A method for cultivating and bottle planting of a blood ear fungus

Abstract

The application belongs to the technical field of blood ear culture and cultivation, and particularly discloses a blood ear strain cultivation and bottle cultivation method, which comprises the following steps: obtaining blood ear basidiospores by separating and purifying culture of basidocarps of the blood ear based on a spore shooting method; obtaining associated bacteria hyphae by separating and purifying culture of ear wood tissues of the blood ear; obtaining blood ear hyphae by pairing the blood ear basidiospores in a culture medium based on a spore pairing method; inoculating the blood ear hyphae and the associated bacteria hyphae into culture media respectively for light-avoiding activation culture to obtain cultivation mother strains respectively; loading culture material into a wide-mouth bottle, compacting, centrally inoculating a hole, covering a bottle cap with a breathable film, high-pressure sterilizing, inoculating mixed cultivation mother strains under aseptic conditions, and carrying out dark culture until the hyphae grow to fill the bottle, and then carrying out ear management to obtain blood ear basidiocarps. The method has the advantages of early ear emergence, short growth cycle, ear cultivation through replacement material, stable strain, and pure blood ear hyphae, etc.

Description

Technical Field

[0001] This invention relates to the field of Hematoxylin and erythrorhizon culture and cultivation technology, specifically to a method for cultivating and bottle-culturing Hematoxylin and erythrorhizon strains. Background Technology

[0002] In recent years, due to the value of edible fungi, people have been collecting wild edible fungi more and more, resulting in a significant reduction in edible fungi germplasm resources. In order to protect my country's edible fungi germplasm resources and species diversity, it is very necessary to carry out research on the isolation and identification of wild edible fungi.

[0003] Tremella sanguinea, also known as blood ear fungus, red ear fungus, and blood wood ear fungus, is a rare edible fungus belonging to the Tremella genus. It possesses extremely high nutritional and medicinal value; however, due to its limited distribution and environmental conditions, wild resources of Tremella sanguinea are extremely scarce, and related research is limited. Tremella sanguinea exhibits the symbiotic phenomenon common to Tremella fungi, requiring the presence of symbiotic fungi for its growth. Currently, isolation techniques for Tremella sanguinea are not yet mature; only yeast-like basidiospores and mycelia of symbiotic fungi can be isolated, resulting in unstable fruiting yields when used as spawn. Patent CN 105154335 B discloses a method for cultivating a mixed strain of *Hymenochaetum rubrum*, specifically: isolating *Auricularia auricula-judae* and *Auricularia auricula-judae* basidiospores from wild *Hymenochaetum rubrum*, purifying and initially culturing them separately to obtain two strains; scaling up the two strains separately in liquid culture medium, mixing the two strains after scaling up to obtain a mixed liquid strain; inoculating the mixed liquid strain onto a solid sawdust and bran culture medium for cultivation to obtain a solid mixed strain of *Hymenochaetum rubrum* for *Hymenochaetum rubrum* cultivation; and using the mature mixed strain for log cultivation to obtain the finished *Hymenochaetum rubrum*. However, this method has drawbacks such as the *Auricularia auricula-judae* basidiospores failing to germinate mycelium, resulting in few or no fruiting fruits, and also the limitation that *Hymenochaetum rubrum* can only be cultivated using logs, and substrate-based cultivation has not yet been achieved. This invention studies the isolation and substrate-based cultivation of wild *Hymenochaetum rubrum* strains to protect *Hymenochaetum rubrum* germplasm resources and lay a foundation for future development and application. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for cultivating and bottle-growing *Tremella fuciformis* strains. This invention solves the problems of not being able to obtain pure *Tremella fuciformis* mycelia and unstable strains in existing technologies. It also solves the problems that *Tremella fuciformis* can only be cultivated using logs, has a late fruiting time, a long growth cycle, and cannot be cultivated using substrate. This invention fills the gap in previous research on the isolation and substrate cultivation techniques of *Tremella fuciformis*, and provides a theoretical basis for the study of *Tremella* fungi.

[0005] To achieve the above objectives, the present invention provides a method for cultivating and culturing *Hypericum auriculatum* in bottles, comprising the following steps:

[0006] S1. Basidiospores of *Hypericum erythrorhizon* were obtained by isolating the fruiting bodies of *Hypericum erythrorhizon* using the spore ejection method and purifying them using a modified PDA medium and peanut solid medium. Mycelia of the associated fungus were obtained by isolating the woody tissue of *Hypericum erythrorhizon* and purifying it using a modified PDA medium and peanut solid medium.

