Pleocidaris abies ZJLS001 and cultivation method thereof

Abstract

The invention discloses a pholiota abietis ZJLS001 and a cultivation method thereof. The classification name of the pholiota abietis is that the preservation number of the pholiota abietis is CGMCC (China General Microbiological Culture Collection Center) NO.41797, and in the cultivation process, the proportion of a cultivation bag in which the pholiota abietis ZJLS001 does not run spawn due to contamination of infectious microbes is 0; 5-9 days after mycelium stimulation, a pale yellow rice-grain primordium is formed, the average bud number of the primordium is 28, and the average diameter of the primordium is 1-2 mm; 290-350 g of fresh mushrooms are produced by two crops of the pholiota abies ZJLS001, and the biological efficiency is 82-87.5%. The strain is suitable for a liquid strain inoculation cultivation artificial propagation method, the cultivation method is simple and convenient, and the strain has the advantages of being fast in spawn running, little in pollution and strong in hypha; sporocarp is thick in pulp, yellow in color and stable in character; the strain has the advantages of high yield, realization of large-scale and industrial cultivation, high application value, short production period, low cost, safety and reliability.

Description

Technical Field

[0001] The present application relates to the technical field of planting Abies polyphylla, in particular to a Abies polyphylla ZJLS001 and a cultivation method thereof. Background Art

[0002] Pholiota abietis belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricomycetes, order Agaricales, family Strophariaceae, and genus Pholiota. It has a broadly convex shape with involute margins that later become nearly flat, with a few having a blunt central convexity. Its surface is sticky and covered with flat, mottled, yellow-brown scales. The cap surface is yellow-brown due to the concentric ring arrangement of scales, while the spaces between the scales and the cap margin are lemon yellow. The margin initially bears fragments of a veil. The flesh is thick, a light dark yellow, with a mild odor and flavor. The gills are initially off-white, then darken to a cinnamon color. They are medium-wide and dense, gradually narrowing toward the cap margin, and are straight to slightly curved.

[0003] Most of the Agaricaceae family are medicinal or edible fungi, such as the Agaricaceae, Agaricaceae, Agaricaceae, and Agaricaceae. They contain crude protein, fat, crude fiber, calcium, phosphorus, potassium, iron, vitamins, and other essential amino acids. However, wild resources of Agaricaceae are limited, and currently, they have not been domesticated for cultivation. Agaricaceae have great potential for edible applications. However, the low yield of wild Agaricaceae makes commercial cultivation impossible, and there are no established models for domestication and cultivation in China to develop this important edible and medicinal resource.

[0004] The information disclosed in the background section is only intended to enhance understanding of the overall background of the invention and should not be considered as an admission or any form of suggestion that the information constitutes the prior art already known to a person of ordinary skill in the art. Summary of the Invention

[0005] In response to the above technical problems, the present application provides a fir-shaped parasol ZJLS001 and a cultivation method thereof. The strain can be artificially cultivated and after harvesting one batch of mushrooms during the cultivation process, the second batch of mushrooms grows faster than other existing edible strains of the genus Parasol, and the biological efficiency and yield are effectively improved.

[0006] The present application provides a Pholiota abietis ZJLS001, which is classified and named as Pholiota abietis and has a deposit number of CGMCC NO.41797. During the cultivation process, the proportion of cultivation bags of Pholiota abietis ZJLS001 that did not germinate due to contamination by miscellaneous bacteria was 0; 5 to 9 days after scratching, light yellow, rice-grain-shaped primordia were formed, with an average number of buds in the primordia of 28 and an average diameter of 1 to 2 mm;

[0007] Two crops of Abies polyphylla ZJLS001 produced 290-350g of fresh mushrooms, with a biological efficiency of 82-87.5%.

[0008] Another aspect of the present application further provides a cultivation method of Abies serrata ZJLS001 as described above, comprising the following steps:

[0009] The mother seed of Abies serrata ZJLS001 was expanded and inoculated into the primary seed culture solution, and primary seed culture and secondary fermentation culture were performed to obtain the secondary fermentation solution of Abies serrata.

[0010] The secondary fermentation liquid of Abies aegypti was inoculated on the sterilized culture medium, and Abies aegypti ZJLS001 was obtained through mycelium culture, bud induction, mushroom fruiting management and harvesting.

