A chrysosporium ramiferum strain suitable for planting of linden and application and cultivation method thereof

Abstract

The application discloses a naematelia Fr gold ear strain suitable for linden tree planting and an application and cultivation method thereof. The application screens a naematelia Fr gold ear strain 7, which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC NO: M2025827. The cultivation method comprises the following steps: 1. field collection; 2. strain separation; 3. purification; 4. preparation of a liquid strain; 5. inoculation and culture; 6. ear management; and 7. linden tree cultivation. The application obtains a high-yield and high-resistance gold ear strain, and realizes effective planting of the gold ear under a forest.

Description

Technical Field

[0001] This invention belongs to the field of edible fungus cultivation technology, specifically relating to a strain of *Auricularia auricula-judae* suitable for linden wood cultivation, its application, and cultivation methods. Background Technology

[0002] Golden ear fungus (scientific name: *Naematelia aurantialba* (Bandoni & M. Zang) Millanes & Wedin) is a fungus belonging to the family Naemateliaceae and the genus *Naematelia*. Golden ear fungus is not only bright in color and delicious in taste and texture, but it is also rich in essential proteins, colloids, amino acids, fine cellulose, carotene, carbohydrates, various vitamins, and minerals. It has the effects of strengthening the spleen, moistening the lungs, protecting the liver, improving eyesight, regulating qi, anti-oxidation, anti-thrombosis, and anti-radiation. The fruiting body and fermentation broth of golden ear fungus contain abundant golden ear fungus polysaccharides, which have the effects of lowering blood sugar, lowering blood lipids, and improving immunity. Currently, dried golden ear fungus from factory cultivation has yellow-white streaks, and due to its large size and high moisture content, the drying cost is high, resulting in lower market acceptance compared to dried golden ear fungus from log cultivation. Furthermore, logistics and preservation in distant areas present significant challenges and high costs. Therefore, high-quality dried golden ear fungus is necessary for better promotion of the product.

[0003] Golden ear fungus lacks the ability to directly decompose and absorb lignin and cellulose, relying instead on its associated fungus, *Trichoderma harzianum*, to obtain nutrients from the substrate. The mycelium of *Trichoderma harzianum* promotes the absorption of nutrients by its associated fungus from the substrate. Different inoculation methods directly affect the growth and development of *Trichoderma harzianum* and its associated mycelium, thus influencing the quality and yield of the golden ear fungus. Due to the significant differences in growth conditions between understory timber cultivation and substrate-based cultivation of *Trichoderma harzianum*, conventional inoculation methods are unsuitable for understory cultivation. Therefore, researching suitable inoculation methods for understory timber cultivation is particularly important.

[0004] Auricularia auricula-judae requires high growth conditions such as temperature, humidity, and light. The growth conditions vary greatly at different stages, such as the young fruiting stage and the fruiting stage. Therefore, the fruiting management of Auricularia auricula-judae grown under forest cover is more difficult than that of conventional cultivation. Due to the long cultivation cycle of Auricularia auricula-judae grown on linden trees under forest cover, if the strain has poor resistance to diseases and miscellaneous bacteria, it will be unable to achieve fruiting or the fruiting will have low uniformity and poor quality.

[0005] To address the above issues, the team of this invention screened a strain suitable for linden wood cultivation and provided its application and cultivation method in linden wood cultivation. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of *Auricularia auricula-judae* suitable for cultivation on linden trees.

[0007] Another object of the present invention is to provide an application of a *Auricularia auricula-judae* strain suitable for linden tree cultivation. Another objective of this invention is to provide a cultivation method for auricularia auricula strain applicable to linden tree cultivation.

[0008] This invention is achieved through the following technical solution: The auricularia strain described in this invention is Naematelia Fr. strain gold 7, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2025827.