[0007] S2. Based on the spore pairing method, the basidiospores of *Hematoxylin and erythrorhizon* were paired in pairs in a modified PDA + peanut solid medium to obtain *Hematoxylin and erythrorhizon* hyphae.

[0008] S3. The *Hypericum aegyptium* hyphae and the associated fungal hyphae are respectively inoculated into a modified PDA + peanut solid medium for activation culture in the dark to obtain *Hypericum aegyptium* hyphae culture mother culture and associated fungal hyphae culture mother culture, respectively; wherein, the *Hypericum aegyptium* / associated fungal hyphae culture mother culture includes *Hypericum aegyptium* / associated fungal hyphae and the remaining solid medium;

[0009] S4. The culture medium is packed into a wide-mouth bottle, compacted, an inoculation hole is made in the center, a bottle cap with a breathable membrane is placed on it, and the bottle is sterilized by high pressure. Under aseptic conditions, a mixed solid culture is inoculated and cultured in the dark until the mycelium fills the bottle. After the fruiting management, the fruiting bodies of *Hymenochloa spp.* are obtained. The mixed solid culture includes *Hymenochloa spp.* mycelial culture mother culture and associated mycelial culture mother culture.

[0010] According to one aspect of the present invention, the formulation of the PDA-modified + peanut solid culture medium is as follows: 200 g / L potato, 20 g / L glucose, 5 g / L peptone, 3 g / L KH2PO4, 1.5 g / L MgSO4, 10 g / L peanut, 20 g / L agar, and the remainder is distilled water.

[0011] According to one aspect of the present invention, in step S2, the spore pairing method specifically involves placing different individual yeast-like basidiospores in pairs on a PDA+peanut plate culture medium, about 0.5 cm apart, and culturing at 25°C until the two basidiospores grow to the point of contact and mycelium germinates at the edge of the contact point.

[0012] According to one aspect of the present invention, in step S3, the conditions for the light-protected activation culture are: light-protected culture at 25°C for 7-14 days.

[0013] According to one aspect of the present invention, in step S4, the culture medium includes hardwood sawdust, wheat bran, corn flour, and gypsum; the moisture content of the culture medium is 58-60%.

[0014] According to one aspect of the present invention, in step S4, the culture medium contains 80 parts by weight of sawdust, 14 parts of wheat bran, 5 parts of corn flour, and 1 part of gypsum.

[0015] According to one aspect of the present invention, in step S4, after the culture medium is filled into a wide-mouth bottle, it is compacted to 1 / 3 to 2 / 3 of the bottle height; the autoclaving temperature is 121°C and the time is 3 hours.

[0016] According to one aspect of the present invention, in step S4, the mass ratio of the mother culture of *Hypericum aegyptium* and the mother culture of associated fungi in the mixed solid culture is 1:1.

[0017] According to one aspect of the present invention, in step S4, the temperature of the dark culture is 25°C, the humidity is 55-65%, and it is kept away from light.

[0018] According to one aspect of the present invention, in step S4, the ear management specifically involves: lowering the temperature to 20°C, increasing the humidity to 85-95°C, and ventilating twice a day.

[0019] The innovative aspects of this invention:

[0020] (1) This invention obtains basidiospores of *Hypericum spp.* by using the spore ejection method and pure hyphae of *Hypericum spp.* by the *Hypericum spp.* separation method. Then, the basidiospores of *Hypericum spp.* are paired on PDA+peanut plate medium to find basidiospore combinations that can germinate *Hypericum spp.* hyphae, thus obtaining pure hyphae of *Hypericum spp.*. Finally, the species status is determined by a combination of morphological and molecular biological identification, thus obtaining a *Hypericum spp.* strain that can be used for production.

[0021] (2) This invention involves simultaneously inoculating the pure mycelium of Blood Ear and its associated fungus Blood Scar Leather Fungi into bottled culture medium. After about 40 days of cultivation, when red water droplets appear on the surface, the fruiting bodies of Blood Ear are obtained at 20°C (about 30 days). This invention has initially realized the artificial domestication and cultivation of Blood Ear using a substitute material and found a method for bottle cultivation of Blood Ear.

