Agrocybe aegerita strain CXG003 fruiting early as well as culture and cultivation method and application thereof

Abstract

The invention belongs to the technical field of edible mushrooms, and discloses an agrocybe cylindracea strain CXG003 with early fruiting as well as a cultivation method and application thereof. The agrocybe cylindracea strain with early fruiting is CXG003 and is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M 20251057, the preservation date is May 14, 2025, and the classification name is Cyclocis chaxingu CXG003. The culture method comprises the following steps: culture medium preparation: preparing a PDA culture medium, a PDB culture medium and a regeneration culture medium; preparing protoplast, and selecting Agrocybe aegerita monokaryon strains; and hybridizing the agrocybe aegerita monokaryons to obtain the agrocybe aegerita The concentration of the protoplast can reach 1.07 * 10 < 8 > / mL, and the fruiting time is early.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungi, and in particular relates to an early-fruiting Agrocybe chaxini strain CXG003, a cultivation method and an application thereof. Background Art

[0002] Agrocybe chaxin Cyclocybe chaxingu , trade name Agrocybe tumefaciens, is one of the main cultivated edible fungi, belonging to the Basidiomycota, Agaricales, Tubariaceae, and Cyclops. Cyclocybe Agrocybe aegerita is rich in nutritional value, delicious, fragrant, with a crisp and tender stem, thick cap and unique flavor. It is an edible fungus that is high in protein, low in fat, rich in fungal polysaccharides and relatively high in mineral content.

[0003] Cultivation of Agrocybe chaxin takes a long time, with multiple fruiting periods, typically lasting from around April to October. This long fruiting cycle leads to serious pest and disease problems, making industrial-scale production impossible. This is a major bottleneck in the development of the Agrocybe chaxin industry. Therefore, cultivating varieties of Agrocybe chaxin with a shorter fruiting cycle is an urgent issue.

[0004] Agrocybe chaxin (tea-firewood mushroom) is an edible fungus. However, the long production cycle of existing varieties leads to serious pest and disease infestations, and the inability to scale up production has hindered the development of the Agrocybe chaxin (tea-firewood mushroom) industry. For years, researchers have been eager to develop varieties of Agrocybe chaxin (tea-firewood mushroom) with a shorter fruiting cycle, reducing pest and disease risks and enabling scaled-up production. However, due to technical difficulties and long R&D cycles, a variety with earlier fruiting has remained elusive. The strain presented in this paper, with its early fruiting and shorter production cycle, addresses this technical challenge that has eluded the Agrocybe chaxin (tea-firewood mushroom) industry.

[0005] Agrocybe chaxin is a traditional edible fungus. Most practitioners and researchers in related fields generally believe that its biological characteristics are characterized by a long fruiting cycle, which is difficult to change. This invention utilizes advanced protoplast technology, and through experimental development and extensive testing, overcomes this technical bias and cultivates a high-quality Agrocybe chaxin strain that produces early fruiting in a short cycle. This strain transforms Agrocybe chaxin from traditional, outdated, simple, and scattered greenhouse cultivation to large-scale, factory-based cultivation. Summary of the Invention

[0006] To overcome the problems existing in the related art, the disclosed embodiments of the present invention provide an early-fruiting Agrocybe chaxini strain CXG003, a cultivation method and an application thereof, and specifically relate to a strain CXG003 with a short fruiting period.

[0007] The technical solution is as follows: an early-fruiting Agrocybe chaxini strain, the early-fruiting Agrocybe chaxini strain is CXG003, which is deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M20251057, a deposit date of May 14, 2025, and a classification name of: Cyclocybe chaxingu CXG003.

[0008] Another object of the present invention is to provide a method for culturing an early-fruiting Agrocybe chaxini strain, the method comprising: S1, culture medium preparation, including preparation of PDA medium, PDB medium and regeneration medium; S2, protoplast preparation, picking the monokaryon strain of Agrocybe chaxingensis; S3, hybrid strains of Agrocybe chaxing were obtained by hybridization of Agrocybe chaxing monokaryons.

[0009] In step S1, the preparation of the PDA culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, boiling them in an induction cooker, pouring the cooked potatoes into a beaker, filtering them with gauze to obtain a cooked potato aqueous solution; weighing glucose and agar, adding the filtered potato water, stirring evenly with a glass rod, then pouring them into a beaker and then packaging them, and sterilizing them; cooling, pouring the liquid culture medium into a sterile culture dish in a clean bench, cooling the solution in the culture dish until it cools and solidifies, and wrapping it with sealing film for later use; The preparation of PDB culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, and boiling them in an induction cooker. After the potatoes are cooked, pouring them into a beaker and filtering them with gauze to obtain a cooked potato solution; weighing glucose, adding the filtered potato water, stirring it evenly with a glass rod while adding, pouring it into a beaker, and making up the volume. The prepared solution is then dispensed into conical flasks, sterilized, and cooled for later use; The preparation of the regeneration culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, and boiling them in an induction cooker; pouring the potato water into a beaker after it is boiled, filtering it with gauze to obtain a cooked potato solution; weighing glucose, agar, and mannitol, adding them to the filtered potato water, stirring them evenly with a glass rod, pouring them into a beaker, fixing the volume, dispensing them into conical flasks, sterilizing them, cooling them, and pouring the liquid culture medium into a sterile culture dish in an ultra-clean workbench until the solution in the culture dish cools and solidifies, and wrapping them with sealing film for later use.