[0009] The application described in this invention is the application of Naematelia Fr. strain 7 in linden tree cultivation. The cultivation method described in this invention includes the following steps: Step 1: Field collection: Collect whole pieces of wood with golden ear fungus and its associated fungus, Leuciscus truncatus. Step 2: Strain isolation: Except for the area with fruiting bodies, spray with 75% alcohol for preliminary disinfection, cut the golden ear fungus along the base, take out the internal fruiting bodies and put them into the golden ear fungus special culture medium; Isolate the hairy leather fungus: Except for the area with fruiting bodies, spray with 75% alcohol for preliminary disinfection, cut the leather fungus along the base, take out the internal fruiting bodies and put them into PDA culture medium. Step 3: Purification: Under a microscope, distinguish the mixed hyphae growing from the fruiting bodies of Auricularia auricula-judae. The strong ones are Mycorrhizal trichomoniasis and the weak ones are Auricularia auricula-judae hyphae. Select newly grown Auricularia auricula-judae hyphae and culture them on Auricularia auricula-judae special medium at 19-21℃ for 7 days for later use. Select Mycorrhizal trichomoniasis hyphae and culture them on PDA medium at 19-21℃ for 7-8 days for later use. Step 4: Prepare liquid culture: Inoculate the purified auricularia auricula mycelium into liquid culture medium and incubate in the dark for 6 days; Step 5: Inoculation and culture: Inoculate the liquid culture medium, cover with a film after inoculation, and culture. Step 6: Ear growth management: After cultivation, remove the membrane and apply diffused light to stimulate ear base formation. When the ear base grows to 3-4 cm in size, it can open and produce fruiting bodies. Step 7: Linden wood cultivation: When the ear base is cultured to a size of 5-6cm, it is cut into small fruit pieces of 0.5cm under aseptic conditions for later use; after the steps of preparing linden wood, drilling holes for inoculation, stacking for mycelial growth, arranging for ear emergence, harvesting and processing, golden ear is obtained.

[0010] The golden ear fungus described in step 1 of this invention has a yellow fruiting body, a small size, 1-2 cm in length, irregular shape, and thin flesh. The hairy leather fungus is a white leathery fruiting body that grows 5 cm away from the golden ear fungus.

[0011] The components of the auricularia auricula cultivation medium in step 2 of this invention are: 100g of auricularia auricula cultivation material boiled in water and filtered, 15g of glucose, 2g of peptone, 1g of soybean powder, 2g of potassium dihydrogen phosphate, 18g of agar, and water added to make up to 1L.

[0012] The culture conditions for Auricularia auricula-judae and Leuciscus fasciatus described in step 2 of this invention are a temperature of 19-21℃ and a culture time of 7-8 days.

[0013] The liquid culture medium in step 4 of this invention consists of: 100g of golden ear fungus cultivation material boiled in water and filtered, 15g of glucose, 2g of peptone, 1g of soybean powder, 2g of potassium dihydrogen phosphate, and water added to make up to 1L.

[0014] The cultivation conditions described in step 4 of this invention are: temperature 20-22℃ and rotation speed 180r / min.

[0015] The cultivation medium in step 5 of this invention consists of: 30% cottonseed hulls, 30% sawdust, 25% corn cob, 13% wheat bran, and 2% gypsum; the moisture content of the medium is 57-59%.

[0016] The inoculation method described in step 5 of this invention is as follows: each stick is inoculated into 4 wells, and 1-1.5 mL is inoculated into each well. After inoculation, the membrane is covered. The culture conditions are as follows: cultured at 22-24℃, carbon dioxide concentration of 1500-2000 PPM, and in the dark for 12-15 days.

[0017] The scattered light stimulation conditions described in step 6 of this invention are: 300-500 lx, with the light turned on for 5-8 minutes every half hour.

