidisciplinary comprehensive development should conduct simultaneous research in different fields such as basic theory, clinical application, and product development. Inonotus obliquus polysaccharides, triterpenes, and polyphenols are natural anti-cancer drug substances that can be used as the main raw materials of health drugs, health food, health drinks, and microcapsules. Its natural pigments can be used in food additives, deep processing of tea, and cosmetics. , In addition, Inonotus obliquus will also have very broad application prospects in the research and product development of marginal subjects such as the tobacco industry, textile industry, and feed industry.

The price of wild Inonotus obliquus is extremely expensive, because the proportion of this wood-rot fungus in birch forests is only about 1/10,000. Due to a large number of collections, the resources of wild Inonotus obliquus are becoming increasingly exhausted. Therefore, the cultivation technology of Inonotus obliquus has become important research field.

At present, due to insufficient understanding of the growth and development mechanism of the fruiting body of Inonotus obliquus, the artificially created conditions are very different from the environment of microorganisms in nature, which has caused certain difficulties in artificial domestication and cultivation. The artificial domestication of fungus sclerotia has achieved certain results and scale. But so far, there is no precedent for the formation of fruiting bodies. Inonotus obliquus cultivation technology still has some problems that need to be overcome urgently. The existing technology can only promote the production of sclerotia without forming fruiting bodies.

Contents of the invention

The purpose of the present invention is to overcome the defects of the above-mentioned technologies, provide a method for artificial cultivation of Inonotus obliquus, optimize the cultivation formula, and successfully promote the formation of fruiting bodies by using variable temperature treatment according to the biological characteristics of wild Inonotus obliquus . Its specific technical plan is:

A method for artificial cultivation of Inonotus obliquus, comprising the steps of:

1) Isolation and purification of Inonotus obliquus strains: first use 1:14 calcium hypochlorite supernatant to sterilize the surface of fruiting bodies, then use high-temperature sterilized tweezers to take a small square tissue of about 5 mm in the middle, and transfer it to Connect it to the center of the PDA medium plate. When cutting the tissue, inoculate it for 1 day and then culture it in a constant temperature dark room at 25°C. Check the growth of the strain at any time, and remove the contaminated plate in time. After the mycelium is full of the plate, transfer it to PDA Preserve the pure species on the slant test tube culture medium, and then store it in a refrigerator at 4°C for later use;

2) With carbon-free medium and nitrogen-free medium as the basic medium, the carbon source for the growth of Inonotus obliquus mycelium is screened out as sucrose, the nitrogen source is soybean flour, the carbon-nitrogen ratio is 20:1, the pH is 7, and the temperature 30°C;

3) Artificial domestication and cultivation of Inonotus obliquus:

On the PDA medium, it was cultured at 35°C for 15 days, and it was cultured at room temperature for 20 days. Inonotus obliquus fruiting bodies grew on the center right of the medium;

Cultivation medium: birch wood chips 78%, soybean powder 20%, gypsum 1%, sucrose 1%, the moisture content of the medium is 65%, cultivated in a 35°C incubator for 30 days, and placed at room temperature without opening for 20-50d , Fruiting bodies were also produced at the mouth and base of the culture bag.

Further preferably, the nucleotide sequence of the Inonotus obliquus strain is shown in SEQ ID NO.1.

Compared with the prior art, the beneficial effects of the present invention are: the present invention can form the fruiting body of Inonotus obliquus, and after large-scale production, it will produce obvious economic benefits.

Description of drawings

Figure 1 shows the growth of Inonotus obliquus under different nitrogen sources, Figure 1a is soybean flour, Figure 1b is yeast powder, Figure 1c is peptone, Figure 1d is glycine, Figure 1e is ammonium sulfate, Figure 1f is nitrogen-free on Chaga The effect of porosi growth;

Figure 2 shows the growth of Inonotus obliquus with different carbon-nitrogen ratios. Figure 2a is a carbon-nitrogen ratio of 5:1, Figure 2b is 10:1, Figure 2c is 20:1, Figure 2d is 30:1, and Figure 2e is 40 : 1, Fig. 2 f is the influence of 50: 1 on the growth of Inonotus obliquus;