[0010] In step S2, the protoplast preparation is performed by selecting the monokaryon strain of Agrocybe chaxingensis, including: The protoplast preparation includes first preparing protoplasts of Agrocybe ND5 and LJH001, specifically comprising: inoculating Agrocybe ND5 and LJH001 strains into the center of a disposable plate containing PDA culture medium; when mycelium grows to two-thirds of the plate, punching holes in the edge of the solid bacterial block with a sterilized puncher, inoculating the solid bacterial block into PDB culture medium for cultivation; filtering the liquid mycelium obtained by the culture through gauze and collecting it in a centrifuge tube for later use; enzymatically hydrolyzing the obtained mycelium with a wall-lytic enzyme and performing the enzymatic hydrolysis in a water bath to obtain protoplasts; The method of selecting the monokaryon strain of Agrocybe chaxingensis includes: diluting the protoplast quantity with mannitol-stabilized osmotic fluid, applying the protoplast onto the regeneration medium with a spreader, and placing the protoplast on the regeneration medium in a constant temperature incubator for dark culture; When regenerated colonies grow on the regeneration plate, pick out the regenerated colonies one by one with a sterilized needle, transfer them to PDA culture medium, and place them in an incubator for separate culture; after culture, pick the regenerated hyphae and prepare them on a glass slide, and place them under an inverted microscope to observe the lock-like union; transfer the strains without lock-like union to a new PDA culture medium, place them in an incubator for separate culture, and observe again whether they have lock-like union for secondary verification; transfer the monokaryon hyphae without lock-like union to a new PDA culture medium for culture.

[0011] The obtained mycelium was enzymatically hydrolyzed with 2% lytic enzyme, the water bath enzymatic hydrolysis temperature was 29-31 °C, the enzymatic hydrolysis time was 3.5-4.5 h, and the protoplast concentration was 1.07×10 8 / mL, dilute with 0.6mol / L mannitol-stabilized water until the number of protoplasts is 1×10 2 pieces / mL.

[0012] In step S3, hybridization of Agrocybe chaxing monokaryons to obtain Agrocybe chaxing hybrid strains includes: Step 1: Using Agrocybe chaxingu ND5 and LJH001 as parents, the monokaryon strains of ND5 and LJH001 were inoculated onto a PDA medium and cultured at a constant temperature. After culture, multiple clumps were punched out using a cork hole and cultured on the PDA medium in a standoff manner. The clumps were then placed in an incubator for dark culture. As hyphae on the two clumps continued to grow, the hyphae at both ends intertwined to form a grooved or raised antagonistic line. Multiple clumps were selected from the antagonistic line and transferred to new PDA medium, which were then cultured in a constant temperature incubator in the dark. Step 2, preliminary screening of Agrocybe chaxini hybrid strains; after hybridization of Agrocybe chaxini monokaryon strains, mycelia of hybrid strains with lock-like union are obtained, i.e., Agrocybe chaxini hybrid strains; Step 3: SRAP molecular marker analysis of hybrid strains, including SRAP primer design, SRAP-PCR amplification and horizontal agarose gel electrophoresis.

[0013] Another object of the present invention is to provide an application of the early-fruiting Agrocybe chaxini strain in breeding early-fruiting hybrid Agrocybe chaxini.

[0014] Another object of the present invention is to provide a method for cultivating an early-fruiting Agrocybe chaxini strain, which is obtained by cultivating the early-fruiting Agrocybe chaxini hybrid strain.

[0015] The cultivation method of the early-fruiting Agrocybe chaenomeles strain comprises: The hybrid strain of Agrocybe chaxingensis and its parent strain were removed and transferred to PDA culture medium in a clean bench. The culture medium was sealed and placed in a constant temperature incubator in the dark for static cultivation. After the mycelium covered the entire plate, the culture medium was removed and set aside. The culture medium formula included: 51-52% corn cobs, 21-22% cottonseed hulls, 16.5-17.5% wheat bran, 6-7% corn flour, 0.5-1.5% gypsum, and 0.5-1.5% lime.

