An Ophiocordyceps sinensis strain FDCUXZ-3 and its domestication culture method and application

Abstract

The present invention relates to a Ganoderma spore blood mushroom strain FDCU XZ-3 and its domestication culture method and application, belonging to the field of microbial technology. This strain was deposited at the Guangdong Provincial Culture Collection of Microorganisms on September 11, 2024, with the deposit number GDMCC No. 65120. For the Ganoderma spore blood mushroom strain FDCU XZ-3 of the present invention, compared with the currently cultivated Ganoderma rimosum and Ganoderma atrum, the cap of Ganoderma spore blood mushroom is larger, the fruiting body is larger and thicker, the color is yellowish-brown, the pores turn blood-red after being injured, the stipe is short or absent, which is somewhat different from the wild fruiting body. Overall, the mushroom fruiting is neat, the fruiting rate is high, it is heat-resistant and has strong anti-contamination ability.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology. Specifically, it relates to an oospore ganoderma strain FDCU XZ-3, its cultivation method and application. Background Art

[0002] The genus Ganoderma Sanguinoderma belongs to the family Ganodermataceae Ganodermataceae and mainly grows on the surface of soil humus, distributed in tropical and subtropical regions. There are 21 species of Ganoderma reported so far, and 15 species are distributed in China.

[0003] Compendium of Materia Medica records many Ganoderma species in previous dynasties and classifies them into six types, namely Ganoderma lucidum, Ganoderma sinense, Ganoderma applanatum, Ganoderma leucocontextum, Ganoderma atrum, and Ganoderma tsugae. Among them, the description of Ganoderma atrum is relatively similar to the current Ganoderma sanguineum. Ganoderma sanguineum is regarded as a precious medicinal material and is effective in preventing and treating cancer. Ganoderma sanguineum is rich in various active substances such as sterols, polysaccharides, triterpenoids, phenols, esters, etc. In terms of medicinal value, Ganoderma sanguineum has antioxidant, anti-inflammatory, anti-tumor, anti-hyperlipidemia, nerve protection, anti-microbial and other effects.

[0004] With the gradual understanding of the medicinal functions of Ganoderma sanguineum, the future demand for Ganoderma sanguineum will gradually increase. However, the yield of wild Ganoderma sanguineum is low, and there are few relevant cultivation reports, which cannot meet the current market demand. Moreover, the Ganoderma sanguineum on the market is mainly Ganoderma pseudoferreum and Ganoderma conicum. At present, there is no Ganoderma sanguineum variety for large-scale cultivation. In addition, the problem of single germplasm also seriously restricts the development of Ganoderma sanguineum.

[0005] Yunnan has rich resources of Ganoderma sanguineum, but there are no relevant reports on the domestication and cultivation of Ganoderma sanguineum in Yunnan. Summary of the Invention

[0006] In order to overcome the problems in the background art, the present invention provides an oospore ganoderma strain FDCU XZ-3, its domestication and cultivation method and application. The oospore ganoderma strain FDCU XZ-3 is obtained by domesticating the wild Ganoderma sanguineum strain collected from Sawu Village, Mangdong Town, Lianghe County, Dehong Prefecture, Yunnan Province. The obtained strain has a fast mycelial growth rate, white and thick mycelia. The fruiting bodies cultivated by domestication are thick, brightly colored, have a short growth cycle, and can be picked multiple times.

[0007] To achieve the above object, the present invention is realized by the following technical solutions:

[0008] The oospore ganoderma strain FDCU XZ-3 Sanguinoderma ovisporum was deposited in the Guangdong Provincial Culture Collection of Microorganisms on September 11, 2024, with the deposit number GDMCC No.65120.

[0009] The oospore ganoderma strain Sanguinoderma ovisporumFDCU XZ-3 has characteristic sequences as described in SEQ ID NO.1 to SEQ ID NO.6.

[0010] The Ganoderma lingzhi strain Sanguinoderma ovisporum The domestication and cultivation method of FDCU XZ-3 comprises the following steps:

[0011] (1) Take the Ganoderma lingzhi strain Sanguinoderma ovisporum After the mycelium of FDCU XZ-3 is activated at room temperature, it is inoculated on a PDA medium and placed in an incubator at 24 - 28 °C for constant-temperature cultivation until the colony fills the culture dish to obtain the mother culture.

[0012] (2) Transfer the mother culture obtained in step (1) to a liquid medium and place it in a constant-temperature shaker for light-avoiding cultivation; when the number of mycelial pellets in the liquid shaker flask accounts for two-thirds of the total liquid volume, the liquid spawn is obtained.

[0013] The liquid medium: 20 g of glucose, 3 g of soy peptone, 0.5 g of magnesium sulfate, 1000 mL of water, pH: natural.

[0014] (3) Inoculate the liquid spawn in step (2) into the cultivation spawn bag and place it in a dark cultivation room at 22 - 26 °C to obtain the cultivation spawn of the Ganoderma lingzhi strain FDCU XZ-3.

[0015] Further, in the PDA medium described in step (1), fructose is added in an amount of 20 g / L, soy peptone is added in an amount of 3 g / L, and sodium chloride is added in an amount of 0.5 g / L.

[0016] Further, the pH value of the medium in step (1) is adjusted to 10 with sodium hydroxide solution or hydrochloric acid solution.

[0017] Further, the constant-temperature shaker in step (2) is at 26 °C and 160 r / min.

[0018] Further, the culture medium used for the cultivation spawn bag in step (3) is composed of the following raw materials according to the dry matter weight parts: 78 - 81 parts of broad-leaved tree sawdust, 17 - 22 parts of wheat bran, 0.9 - 1.2 parts of gypsum, 1.9 - 2.2 parts of white sugar; water is added until the water content of the culture medium is 50 - 55 wt%, and the pH value of the culture medium is 5 - 7.