Fig. 3 is different pH levels to the growth situation of Inonotus obliquus; Fig. 3a-Fig. 3f is successively the influence of pH3, 8, 5, 6, 7 and 4 on the growth of Inonotus obliquus;

Figure 4 is the fruiting body produced on the artificial medium. Figure 4a-figure 4c are the formation of fruiting bodies, the formation of fruiting bodies, and the growth of mycelia in sequence.

detailed description

The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

The test materials were collected in May 2011 and May 2012 from the wild Inonotus obliquus in Huzhong Nature Reserve, Daxing'anling, Heilongjiang Province. The cut surface feels moist to the touch. Fungus corky, with indistinct ring patterns, light yellowish brown. The mycelium on the colony cultured on the PDA solid plate for 7 days is thick and neat, and grows from the center to the surrounding; the color of the center of the colony is yellowish brown, and the yellow gradually fades from the center to the edge, and the periphery is pure white; the colony on the back of the plate has obvious light Brown rings. The isolated and purified strains were transferred to the PDA slant, and the tube was full of culture for 5 days. The colony was pure white at the beginning, and then gradually turned light yellow, and gradually turned yellowish brown. The strains were connected to PDA liquid medium, and cultured in a cabinet-type constant temperature shaker at 120rpm at 28°C for 7 days. In the liquid; the color of the liquid in the shake flask gradually changes from light yellow to dark brown, and the exfoliated mycelia combine into round or oval mycelium balls. The vegetative mycelium of the flat plate is picked and pressed and observed under an optical microscope. The mycelium is semi-curved, transparent and filamentous. The vegetative hyphae are multi-branched, and the mycelial septum can be seen without lock-like joints. The ITS-rDNA of the wild strain was sequenced to obtain a target fragment length of 761bp, including 18SrDNA partial sequence, 306bp full-length ITS1 sequence, 160bp5.8SrDNA full-length, 243bp ITS2 full-length, and part of 28SrDNA sequence. According to BLAST comparison on GeneBank, it has 99% similarity with the ITS-rDNA sequence of Inonotus obliquus. Combining with morphological observation, it is confirmed that the wild strain collected is Inonotus obliquus. The sequence has been submitted to the EST library on NCBI, and its accession number on GenBank is: KC312697 (GI: 461725870).

Through the analysis of BLAST software, the sequence measured by Inonotus obliquus strain and the ITS-rDNA sequence downloaded in GenBank are clustered and analyzed. The bacterial classification (targeted sequence) collected by the present invention has the closest homology with Inonotus obliquus isolate FS656163 and Inonotus obliquus strain MDJCBS88. One is a different strain of Inonotus obliquus, and the most distant relatives are Inonotus linteus strain PL08121 and Inonotus rick iisolate PF241, both of which belong to the genus Inonotus linteus. ) Sampling place of Inonotus obliquus was in the natural mature and over-mature forest of Birch, and was isolated from the forest pathology laboratory of Northeast Forestry University. Bacterial strains were isolated using conventional tissue separation techniques, first using 1:14 calcium hypochlorite supernatant to sterilize the surface of fruiting bodies. Then use high-temperature sterilized tweezers to take a small square tissue of about 5 mm in the middle, and transfer it to the center of the PDA medium plate. When cutting the tissue, culture it in a constant temperature darkroom at 25°C after 1 day of inoculation, check the growth of the strain at any time, and remove the contaminated plate in time. After the mycelium is overgrown on the plate, transfer it to the PDA slant test tube culture medium to preserve the pure species, and then store it in a refrigerator at 4°C for later use.

Carbon-free basal medium: glycine 1g, KH ₂ PO ₄ 0.5g, magnesium sulfate 0.25g, agar 10g, water 500ml.

Nitrogen-free basal medium: glucose 10g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 0.25g, agar 10g, water 500ml.