[0016] Weigh the raw materials according to the cultivation formula ratio, stir the raw materials evenly, add water and mix well, stir the mixed culture medium evenly and put it into a medium-sized polypropylene plastic bag, place the bagged bacteria bag in a high-temperature and high-pressure sterilizer for sterilization at 121°C for 2 hours; after sterilization, remove the bacteria bag from the high-temperature and high-pressure sterilizer when the temperature drops below 50°C; place the sterilized bacteria bag in a pre-sterilized incubator to cool, place the cooled bacteria bag in a sterile clean bench, inoculate the prepared bacteria with an inoculation spatula, and inoculate each bacteria bag with a bacterial block; Place the inoculated bags in a constant temperature incubator for light-proof cultivation until the mycelium completely fills the bags; move the bags into the fruiting room, open the bags for fruiting, cut off the plastic film on the upper half of the empty bags, and harvest when the fruiting bodies grow to the edge of the cap and the film under the cap is about to break.

[0017] The conditions for fruiting are: temperature set at 24-26℃ and relative humidity at 90-95%.

[0018] In combination with all the above technical solutions, the beneficial effects of the present invention are as follows: Compared with the prior art, the present invention has a good improvement in the biological efficiency of the first batch of mushrooms. The protoplast concentration of the present invention can reach 1.07×10 8 The present invention has improved the breeding method of Agrocybe chaxingensis and increased breeding efficiency. This strain produces mushrooms early. The aroma and flavor of the present invention's Agrocybe chaxingensis CXG003 are significantly superior to existing commercially available mushrooms, further demonstrating that the present invention has the effect of increasing consumer acceptance.

[0019] The strain of the present invention has early mushroom fruiting, shortens the cycle, can save manpower and cultivation costs, is suitable for factory production, and can carry out large-scale and automated production.

[0020] Existing varieties of Agrocybe chaxin have the disadvantage of a long fruiting cycle, which has led to problems such as the inability of the Agrocybe chaxin industry to produce mushrooms in factories and excessive pesticide residues. The strain of the present invention has early fruiting and a short fruiting cycle, which not only reduces pests and diseases but is also suitable for factory production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure; Figure 1 This is a flow chart of a method for cultivating an early-fruiting Agrocybe chaxini strain provided by an embodiment of the present invention; Figure 2 This is a rendering of the hyphae of the ND5 monokaryon strain without lock-like association provided by an embodiment of the present invention; Figure 3 This is a rendering of the hyphae of the LJH001 monokaryon strain without lock-like union provided by an embodiment of the present invention; Figure 4 This is a diagram showing the lock-shaped combined effect of the successful hybridization strains provided by the embodiment of the present invention; Figure 5 This is an electrophoresis pattern obtained by running horizontal agarose gel electrophoresis after PCR amplification provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0022] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0023] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0024] Example 1: An early-fruiting Agrocybe chaxin strain provided by the present invention is CXG003, which is deposited in the China Center for Type Culture Collection (Wuhan, China) with a deposit number of CCTCC NO: M20251057 and a deposit date of May 14, 2025. The test result is: survival; the classification name is: Cyclocybe chaxingu CXG003.

[0025] Example 2, as Figure 1As shown, a method for culturing an early-fruiting Agrocybe chaxini strain is applied to the cultivation of an early-fruiting Agrocybe chaxini strain, and the method comprises: S1, culture medium preparation, including preparation of PDA medium, PDB medium and regeneration medium; S2, protoplast preparation, picking the monokaryon strain of Agrocybe chaxingensis; S3, hybrid strains of Agrocybe chaxing were obtained by hybridization of Agrocybe chaxing monokaryons.

[0026] Exemplarily, in step S1, the preparation of the PDA culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, and boiling them in an induction cooker; pouring the cooked potatoes into a beaker, filtering them with gauze to obtain a cooked potato aqueous solution; weighing glucose and agar, adding the filtered potato water, stirring evenly with a glass rod, then pouring them into a beaker, packaging them, and sterilizing them; cooling, pouring the liquid culture medium into a sterile culture dish in a clean bench, waiting for the solution in the culture dish to cool and solidify, and wrapping them with sealing film for later use; The preparation of PDB culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, and boiling them in an induction cooker. After the potatoes are cooked, pouring them into a beaker and filtering them with gauze to obtain a cooked potato solution; weighing glucose, adding the filtered potato water, stirring it evenly with a glass rod while adding, pouring it into a beaker, and making up the volume. The prepared solution is then dispensed into conical flasks, sterilized, and cooled for later use; The preparation of the regeneration culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, and boiling them in an induction cooker; pouring the potato water into a beaker after it is boiled, filtering it with gauze to obtain a cooked potato solution; weighing glucose, agar, and mannitol, adding them to the filtered potato water, stirring them evenly with a glass rod, pouring them into a beaker, fixing the volume, dispensing them into conical flasks, sterilizing them, cooling them, and pouring the liquid culture medium into a sterile culture dish in an ultra-clean workbench until the solution in the culture dish cools and solidifies, and wrapping them with sealing film for later use.