[0019] The present invention also provides the application of the mycelium or fruit body of the above-mentioned Ganoderma lingzhi strain FDCU XZ-3 in the preparation of drugs for anti-tumor, antioxidant and epilepsy treatment.

[0020] The beneficial effects of the present invention:

[0021] The Ganoderma lingzhi strain obtained by the present invention Sanguinoderma ovisporumFDCU XZ-3 was obtained by domesticating the wild Ganoderma sichuanense collected from Sawu Village, Mangdong Town, Lianghe County, Dehong Prefecture, Yunnan Province. The obtained strain has a fast mycelial growth rate, with white and thick mycelia. After domesticated cultivation, the cap is 10.2 ± 1.94 cm long, the cap width is 7.36 ± 2.43 cm, the dry weight is 12.6 ± 4.79 g per fruit body, and 2 - 3 flushes can be harvested per bag. Compared with the currently cultivated Ganoderma tsugae and Ganoderma atrum, Ganoderma ovoideosporum has a larger cap, a larger and thicker fruiting body, with a yellowish-brown color. The pores turn blood-red after being injured, and the stipe is short or absent, showing certain differences from the wild fruiting body. The overall mushroom emergence is neat, with a high mushroom emergence rate, high temperature tolerance, and strong anti-contamination ability. Description of the Drawings

[0022] Figure 1 It is an illustration of the grouping of the nine strains in Example 2 of the present invention;

[0023] Figure 2 It is a growth situation diagram of the nine strains in Example 2 of the present invention after 10 days of cultivation;

[0024] Figure 3 It is the maximum likelihood (ML) phylogenetic tree of Ganoderma constructed by the present invention based on ITS + LSU + rpb 2 + tef l–α + mtSSU + nSSU multiple genes; the nodes with a maximum likelihood support rate equal to or higher than 75% and a Bayesian posterior probability value equal to or higher than 0.95 are marked on the branches, and Ganoderma ovoideosporum is marked in bold red;

[0025] Figure 4 It is the result of the effect of carbon source on the mycelial growth of Ganoderma ovoideosporum strain;

[0026] Figure 5 It is the result of the effect of nitrogen source on the mycelial growth of Ganoderma ovoideosporum strain;

[0027] Figure 6 It is the result of the effect of inorganic salts on the mycelial growth of Ganoderma ovoideosporum strain;

[0028] Figure 7 It is the result of the effect of temperature on the mycelial growth of Ganoderma ovoideosporum strain;

[0029] Figure 8 It is the result of the effect of pH on the mycelial growth of Ganoderma ovoideosporum strain;

[0030] Figure 9 It is the cultivation process diagram of Ganoderma ovoideosporum of the present invention; a - b: primordium stage; c: differentiation stage; d - e: growth stage; f - g: maturity stage.

[0031] Figure 10It is the morphological identification diagram of Ganoderma sichuanense FDCU XZ-3 of the present invention. In the figure, a and b are fruiting bodies, c is the cut surface of the pileus, d is the pores, e is the pileus epidermal cells, f is the generative hyphae, g is the skeletal hyphae, h is the binding hyphae and skeletal hyphae, i-j are the basidioles, k is the basidium, and l is the spore; Scale bar: 5 cm (a, b), 5 mm (c), 1 mm (d), 20 μm (e-h), 15 μm (i-k), 10 μm (l).

[0032] The Ganoderma sichuanense strain FDCU XZ-3 of the present invention ( Sanguinoderma ovisporum ) was deposited at the Guangdong Provincial Culture Collection of Microorganisms on September 11, 2024, with the deposit number GDMCC No. 65120, and the deposit address is the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. Detailed implementation manners

[0033] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0034] To illustrate the present invention more clearly, the following detailed description is provided through the following embodiments. Embodiment

[0035] A Ganoderma sichuanense strain Sanguinoderma ovisporum FDCU XZ-3 was deposited at the Guangdong Provincial Culture Collection of Microorganisms on September 11, 2024, with the deposit number GDMCC No. 65120.

[0036] The Ganoderma sichuanense strain Sanguinoderma ovisporum The characteristic sequences ITS, mtSSU, nLSU, nSSU, rpb 2, tef 1–α of FDCU XZ-3 are shown in SEQ ID NO.1 to SEQ ID NO.6 respectively.

[0037] Embodiment 2 Domestication and cultivation method of Ganoderma sichuanense strain FDCU XZ-3

[0038] (1) Strain selection

[0039] Nine Ganoderma sichuanense strains collected from different regions, and the sources of the nine strains are as follows in the table

[0040] Table 1 Sources of nine wild strains

[0041]

[0042] The nine strains collected above were grouped into three groups of three (the grouping method is as Figure 1 ), inoculated on solid PDA medium, with a total of 16 groups, and placed in an incubator at 26 °C for constant temperature culture for antagonistic screening.

[0043] After 7 days of culture, the growth pictures of the 16 groups of strains are as Figure 2 shown. Select the strain with the densest mycelium and the fastest growth rate, and name this strain the Ganoderma sanguinolentum strain Sanguinoderma ovisporum FDCU XZ-3. This strain is the strain with the preservation number GDMCC No. 65120 in Example 1, and the original strain of this strain was collected from Sawu Village, Mangdong Town, Lianghe County, Dehong Prefecture, Yunnan Province.

[0044] (2) Strain activation, rejuvenation and propagation

[0045] Take the screened Ganoderma sanguinolentum strain Sanguinoderma ovisporum FDCU XZ-3 in step (1). After activating the mycelium at room temperature, inoculate it on PDA medium and place it in an incubator at 24 - 28 °C for constant temperature culture until the colony fills the petri dish.