[0011] Preferably, the first-level seed liquid culture medium used for the first-level seed culture is prepared by: boiling potato slices, filtering, and collecting the filtrate; adding glucose, corn flour, potassium dihydrogen phosphate, magnesium sulfate, yeast powder, peptone, and VB to the filtrate, and sterilizing to obtain the first-level seed liquid culture medium;

[0012] The secondary fermentation liquid used in the secondary fermentation culture includes: soybean powder, corn powder, glucose, potassium dihydrogen phosphate, magnesium sulfate, yeast powder, VB;

[0013] Preferably, the secondary fermentation broth used in the secondary fermentation culture is prepared by: grinding soybeans with water, collecting the filtrate, crushing the filter residue again, collecting and combining the filtrates; taking glucose, potassium dihydrogen phosphate, magnesium sulfate, yeast powder, VB and the filtrate and sterilizing them to obtain the secondary fermentation broth.

[0014] Preferably, the first-level seed culture is to inoculate the mother strain of Abies serrata ZJLS001 into the first-level seed liquid culture medium, and then culture the culture in a shake flask at 25-28° C. for 5-10 days to obtain the first-level liquid strain;

[0015] The secondary fermentation culture is as follows: the primary liquid strain is inoculated into the secondary fermentation liquid, and cultured under ventilation conditions of 25-28° C. and 0.05-0.07 MPa for 6-8 days to obtain the secondary fermentation liquid strain of Abies abies.

[0016] Preferably, the cultivation material is composed of 40-55 parts by weight of sawdust, 15-22 parts by weight of wheat bran, 16-20 parts by weight of cottonseed hulls, 8 parts by weight of corn flour, 8 parts by weight of corn cobs, 1 part by weight of lime, and 1 part by weight of sucrose.

[0017] Preferably, the inoculation amount is: at least 15 mL of the secondary fermentation liquid of Abies abies per 300 g of the culture medium;

[0018] Preferably, the inoculation amount is: 15 to 21 mL of the secondary fermentation liquid of Abies abies per 300 g of the culture medium.

[0019] Preferably, the mycelium culture is to inoculate the culture material with the secondary fermentation liquid of Abies aegyptifolia in the dark at 22-25° C., culture with ventilation for 30 days, after-ripen for 10 days, and induce budding after scratching.

[0020] Preferably, the bud-inducing operation is: bud-inducing is carried out in an environment of 12-22°C, 80-95% humidity, 1000-1100 ppm carbon dioxide concentration, and 400-500 lx light intensity, ventilation increases the temperature difference to promote primordium differentiation, and mushroom fruiting management is carried out after rice-grain-shaped primordium is formed in 5-9 days.

[0021] Preferably, the mushrooms are cultivated at a temperature of 18°C, a humidity of 95%, a carbon dioxide concentration of 700 ppm, and scattered light conditions after the primordium appears, and are harvested when most of the fruiting bodies are 70% mature and the cap expansion reaches 50%.

[0022] Preferably, after harvesting the first batch of mushrooms, stop humidification, remove the remaining mushroom roots, mushroom feet and dead mushrooms, and after scratching the mushrooms, repeat the mushroom fruiting management for 5 to 8 days before harvesting the second batch of mushrooms.

[0023] The beneficial effects of this application include:

[0024] 1) The present application provides a strain of Abies polypore ZJLS001 and a cultivation method thereof. The strain is suitable for liquid spawn inoculation cultivation and artificial propagation methods, and the cultivation method is simple. The strain has a fast spawning time and can form rice-grain-shaped primordia in just 5 days; it has little pollution, and the proportion of cultivation bags that fail to spawn due to contamination by foreign bacteria during the cultivation process is 0, indicating that the strain has a good effect of inhibiting the growth of foreign bacteria; 5 to 9 days after scratching, light yellow, rice-grain-shaped primordia are formed, the average number of primordium buds can reach 28, the average primordium diameter is 1 to 2 mm, and the primordium size is uniform, indicating that the strain has a higher growth rate; the fruiting body is thick, yellow in color, and has stable properties; the strain has a high yield, can be cultivated on a large scale and in a factory, has high application value, a short production cycle, low cost, and is safe and reliable. The strain can produce 290 to 350 g of fresh mushrooms in two crops, and the biological efficiency can reach 82 to 87.5%. The control strain produced 260-305 g in two crops, with a biological efficiency of 74.3-76.3%, indicating that the biological efficiency and yield of the strain provided in the present application were higher than those of the control strain.

[0025] 2) The cultivation method of Abies adiposa ZJLS001 provided in this application shows that after the strain is cultivated using this cultivation method, its growth rate after one harvest is faster than that of the existing control strain of the same genus, Pholiota adiposa, which can effectively shorten the fruiting management time to 5-8 days and effectively improve the strain and increase yield.