[0027] In step S2, the protoplast preparation is performed by selecting the monokaryon strain of Agrocybe chaxingensis, including: The protoplast preparation includes first preparing protoplasts of Agrocybe ND5 and LJH001, specifically comprising: inoculating Agrocybe ND5 and LJH001 strains into the center of a disposable plate containing PDA culture medium; when mycelium grows to two-thirds of the plate, punching holes in the edge of the solid bacterial block with a sterilized puncher, inoculating the solid bacterial block into PDB culture medium for cultivation; filtering the liquid mycelium obtained by the culture through gauze and collecting it in a centrifuge tube for later use; enzymatically hydrolyzing the obtained mycelium with a wall-lytic enzyme and performing the enzymatic hydrolysis in a water bath to obtain protoplasts; The method of selecting the monokaryon strain of Agrocybe chaxingensis includes: diluting the protoplast quantity with mannitol-stabilized osmotic fluid, applying the protoplast onto the regeneration medium with a spreader, and placing the protoplast on the regeneration medium in a constant temperature incubator for dark culture; When regenerated colonies grow on the regeneration plate, pick out the regenerated colonies one by one with a sterilized needle, transfer them to PDA culture medium, and place them in an incubator for separate culture; after culture, pick the regenerated hyphae and prepare them on a glass slide, and place them under an inverted microscope to observe the lock-like union; transfer the strains without lock-like union to a new PDA culture medium, place them in an incubator for separate culture, and observe again whether they have lock-like union for secondary verification; transfer the monokaryon hyphae without lock-like union to a new PDA culture medium for culture.

[0028] The obtained mycelium was enzymatically hydrolyzed with 2% lytic enzyme, the water bath enzymatic hydrolysis temperature was 29-31°C, the enzymatic hydrolysis time was 3.5-4.5h, and the protoplast concentration was 1.07×10 8 / mL, diluted with 0.6mol / L mannitol-stabilized water until the number of protoplasts was 1×10 2 pieces / mL.

[0029] In step S3, hybridization of Agrocybe chaxing monokaryons to obtain Agrocybe chaxing hybrid strains includes: Step 1: Using Agrocybe chaxingu ND5 and LJH001 as parents, the monokaryon strains of ND5 and LJH001 were inoculated onto PDA medium and cultured at a constant temperature. After culture, multiple clumps were punched out using a cork hole and cultured on PDA medium in a standoff manner. The clumps were then placed in an incubator for dark incubation. As hyphae on the two clumps continued to grow, the hyphae at both ends intertwined to form a grooved or raised antagonistic line. Multiple clumps were selected from the antagonistic line and transferred to new PDA medium, which were then cultured in a constant temperature incubator in the dark. Step 2, preliminary screening of Agrocybe chaxini hybrid strains; after hybridization of Agrocybe chaxini monokaryon strains, mycelia of hybrid strains with lock-like union are obtained, i.e., Agrocybe chaxini hybrid strains; Step 3, SRAP molecular marker analysis of hybrid strains, including SRAP primer design, SRAP-PCR amplification and horizontal agarose gel electrophoresis.

[0030] Example 3: Application of an early-fruiting Agrocybe chaxini strain in breeding early-fruiting hybrid Agrocybe chaxini.

[0031] Example 4: A method for cultivating an early-fruiting Agrocybe chaxini strain, obtained by cultivating an early-fruiting Agrocybe chaxini strain.

[0032] The cultivation method of the early-fruiting Agrocybe chaenomeles strain comprises: The hybrid strain of Agrocybe chaxingensis and its parent strain were removed and transferred to PDA culture medium in a clean bench. The culture medium was sealed and placed in a constant temperature incubator in the dark for static cultivation. After the mycelium covered the entire plate, the culture medium was removed and set aside. The culture medium formula included: 51-52% corn cobs, 21-22% cottonseed hulls, 16.5-17.5% wheat bran, 6-7% corn flour, 0.5-1.5% gypsum, and 0.5-1.5% lime.

[0033] Weigh the raw materials according to the cultivation formula ratio, stir the raw materials evenly, add water and mix well, stir the mixed culture medium evenly and put it into a medium-sized polypropylene plastic bag, place the bagged bacteria bag in a high-temperature and high-pressure sterilizer for sterilization at 121°C for 2 hours; after sterilization, remove the bacteria bag from the high-temperature and high-pressure sterilizer when the temperature drops below 50°C; place the sterilized bacteria bag in a pre-sterilized incubator to cool, place the cooled bacteria bag in a sterile clean bench, inoculate the prepared bacteria with an inoculation spatula, and inoculate each bacteria bag with a bacterial block; Place the inoculated bags in a constant temperature incubator for light-proof cultivation until the mycelium completely fills the bags; move the bags into the fruiting room, open the bags for fruiting, cut off the plastic film on the upper half of the empty bags, and harvest when the fruiting bodies grow to the edge of the cap and the film under the cap is about to break.

[0034] The conditions for fruiting are: temperature set at 24-26 ℃ and relative humidity 90-95%.

[0035] Illustratively, through the above technical solution of the present invention, protoplasts and / or spores and / or mycelium and / or fruiting bodies and / or mushroom sticks can be obtained.