[0022] The beneficial effects of this invention are:

[0023] (1) Compared with the prior art, the present invention obtains pure hyphae of *Hypericum halophilum* by germinating hyphae by pairing two *Hypericum halophilum* basidiospores, which have good vitality and stability;

[0024] (2) The present invention uses a method of cultivating blood ear fungus by substitute material, and grows blood ear fungus fruiting bodies by bottle cultivation, which shortens the time for blood ear fungus to emerge (about 30 days). Attached Figure Description

[0025] Figure 1 This is a photograph of Hemorrhage basidiospores collected by the spore ejection method in Example 1 of the present invention.

[0026] Figure 2 This is a photograph of the mycelium of the associated fungus obtained by the ear fungus isolation method in Example 1 of the present invention;

[0027] Figure 3 This is a photograph of a single yeast-like basidiospore in Example 1 of the present invention, showing that after they grow in pairs and come into contact, hyphae will germinate at the edge of the contact point.

[0028] Figure 4 This is a photograph of pure hyphae of *Hygrophora haematocephala* obtained by pure culture of hyphae germinating from paired *Hygrophora haematocephala* basidiospores in Example 1 of the present invention.

[0029] Figure 5 This is a photograph of the culture bottle in Example 1 of the present invention, after being inoculated with Haematomyces mycelium and associated mycelial blocks and cultured at 25°C in the dark until the mycelium completely covered the bottle.

[0030] Figure 6 This is a photograph of the actual product of the present invention, in Example 1, showing red water droplets appearing on the surface of the cultivation bag after the mycelium has covered the bag.

[0031] Figure 7 This is a physical image of the process from ear emergence management to the formation of a blood ear fruiting body in Embodiment 1 of the present invention;

[0032] Figure 8 This is a food illustration for comparative example 3 of the present invention, showing that the method of obtaining mycelium of Auricularia auricula-judae or Auricularia spp. from the blood ear fungus could not germinate. Detailed Implementation

[0033] To make the present invention easier to understand, specific embodiments are described below to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art; unless otherwise specified, the raw materials and reagents involved herein can be purchased commercially or obtained by known methods.

[0034] To address the problems of obtaining pure mycelia and unstable strains in existing technologies, this invention solves the issues of *Hypericum aegypti* only being able to be cultivated on logs, having a late fruiting time, a long growth cycle, and being unable to be cultivated using substrate substitutes. It fills a gap in previous research on the isolation and substrate substitute cultivation techniques of *Hypericum aegypti* and provides a theoretical basis for the study of *Tremella fuciformis* fungi. This invention provides a method for cultivating and bottle-culturing *Hypericum aegypti* strains, including the following steps:

[0035] S1. Basidiospores of *Hypericum erythrorhizon* were obtained by isolating the fruiting bodies of *Hypericum erythrorhizon* using the spore ejection method and purifying them using a modified PDA medium and peanut solid medium. Mycelia of the associated fungus were obtained by isolating the woody tissue of *Hypericum erythrorhizon* and purifying it using a modified PDA medium and peanut solid medium.

[0036] S2. Based on the spore pairing method, the basidiospores of *Hematoxylin and erythrorhizon* were paired in pairs in a modified PDA + peanut solid medium to obtain *Hematoxylin and erythrorhizon* hyphae.

[0037] S3. The *Hypericum aegyptium* hyphae and the associated fungal hyphae are respectively inoculated into a modified PDA + peanut solid medium for activation culture in the dark to obtain *Hypericum aegyptium* hyphae culture mother culture and associated fungal hyphae culture mother culture, respectively; wherein, the *Hypericum aegyptium* / associated fungal hyphae culture mother culture includes *Hypericum aegyptium* / associated fungal hyphae and the remaining solid medium;

[0038] S4. The culture medium is packed into a wide-mouth bottle, compacted, an inoculation hole is made in the center, a bottle cap with a breathable membrane is placed on it, and the bottle is sterilized by high pressure. Under aseptic conditions, a mixed solid culture is inoculated and cultured in the dark until the mycelium fills the bottle. After the fruiting management, the fruiting bodies of *Hymenochloa spp.* are obtained. The mixed solid culture includes *Hymenochloa spp.* mycelial culture mother culture and associated mycelial culture mother culture.

[0039] It should be noted that the aforementioned associated bacteria is *Hemiberlesia lataniae*.

[0040] In some specific embodiments of the present invention, the formula of the PDA-modified + peanut solid culture medium is as follows: 200 g / L potato, 20 g / L glucose, 5 g / L peptone, 3 g / L KH2PO4, 1.5 g / L MgSO4, 10 g / L peanut, 20 g / L agar, and the remainder is distilled water.