[0046] (3) Preparation of liquid strain

[0047] Transfer the mother strain cultured in step (2) to a liquid shake flask of liquid strain medium, and culture it in a constant temperature shaker at 26 °C and 160 r / min in the dark; when the number of mycelial pellets in the liquid shake flask accounts for two-thirds of the total liquid volume, liquid strain is obtained.

[0048] Liquid strain medium: 20 g of glucose, 3 g of soy peptone, 0.5 g of magnesium sulfate, 1000 mL of water, and the pH is natural (that is, the pH is not adjusted).

[0049] (4) Preparation of cultivated strain

[0050] Transfer the liquid strain in step (3) into the cultivated strain bag, place it in a dark culture room at 22 - 26 °C, keep the air humidity at 60 - 65%, and wait until the mycelium fills the strain bag to obtain the cultivated strain.

[0051] The culture medium used for the cultivated strain bag is composed of the following dry-based raw materials and water by dry matter weight parts: 79 parts of broad-leaved tree sawdust, 18 parts of wheat bran, 1 part of gypsum, 2 parts of white sugar; add water to make the moisture content of the substrate 50 - 55 wt%, and measure the pH value of the culture medium to be 6.5.

[0052] Molecular biology research on the Ganoderma sanguinolentum strain Sanguinoderma ovisporum FDCU XZ-3:

[0053] Take the Ganoderma sanguinolentum strain in step (1) Sanguinoderma ovisporumThe mycelium of FDCU XZ-3 was ground in liquid nitrogen, and DNA was extracted using the Plant Genome Kit of Beijing Tsingke New Industry Biotechnology Co., Ltd. Six gene fragments were selected: ITS, LSU, tef 1-α, mtSSU, nSSU, and rpb 2. The base compositions of the specific primers are shown in Table 2. The RNA polymerase chain reaction (PCR) technique was used to amplify the six gene fragments of ITS, LSU, tef -1α, mtSSU, nSSU, and rpb 2. The PCR reaction system (25 μl) included: 12.5 μl mix, 9.5 μl sterile ddH2O, 1 μl each of the upstream and downstream primers with a concentration of 100 pmol / μl, and 1 μl DNA solution. The primers used for amplifying the six gene fragments are as follows in the table.

[0054] Table 2 Primers and base compositions used in PCR amplification

[0055]

[0056] Note: The bases include a, t, c, g. Other letters appearing in the primers represent degenerate bases.

[0057] The amplification procedures for the six gene fragments are as follows:

[0058] The PCR cycles for ITS and mtSSU are as follows: initial denaturation at 95 °C for 3 min, 94 °C for 40 s, 54 °C for 45 s, 72 °C for 1 min, repeated for 34 cycles, and extension at 72 °C for 10 min.

[0059] The PCR cycles for nLSU are as follows: initial denaturation at 94 °C for 1 min, 94 °C for 30 s, 53 °C for 60 s, 72 °C for 1.5 min, repeated for 35 cycles, and extension at 72 °C for 10 min.

[0060] The PCR cycles for nSSU are as follows: initial denaturation at 94 °C for 1 min, 94 °C for 1 min, 54 °C for 60 s, 72 °C for 1.5 min, repeated for 35 cycles, and extension at 72 °C for 10 min.

[0061] tef The PCR cycles for 1–α are as follows: initial denaturation at 94 °C for 5 min, 94 °C for 30 s, 55 °C for 30 s, 72 °C for 50 s, repeated for 35 cycles, and extension at 72 °C for 10 min.

[0062] rpbThe PCR cycle of 2 was as follows: initial denaturation at 95 °C for 5 min, 95 °C for 1 min, 51 °C for 2 min, 72 °C for 1.5 min, repeated for 35 cycles, and extension at 72 °C for 10 min.

[0063] The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the characteristic sequences of strain FDCU XZ-3 were obtained: ITS, mtSSU, nLSU, nSSU, rpb 2, tef 1–α were as follows.

[0064] Table 3 Serial numbers corresponding to characteristic sequences

[0065]

[0066] The sequence results were searched by Blast in the genBank database of the NCBI website and compared with the nucleotide homology of the 5.8S ribosomal RNA gene, small subunit ribosomal RNA gene, 28S ribosomal RNA gene, 18S rRNA gene, and the second largest subunit gene of RNA polymerase II that had been logged in. It was found that the sequence of strain FDCU XZ-3 obtained from the instance had a relatively high similarity with Sanguinoderma. The sequences of the 5.8S ribosomal RNA gene, small subunit ribosomal RNA gene, 28S ribosomal RNA gene, 18S rRNA gene, and the second largest subunit gene of RNA polymerase II with relatively high homology to strain FDCU XZ-3 were downloaded. The MAFFT v.7 (https: / / mafft.cbrc.jp / alignment / server / ) online version was used to align the sequences, and trimal v1.2 was used to trim the aligned sequences. Based on the combined dataset, maximum likelihood (ML) analysis was performed in RAxML-HPC2 v.8.2.3 (https: / / www.phylo.org / portal2 / login.action) to construct a phylogenetic tree (as Figure 5 )). Bayesian analysis was performed in MrBayes 3.2, and MrModeltest 2.3 was used to estimate the best model for sequence evolution; the selected models were: HKY+G model for ITS, GTR+I model for LSU and nSSU, GTR+I+G model for mtSSU and rpb 2, tefThe HKY+I+G model was used in the 1-α region. The posterior probability (PP) was calculated using the Markov chain Monte Carlo (MCMC) sampling method. Bayesian analysis was performed for 10,000,000 generations with six Markov chains running simultaneously, and the tree was sampled every 1000 generations. The first 5000 trees represented the burn-in phase of the analysis and were discarded, and the remaining 1500 trees were used to calculate the posterior probability in the majority rule consensus tree (the critical value for topological convergence diagnosis was 0.01). Phylogenetic tree analysis showed that FDCU XZ-3 of Ganoderma ovisporum clustered with another Ganoderma ovisporum specimen, with high bootstrap support, and formed a sister branch with Ganoderma schizodermum with high support (ML-BS = 99%, BPP = 0.82), and strain FDCU XZ-3 was identified as Ganoderma ovisporum Sanguinoderma ovisporum.