[0026] Collection information:

[0027] The Pholiota abietis strain ZJLS001 was deposited in the General Microbiology Center of the China General Culture Collection Administration (CGMCC); the deposit number is CGMCC NO.41797; the deposit date is March 17, 2025; the strain CGMCC NO.41797 was used to prepare the fungus; the classification name is: Pholiota abietis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a photo taken of the wild Abies abies fruiting body in Example 1 of the present application;

[0029] Figure 2 This is a photo of the fruiting of the Abies aegypti strain ZJLS001 in Example 2 of this application;

[0030] Figure 3 These are photos of the culture medium after 20 days of inoculation with Abies abies ZJLS001 and the control strain in Experimental Example 1 of this application; a) is a photo of the front side of the culture medium; b) is a photo of the back side of the culture medium;

[0031] Figure 4 This is the developmental tree of the Abies abies ZJLS001 strain obtained in Experimental Example 1 of this application. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0033] Example

[0034] Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0035] Example 1 Obtaining the Abies abies strain ZJLS001

[0036] The strain was isolated from wild Abies serrata fruiting bodies collected from Donggang Town, Fusong County, Changbaishan City, Jilin Province ( Figure 1 ), collection time: September 2024, strain isolation and domestication cultivation.

[0037] 1. Preparation and production of mother culture medium

[0038] (1) Commonly used mother culture medium

[0039] ① Potato glucose medium: 200g potatoes, 20g glucose, 20g agar, add water to make up to 1000mL;

[0040] ② Potato comprehensive culture medium: 200g potatoes, 20g glucose, 20g agar, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 10-20mg VB1, add water to make up to 1000mL;

[0041] (2) Preparation of mother culture medium

[0042] ① Process flow of mother culture medium production:

[0043] Material selection → accurate weighing (according to the formula) → material processing → quantitative preparation → filling in test tubes → sterilization → placing on the inclined surface.

[0044] ② Preparation of potato glucose medium

[0045] A. Boil potatoes: Peel, wash, and slice. Weigh 200g of potatoes and add 1000mL of water. Boil until the potatoes are crispy but not mushy. Filter through four layers of cheesecloth.

[0046] B. Add 20g glucose and 20g agar to the filtrate, add water to make up to 1L, and stir to dissolve;

[0047] C. Aliquot and sterilize. Dissolve and dispense into test tubes while still hot, typically filling 1 / 5 to 1 / 4 the length of the tube. After aliquoting, insert cotton plugs (5 in a pack). Wrap one end of the tube tightly with kraft paper. Place the wrapped tube in an autoclave at 121°C for 30 minutes.

[0048] D. Inclined placement: After sterilization, take out the test tube while it is still hot and place it on the workbench to form an inclined surface. The length of the inclined surface is usually 1 / 2 of the test tube. After solidification, place it in a clean and dry place for later use.

[0049] (3) Mother seed production

[0050] Cut a small piece of the fungus and inoculate it aseptically on the PDA test tube culture medium. Culture it in the dark at 25℃. It will cover the slant culture medium in 10 to 14 days. The mycelium will be white, dense, and have velvety aerial mycelium.

[0051] 2. Preparation of primary seed liquid culture medium and its strains

[0052] ①First-level seed liquid culture medium: 200g potatoes, 20g glucose, 3g yeast powder, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, 110mg VB, 1000mL water.

[0053] ②Preparation of primary seed liquid culture medium

[0054] A. Recipe: 12g sugar, 3g soy flour, 0.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 600mL water.

[0055] B. Aliquoting and Sterilization: Weigh and dissolve the solution according to the formula, then dispense into 1000mL Erlenmeyer flasks while still hot. After aliquoting, seal the flasks with parafilm, then wrap the film tightly with kraft paper. Place the wrapped flasks in an autoclave at 121°C for 30 minutes.

[0056] ③ Inoculation: Place the sterilized conical flask in the ultra-sterilization table, cool it, and sterilize it with ultraviolet light. Then, cut the mother culture test tube into pieces and place them into the conical flask;

[0057] ④ Oscillating culture: Place the inoculated conical flask in an oscillating shaker at 24°C and 160 rpm. Oscillating culture for about 8 days is ready for use.

[0058] 3. Mushroom bag production

[0059] (1) Preparation of cultivation medium

[0060] Mix the culture medium in the correct proportions, adjust the moisture content to 65-68%, and the pH to a natural value. Place the mixture into a 18cm*3cm*0.005cm heat-resistant polypropylene bag. The formula is: 35% corn cobs, 8% bran, 35% cottonseed hulls, 20% cornmeal, 1.3% calcium hydrogen, and 0.7% lime.