[0041] In some specific embodiments of the present invention, in step S2, the spore pairing method is specifically as follows: different individual yeast-like basidiospores are placed in pairs on PDA+peanut plate culture medium, about 0.5 cm apart, and cultured at 25°C until the two basidiospores grow to the point of contact and mycelium germinates at the edge of the contact point.

[0042] In some specific embodiments of the present invention, in step S3, the conditions for the light-protected activation culture are: light-protected culture at 25°C for 7-14 days.

[0043] In some specific embodiments of the present invention, in step S4, the culture medium includes hardwood sawdust, wheat bran, corn flour, and gypsum; the moisture content of the culture medium is 58-60%.

[0044] In some specific embodiments of the present invention, in step S4, the culture medium contains 80 parts by weight of sawdust, 14 parts of wheat bran, 5 parts of corn flour, and 1 part of gypsum.

[0045] In some specific embodiments of the present invention, in step S4, after the culture medium is filled into a wide-mouth bottle, it is compacted to 1 / 3 to 2 / 3 of the bottle height; the high-pressure sterilization temperature is 121°C and the time is 3 hours.

[0046] In some specific embodiments of the present invention, in step S4, the mass ratio of the mother culture of *Hypericum aegyptium* and the mother culture of associated fungi in the mixed solid culture is 1:1.

[0047] In some specific embodiments of the present invention, in step S4, the temperature of the dark culture is 25°C, the humidity is 55-65%, and it is kept away from light.

[0048] In some specific embodiments of the present invention, in step S4, the ear management specifically involves: lowering the temperature to 20°C, increasing the humidity to 85-95°C, and ventilating twice a day.

[0049] The following examples and comparative models further illustrate this point.

[0050] Example 1

[0051] A method for cultivating and culturing a strain of *Hypericum auriculatum* in bottles, comprising the following steps:

[0052] (1) Isolation and purification of strains: In a clean bench, the freshly collected *Hymenochaeces rubra* fruiting bodies were wiped clean with 75% alcohol, and the surface moisture was absorbed. The fruiting bodies were then suspended with sterilized hooks in Erlenmeyer flasks containing a thin layer of PDA medium (200g potato, 20g glucose, 15g agar, 1L water). The flasks were sealed with a breathable sealing film and incubated at 25℃. After one day, the fruiting bodies were removed. After about two days, the spores began to germinate, appearing as white yeast. Individual basidiospores were picked and purified in modified PDA + peanut solid medium (200 g / L potato, 20 g / L glucose, 5 g / L peptone, 3 g / L KH2PO4, 1.5 g / L MgSO4, 10 g / L peanut, 20 g / L agar, 1L distilled water). The basidiospores of *Hymenochaeces rubra* ( Figure 1 Collect wood from the base of the ear of *Hypericum aegyptium*. First, disinfect the surface with 75% alcohol. In a clean bench, cut the wood into small pieces the size of rice grains, soak them in 75% alcohol for 30 seconds, then rinse three times with sterile water. Next, blot the surface moisture with sterile gauze or filter paper. Place the wood on a modified PDA + peanut solid medium (potato 200 g / L, glucose 20 g / L, peptone 5 g / L, KH2PO4 3 g / L, MgSO4 1.5 g / L, peanut 10 g / L, agar 20 g / L, distilled water 1 L) and incubate at 25℃. Mycelia will germinate after 3 days. Select terminal mycelia and incubate them on a modified PDA + peanut solid medium (potato 200 g / L, glucose 20 g / L, peptone 5 g / L, KH2PO4 3 g / L, MgSO4 1.5 g / L, peanut 10 g / L, agar 20 g / L, distilled water 1 L). Purification and culture were performed using (g / L, 1L distilled water) to obtain associated fungal mycelia ( Figure 2).

[0053] (2) Spore pairing: The collected single basidiospores were paired in pairs. Different basidiospores were selected and, using the spore pairing method, different single yeast-like basidiospores were placed in pairs on PDA modified + peanut solid medium (potato 200 g / L, glucose 20 g / L, peptone 5 g / L, KH2PO4 3 g / L, MgSO4 1.5 g / L, peanut 10 g / L, agar 20 g / L, distilled water 1L), about 0.5 cm apart, and cultured at 25℃. When the two basidiospores grew to the point of contact, mycelium would germinate at the edge of the contact point ( Figure 3 ), observe the mycelial germination, record the pairing combinations of germinating hyphae, and then select germinating hyphae for pure culture to obtain pure mycelia of *Hypericum erythropterum* ( Figure 4 (Be careful not to touch the yeast-like spores when picking them up.)