[0067] Example 3 Domestication and Cultivation of Ganoderma ovisporum Strain FDCU XZ-3

[0068] Select the Ganoderma ovisporum strain of Example 2 Sanguinoderma ovisporum The mycelium of FDCU XZ-3, with the basic medium containing 20 g of glucose, 3 g of soy peptone, 0.5 g of magnesium sulfate, 20 g of agar, 1000 mL of water, and natural pH (the pH value of the substrate is not adjusted additionally). The effects of carbon source, nitrogen source, inorganic salts, culture temperature, and medium pH value on mycelial growth were investigated. When investigating the carbon source, only the carbon source was replaced, and other components remained unchanged. The investigation methods for nitrogen source and inorganic salts were the same as those for the carbon source.

[0069] Single-factor screening test for carbon source: Using the basic medium as a control, 5 carbon sources were selected respectively: glucose, sucrose, maltose, lactose, fructose, with a concentration of 20 g / L (20 g of carbon source was added to each liter of medium), and a group without carbon source was used as a blank control. A 7-mm diameter punch was used to cut out fungal blocks and inoculate them in the center of the experimental medium (petri dish diameter 90 mm). Each experimental group was repeated 7 times. The cultures were incubated in the dark in a constant temperature incubator at 26 °C. The mycelial length was measured every 24 h using the cross-streaking method. The measurement was stopped after the medium was completely covered with mycelium, and the mycelial growth rate, mycelial density, color were recorded and photographed. The growth of mycelium under different carbon sources was obtained (Table 4 and Figure 4 , Figure 4Among them, a: glucose; b: sucrose; c: lactose; d: fructose; e: maltose; f: blank), the growth rates from fast to slow are fructose > glucose > sucrose > maltose > lactose > blank. Among them, when fructose is used as the carbon source, the mycelial growth rate is the fastest, reaching 6.971 ± 0.058 mm / d, and there are significant differences in the growth rates compared with other carbon sources. When lactose and maltose are used as carbon sources, the mycelial growth rates are slower, being 4.485 ± 0.087 mm / d and 5.100 ± 0.058 mm / d respectively. The mycelial growth rate of the blank group is the slowest, being 3.035 ± 0.086 mm / d, and there are significant differences in the growth rates compared with the mycelia containing carbon sources. Considering the mycelial growth rate and the growth trend of the mycelia under different carbon source conditions, fructose is the optimal carbon source.

[0070] Table 4 Effects of Different Carbon Sources on Mycelial Growth

[0071]

[0072] Note: +: Sparse mycelia; ++: Normal mycelia; +++: Dense mycelia; Different lowercase and uppercase letters in the same column represent significant differences (P < 0.05) and extremely significant differences (P < 0.01) respectively.

[0073] Single-factor screening test for nitrogen sources: Taking the basal medium as the control, five nitrogen sources were selected respectively: yeast extract, ammonium chloride, ammonium sulfate, urea, and soy peptone, with the addition amount of 3 g / L each (3 g of nitrogen source was added to each liter of the medium), and a group without nitrogen source was used as the blank control. Using a punch with a diameter of 7 mm, the mycelial blocks were inoculated in the center of the experimental medium (the diameter of the petri dish is 90 mm). Each experimental group was repeated 7 times, and they were cultured in the dark in a constant temperature incubator at 26 °C. The mycelial length was measured every 24 h by the cross-streaking method, and the measurement was stopped after the medium was covered with mycelia. The mycelial growth rate, the density of the mycelia, the color were recorded and photographed. The growth conditions of the mycelia under different nitrogen sources were obtained (Table 5 and Figure 5 , Figure 5 Among them, a: yeast extract; b: ammonium chloride; c: ammonium sulfate; d: urea; e: soy peptone; f: blank), the mycelial growth rate and the growth trend of Oospora sanguinea vary greatly under different nitrogen source conditions. The mycelial growth rates from fast to slow are soy peptone > yeast extract > sodium sulfate > ammonium sulfide, and the mycelia of the urea and blank groups could not grow. When soy peptone was used as the nitrogen source, the mycelia were dense and the growth rate was the fastest, reaching 5.987 ± 0.046 mm / d, which was significantly higher than other nitrogen sources. When ammonium chloride and ammonium sulfate were used as nitrogen sources, the mycelial growth rates were slower, being 0.328 ± 0.021 mm / d and 0.656 ± 0.019 mm / d respectively. Considering the mycelial growth rate and the growth trend of the mycelia under different nitrogen sources, soy peptone is the most suitable nitrogen source.

[0074] Table 5 Effects of Different Nitrogen Sources on Mycelial Growth

[0075]

[0076] Note: +: Sparse mycelium; ++: Moderate mycelium; +++: Dense mycelium. Different lowercase and uppercase letters in the same column represent significant difference (P<0.05) and extremely significant difference (P<0.01), respectively.