[0061] (2) High temperature and high pressure sterilization

[0062] Sterilize at 121°C, 0.15 MPa for 120 min.

[0063] (3) Cooling and inoculation

[0064] Place the sterilized cultivation bag in a cooling room to cool down, and move it to the inoculation room after it cools to room temperature; treat the inoculation room with ozone or ultraviolet light 2 to 3 hours in advance to achieve sterile conditions; inoculate the above-mentioned liquid bacteria onto the sterilized culture medium, with an inoculation volume of 20 to 25 mL per bag.

[0065] (4) Mycelium culture

[0066] Move the cultivation bottle with liquid fungus into the culture room, culture it at 18℃~20℃, dark conditions, ventilate occasionally, and culture it for 20~30 days. After the mycelium fills the cultivation bag, ripen it for 10 days and move it to the intelligent fruiting room for fruiting management.

[0067] 4. Mushroom production management

[0068] (1) Scratching fungi

[0069] Use a scratching machine (or manually) to remove old fungus blocks and fungus skins so that the fruiting bodies emerge neatly from the surface of the culture medium.

[0070] (2) Bud-inducing

[0071] Move the bags (bottles) after scratching to the fruiting room, and promote budding under the conditions of temperature 13-15℃, humidity 90-99%, CO2 concentration 300-3000ppm. Buds will begin to appear after 5-8 days.

[0072] (3) Development

[0073] When the mushroom buds grow 0.5cm to 1.0cm out of the bag mouth, adjust the temperature in the mushroom room to 16℃ to 21℃ and the humidity to 80% to 85% to make the stipes grow appropriately.

[0074] (4) Harvesting

[0075] When the fruiting body grows to 80% maturity, the cap is hemispherical, and spores have not yet been ejected, it is the right time to harvest. At this time, the mushroom stem is about 10 to 12 cm long and the cap is inward-turned.

[0076] After collecting wild Abies cylindrica fruiting bodies and drying them slightly in a well-ventilated area, tissue dissection was performed in a cleanroom. The dissociated tissue was transferred to slant tubes of PDA culture medium and incubated in the dark at 25°C for 14 days. The selection criteria for strains were: clustered fruiting bodies, with an average cap diameter of 3–8 cm, broadly convex, with involute margins in immature, a sticky surface, and mottled, white to yellowish-brown scales. Mature caps display concentric rings of yellowish-brown scales, with lemon-yellow between scales and at the cap margin. The flesh was thick and pale dark yellow, with straight to slightly curved gills, off-white to dark cinnamon in color, tapering toward the cap margin. The stipe was 4–10 cm long and 6–15 mm thick, moderately solid, of equal thickness to slightly swollen at the base, with a pale yellow surface. Above the ring, the stem had a powdery to smooth surface, while below the ring, it had dense, fibrillar scales. The spore print is cinnamon rust-colored; basidiospores are 6–7.5 × 3.5–4.5 μm, smooth, oblong on the front, rarely oval, and oval or nearly obtusely unequal on the sides, with a cinnamon-brown color. Basidiophores are 22–27 × 5–6.5 μm, rod-shaped, with four peduncles, and transparent to light yellow. The cysts are numerous, rod-shaped, spindle-shaped, and rarely irregular in shape. The hyphae are present in all parts of the fruiting body with lock-like unions. The selected strains were used to prepare liquid spawn for cultivation and fruiting experiments.

[0077] Liquid spawn herein refers to a spawn produced by rapidly growing and multiplying edible fungus mycelium in a container filled with liquid culture medium under specific culture conditions.

[0078] The liquid culture preparation method here is:

[0079] (1) Preparation of primary seed liquid culture medium and its strains

[0080] ① Primary seed liquid culture medium: 200g potatoes, 20g glucose, 3g yeast powder, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, 110mg VB1, 1000mL water.

[0081] ②Preparation of primary seed liquid culture medium

[0082] A. Recipe: 12g sugar, 3g cornstarch, 0.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 600mL water.

[0083] B. Aliquoting and Sterilization: Weigh and dissolve the solution according to the formula, then dispense into 500mL Erlenmeyer flasks while still hot. After aliquoting, seal the flasks with parafilm, then wrap the film tightly with kraft paper. Place the wrapped flasks in an autoclave at 121°C for 30 minutes.

[0084] ③ Inoculation: Place the sterilized conical flask in a clean bench, cool it, and sterilize it with ultraviolet light. Then, cut the mother test tube into pieces and place them into the conical flask.