[0018] Step 7 of this invention describes the cultivation of linden trees, which includes the following steps: Step 7-1: Linden wood preparation: Apply 20% lime water to the cut surface until the cut surface turns white, and let it dry until no more lime water drips before inoculation; Step 7-2: Drilling and Inoculation: Before inoculation, spray water on the linden wood until the surface is moist but not waterlogged. Drill holes with a longitudinal spacing of 12-20cm, a row spacing of 5-10cm, and a hole depth of 2-3cm. When inoculating, place the wood slightly below the hole opening. After inoculation, seal the opening with paraffin wax. Step 7-3: Mycelial Growth in Pile: After inoculation, pile the logs in a clean, sunny location for mycelial growth. Pre-wet the ground, then sprinkle lime powder until the lime is clearly visible and white. Place stones or sleepers on the ground, 10cm above the ground. Pile the inoculated logs in a "well" shape, up to a height of 1.5m. First cover with a plastic film, then cover with straw or corn stalks. After 15 days of inoculation, uncover the film and check. If cracks are found in the cross-section, spray water once while turning the pile. Turning the pile involves misaligning the logs vertically and horizontally to ensure even mycelial growth. Turn the pile again 10-15 days after the first turn. After 4-5 turns, when golden ear fruiting bodies appear on the logs, move them to the greenhouse for fruiting. Step 7-4: Arranging for Fruiting: Set up a 70-90cm high support frame inside the shed. Place the linden logs diagonally on the crossbeams of the support frame in an "A" shape, with 6-8 logs per square meter. During the fruiting period, ensure the air humidity is 80-90%. In windy or dry weather, spray water twice a day, morning and evening, until the surface of the linden logs is moist and water seeps out when the bark is squeezed. On rainy days, spray less or not at all. During the early fruiting stage, spray less (there should be no standing water on the surface of the mushrooms, and the linden logs should be moist), spray finely (use a sprayer or atomized water to make the water droplets as small as possible, or even a mist), and spray frequently (3-5 times a day). Gradually increase the amount of water as the fruiting bodies grow. Stop spraying water the day before harvesting and increase ventilation. Stop spraying water for two days after harvesting. Step 7-5: Harvesting and processing: When the fruiting bodies of the golden ear are mature, cut them off near the base of the ear. After harvesting, dry them in a dry or sun-drying manner. The moisture needs to be removed quickly and the temperature should be set at 50-60℃.

[0019] When inoculating as described in step 7-2 of this invention, select white flocculent material that is connected to the fruiting body of Auricularia auricula-judae and the culture medium, but it should not contain the fruiting body of Auricularia auricula-judae. Take a pea-sized amount of inoculum and place it at the bottom of the hole, then fill it tightly with culture medium, but do not fill the entire inoculation hole.

[0020] When turning the pile as described in step 7-3 of this invention, if miscellaneous bacteria are found, they can be sprayed with carbendazim or green mold fungicide, and ventilation should be increased; wet and coarse linden wood can be sun-dried more, while wet and fine linden wood should be sun-dried less.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention solves the problems of conventional inoculation methods being unsuitable for understory cultivation of *Auricularia auricula-judae*, the cultivation of *Auricularia auricula-judae* on dead wood, pest and disease management in fruiting management, and fruiting induction. The obtained *Auricularia auricula-judae* strain Jin 7 has firm, moderately shaped fruit bodies, golden yellow color, and clear texture. After drying, its color and characteristics are well preserved. The log inoculation experiment showed that the yield of strain Jin 7 of this invention was 187g / tree, 190.5g / tree, and 210.5g / tree, respectively, all higher than other strains. The infection rates were 0.02%, 0.036%, and 0.024%, respectively, all lower than other strains. The cultivation cycle is shorter, realizing effective understory cultivation of *Auricularia auricula-judae*.

[0022] Preservation Information: Strain naming: Naematelia Fr gold 7 strain; Preservation institution: China Center for Type Culture Collection (CCTCC); Location of collection: Wuhan University, Wuhan, China; Deposit date: April 18, 2025; The accession number is CCTCCNO: M2025827. Attached Figure Description

[0023] Figure 1Schematic diagram of field collection in Embodiment 1 of the present invention. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0025] Example 1 1. Field Collection. In March 2024, *Auricularia auricula-judae* and its associated fungus *Trichoderma pubescens* were found growing on dead cherry wood in the Zunyi Botanical Garden, Guizhou Province. The fruiting bodies of *Auricularia auricula-judae* were yellow, small (1-2 cm long), irregular, and thin-fleshed. White, leathery fruiting bodies, identified as *Trichoderma pubescens*, grew 5 cm away from the *Auricularia auricula-judae*. The entire piece of wood was brought back to the laboratory for timely isolation and purification of the fungal strain. A diagram illustrating the field collection is shown below. Figure 1 .