[0054] (3) Mother culture: The above-mentioned associated fungal mycelia and pure mycelia of Hemlock were inoculated onto PDA modified + peanut solid medium (potato 200 g / L, glucose 20 g / L, peptone 5 g / L, KH2PO4 3 g / L, MgSO4 1.5 g / L, peanut 10 g / L, agar 20 g / L, distilled water 1L) and placed at 25℃ for 10 days in the dark to obtain the cultivation mother culture.

[0055] (4) Preparation of cultivation bottles: Soak sawdust, wheat bran, corn flour and gypsum in water in advance. After soaking thoroughly, place the materials in a cool place to drain the water and set aside. Weigh each material according to the proportion in the formula (80 parts sawdust, 14 parts wheat bran, 5 parts corn flour and 1 part gypsum). After weighing, pile the materials together, mix them evenly, add water to adjust the moisture content of the cultivation material to 60%. The moisture content is measured by grabbing the cultivation material with your hand and squeezing it tightly. When you open it, the cultivation material will clump together and fall apart when it hits the ground. There will be water stains on your hand but no water dripping. Then, the prepared culture medium is divided into 1000ml glass bottles (9.2cm × 18.9cm), with each bottle containing 0.3-0.4kg (about half a bottle). The culture medium is pressed firmly by hand, and an inoculation hole is made in the center. The bottles are then covered with a lid with a breathable membrane. The surface of the bottles is wiped clean, and they are placed in an autoclave at 121℃ for 3 hours. After sterilization, the bottles are removed and placed in the inoculation room. Once the bottles have cooled to room temperature, they are placed in a clean bench and inoculated with the same proportion of *Hymenochloa spp.* mycelium and associated mycelium blocks (the mass ratio of *Hymenochloa spp.* mycelium to associated mycelium is 1:1; wherein the *Hymenochloa spp.* mycelium / associated mycelium includes *Hymenochloa spp.* mycelium / associated mycelium and the remaining solid culture medium). After inoculation, the bottles are placed at 25℃ in the dark and cultured until the mycelium has fully colonized the bottle. Figure 5 ).

[0056] (5) Ear emergence management: After the mycelium has covered the cultivation bag, red water droplets appear on the surface ( Figure 6 Lower the temperature to 20℃, increase the humidity to 85%-95%, ventilate twice a day, and cultivate for about 30 days until the fruiting bodies of *Hymenochloa chinensis* are formed. Figure 7 Harvesting.

[0057] Comparative Example 1

[0058] A method for obtaining Tremella fuciformis mycelium includes the following steps:

[0059] (1) Select healthy, mature tremella ear pieces free from diseases and pests, wash them thoroughly with sterile water in a clean bench, then dry them with sterile filter paper, and collect tremella spores in a small triangular flask using the hook suspension method.

[0060] (2) The collected single spores of Tremella fuciformis were inoculated onto a germination-promoting medium plate and cultured at 25℃ for 7-10 days. Germinating mycelia were selected. The formula of the germination-promoting medium (1L) was: MgSO4·7H2O 1g, (NH4)2SO4 1g, KH2PO4 1.5g, glucose 10g, maltose 10g, agar powder 30g, 400mL of ash fungus extract, and ddH2O to a final volume of 1L. The preparation method of *Clerodendrum inerme* extract is as follows: Using a sterile inoculation hook, a piece of *Clerodendrum inerme* inoculated from a test tube is placed in the center of a PDA-enriched solid medium (200g peeled potato, 20g glucose, 20g agar, 5g yeast powder, 2g peptone, 1L water). Activate at 24℃ for 7 days. Use a 6mm punch to make holes in the PDA-enriched solid medium containing the *Clerodendrum inerme*. Then, using a sterile inoculation hook, pick up one piece and place it in the center of a sawdust medium (80 parts sawdust, 14 parts wheat bran, 5 parts corn flour, 1 part gypsum, 60% water content). Incubate at 24℃ for 30 days. Adjust the water content of the medium to 60%. Accurately weigh 300g of sawdust medium containing *Clerodendrum inerme*, add 1500mL of water, and boil in a water bath for 30 minutes. Filter twice through four layers of gauze, and then add water to bring the volume to 1L.