[0077] Effects of Inorganic Salts on Mycelial Growth Characteristics:

[0078] Taking the basal medium as the control, five inorganic salts were selected: ferric chloride, ferrous sulfate, sodium chloride, calcium carbonate, and magnesium sulfate, and the addition amount was 0.5 g / L (0.5 g of inorganic salt was added to each liter of the medium). One group without inorganic salt was used as the blank control. A 7-mm-diameter punch was used to cut out fungal blocks and inoculate them in the center of the experimental medium (the diameter of the petri dish was 90 mm). Each experimental group was repeated 7 times. The cultures were incubated in the dark in a constant-temperature incubator at 26 °C. The mycelial length was measured every 24 h using the cross-streak method until the medium was fully covered with mycelium, and then the measurement was stopped. The mycelial growth rate, density, color were recorded and photographed. The growth conditions of the mycelium under different inorganic salts were obtained (in Table 6 and Figure 6 , Figure 6 , where a: ferric chloride; b: ferrous sulfate; c: sodium chloride; d: calcium carbonate; e: magnesium sulfate; f: blank). The results showed that the mycelium of Ganoderma tropicum could grow normally under different inorganic salt conditions, but there were significant differences in the mycelial growth rate and growth trend. The mycelial growth rate from fast to slow was sodium chloride > magnesium sulfate > blank > calcium carbonate > ferrous sulfate > ferric chloride. When sodium chloride was used as the inorganic salt, the mycelium grew most densely and had the fastest growth rate, reaching 6.258±0.071 mm / d, and there was no extremely significant difference compared with magnesium sulfate as the inorganic salt. When ferric chloride, ferrous sulfate, and calcium carbonate were used as inorganic salts, the mycelial growth rates were relatively slow, 1.500±0.018 mm / d, 2.381±0.060 mm / d, and 3.850±0.087 mm / d, respectively, which were lower than that of the blank group (5.233±0.045 mm / d). Considering the mycelial growth rate and growth trend of different inorganic salts, sodium chloride was the most suitable inorganic salt.

[0079] Table 6 Effects of Different Inorganic Salts on Mycelial Growth

[0080]

[0081] Note: +: Sparse mycelium; ++: Moderate mycelium; +++: Dense mycelium. Different lowercase and uppercase letters in the same column represent significant difference (P<0.05) and extremely significant difference (P<0.01), respectively.

[0082] Effect of temperature on the growth characteristics of mycelium: Using a punch with a diameter of 7 mm, take the mycelium block and inoculate it in the center of the experimental medium (the diameter of the petri dish is 90 mm). Place the medium in a constant temperature incubator at 22 °C, 24 °C, 26 °C, 28 °C, 30 °C, and 32 °C respectively for dark cultivation. Examine the effect of temperature on the growth of Ganoderma sanguineum mycelium. Use the cross-streaking method to measure the mycelium length every 24 h. Stop the measurement after the medium is covered with mycelium. Record the mycelium growth rate, mycelium density, color, and take pictures. Obtain the growth conditions of mycelium under different inorganic salts and different temperatures. The results show that there are significant differences in the growth trend and growth rate of Ganoderma sanguineum mycelium under different temperature conditions (in Table 7 and Figure 7 , Figure 7 Figure 1, a: 22 °C; b: 24 °C; c: 26 °C; d: 28 °C; e: 30 °C; f: 32 °C). The mycelium growth rates at different temperatures from fast to slow are 28 °C > 26 °C > 24 °C > 22 °C > 30 °C (Table 5). The mycelium cannot grow at 32 °C. The mycelium growth rates are relatively fast at 28 °C and 26 °C, which are 6.150 ± 0.038 mm / d and 5.987 ± 0.046 mm / d respectively, and there are significant differences from the mycelium growth rates at other temperatures. The mycelium density at 28 °C is better than that at 26 °C. The mycelium growth rate decreases significantly at 30 °C, and the mycelium is extremely sparse. When the temperature reaches 32 °C, the mycelium stops growing. Considering the mycelium growth rate and growth trend at different temperatures, 28 °C is the optimal cultivation temperature.

[0083] Table 7 Effect of different temperatures on mycelium growth

[0084]

[0085] Note: +: Sparse mycelium; ++: General mycelium; +++: Dense mycelium; Different lowercase and uppercase letters in the same column represent significant difference (P < 0.05) and extremely significant difference (P < 0.01) respectively

[0086] Effect of pH value on the growth characteristics of mycelium:

[0087] The pH of the basal medium was adjusted to 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 using 1 mol / L HCl and 1 mol / L NaOH aqueous solutions. A 7-mm-diameter punch was used to cut out fungal blocks and inoculate them in the center of the experimental medium (petri dish diameter 90 mm). Each experimental group was repeated 7 times. The cultures were incubated in the dark in a constant temperature incubator at 26 °C. The mycelial length was measured every 24 h using the cross-streaking method until the medium was completely covered with mycelium, and then the measurement was stopped. The mycelial growth rate, density, color were recorded and photographed. The growth of mycelium under different inorganic salts and different pH values was obtained. The results showed that under different pH conditions, the mycelium of Ganoderma sanguinolentum var. ovoides could grow, and there were significant differences in the mycelial growth rate, while the differences in mycelial growth vigor were relatively small (in Table 8 and Figure 8 , Figure 8 , a: 5.0; b: 6.0; c: 7.0; d: 8.0; e: 9.0; f: 10.0). The mycelial growth rate from fast to slow at different pH values was 10 > 9 > 8 > 6 > 5 > 7. When pH = 10, the mycelial growth rate was the fastest, reaching 7.800 ± 0.023 mm / d, and there was no extremely significant difference compared with pH = 9, and the mycelial growth vigor was the same. When pH was 7, 6, and 5, the mycelial growth rates were slower, 4.735 ± 0.106 mm / d, 4.850 ± 0.075 mm / d, and 4.814 ± 0.096 mm / d respectively, and there was no significant difference. Considering the mycelial growth rate and growth vigor at different pH values, the optimal pH was 10.

[0088] Table 8 Effects of different temperatures on mycelial growth

[0089]

[0090] Note: +: Sparse mycelium; ++: Moderate mycelium; +++: Dense mycelium; Different lowercase and uppercase letters in the same column represent significant difference (P < 0.05) and extremely significant difference (P < 0.01) respectively

[0091] Optimal solid culture conditions for FDCU XZ-3 strain: The medium selects fructose as the carbon source, soy peptone as the nitrogen source, sodium chloride as the inorganic salt, pH is 10, and cultivation is carried out at an environmental temperature of 28 °C.