[0085] ④ Oscillating culture: Place the inoculated conical flask in an oscillating shaker at 20°C and 180 rpm. Oscillating culture for about 7 days is ready for use.

[0086] The fruiting bodies of Psoralea abies that meet the above requirements are separated and cultivated again. The screening criteria for each time are the above-mentioned "Standards for Selecting Strain".

[0087] After repeating the cultivation experiment three times, the mycelium was dense, grew quickly, had a short cultivation cycle (mycelium could germinate 1 day after inoculation and could be harvested 50 days later), and had a high yield (290 to 350 g of fresh mushrooms were produced in two crops, and the biological efficiency could reach 82 to 87.5%). The strain ZJLS001 was named "Zhongjun Agaricus No. 1".

[0088] Example 2 Identification and preservation of strain ZJLS001

[0089] 1. Molecular identification method: The total DNA of the mycelium of “Zhongjun Abies cylindrica No. 1” was extracted by CTAB method and detected by 1% agarose gel electrophoresis; the extracted total DNA was used as template and Mix reagent (Hunan Qingke Biotechnology Co., Ltd.) was used to identify the mycelium with ITS4

[0090] (5'-TCCTCCGCTTATTGATATGC-3') and ITS5

[0091] The internal transcribed spacer (ITS) sequence was amplified using primers (5'-GGAAGTAAAAGTCGTAACAAGG-3'). The PCR amplification system (25 μL) consisted of 12.5 μL of 2× Mix, 0.5 μL of each 10 μM primer, 11 μL of ddH2O, and 0.5 μL of DNA template. The reaction procedure was as follows: 95°C pre-denaturation for 5 min, 35 amplification cycles: 95°C denaturation for 40 sec, 50°C annealing for 40 sec, 72°C extension for 1 min, then 72°C extension for 10 min, and storage at 4°C. After detection by 1% agarose gel electrophoresis, the amplified DNA was sent to Hunan Qingke Biotechnology Co., Ltd. for sequencing. The resulting ZJLS001-mycelium nucleotide sequence is SEQ ID NO. 1:

[0092]

[0093] 2. Development tree:

[0094] The amplified ITS fragments were compared in the NCBI database, and the sequences with high similarity were downloaded. The NJ (Neighbor-Joining) phylogenetic tree was constructed using MEGA5.1 software ( Figure 4 ) and confirmed that the "Zhongjun Abies abietis No. 1" is Abies abietis (Pholiota abietis).

[0095] Example 3 Factory cultivation method of Abies abies ZJLS001 strain

[0096] 1. Liquid culture preparation

[0097] 1. Preparation and production of mother culture medium

[0098] (1) Commonly used mother culture medium

[0099] ① Potato glucose medium: 200g potatoes, 20g glucose, 20g agar, add water to make up to 1000mL;

[0100] ② Potato comprehensive culture medium: 200g potatoes, 20g glucose, 20g agar, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 10-20mg VB1, add water to make up to 1000mL;

[0101] (2) Preparation of mother culture medium

[0102] ① Process flow of mother culture medium production:

[0103] Material selection → accurate weighing (according to the formula) → material processing → quantitative preparation → filling in test tubes → sterilization → placing on the inclined surface.

[0104] ② Preparation of potato glucose medium

[0105] A. Boil potatoes: Peel, wash, and slice. Weigh 200g of potatoes and add 1000mL of water. Boil until the potatoes are crispy but not mushy. Filter through four layers of cheesecloth.

[0106] B. Add 20g glucose and 20g agar to the filtrate, add water to make up to 1L, and stir to dissolve;

[0107] C. Aliquot and sterilize. Dissolve and dispense into test tubes while still hot, typically filling 1 / 5 to 1 / 4 the length of the tube. After aliquoting, insert cotton plugs (5 in a pack). Wrap one end of the tube tightly with kraft paper. Place the wrapped tube in an autoclave at 121°C for 30 minutes.

[0108] D. Inclined placement: After sterilization, take out the test tube while it is still hot and place it on the workbench to form an inclined surface. The length of the inclined surface is usually 1 / 2 of the test tube. After solidification, place it in a clean and dry place for later use.

[0109] (3) Mother seed production

[0110] Cut a small piece of the fungus and inoculate it aseptically on the PDA test tube culture medium. Culture it in the dark at 25℃. It will cover the slant culture medium in 10 to 14 days. The mycelium will be white, dense, and have velvety aerial mycelium.