[0036] The application of the present invention is further described below in conjunction with specific data and specific experimental data.

[0037] Application Example 1, culture medium preparation method.

[0038] PDA medium: Weigh 200 g of fresh potatoes and cut them into 1 cm x 1 cm pieces. Add distilled water and boil on an induction cooker for 10-15 minutes, until the pieces can be gently pierced with tweezers. Once cooked, pour the potatoes into a beaker and filter through four layers of gauze to obtain 1 L of cooked potato-water solution. Weigh 20 g of glucose and 20 g of agar, add the filtered potato water, and stir thoroughly with a glass rod. Pour the mixture into the beaker and bring the volume up to 1000 mL. Dispense into conical flasks, adjust the pH to natural, and sterilize at 121°C for 20 minutes. Cool to approximately 50°C. Pour the liquid medium into sterile Petri dishes in a laminar flow hood. Allow the solution to cool and solidify in the dish, then wrap with parafilm and set aside.

[0039] PDB medium: Weigh 200 g of fresh potatoes and cut them into 1 cm x 1 cm pieces. Add distilled water and boil on an induction cooker for 10-15 minutes, until the pieces can be gently broken apart with tweezers. Once cooked, pour the potatoes into a beaker and filter through four layers of gauze to obtain 1 L of cooked potato solution. Weigh 20 g of glucose and add the filtered potato solution, stirring with a glass rod. Pour the mixture into a beaker and bring the volume up to 1000 mL. Aliquot the solution into Erlenmeyer flasks, adjust the pH to natural, sterilize at 121°C for 20 minutes, and cool until ready for use.

[0040] Regeneration medium: Weigh 200 g of fresh potatoes and cut them into 1 cm x 1 cm pieces. Add distilled water and boil on an induction cooker for 10-15 minutes, until the potato pieces can be gently pierced with tweezers. Once the potato water is cooked, pour it into a beaker and filter through four layers of gauze to obtain 1 L of cooked potato solution. Weigh 20 g of glucose, 20 g of agar, and 109.3 g of mannitol and add them to the filtered potato water. Stir thoroughly with a glass rod, then pour into a beaker and bring the volume up to 1000 mL. Dispense into Erlenmeyer flasks, adjust the pH to natural, and sterilize at 121°C for 20 minutes. Cool to approximately 50°C. Pour the liquid medium into sterile Petri dishes in a laminar flow hood and incubate until the solution in the dish cools and solidifies. Wrap with parafilm and set aside.

[0041] Application Example 2: Protoplast preparation, picking the monokaryon strain of Agrocybe chaxingensis.

[0042] The parent strains ND5 and LJH001 were purchased from Jiangxi Licai Edible Fungi Co., Ltd.

[0043] First, prepare protoplasts from Agrocybe ND5 and LJH001. The following steps are used: Inoculate Agrocybe ND5 and LJH001 into the center of a disposable plate containing PDA medium. Once the mycelium has grown to two-thirds of the plate, use a sterile hole punch to punch three 6-10 mm solid blocks along the edge. These blocks are then inoculated into PDB medium and cultured for 11 days. The resulting liquid mycelium is filtered through a double layer of gauze and collected in a 10 mL centrifuge tube for later use. The resulting mycelium is then enzymatically hydrolyzed with 2% lytic enzyme (purchased from the Guangdong Institute of Microbiology). The hydrolysis temperature is maintained at 29°C-31°C, preferably 29.5°C, and the hydrolysis time is 3.5-4.5 hours, preferably 4 hours. The resulting protoplast concentration can reach 1.07×10 8 The protoplasts were diluted with 0.6 mol / L mannitol (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) to a concentration of 1 × 102 / mL, apply it to the regeneration culture medium with a spreader, place it in a constant temperature incubator at 25℃ for dark culture, and observe it 1-2 times a day.

[0044] When regenerated colonies grow on the regeneration plate, pick out the regenerated colonies one by one with a syringe, transfer them to PDA culture medium, and place them in a 25 ℃ incubator for separate culture. After 3-5 days of culture, pick the regenerated hyphae and make a slide on a glass slide, and place it under an inverted microscope to observe the lock-like union. Transfer the strains without lock-like unions to a new PDA culture medium, place them in a 25 ℃ incubator for separate culture for 3-5 days, and observe again whether they have lock-like unions for secondary verification. Transfer the monokaryon hyphae without lock-like unions to a new PDA culture medium for culture. Figure 2 The effect of the hyphae of the ND5 monokaryon strain without lock-like union is shown. Figure 3 The effect of the hyphae of the LJH001 monokaryon strain without locking union is shown.

[0045] The present invention prepares protoplasts from Agrocybe ND5 and LJH001. Compared with the method used in the prior art, the protoplast output reaches 2.50×10 7 / mL, while the protoplast concentration of the present invention can reach 1.07×10 8 / mL or more, which can reflect the advantages of the present invention in preparing protoplasts.