[0061] (3) Randomly select hyphae from the single spores of Tremella fuciformis for pairing and hybridization. Inoculate them on the same plate at a distance of 0.5 cm and culture at 25℃. When the two monokaryotic hyphae come into contact with the germinating hyphae, microscopic examination shows that there are clamp-like hyphae (indicating successful pairing).

[0062] Comparative Example 2

[0063] A method for obtaining Auricularia auricula-judae mycelium includes the following steps:

[0064] (1) Select healthy and mature golden ear pieces free from diseases and pests, wash them thoroughly with sterile water in a clean bench, then dry them with sterile filter paper, and collect golden ear spores in a small triangular flask using the hook suspension method.

[0065] (2) The collected auricularia auricula var. ... The preparation method of the concomitant bacteria extract is as follows: Using a sterile inoculation hook, take a piece of concomitant bacteria inoculation from the test tube and place it in the center of a PDA-enriched solid medium (200g peeled potato, 20g glucose, 20g agar, 5g yeast powder, 2g peptone, 1L water). Activate at 24℃ for 7 days. Use a 6mm punch to make holes in the PDA-enriched solid medium with concomitant bacteria. Then, under sterile conditions, pick up a piece of PDA-enriched solid medium containing concomitant bacteria and place it in the center of a sawdust medium (80 parts sawdust, 14 parts wheat bran, 5 parts corn flour, 1 part gypsum, 60% water content). Incubate at 24℃ for 30 days. Adjust the water content of the medium to 60%. Accurately weigh 300g of sawdust medium containing concomitant bacteria, add 1500mL of water, boil in a water bath for 30 minutes, filter twice with 4 layers of gauze, and add water to make up to 1L.

[0066] (3) Randomly select hyphae from the single spores of Auricularia auricula-judae for pairing and hybridization. Inoculate them on the same plate at a distance of 0.5 cm and culture at 25℃. When the two monokaryotic hyphae come into contact with the germinating hyphae, microscopic examination shows that there are clamp-like hyphae (indicating successful pairing).

[0067] Comparative Example 3

[0068] An experiment to obtain blood ear fungus using a method for obtaining Tremella fuciformis mycelium (Comparative Example 1) or Auricularia auricula-judae mycelium (Comparative Example 2):

[0069] (1) Select healthy, mature blood ear pieces free from diseases and pests, wash them thoroughly with sterile water in a clean bench, then dry them with sterile filter paper, and collect blood ear spores in a small triangular flask using the hook suspension method.

[0070] (2) The collected single spores of *Hypericum spp.* were inoculated onto a germination-promoting medium plate and cultured at 25°C for 7-10 days. Germinating hyphae were selected. The germination-promoting medium (1L) was formulated as follows: 200 g / L potato, 20 g / L glucose, 5 g / L peptone, 3 g / L KH2PO4, 1.5 g / L MgSO4, 10 g / L peanut, 300 ml *Hypericum spp.* extract, 20 g / L agar, and distilled water was added to bring the volume to 1L. The preparation method of *Brachys rubrum* extract is as follows: Using a sterile inoculation hook, a piece of *Brachys rubrum* inoculated from a test tube is placed in the center of a PDA-enriched solid medium (200g peeled potato, 20g glucose, 20g agar, 5g yeast powder, 2g peptone, 1L water). Activate at 24℃ for 7 days. Use a 6mm punch to make holes in the PDA-enriched solid medium containing *Brachys rubrum*. Then, using a sterile inoculation hook, pick up one piece and place it in the center of a sawdust medium (80 parts sawdust, 14 parts wheat bran, 5 parts corn flour, 1 part gypsum, 60% water content). Incubate at 24℃ for 30 days. Adjust the water content of the medium to 60%. Accurately weigh 300g of sawdust medium containing *Brachys rubrum*, add 1500mL of water, and boil in a water bath for 30 minutes. Filter twice through four layers of gauze, and then add water to a final volume of 1L.