Carbon source: According to the instructions of the experimental medicine purchased, the carbon content of glucose, maltose, fructose, sucrose, and soluble starch are 40%, 42.1%, 40%, 42.1%, and 44.4%, respectively. Glucose, maltose, fructose, sucrose, and soluble starch were used as carbon sources, and 10g, 9.5g, 10g, 9.5g, and 9.01g were added to the carbon-free basal medium respectively, and another sugar-free control was set, and a total of 36 treatments were repeated 6 times , at 25°C, under dark conditions, using the plate culture method, quantitatively inoculated with a 0.5mm hole punch in the center of the petri dish, and cultured upside down in a 25°C incubator. On the 3rd day after inoculation, use the cross method to record the growth diameter of the colony every 24 hours, and pay attention to the color of the mycelia, the thickness of the lawn, and the characteristics of the edge of the colony, and then use the Dunca test in the Spssl1. Sexual analysis, see Table 1-2.

Nitrogen source: According to the instructions of the experimental medicine purchased, the nitrogen content of glycine, yeast extract powder, peptone, and ammonium sulfate are 18.65%, 8-9%, 14.5%, and 21%, respectively. The protein content of soybean flour and corn flour is 32.7% and 8.7%. According to the Kjeldahl method, in any biological sample, every gram of nitrogen is equivalent to 6.25g of protein, and the nitrogen content of soybean flour and corn flour can be calculated as 5.232% and 1.392%. At 25°C, under dark conditions, weigh 1.10g of yeast powder, 1.7823g of soybean powder, 0.6431g of peptone, 0.5g of glycine, and 0.444g of ammonium sulfate with an analytical balance, and add them to the nitrogen-free basal medium. For comparison. The inoculation and measurement methods are the same as the carbon source treatment experiment above, and the experimental results are shown in Table 1-3.

Carbon-to-nitrogen ratio: under 25°C and no light conditions, fix the content of glucose in the basal medium, change the content of soybean flour, and prepare C/N ratios of 5:1, 10:1, 20:1, and 30: 1, 40:1, 50:1 medium. Each treatment was quantitatively inoculated with a 0.5mm hole puncher into the medium with different carbon-nitrogen ratios, and repeated 6 times for a total of 36 treatments. The inoculation and measurement methods were the same as the carbon source treatment experiment above. The experimental results are shown in Table 1-4.

pH value: On the medium PDA, adjust the pH value of the medium with disodium hydrogen phosphate-citric acid buffer solution: 3, 4, 5, 6, 7, 8, repeat 6 times for a total of 36 treatments, inoculation and The measurement method is the same as the carbon source treatment experiment above, and the experimental results are shown in Table 1-5.

Temperature: On the medium PDA, Inonotus obliquus was inserted, respectively set at 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, repeated 7 times for a total of 42 treatments, inoculation and measurement methods Same as the carbon source treatment experiment above, the experimental results are shown in Table 1-6.

Screening of parent species: Purify and expand the strains from different collection locations or different collection times for future use. Inoculate on the PDA medium, the method is the same as the carbon source treatment experiment above, and the result measurement is also the same as the carbon source treatment experiment above. The experimental results are shown in Table 1-6.

Cultivation material screening: Both the mother species and the original species medium use conventional PDA medium; the cultivated species use two active strains obtained in the experiment of screening the mother species, take the 1-2cm long PDA slant as the cultivated species, and white birch Wood chips are the main material, and soybean flour, corn flour, white sugar, gypsum, lime, etc. are used as auxiliary materials to form compost for cultivated species in different proportions. The compost formula for cultivation bags is screened. The composition and number of each formula are shown in Table 1-1. The main ingredients and auxiliary materials were packed into bags with conventional mixing materials, and polyethylene knuckle bags with specifications of 17×33 cm were selected. After bagging, they were sterilized in an autoclave (121°C) for 120 minutes. 30 treatments per formula for each strain, for a total of 180 bacteria bags. After inoculation, place it in a constant temperature incubator at 25°C for dark cultivation, observe the condition of the bacteria bags regularly, clean out the contaminated bacteria bags at any time, and observe the growth of the mycelium after the mycelium colonizes, and the size, density, and number of sclerotia appear. According to data analysis and growth conditions, the best compost formula was selected for further domestication research.