[0092] The diameter of generative hyphae is 3 - 6 μm, colorless, with thin cell walls and clamp connections. The diameter of skeletal hyphae is 5 - 8 μm, with thick cell walls, and the cell cavity is from narrow to thick, sub-solid, and curved like a tree. The diameter of binding hyphae is 2 - 5 μm, light yellow, and curved like a tree. All hyphae are IKI– and CB+, and the hyphae darken in KOH.

[0093] Example 4 Ganoderma sanguinolentum var. ovoides strain Sanguinoderma ovisporumCultivation of FDCU XZ-3

[0094] Inoculate the liquid spawn of Example 2 into the Ganoderma sinense cultivation bags. The substrate of the cultivation bags: 79 parts of broad-leaved tree sawdust, 18 parts of wheat bran, 1 part of gypsum, and 2 parts of white sugar; the pH value of the culture substrate is 6.5, and the water content is 50-55 wt%.

[0095] Dig planting grooves with a width of 0.60-0.80 m and a depth of 0.15-0.20 m. Cut a hole with a diameter of 1-2 cm in the plastic of the fungus bag at the upper rod shoulder of the Ganoderma sinense cultivation bag as a small hole. Place the Ganoderma sinense cultivation bags with small holes vertically in the planting grooves, with the open end facing up. Keep a spacing of 5 cm between the cultivation bags and a row spacing of 10 cm; cover the soil on the planting grooves with a soil covering thickness of 2-3 cm, and then carry out the mushroom emergence management of Ganoderma sinense.

[0096] Primordia begin to grow out after about 15 days. About 7 days after the appearance of the primordia, they begin to differentiate into pilei. After that, the fruiting bodies mature in about 20 days. There is no obvious spore ejection phenomenon after the fruiting bodies mature. Two to three crops can be picked from a single fungus bag. Compared with the currently cultivated Ganoderma rimosum and Ganoderma atrum, Ganoderma sinense has a shorter cycle of primordium formation, pileus differentiation, and fruiting body maturity, more picking times, and the fruiting bodies are brightly colored.

[0097] The primordia of the domesticated cultivated fruiting bodies are grayish-white. At the initial stage of primordium differentiation, they are multi-branched finger-shaped, with a light red base, a grayish-white surface. Later, the multi-branched finger-shaped gradually forms a nearly fan-shaped shape, with fine villi on the surface, a grayish-white edge, a yellowish-brown middle, and a reddish-brown base. After maturity, the whole pileus turns yellowish-brown, with obvious concentric rings, no lacquered luster, no stipe or a short stipe. The average size of the pileus is 10.2±1.94 cm × 7.36±2.43 cm, the average thickness is 0.4±0.1 cm, and the average dry weight is 12.6±4.79 g / fruit body. See the growth state diagram of the Ganoderma sinense strain FDCU XZ-3 during the cultivation process in Figure 9 , Ganoderma sinense has a larger pileus compared with the currently cultivated Ganoderma rimosum and Ganoderma atrum. The fruiting bodies are larger and thicker, with a yellowish-brown color. The pores turn blood-red after being injured. The stipe is short or absent, which is somewhat different from the wild fruiting bodies. During the process of fungus bag cultivation, the contamination rate of the fungus bags produced in the same batch is only 5%, which is significantly increased by 5% compared with the common cultivated Ganoderma in terms of the ability to resist miscellaneous bacteria. The cultivated fruiting bodies can still maintain a good growth state at a temperature of 28-30°C and a humidity of 80%. The high-temperature tolerance is increased by 2-4°C compared with the currently cultivated Ganoderma, and the humidity requirement is reduced by 5%-10%. The diameter of the pileus of the currently cultivated Ganoderma fruiting bodies is generally about 7 cm. After domestication and cultivation, the diameter of the Ganoderma sinense fruiting bodies is 10 cm, which is significantly larger and has a better commercial shape. The mushroom production rate is 90%. The overall size of the fruiting pilei is 10.2 × 7.36 cm, and the standard deviations of the length and width of the pilei are ±1.94 cm and ±2.43 cm respectively. Therefore, the overall mushroom emergence is neat.

[0098] Ganoderma sanguinolentum var. ovoidosporum is characterized by having a one-year-old fruiting body. The stipe is laterally attached, solitary, occasionally overlapping, and ranges from soft to hard woody. The pileus is solitary, kidney-shaped, 11.6 cm in diameter, and 4 mm thick. The surface of the pileus is orange, dull, hairy, and has concentric rings alternating from orange to dark orange, with dense undulating folds. The margin of the pileus is greyish orange, entire, and irregularly curved. The context is 2 mm thick, with non-uniform texture. The upper layer is dark greyish yellow, the middle layer is greyish yellow, and the lower layer is grey, with a texture ranging from woody to fibrous. The tubes are 2 mm thick, pale orange, bright grey near the pores, hard woody, and non-stratified. There are 3 - 5 pores per millimeter, oblong-ellipsoid to sub-rhomboid, bright grey. When the pores of the fresh fruiting body are injured, they turn red and then quickly turn black, and do not change color after drying. The pore septa are intact. The stipe is 7 cm long, 2 mm in diameter, cylindrical and hollow, slightly swollen at the base, and dark greyish orange. (See Figure 10 , where a and b are the fruiting bodies, c is a cross-section of the pileus, d are the pores, e are the epidermal cells of the pileus, f are the generative hyphae, g are the skeletal hyphae, h are the binding and skeletal hyphae, i - j are the promycelia, k are the basidia, and l are the spores; scale bars: 5 cm (a, b), 5 mm (c), 1 mm (d), 20 μm (e - h), 15 μm (i - k), 10 μm (l)). Compared with the common Ganoderma sinense and Ganoderma rimosum, the pileus color of Ganoderma sanguinolentum var. ovoidosporum is dark orange, the pileus is hairy, with obvious concentric rings, and the context color shows stratification, with the upper layer being dark greyish yellow, the middle layer being greyish yellow, and the lower layer being grey. The largest number of pores reported for Ganoderma sinense is 1 per millimeter, and the smallest is 8 per millimeter. The pores of the Ganoderma sanguinolentum var. ovoidosporum in this patent are 3 - 5 per millimeter, of medium size.