[0046] Application Example 3: Agrocybe chaxini strains were obtained by hybridization of Agrocybe chaxini monokaryons.

[0047] Step 1: Using Agrocybe chaxingu ND5 and LJH001 as parent strains, inoculate the monokaryon strains of ND5 and LJH001 onto PDA medium and culture at 25°C. After 5-10 days of culture, use a cork punch to punch out 0.6 cm diameter plugs from each culture medium and culture them face-to-face on 60 mm PDA medium, 3 cm apart. These plugs are then placed in a dark incubator at 25°C for 10-15 days. As hyphae from the two plugs continue to grow, the hyphae at both ends will intertwine to form a grooved or raised antagonistic line. Three 0.3 cm × 0.3 cm plugs from this line are transferred to fresh PDA medium and cultured in a dark incubator at 25°C for 3-7 days.

[0048] Step 2: Initial screening of Agrocybe chaxing hybrid strains.

[0049] Because the mycelium of the monokaryon strain is a single mycelium when observed under a microscope, there is no lock-like union phenomenon. However, after hybridization, the mycelium of the hybrid strain will show lock-like union phenomenon. The lock-like union phenomenon can be observed through microscopic examination. Figure 4 The successful hybridization strains show a lock-shaped joint effect.

[0050] Step 3: SRAP molecular marker analysis of hybrid strains.

[0051] Step 3.1, SRAP primer design.

[0052] me2 (upstream primer sequence 5′-3′): TGAGTCCAAACCGGAGC (SEQ ID NO: 1); em3 (downstream primer sequence 5′-3′): GACTGCGTACGAATTGAC (SEQ ID NO: 2); Step 3.2, then perform subsequent SRAP-PCR amplification; Amplification reaction system: template DNA 1 μL, upstream primer 1 μL; downstream primer 1 μL; 2 × Tap PCR Master Mix 12.5 μL; ddH2O 9.5 μL; Amplification procedure: Pre-denaturation at 94°C for 5 min; 5 cycles of denaturation at 94°C for 1 min, annealing at 35°C for 1 min, and annealing at 72°C for 1 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min; extension at 72°C for 10 min, and storage at 12°C.

[0053] The above primer sequences are from the literature: LIU JH, DING FH, SONG HY, et al. Analysis of genetic diversity among Chinese Cyclocybe chaxingu strains using ISSR and SRAP markers [J]. PeerJ, 2022, 10:e14037.

[0054] Step 3.3, horizontal agarose gel electrophoresis; After PCR amplification is completed, the electrophoresis pattern is obtained by running horizontal agarose gel electrophoresis and photographed. Figure 5 ; Among them, P1 is ND5, P2- is LJH001, and 1 represents the strain CXG003 with a short fruiting period of the present invention.

[0055] In order to prove that the present invention has a good effect, it is necessary to verify its advantages through control experiments: Experimental Example 1, a cultivation experiment of a hybrid strain of Agrocybe chaxingu.

[0056] Remove the hybrid strain of Agrocybe chaxingensis and the parent strain, transfer them to PDA culture medium in a clean bench, seal them, and place them in a 25°C constant temperature incubator in the dark for static cultivation. Wait until the mycelium has covered the plate and then remove them for use. The cultivation medium formula is as follows: Corn cob 51-52%, cottonseed hull 21-22%, wheat bran 16.5-17.5%, corn flour 6-7%, gypsum 0.5-1.5%, lime 0.5-1.5%, preferably corn cob 51.8%, cottonseed hull 21.7%, wheat bran 17.4%, corn flour 6.7%, gypsum 1.2%, lime 1.2%, total dry material weight 200 g.

[0057] Weigh the raw materials according to the cultivation formula, mix them thoroughly, and then add water to mix thoroughly. Keep the moisture content of the bag at around 60%. Grab the compost and squeeze it tightly, ensuring that no water droplets remain. Stir the compost thoroughly and place it into a medium-sized polypropylene plastic bag (14 x 28 cm). Press firmly with your fist until the compost slightly springs back. Seal the bag with a double-ring. Each bag weighs 0.5 kg. Sterilize the bag in an autoclave. Before sterilization, inspect the bag for damage. Sterilize at 121°C for 2 hours. After sterilization, remove the bag from the autoclave when the temperature drops below 50°C. Place the sterilized bag in a pre-sterilized incubator to cool to below 10°C before inoculation. Place the cooled fungus bag in a sterile clean bench and inoculate the prepared fungus with an inoculation shovel. For example, inoculate 5-10 fungus blocks of 0.5 cm × 0.5 cm in size into each fungus bag. Maintain sterile operation during the inoculation process.