Table 1 Composition of compost (dry material %)

results and analysis

Effects of Different Carbon Sources on Mycelia Growth of Inonotus obliquus

Carbon source is the most important nutrient for microorganisms. As an important component of carbohydrates and proteins, it provides energy for fungi and forms the skeleton of cells. Inonotus obliquus is an organotrophic heterotroph in terms of nutrition type, and cannot use direct carbon dioxide for photosynthesis to maintain normal life activities. Under natural growth conditions, plant-derived carbohydrates lignin, starch and various sugars provide a large number of rich carbon sources for Inonotus obliquus. Almost all fungi can absorb polysaccharides such as starch, dextrin, cellulose, monosaccharides such as glucose, fructose, galactose, etc., disaccharides such as maltose, etc.

Table 2 Effects of different carbon sources on the culture characteristics of Inonotus obliquus mycelium

From the experimental results, it can be observed that the mycelia of Inonotus obliquus grow fastest on the medium with maltose as the carbon source, followed by soluble starch and sucrose; but the difference between the three is not significant; fructose is the most beneficial to Inonotus obliquus development. In the medium with sucrose as the carbon source, the color of the colony is dark in the middle, light on both sides, and thick in texture. This is different from the conclusion of Jiang Yuji et al. (2004) that rice flour is the best carbon source. The author believes that the reason for this difference is due to the difference of strains and the difference of reference samples. Jiang Yuji's strain specimens in Wenzhong came from South Korea, and the reference standard was the dry weight of mycelia; on the other hand, the experimental results should confirm Chen Yanqiu's (2005) conclusion that maltose and glucose are the best carbon sources. It can be seen that different bacterial strains and different reference standards have an impact on the experimental results.

Effects of Different Nitrogen Sources on Mycelia Growth of Inonotus obliquus

The role of nitrogen source is second only to nitrogen source for microbial growth, and it is the main element that constitutes the necessary proteins, vitamins, amino acids and nucleic acids in organisms. Nitrogen is divided into organic nitrogen and inorganic nitrogen. Nitrate and ammonium salt are commonly used for inorganic nitrogen. Generally, fungi have a high utilization rate of ammonium salt, but they show adaptation to the utilization of nitrate. Some fungi can absorb nitrite, while others cannot. Organic nitrogen commonly used peptone, beef extract, yeast extract. The nitrogen content in each wood is very small, about 0.03-0.1%, which is not enough for the growth of fungi.

Table 3 Effects of different nitrogen sources on the culture characteristics of Inonotus obliquus mycelium

It can be seen from the table that the growth rate of mycelium is the fastest in the medium with soybean flour as the nitrogen source, followed by yeast powder. It is deep, dense and robust, and the bacterial lawn is thick. According to the experimental results, it is known that soybean flour is the best nitrogen source; followed by yeast powder, peptone, glycine, and ammonium sulfate. The mycelial growth of Inonotus obliquus must have a nitrogen source, and Inonotus obliquus hardly grows under nitrogen-free conditions; the utilization effect of Inonotus obliquus on the mycelial growth and growth speed is greater than that on inorganic nitrogen Utilization effect, ammonium sulfate is the most difficult to utilize. Chen Yanqiu et al. (2005) experimented that the nitrogen sources of different treatments had no significant effect on the growth of mycelia; Jiang Yuji et al. (2004) believed that wheat bran was the best nitrogen source; The differences of the experimental strains are related to the judging criteria. On the other hand, Zhang Liqiu (2006) found that the best nitrogen source was also soybean flour. This phenomenon once again verified the idea that different strains have different growth characteristics.

Effects of different carbon-nitrogen ratios on mycelial growth of Inonotus obliquus

Lignocellulose in wood accounts for more than 90% of the dry weight of wood. It is rich in carbon and extremely poor in nitrogen. Its carbon-nitrogen ratio can reach 350:1-1250:1. Low nitrogen level in birch wood is a factor that limits the colonization and growth of Inonotus obliquus. At present, there is a lack of research on the optimal carbon-nitrogen ratio of Inonotus obliquus.