[0111] 2. Preparation of PDA solid plate culture medium

[0112] a. Recipe: 200g potatoes, 20g glucose, 20g agar, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 10-20mg vitamin B1. Add water to 1000mL. Dispense the culture medium into shake flasks, filling them between 2 / 5 and 3 / 5 full. Seal the flasks with parafilm and wrap with kraft paper.

[0113] b. Sterilization and Subpackaging: Place the subpacked shake flasks in an autoclave and sterilize at 121°C for 30 minutes. After sterilization, move the shake flasks to a clean bench and irradiate with UV light for 15 minutes. Once the shake flasks have cooled to approximately 40°C, use 70mm plates for subpackaging.

[0114] c. Inoculation: After the culture medium has cooled, quickly transfer a 2-3 mm x 2-3 mm mother seed block to a PDA plate in a clean bench. Incubate in the dark at 25°C. After 10 to 14 days, the mycelium will grow completely over the entire plate, becoming dense, white, and characterized by fuzzy aerial hyphae.

[0115] 1. Preparation and production of mother culture medium

[0116] (1) Commonly used mother culture medium

[0117] ① Potato glucose medium: 200g potatoes, 20g glucose, 20g agar, add water to make up to 1000mL;

[0118] ② Potato comprehensive culture medium: 200g potatoes, 20g glucose, 20g agar, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 10-20mg VB1, add water to make up to 1000mL;

[0119] (2) Preparation of mother culture medium

[0120] ① Process flow of mother culture medium production:

[0121] Material selection → accurate weighing (according to the formula) → material processing → quantitative preparation → filling in test tubes → sterilization → placing on the inclined surface.

[0122] ② Preparation of potato glucose medium

[0123] A. Boil potatoes: Peel, wash, and slice. Weigh 200g of potatoes and add 1000mL of water. Boil until the potatoes are crispy but not mushy. Filter through four layers of cheesecloth.

[0124] B. Add 20g glucose and 20g agar to the filtrate, add water to make up to 1L, and stir to dissolve;

[0125] C. Aliquot and sterilize. Dissolve and dispense into test tubes while still hot, typically filling 1 / 5 to 1 / 4 the length of the tube. After aliquoting, insert cotton plugs (5 in a pack). Wrap one end of the tube tightly with kraft paper. Place the wrapped tube in an autoclave at 121°C for 30 minutes.

[0126] D. Inclined placement: After sterilization, take out the test tube while it is still hot and place it on the workbench to form an inclined surface. The length of the inclined surface is usually 1 / 2 of the test tube. After solidification, place it in a clean and dry place for later use.

[0127] (3) Mother seed production

[0128] Cut a small piece of the fungus and inoculate it aseptically on the PDA test tube culture medium. Culture it in the dark at 25℃. It will cover the slant culture medium in 10 to 14 days. The mycelium will be white, dense, and have velvety aerial mycelium.

[0129] 2. Expanding the mother culture

[0130] Inoculate the mother stock of Abies abies ZJLS001 onto a PDA solid plate. Place the plate in a constant temperature incubator and incubate in the dark at 25°C for 10 days. Expand the culture (mother stock propagation) by rapidly inoculating 7-8 pieces of mother stock, 2-3 mm x 2-3 mm, onto the inoculated medium within a clean bench. Typically, one tube of mother stock can be propagated into 20-30 tubes. After inoculation, label the tubes promptly to prevent contamination.

[0131] 3. Preparation of primary seed culture medium

[0132] Weigh 200 parts of potatoes, slice them, boil them for 20 minutes, filter them through 4 layers of gauze, and collect the filtrate in a conical flask; add 20 parts of glucose, 2 parts of corn flour, 3 parts of potassium dihydrogen phosphate, 2 parts of magnesium sulfate, 3 parts of yeast powder, 1 part of peptone, and 0.01 part of VB1 to the filtrate, and sterilize them at 121°C and 0.15 MPa for 30 minutes to obtain a primary seed liquid culture medium.

[0133] After cooling, use a 5 mm diameter circular puncher to punch holes in the aforementioned mother culture expansion medium to obtain circular mushroom blocks; the obtained mushroom blocks are inoculated into the primary seed culture solution, placed on a shaker at 25°C and 160 rpm, and cultured for 10 days to obtain the primary liquid fungus of Abies abies.

[0134] 4. Preparation of secondary fermentation broth

[0135] Weigh 300 parts of soybean flour, 300 parts of corn flour, 2000 parts of glucose, 120 parts of potassium dihydrogen phosphate, 100 parts of magnesium sulfate, 150 parts of yeast powder, and 11 parts of VB, add them into a 100L fermentation tank, and sterilize at 121°C and 0.15MPa for 40 min to obtain the secondary fermentation liquid.