[0099] The above has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

[0100] Sequence number SEQ ID NO.1

[0101] Sequence name ITS

[0102] Length 601

[0103] Molecular type DNA

[0104] ttatcgagtc ttgactgggt tgtagctggc cttccgaggc atgtgcacgc cctgctcatc 60

[0105] cgctctacac ctgtgaactt actgtgggtt tcatatcgtg aagcgtgccg tttaacggct 120

[0106] cgtgaagcgg tctgtgcctg cgtttattac aaacccttgt aaagtaatag aacgtgtatt 180

[0107] gcgatgtaac gcatctatat acaactttca gcaacggatc tcttggctct cgcatcgatg 240

[0108] aagaacgcag cgaaatgcga taagtaatgt gaattgcaga attcagtgaa tcatcgaatc 300

[0109] tttgaacgca ccttgcgctc cttggtattc cgaggagcat gcctgtttga gtgtcatgaa 360

[0110] atcttcaacc taaaagcctt tgcgggtttg ttaggcttgg atttggaggc ttgtcggctc 420

[0111] attagtcggc tcctcttaaa tgcattagct tggttccttg cggatcggct gtcggtgtga 480

[0112] taattgtcaa tgccgcgacc gtgaagcgtt tggcgagctt ctaaccgtct cgttagagac 540

[0113] agattattga cctctgacct caaatcaggt aggactaccc gctgaactta agcatatcaa 600

[0114] a

[0115] Sequence number SEQ ID NO.2

[0116] Sequence name mtSSU

[0117] Length 511

[0118] Molecular type: DNA

[0119] gaatttcttt aaattcatga tgtcgtaagg gaaaataatg atattacctt actatgagtg 60

[0120] tcgtccaaag ctggtgccag aagactcggt aagaccagag acgcaaacgt taatcatctt 120

[0121] aaacaggcgt aaagggtttg taggcagctt tcataaaatt attttacaaa aataaagaag 180

[0122] tggtcttatt gaaagctaga atcaaaaaga ggttatagtg tataacgcct agaggagggc 240

[0123] tgatatcctt agatcctagg cagaatactt agggcgaagg ccactctcca ctaatgattg 300

[0124] acgctgagaa acgaaggtaa gggtaggaaa taggattaga taccccggta ctcctttctg 360

[0125] taaacgatga atggtagtca ttagtaaaat taaaaattac tagagacgaa gttaacacaa 420

[0126] taaccattcc gccttgtgag tactactgca aagtagaaaa caaaaaaatt agtcggtctc 480

[0127] gaagcaaacg gagtgaagca tgttatttaa t 511

[0128] Sequence number: SEQ ID NO.3

[0129] Sequence name: nLSU

[0130] Length: 885

[0131] ctcaaagagt cgagttgttt gggaatgcag ctcaaaatgg gtggtgaatt ccatctaaag 240

[0132] ctaaatattg gcgagagacc gatagcgaac aagtaccgtg agggaaagat gaaaagcact 300

[0133] ttggaaagag agttaaacag tacgtgaaat tgctgaaagg gaaacgcttg aagtcagtcg 360

[0134] cgtcgtccag aactcagcct tgctttcgct tggtgcactt tctggatgac gggtcagcat 420

[0135] cgattttgac cgtcggaaaa gggctggagt aatgtggcac ctccgggtgt gttatagact 480

[0136] tcagtcgcat acggcggttg ggatcgagga acgcagcgcg ccgcaaggca ggggttcgcc 540

[0137] cactttcgcg cttaggatgc tggcataatg gctttaaacg acccgtcttg aaacacggac 600

[0138] caaggagtct aacatacctg cgagtgtttg ggtggaaaac ccgagcgcgt aatgaaagtg 660

[0139] aaagttgaga cctctgtcgt ggagggcatc gacgcccgga cctgacgttc tctgacggat 720

[0140] ccgcggtaga gcatgtatgt tgggacccga aagatggtga actatgcctg aatagggtga 780

[0141] agccagagga aactctggtg gaggctcgta gcgattctga cgtgcaaatc gatcgtcaaa 840

[0142] tttgggtata ggggcgaaag actaatcgaa ccatctagta gctgg 885

[0143] Sequence number SEQ ID NO.4

[0144] Sequence name nSSU

[0145] Length 865

[0146] Molecular type DNA

[0147] ttggtgattc ataataactt ctcgaatcgc atggccttgc gccggcgatg cttcattcaa 240

[0148] atatctgccc tatcaacttt cgatggtagg atagaggcct accatggttt caacgggtaa 300

[0149] cggggaataa gggttcgatt ccggagaggg agcctgagaa acggctacca catccaagga 360

[0150] aggcagcagg cgcgcaaatt acccaatccc gacacgggga ggtagtgaca ataaataaca 420

[0151] atatggggct ctttcgggtc tcataattgg aatgagtaca atttaaatct cttaacgagg 480

[0152] aacaattgga gggcaagtct ggtgccagca gccgcggtaa ttccagctcc aatagcgtat 540

[0153] attaaagttg ttgcagttaa aaagctcgta gttgaacttc agacctggcc gggcggtctg 600

[0154] cctaacggta tgtactgtct ggctgggtct tacctcttgg tgagccggca tgcccttcac 660

[0155] tgggtgtgtc ggggaaccag gacttttacc ttgagaaaat tagagtgttc aaagcaggcc 720

[0156] tatgcccgaa tacattagca tggaataata aaataggacg tgcggttcta ttttgttggt 780

[0157] ttctagagtc gccgtaatga ttaataggga tagttggggg cattagtatt cagttgctag 840

[0158] aggtgaaatt cttggattta ctgaa 865

[0159] Sequence number SEQ ID NO.5

[0160] Sequence name rpb2

[0161] Length 725

[0162] Molecule type DNA

[0163] gagacatatt tgcatcactg gagcggaaac tcagtagatc atttttagtg cgtcgagaca 120

[0164] cacaaggagt tcaacatctc gcttgcggtc aagcacaaca cgatcaccaa tggcctcaag 180