Table 4 Effects of different carbon-nitrogen ratios on the culture characteristics of Inonotus obliquus mycelium

From the experimental results, it can be obtained that there is no significant difference in the color of the colonies in all 6 proportions, basically all of them are yellowish brown. From the daily growth rate of mycelia, when the carbon-nitrogen ratio is 5:1, the mycelium of Inonotus obliquus grows fastest, and the ratio of 10:1, 20:1, 30:1, 40:1 and 50:1 is 0.01 There is no significant difference at the level of 0.05, the first four have no significant difference at the 0.05 level, and there is no significant difference between 40:1 and 50:1 at the 0.05 level. From the thickness of the lawn and the color of the colony edge, the lawn of 20:1 is the thickest, the edge of the colony is the most tidy, and the mycelium is strong. The optimum carbon-nitrogen ratio is 20:1. The sparser the colonies, the more irregular the edges of the colonies. Below 20:1, the larger the carbon-nitrogen ratio, the stronger the colony. Thus, the smaller the carbon-to-nitrogen ratio, the faster the growth rate of mycelia; but from the perspective of the thickness of the lawn and the robustness of the mycelium: 20:1 is the best carbon-nitrogen ratio. This is consistent with the optimal carbon-nitrogen ratio of 20:1 obtained by Jiang Yuji (2005) from the perspective of mycelium dry weight.

Effects of different pH values on mycelial growth of Inonotus obliquus

Fungi can adapt to a wide range of acid and alkali in life, and generally can survive at a pH value of 3-9. However, for most bacterial species, the optimum pH value for their growth is generally acidic, between 5.5 and 6.5. The survival of most wood-damaging fungi is obviously suitable for living in acidic medium no matter in the spore development stage or the mycelial growth period (Lv Wenhua et al., 2002; Zhou Huiming, 1991).

Table 1-5 Effects of different pH values on the culture characteristics of Inonotus obliquus mycelium

At different initial pH, the colony diameter and hyphal growth of Inonotus obliquus showed different growth conditions. When the pH is 7, the mycelial growth rate is the fastest, followed by pH 8; there is no significant difference between pH 6 and pH 5, and the mycelial growth rate is the worst when pH is 4 or 3; in terms of colony texture, pH The higher, the thinner the texture. From the experimental results, it can be concluded that the optimal growth pH of Inonotus obliquus is 7, acidic conditions are conducive to the formation of bacterial lawns, and neutral conditions are conducive to the growth rate of mycelia. This result is the same as that of Ji Hong, Chen Yanqiu, Zhang Liqiu, Jiang Yuji ^[21-24] .

Effects of Different Temperatures on Mycelia Growth of Inonotus obliquus

Inonotus obliquus has strong cold resistance and can survive in a low temperature environment of -40°C. The germination of basidiospores and the entire growth cycle of mycelia are limited and restricted by temperature. At the optimum temperature, its life activities are also the most vigorous. The life activity is the slowest at the lowest or highest temperature.

Table 6 Effects of different temperatures on the culture characteristics of Inonotus obliquus mycelium

From the experimental results, it can be obtained that the growth rate of mycelium is the fastest when the temperature is 30°C, followed by 25°C and 20°C, and there is no significant difference between the two at the level of 0.05 and 0.01, followed by growth at 15°C, 35°C The speed is very slow; it hardly grows at 10°C; it can be seen that the suitable temperature for the growth of Inonotus obliquus is 30°C, and it hardly grows at too high or too low temperature. In terms of mycelial pigment formation, there was no significant difference between 30°C and 25°C. Temperatures higher than 30°C or lower than 25°C would affect the formation of pigments in Inonotus obliquus. This conclusion is consistent with the research results of Zhang Xuanqiu and Chen Yanqiu of the National Agricultural College of Korea.