[0071] (3) Randomly selected hyphae germinating from single spores of *Hypericum erythrorhizon* were paired and hybridized in pairs. They were inoculated on the same plate at a distance of 0.5 cm and cultured at 25℃. It was found that *Hypericum erythrorhizon* basidiospores could not germinate into single spore hyphae on a culture medium containing extracts of their respective symbiotic fungi, unlike *Auricularia auricula-judae* or *Auricularia auricula-judae*. Figure 8 Instead, as in Example 1 of this application, the collected individual basidiospores of *Hydroxella spp.* need to be paired up in pairs. Different *Hydroxella spp.* basidiospores are selected, and using the spore pairing method, different individual yeast-like basidiospores are placed in pairs on PDA+peanut plate culture medium, about 0.5 cm apart, and cultured at 25°C. When the two basidiospores grow to the point of contact, mycelium will germinate at the edge of their contact point, resulting in pure *Hydroxella spp.* mycelium.

Claims

1. A method for cultivating and culturing *Hypericum auriculatum* in bottles, characterized in that, Includes the following steps: S1. Basidiospores of *Hypericum halophilum* were obtained by isolating the fruiting bodies of *Hypericum halophilum* using the spore ejection method and purifying them using a modified PDA medium plus peanut solid medium. Mycelia of the associated fungus were obtained by isolating the woody tissue of *Hypericum halophilum* and purifying it using a modified PDA medium plus peanut solid medium. The formulation of the modified PDA medium plus peanut solid medium was: 200 g / L potato starch, 20 g / L glucose, 5 g / L peptone, 3 g / L KH₂PO₄, 1.5 g / L MgSO₄, 10 g / L peanut starch, 20 g / L agar, with the remainder being distilled water. S2. Based on the spore pairing method, the *Heterophyllum basidiospores* are paired in pairs on a modified PDA + peanut solid medium to obtain *Heterophyllum basidiospores* hyphae; wherein, the spore pairing method is specifically as follows: different single yeast-like basidiospores are placed in pairs on a PDA + peanut plate medium, 0.5 cm apart, and cultured at 25°C until the two basidiospores grow to the point of contact and hyphae germinate at the edge of the contact point; S3. The *Hypericum aegyptium* hyphae and the associated fungal hyphae are respectively inoculated into a modified PDA + peanut solid medium for activation culture in the dark to obtain *Hypericum aegyptium* hyphae culture mother culture and associated fungal hyphae culture mother culture, respectively; wherein, the *Hypericum aegyptium* / associated fungal hyphae culture mother culture includes *Hypericum aegyptium* / associated fungal hyphae and the remaining solid medium; S4. The culture medium is packed into a wide-mouth bottle, compacted, an inoculation hole is made in the center, a bottle cap with a breathable membrane is placed on top, and the bottle is sterilized by autoclaving. Under aseptic conditions, a mixed solid inoculum is inoculated and cultured in the dark until the mycelium fills the bottle. After fruiting management, the fruiting bodies of *Hymenochae rubra* are obtained. The mixed solid inoculum includes *Hymenochae rubra* mycelial culture mother culture and associated mycelial culture mother culture. The mass ratio of *Hymenochae rubra* mycelial culture mother culture to associated mycelial culture mother culture in the mixed solid inoculum is 1:

1.

2. The method for cultivating and culturing *Hypericum auriculatum* strain according to claim 1, characterized in that, In step S3, the conditions for the light-protected activation culture are: light-protected culture at 25℃ for 7-14 days.

3. The method for cultivating and culturing *Hypericum auriculatum* strain according to claim 1, characterized in that, In step S4, the culture medium includes hardwood sawdust, wheat bran, corn flour, and gypsum; the moisture content of the culture medium is 58-60%.

4. The method for cultivating and culturing *Hypericum auriculatum* strain according to claim 3, characterized in that, In step S4, by weight, the culture medium contains 80 parts of sawdust, 14 parts of wheat bran, 5 parts of corn flour, and 1 part of gypsum.

5. The method for cultivating and culturing *Hypericum auriculatum* strain according to claim 1, characterized in that, In step S4, after the culture medium is filled into a wide-mouth bottle, it is compacted to 1 / 3 to 2 / 3 of the bottle height; the autoclaving temperature is 121℃ and the time is 3 hours.

6. The method for cultivating and culturing *Hypericum auriculatum* strain according to claim 1, characterized in that, In step S4, the temperature for dark culture is 25°C, the humidity is 55-65%, and it is kept away from light.

7. The method for cultivating and culturing *Hypericum auriculatum* strain according to claim 1, characterized in that, In step S4, the ear management specifically involves: lowering the temperature to 20°C, increasing the humidity to 85-95°C, and ventilating twice a day.

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