[0136] The primary liquid strain was inoculated into a fermentation tank loaded with secondary fermentation liquid, and cultured with aeration at 25° C. and 0.05 MPa for 8 days to obtain the secondary fermentation liquid strain of Abies abies.

[0137] 2. Factory cultivation

[0138] 1. Cultivated seed preparation

[0139] The ingredients of the cultivation material are as follows: 50 parts by weight of sawdust, 18 parts by weight of wheat bran, 16 parts by weight of cottonseed hulls, 8 parts by weight of soybean flour, 1 part by weight of lime, and 1 part by weight of sucrose;

[0140] The raw materials of the cultivation materials were mixed evenly, the moisture content of the cultivation materials was 55%, the pH was natural, and the materials were placed in cultivation bags. Each bag was filled with 300g of materials (based on dry materials) and sterilized at 121°C and 0.15MPa for 2h. The specifications of the cultivation bags were 18×33×0.005cm.

[0141] 2. Cooling and inoculation

[0142] The sterilized cultivation bags were moved into the cooling room. After the cultivation bags cooled to room temperature, they were moved into the inoculation room. The above-mentioned Abies aegypti secondary fermentation liquid was inoculated onto the surface of the material. The inoculation volume per bag was 15 mL.

[0143] 3. Mycelium culture

[0144] Move the inoculated cultivation bag into the culture room and culture it in the dark at 25℃ with proper ventilation for 30 days. After the mycelium is fully grown, ripen it for 10 days and then remove it from the culture room after scratching.

[0145] 4. Induce budding

[0146] Under the conditions of temperature 12-16℃, humidity 80%, carbon dioxide concentration 1100ppm, and light intensity 400lx, the primordium will appear after 6-9 days of bud induction.

[0147] 5. Mushroom production management

[0148] After the primordium appeared, the cells were cultured at 16°C, 85% humidity, 600 ppm carbon dioxide concentration, and diffuse light.

[0149] 6. Harvesting

[0150] After 7-9 days of fruiting management, most of the fruiting bodies have grown to 70% maturity and the cap expansion has reached 50%. Figure 2 As shown, harvest the first batch of mushrooms. After harvesting the first batch of mushrooms, stop humidification, promptly remove the remaining mushroom roots, mushroom feet and dead mushrooms, and scratch the fungi.

[0151] Harvest again after 9-11 days, and manage the mushrooms in the same way as the first crop. The two crops together yield 290-350g of fresh mushrooms, with a biological efficiency of 82-87.5.

[0152] Using this cultivation method, the biological efficiency of "Zhongjun Abies 1" reached 86%, indicating that this strain has better biological efficiency and growth. The first crop of mushrooms can be harvested after 10 to 14 days of growth after scratching, and the interval between the first and second crops is relatively short.

[0153] Comparative Example 1

[0154] Commercially available strains marketed under the trade names of Pholiota adiposa or Tiger Tricholoma matsutake were used as control strains for identification using the same method used for ZJLS001. Total DNA from the mycelium of the "control strain" was extracted using the CTAB method and sent to Qingke Biotechnology Co., Ltd. for sequencing. The amplified ITS fragments were subjected to a homology search using BLAST (www.ncbi.nlm.nih.gov / BLAST) and similarity analysis with sequences from various strains in the database. The identities (identities) with the corresponding sequences of various strains of Pholiota adiposa in the database reached 98.83-100%. Sequencing results using the method described in Example 2 indicated that the commercially available strain was Pholiota adiposa. The control strains used in the following examples were all Pholiota adiposa. Because Pholiota adiposa and Pholiota abies are strains of the same genus but different species, this was used as the control strain for performance comparison.

[0155] The control bacteria were cultivated using the above method, and the control bacteria were prepared using the mother culture preparation method in Example 3.

[0156] The time interval between the first and second harvests of the control strain was 10 to 13 days.

[0157] Under this method, the bacterial strain grows faster between the first and second crops, which is beneficial to shortening the harvest time.

[0158] During the cultivation of Abies cylindrica ZJLS001, the number of mushroom bags that failed to yield due to contamination by foreign fungi was 0.

[0159] Example 3 Factory cultivation method of Abies serrata ZJLS001

[0160] 1. Liquid culture preparation

[0161] 1. Expanding the mother culture

[0162] The mother strain of Abies abies ZJLS001 was inoculated onto PDA solid plate medium and cultured in the dark at 25°C for 10 days to expand the culture.