Production of fruiting bodies of Inonotus obliquus on artificial medium

In the experiment of exploring the influence of different temperatures on Inonotus obliquus, the hz3 strain of Inonotus obliquus was placed on PDA medium, cultured at 35°C for 15 days, and then cultured at room temperature for 20 days. Inonotus obliquus fruiting bodies grow from the center right. This is the first time in the related research of Inonotus obliquus that fruiting bodies are produced under artificial culture conditions. Under natural conditions, the fruiting bodies of Inonotus obliquus are generally rare, and they are completely flat under the bark of living trees or on the sapwood of dead trees, and it is difficult to separate them from the substrate. At home and abroad, there are no reports about the production of fruiting bodies of Inonotus obliquus on artificial medium. In this paper, the fruiting body of Inonotus obliquus was found for the first time on the artificial culture substrate. The fruiting body was measured to be about 5mm thick and light brown; the fungal tube was shorter than that under natural conditions, only 3-5mm, and the front end of the fungal tube was also in a state of cracking. Larger than under natural conditions, reaching 1 to 3 per mm, generally light white or bright yellow, and then dark brown; bristles can be observed under a microscope (see Figure 4).

Under table 7 natural conditions and the fruiting body contrast on the artificial medium

Strain Screening Results

There were certain differences in the activity of strains from different sources and different collection times. Five strains were obtained through field collection, indoor strain isolation and strain purification, and the experimental results of each strain were as follows: hz3 and th1 mycelia grew better, followed by th2, hz1, and hz2. There was no significant difference in colony color.

Table 8 Effects of different bacterial strains on the culture characteristics of Inonotus obliquus mycelium

The preserved hz3 PDA test tube slopes were used as cultivars, inoculated into different cultivation compost formulations, and cultivated in a 35°C incubator for 30 days. The three formulations were basically covered with mycelium, and there was no significant difference in mycelial growth rate. Large, but the yield of sclerotia in formula 1 and 2 is less, and the size is relatively small, and the sclerotium production is mainly concentrated on the top of the fungus bag; while the growth rate of the fungus bag in formula 3 is fast in the later stage, the number of sclerotia produced is large and the largest, and the largest The diameter of the sclerotium is 4.52cm. The above bacteria bag was placed without opening at room temperature for 20-50 days, and fruiting bodies were also produced at the mouth and base of the culture bag. This is the first report of producing fruiting bodies of Inonotus obliquus on cultivation materials.

The above is only a preferred specific embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any person familiar with the technical field within the technical scope disclosed in the present invention can obviously obtain the simplicity of the technical solution. Changes or equivalent replacements all fall within the protection scope of the present invention.

Claims (2)

1. an Inonotus obliquus artificial cultivation method, is characterized in that, comprises the steps:

1) separation of Inonotus obliquus bacterial strain, purifying: first use the calcium hypochlorite supernatant of 1: 14 to carry out surface sterilization to fruit body, middle about 5mm blockage tissue is got again with the tweezers of high-temperature sterilization, moved the central authorities being connected to PDA culture medium flat plate, during cutting tissue, inoculation 1d is placed in 25 DEG C of constant temperature darkrooms and cultivates, check strain growth situation at any time, the contaminated flat board of timely eliminating, cover with after flat board until mycelia, proceed to and PDA slant tube medium is preserved purebred, the refrigerator being then placed in 4 DEG C after covering with saves backup;

2) medium based on carbon-free medium, nitrogen-free agar, the carbon source filtering out Inonotus obliquus mycelial growth is sucrose, and nitrogenous source is analysis for soybean powder, and C/N ratio 20: 1, pH is 7, and temperature is 30 DEG C;

3) Inonotus obliquus artificial domesticating cultivation:

On PDA medium, under 35 DEG C of conditions, cultivate 15d, cultivate 20d under placing it in room temperature condition, on medium, position to the right, center has grown Inonotus obliquus sporophore;

Culture medium for cultivating: birch wood chip 78%, analysis for soybean powder 20%, gypsum 1%, sucrose 1%, the moisture content of medium is 65%, cultivates 30 days under 35 DEG C of incubators, and under room temperature, opening does not place 20-50d, creates fruit body equally at cultivation sack and base portion.

2. Inonotus obliquus artificial cultivation method according to claim 1, is characterized in that, the nucleotide sequence of described Inonotus obliquus bacterial strain is as shown in SEQIDNO.1.