[0058] After inoculation, place the bags in a constant-temperature incubator for incubation in the dark, set at 25°C and 70% relative humidity. In the early stages of incubation, the mycelium begins to germinate slowly in the bags. After about a week of germination, turn the bags to inspect for contamination. Dispose of any contaminated bags promptly to prevent contamination of other bags. Incubate in the dark until the mycelium completely fills the bags. Move the bags into the fruiting room and wait for primordia to differentiate. Open the bags for fruiting. Fruiting conditions include a temperature of 24-26°C, a relative humidity of 90-95%, and diffuse light. When opening the bags for fruiting, remove the remaining plastic film from the upper half of the bag to prevent water accumulation and ensure the survival of the small mushroom buds. Harvest when the fruiting bodies have grown to a pale color around the edge of the cap and the film beneath the cap is about to break.

[0059] Experimental Example 2: Determination of agronomic traits of Agrocybe chaxingu hybrid strains.

[0060] Determination of the fruiting time of Agrocybe chaxingensis: The fruiting time of the first batch of mushrooms is the interval from the inoculation of the strain into the fungus bag to the first picking of the Agrocybe chaxingensis fruiting bodies.

[0061] Yield determination of Agrocybe chaxing: Weigh and record the fresh weight of the fruiting bodies grown on the first batch of Agrocybe chaxing (see Table 1). Calculate the biological efficiency of Agrocybe chaxing using the formula: Biological efficiency = (fresh yield of mushrooms from one batch / dry weight of culture medium) × 100%.

[0062] Table 1 Yield of monokaryon hybrid strains of Agrocybe chaxingensis

[0063] Experiments have shown that the fruiting period of the existing tea-fired mushroom is about 50-60 days. Compared with the existing technology, the present invention has advanced the fruiting period and significantly improved the biological efficiency of the first batch of mushrooms. Furthermore, the culture medium used in the existing technology is formulated as follows: 63.8% cottonseed hulls, 21% wheat bran, 8% peanut cake, 5% corn flour, 1.4% gypsum, and 0.8% lime. Through screening and improving different culture media and culture processes, the present invention can achieve a protoplast concentration of 1.07×10 8 / mL, which improved the breeding method of Agrocybe chaxing and increased the breeding efficiency.

[0064] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant description of other embodiments.

[0065] The above description is only a preferred specific implementation method of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A tea-fired mushroom strain with early fruiting, characterized in that: The early-fruiting Agrocybe chaxini strain is CXG003, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20251057 and a deposit date of May 14, 2025. It is classified and named as: Cyclocybe chaxingu CXG003.

2. A method for cultivating an early-fruiting Agrocybe chaxini strain, characterized in that: The culture method is applied to the culture of the early-fruiting Agrocybe chaxini strain according to claim 1, and comprises the following steps: S1, culture medium preparation, including preparation of PDA medium, PDB medium and regeneration medium; S2, protoplast preparation, picking the monokaryon strain of Agrocybe chaxingensis; S3, hybrid strains of Agrocybe chaxing were obtained by hybridization of Agrocybe chaxing monokaryons.

3. The method for cultivating the early-fruiting Agrocybe chaxing strain according to claim 2, characterized in that: In step S1, the preparation of the PDA culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, boiling them in an induction cooker, pouring the cooked potatoes into a beaker, filtering them with gauze to obtain a cooked potato aqueous solution; weighing glucose and agar, adding the filtered potato water, stirring evenly with a glass rod, then pouring them into a beaker and then packaging them, and sterilizing them; cooling, pouring the liquid culture medium into a sterile culture dish in a clean bench, cooling the solution in the culture dish until it cools and solidifies, and wrapping it with sealing film for later use; The preparation of PDB culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, and boiling them in an induction cooker. After the potatoes are cooked, pouring them into a beaker and filtering them with gauze to obtain a cooked potato solution; weighing glucose, adding the filtered potato water, stirring it evenly with a glass rod while adding, pouring it into a beaker, and making up the volume. The prepared solution is then dispensed into conical flasks, sterilized, and cooled for later use; The preparation of the regeneration culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, and boiling them in an induction cooker; pouring the potato water into a beaker after it is boiled, filtering it with gauze to obtain a cooked potato solution; weighing glucose, agar, and mannitol, adding them to the filtered potato water, stirring them evenly with a glass rod, pouring them into a beaker, fixing the volume, dispensing them into conical flasks, sterilizing them, cooling them, and pouring the liquid culture medium into a sterile culture dish in an ultra-clean workbench until the solution in the culture dish cools and solidifies, and wrapping them with sealing film for later use.