[0026] 2. Strain Isolation. Except for the areas with fruiting bodies, spray with 75% alcohol for preliminary disinfection. Place on a clean bench and use a sterilized scalpel to cut open the *Auricularia auricula-judae* along its base. Quickly use the tip of the scalpel to remove the internal fruiting bodies and place them on a special *Auricularia auricula-judae* culture medium (100g of *Auricularia auricula-judae* cultivation substrate boiled and filtered, 15g of glucose, 2g of peptone, 1g of soybean powder, 2g of potassium dihydrogen phosphate, 18g of agar, and water added to a final volume of 1L) for cultivation. The cultivation conditions are 19-21℃ for 7-8 days. *Trichoderma harzianum* Isolation: Except for the areas with fruiting bodies, spray with 75% alcohol for preliminary disinfection. Cut open the *Trichoderma harzianum* along its base and remove the internal fruiting bodies, placing them on PDA medium for cultivation. The cultivation conditions are the same as for *Auricularia auricula-judae*.

[0027] 3. Purification. The mycelia growing from the fruiting bodies of *Auricularia auricula-judae* are generally mixed mycelia. Under a microscope, the mixed mycelia can be distinguished: the robust ones are *Trichoderma harzianum*, while the slender ones are *Auricularia auricula-judae* mycelia. *Auricularia auricula-judae* mycelia purification: Newly grown *Auricularia auricula-judae* mycelia are picked and cultured on *Auricularia auricula-judae*-specific medium at 19-21℃ for 7 days before use. *Trichoderma harzianum* mycelia purification: After isolation, the grown mycelia are promptly picked and placed on PDA medium, cultured at 19-21℃ for 7-8 days before use.

[0028] 4. Prepare liquid culture medium. Inoculate the purified auricularia auricula mycelium into the liquid culture medium (100g of auricularia auricula cultivation substrate boiled and filtered, 15g of glucose, 2g of peptone, 1g of soybean powder, 2g of potassium dihydrogen phosphate, and water added to 1L), and place it at a temperature of 20-22℃ and a rotation speed of 180r / min in the dark for 6 days.

[0029] 5. Inoculation and Cultivation. Prepare the cultivation medium in advance (30% cottonseed hulls, 30% sawdust, 25% corn cob, 13% wheat bran, 2% gypsum), with a moisture content of 57-59%, sterilize and cool before use. Inoculate 1-1.5 mL per well (4 wells per stick). After inoculation, cover with a film and cultivate at 22-24℃, carbon dioxide concentration of 1500-2000 PPM, and in the dark for 12-15 days.

[0030] 6. Ear growth management. After cultivation, remove the membrane and stimulate the ear base with diffused light to form. The diffused light stimulation conditions are: 300-500 lx, with the light turned on for 5-8 minutes every half hour. When the ear base grows to 3-4 cm in size, it can open and produce fruiting bodies.

[0031] 7. Linden wood cultivation. When the auricles reach a size of 5-6cm, they should be aseptically cut into 0.5cm pieces for later use.

[0032] 7.1 Preparation of linden wood: Apply 20% lime water to the cut surface until the cut surface turns white, and let it dry until no more lime water drips before inoculation.

[0033] 7.2 Inoculation by Drilling Holes: Before inoculation, spray water on the linden wood until the surface is moist but not waterlogged. Drill holes, the spacing depending on the thickness of the wood, generally 12-20cm between holes, 5-10cm between rows, and 2-3cm deep is suitable. Do not pack the holes too loosely or too tightly; place them slightly below the opening. When inoculating, select white, fluffy material connected to the *Auricularia auricula-judae* fruiting bodies, ensuring it does not contain any fruiting bodies. Place a small amount of spawn, about the size of a soybean, into the hole, then fill it tightly with the substrate, but do not fill the entire hole. After inoculation, seal the hole with paraffin wax.