[0165] tactccctcg ccactggtaa ctggggagac cagaagaaga cgatgtcgtc gaaggcgggt 240

[0166] gtctcacagg tccttaaccg ctacacatat gcgtcgactc tgtcccacct gcgtcgttgc 300

[0167] aacacgcccc tcggtcgcga gggcaagatc gccaagcccc gccagctgca caacacccac 360

[0168] tggggcatgg tctgtcctgc cgaaacgcct gaaggacagg cttgtggtct cgtcaagaac 420

[0169] ttgtcactca tgtcctgcat atccgtcggt acactctcgg cacctgtcat cgagttcttg 480

[0170] gaggagtggg gtctcgagtc tctggaggag aacgctcacg cttcaacccc ttgcacgaag 540

[0171] gtgttcgtga acggcgtctg gatgggtgtc caccgagatc ctgtgaagct cgtcagcacc 600

[0172] ctcaggaagc tccgtcggaa ggacgacatc aactgcgagg tatccgtagt ccgtgatatc 660

[0173] cgggagcgcg aacttcgtct ctacacggat gctggtcgcg tctgccgacc actcttcatc 720

[0174] gtcga 725

[0175] Sequence number SEQ ID NO.6

[0176] Sequence name tef1–α

[0177] Length 548

[0178] Molecular type DNA

[0179] ctgactgtgc aattctcatc atcgccgctg gtaccggcga gttcgaggct ggtatctcca 60

[0180] aggatggcca gacccgcgag cacgccctcc tcgccttcac cctcggtgtc aggcagctca 120

[0181] tcgtcgccgt taacaagatg gacactacca aggttcgccg gcacgtgcaa cgtcgatctt 180

[0182] gcattaggtt ctgacctagg tttacagtgg tccgaggacc gtttcaacga aatcgtcaag 240

[0183] gagacgtcga ccttcatcaa gaaggtcggg tacaacccga aggccgttgc gttcgtcccc 300

[0184] atttcagggt ggcacggcga caacatgctg gaggagtcct caaagtgagt atatgcggtc 360

[0185] ttcttctatg cgccgcagga ctgactcgtt gactttagca tgacctggta caagggctgg 420

[0186] acgaaggaga ccaagggtgg tgtcgtgaag gggaagaccc ttctcgacgc catcgacgcc 480

[0187] atcgaacccc ccgtccgtcc ctccgacaag cccctccgtc tccctctcca ggatgtctac 540

[0188] aagatcgg

Claims

1. A strain of Ganoderma lucidum FDCU XZ-3, characterized in that: The described oospore hemoglobin strain Sanguinoderma ovisporum FDCU XZ-3 was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 11, 2024, with the deposit number GDMCC No.65120.

2. The oospore hemoglobin strain as claimed in claim 1 Sanguinoderma ovisporum The domestication and cultivation method of FDCU XZ-3 is characterized in that: The steps include: (1) Obtain the oosporous blood strain Sanguinoderma ovisporum After the mycelium of FDCU XZ-3 was activated at room temperature, it was inoculated on PDA medium and placed in an incubator at 24-28°C for constant temperature culture until the colonies filled the culture dish to obtain the mother strain; (2) The mother culture obtained in step (1) is transferred to a liquid culture medium and cultured in a constant temperature shaker in the dark; when the number of bacterial balls in the liquid shake bottle accounts for two-thirds of the total volume of the liquid, a liquid culture is obtained; (3) The liquid spawn of step (2) is placed in a cultivation bag and placed in a culture room at 22-26°C for dark culture to obtain a cultivar of the Haematococcus oosporeus strain FDCU XZ-3.

3. The acclimation and cultivation method according to claim 2, characterized in that: In the PDA medium described in step (1), fructose is added in an amount of 20 g / L, soy peptone is added in an amount of 3 g / L, and sodium chloride is added in an amount of 0.5 g / L.

4. The acclimation and cultivation method according to claim 2, characterized in that: The pH value of the culture medium in step (1) is adjusted to 10 using sodium hydroxide solution or hydrochloric acid solution.

5. The acclimation and cultivation method according to claim 2, characterized in that: The constant temperature shaker of step (2) is 26°C, 160 r / min; the liquid culture medium of step (2) includes 20 g glucose, 3 g soy peptone, 0.5 g magnesium sulfate, 1000 mL water, and natural pH.

6. The acclimation and cultivation method according to claim 2, characterized in that: The culture medium used in the cultivation seed bag in step (3) is composed of the following raw materials in terms of dry matter weight: 78-81 parts of broad-leaved wood chips, 17-22 parts of wheat bran, 0.9-1.2 parts of gypsum, and 1.9-2.2 parts of white sugar; water is added until the water content of the culture medium is 50-55wt%, and the pH value of the culture medium is 5-7.

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