4. The method for cultivating the early-fruiting Agrocybe chaxing strain according to claim 2, characterized in that: In step S2, protoplast preparation and selecting the monokaryon strain of Agrocybe chaxingensis include: The protoplast preparation includes first preparing protoplasts of Agrocybe ND5 and LJH001, specifically comprising: inoculating Agrocybe ND5 and LJH001 strains into the center of a disposable plate containing PDA culture medium; when mycelium grows to two-thirds of the plate, punching holes in the edge of the solid bacterial block with a sterilized puncher, inoculating the solid bacterial block into PDB culture medium for cultivation; filtering the liquid mycelium obtained by the culture through gauze and collecting it in a centrifuge tube for later use; enzymatically hydrolyzing the obtained mycelium with a wall-lytic enzyme and performing the enzymatic hydrolysis in a water bath to obtain protoplasts; The method of selecting the monokaryon strain of Agrocybe chaxingensis includes: diluting the protoplast quantity with mannitol-stabilized osmotic fluid, applying the protoplast onto the regeneration medium with a spreader, and placing the protoplast on the regeneration medium in a constant temperature incubator for dark culture; When regenerated colonies grow on the regeneration plate, pick out the regenerated colonies one by one with a syringe, transfer them to PDA culture medium, and place them in an incubator for separate culture; after culture, pick the regenerated hyphae and prepare them on a glass slide, and place them under an inverted microscope to observe the lock-like union; transfer the strains without lock-like union to a new PDA culture medium, place them in an incubator for separate culture, and observe again whether they have lock-like union for secondary verification; transfer the monokaryon hyphae without lock-like union to a new PDA culture medium for culture.

5. The method for cultivating the early-fruiting Agrocybe chaxing strain according to claim 4, characterized in that: The obtained mycelium was enzymatically hydrolyzed with 2% lytic enzyme, the water bath enzymatic hydrolysis temperature was 29-31 °C, the enzymatic hydrolysis time was 3.5-4.5 h, and the protoplast concentration was 1.07×10 8 / mL, dilute with 0.6mol / L mannitol-stabilized water until the number of protoplasts is 1×10 2 pieces / mL.

6. The method for cultivating the early-fruiting Agrocybe chaxing strain according to claim 2, characterized in that: In step S3, hybridization of Agrocybe chaxing monokaryons to obtain Agrocybe chaxing hybrid strains includes: Step 1: Using Agrocybe chaxingu ND5 and LJH001 as parents, the monokaryon strains of ND5 and LJH001 were inoculated onto a PDA medium and cultured at a constant temperature. After culture, multiple clumps were punched out using a cork hole and cultured on the PDA medium in a standoff manner. The clumps were then placed in an incubator for dark culture. As hyphae on the two clumps continued to grow, the hyphae at both ends intertwined to form a grooved or raised antagonistic line. Multiple clumps were selected from the antagonistic line and transferred to new PDA medium, which were then cultured in a constant temperature incubator in the dark. Step 2, preliminary screening of Agrocybe chaxini hybrid strains; after hybridization of Agrocybe chaxini monokaryon strains, mycelia of hybrid strains with lock-like union are obtained, i.e., Agrocybe chaxini hybrid strains; Step 3, SRAP molecular marker analysis of hybrid strains, including SRAP primer design, SRAP-PCR amplification and horizontal agarose gel electrophoresis.

7. Use of the early-fruiting Agrocybe chaxini strain according to claim 1 in breeding early-fruiting hybrid Agrocybe chaxini.

8. A method for cultivating an early-fruiting Agrocybe chaxini strain, characterized in that: The product is obtained by cultivating the early-fruiting hybrid strain of Agrocybe chaxingensis according to claim 1.

9. The cultivation method of the early-fruiting Agrocybe chaxini strain according to claim 8, characterized in that: The cultivation method of the early-fruiting Agrocybe chaxini hybrid strain comprises: Remove the hybrid strain of Agrocybe chaxingensis and its parent strain, transfer it to PDA culture medium in a clean bench, seal it and place it in a constant temperature incubator in the dark for static cultivation. Wait until the mycelium covers the plate and remove it for later use. The cultivation medium formula includes: 51-52% corn cobs, 21-22% cottonseed hulls, 16.5-17.5% wheat bran, 6-7% corn flour, 0.5-1.5% gypsum, and 0.5-1.5% lime. Weigh the raw materials according to the cultivation formula ratio, stir the raw materials evenly, add water and mix well, stir the mixed culture medium evenly and put it into a medium-sized polypropylene plastic bag, place the bagged bacteria bag in a high-temperature and high-pressure sterilizer for sterilization at 121°C for 2 hours; after sterilization, take the bacteria bag out of the high-temperature and high-pressure sterilizer when the temperature drops below 50°C; place the sterilized bacteria bag in a pre-sterilized incubator to cool, place the cooled bacteria bag in a sterile clean bench, inoculate the prepared bacteria with an inoculation spatula, and inoculate each bacteria bag with a bacterial block; Place the inoculated bags in a constant temperature incubator for light-proof cultivation until the mycelium completely fills the bags; move the bags into the fruiting room, open the bags for fruiting, cut off the plastic film on the upper half of the empty bags, and harvest when the fruiting bodies grow to the edge of the cap and the film under the cap is about to break.

10. The cultivation method of the early-fruiting Agrocybe chaxini strain according to claim 9, characterized in that: The conditions for fruiting are: temperature set at 24-26℃ and relative humidity at 90-95%.

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