[0034] 7.3 Mycelial Growth in Pile: After inoculation, pile the logs in a clean, sunny location for mycelial growth. Pre-wet the ground, then sprinkle lime powder until it is clearly visible and white. Place stones or sleepers about 10cm above the ground. Pile the inoculated logs in a crisscross pattern to a height of 1.5m. First cover with a thin film, then cover with straw or corn stalks to increase temperature and moisture, which is conducive to mycelial growth. After 15 days, remove the film to inspect. If cracks are found in the cross-section, spray water once while turning the pile. Turning the pile involves offsetting the logs vertically and horizontally to ensure even mycelial growth. Turn the pile again 10-15 days after the first turn. If contaminating bacteria are found during turning, spray with carbendazim or chlorpyrifos and ensure good ventilation. Wet and coarse logs can be sun-dried more, while wet and fine logs should be sun-dried less. After 4-5 turns, when golden ear fruiting bodies appear on the logs, move them to a greenhouse for fruiting.

[0035] 7.4 Fruiting Stage: Choose a well-ventilated, well-lit location with some canopy cover and a water source. Set up a 70-90cm high support frame inside the shed, placing the logs diagonally in an "A" shape on the crossbeams of the support frame, with 6-8 logs per square meter. During the fruiting stage, maintain an air humidity of 80-90%. In windy or dry weather, spray water twice a day, morning and evening, using a large volume of water. On rainy days, reduce or stop spraying. During the early fruiting stage, spray less (avoid standing water on the mushroom surface, keep the logs moist), spray finely (using a sprayer or atomized water to make the water droplets as small as possible, even a mist), and spray frequently (3-5 times a day). Gradually increase the water volume as the fruiting bodies grow. Three days before harvest, spray heavily to ensure the fruiting bodies fully absorb water; stop spraying the day before harvest and increase ventilation; stop spraying for two days after harvest to allow the wounds at the base of the fruiting body to heal and promote the regeneration of the golden fruiting body.

[0036] 7.5 Harvesting and Processing: When the fruiting bodies of the golden ear are mature, they are orange or orange-yellow in color, with a layer of white frost on the surface. They are shaped like brains, with a thick braid at the top. The texture changes from hard to soft and elastic. This is the best season for harvesting. Use a sharp knife to cut them off close to the base of the ear. After harvesting, dry them in an oven or sun-dry them in time.

[0037] To verify the effectiveness of the invention, the inventive team conducted a series of experiments, as follows: The cultivation of *Auricularia auricula-judae* on linden logs presents challenges due to its long cultivation cycle. If the strain has poor resistance, fruiting may not occur, or the fruiting consistency may be low, resulting in poor quality. To address these issues, a suitable strain for linden log cultivation was obtained through screening. The collected *Auricularia auricula-judae* strain has been deposited at the China Center for Type Culture Collection (CCTCC) on April 18, 2025, with accession number CCTCC NO: M2025827. The experimental number is Jin 7. The mycelial morphology is characterized by a white and yellow intermingled distribution, a velvety appearance, and well-developed aerial hyphae. Both thick and thin hyphae are clearly visible under a microscope. The fruiting bodies on linden logs exhibit firm flesh, moderate size, golden color, and clear texture. After drying, the color and characteristics are well preserved, making it popular with consumers of dried fruit.

[0038] Five strains of Jin 3, Jin 4, Jin 5, Jin 7 and Jin 10 were prepared and inoculated onto oak, walnut and birch trees. Fifty linden trees of each species (8-10cm in diameter and 50cm in length, left to stand naturally for 60 days before use) were used. Under the same environmental conditions, the cycle, yield, infection rate and quality were measured. The experimental process and records are shown in Table 1.

[0039] Table 1 shows that the yield per tree of strain Jin 7 was 187 g / tree, 190.5 g / tree, and 210.5 g / tree on the three tree species, respectively, all higher than other strains; the infection rates were 0.02%, 0.036%, and 0.024%, respectively, all lower than other strains; and the growth cycle was second only to strain Jin 3. Overall, strain Jin 7 performed better, with superior resistance to contamination, yield, and quality compared to other strains.

[0040] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A type of golden ear fungus suitable for cultivation on linden trees ( Naematelia aurantialba ) strain, characterized in that, The strain of *Auricularia auricula* is: *Auricularia* genus (… Naematelia Fr. The strain Jin 7 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025827.

2. The application of the *Auricularia auricula-judae* strain as described in claim 1 in linden tree cultivation.

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