[0163] 2. Preparation of primary seed culture medium

[0164] Weigh 200 parts of potatoes, slice them and boil them for 20 minutes, filter them through 4 layers of gauze, and collect the filtrate in a conical flask; add 20 parts of glucose, 2 parts of corn flour, 2 parts of soybean flour, 3 parts of potassium dihydrogen phosphate, 2 parts of magnesium sulfate, 3 parts of yeast powder, 1 part of peptone, and 0.01 part of VB1 to the filtrate, and sterilize them at 121°C and 0.15 MPa for 30 minutes to obtain a first-level seed culture medium.

[0165] After cooling, the mother culture expansion medium was punched with a 5 mm puncher, and the obtained fungus block was inoculated into the primary seed liquid culture medium, placed at 25° C., 160 rpm shaking table, and cultured for 5 to 7 days to obtain the first-level liquid fungus of Abies abies.

[0166] 3. Preparation of secondary fermentation broth

[0167] Weigh 670 parts of soybeans, add water, and grind using a 100-mesh grinder. Collect the filtrate. Crush the resulting residue again, collect and combine the filtrates, and pour the filtrates into a 100-L fermenter. Then, weigh 2000 parts of glucose, 120 parts of potassium dihydrogen phosphate, 100 parts of magnesium sulfate, 150 parts of yeast powder, and 11 parts of VB into the fermenter. Sterilize at 121°C and 0.15 MPa for 40 minutes to obtain the secondary fermentation broth.

[0168] The first-level liquid strain was inoculated into a fermentation tank loaded with the second-level fermentation liquid, and cultured with aeration at 25° C. and 0.05 MPa for 6 to 8 days to obtain the second-level fermentation liquid strain.

[0169] 2. Factory cultivation

[0170] 1. Cultivated seed preparation

[0171] The cultivation material is mixed and controlled to have a moisture content of 60% and a natural pH, and then placed in cultivation bags, 350 g of the material (based on dry matter) per bag, and sterilized at 121° C. and 0.15 MPa for 2 hours. The cultivation bags are 18×33×0.005 cm in size. The cultivation material is composed of 55 parts by weight of sawdust, 15 parts by weight of wheat bran, 18 parts by weight of cottonseed hulls, 10 parts by weight of soybean flour, 1 part by weight of lime, and 1 part by weight of sucrose.

[0172] 2. Cooling and inoculation

[0173] The sterilized cultivation bag was moved into the cooling room. After the cultivation bag was cooled to room temperature, it was moved into the inoculation room and the secondary fermentation liquid was inoculated to the material surface. The inoculation volume per bottle was 20 mL.

[0174] 3. Mycelium culture

[0175] Move the inoculated cultivation bag into the culture room and culture it in the dark at 22℃ with proper ventilation for 30 days. After the mycelium has grown all over the cultivation bag, after-ripen it for 10 days, scratch the fungus and move it out of the culture room.

[0176] 4. Induce budding

[0177] At a temperature of 15-22℃, humidity of 95%, carbon dioxide concentration of 1000ppm, and light intensity of 500lx, ventilation is used to increase the temperature difference to promote primordium differentiation, and rice-grain-shaped primordia can be formed in 5 days.

[0178] 5. Mushroom production management

[0179] Cultivate at a temperature of 18°C, a humidity of 95%, a carbon dioxide concentration of 700 ppm, and diffuse light conditions;

[0180] 6. Harvesting

[0181] After 7-9 days of fruiting, when most fruiting bodies have grown to 70% maturity and the caps have expanded to 50%, the first batch of mushrooms can be harvested. After harvesting the first batch of mushrooms, stop humidification, promptly remove the remaining mushroom roots, mushroom feet and dead mushrooms, and scratch the mushrooms.

[0182] Harvest again after 7-8 days, using the same management procedures as the first crop. The two crops yielded a total of 290g of fresh mushrooms, with a biological efficiency of 82%. Using this cultivation method, the biological efficiency of "Zhongjun Abies cylindrica No. 1" was 82%.

[0183] The first crop of "Zhongjun Fir Tree Umbrella No. 1" can be harvested 7-8 days after opening the bag. When cultivating a control strain using this method, the interval between the first and second crops is 9-11 days. During this stage, the fungus grows faster than the control strain.

[0184] During the cultivation of Abies cylindrica ZJLS001, the number of mushroom bags that failed to yield due to contamination by foreign fungi was 0.

[0185] Example 4 Factory cultivation of Abies abies ZJLS001

[0186] The difference from Example 2 is: 1. Preparation of liquid bacteria

[0187] 1. Expanding the mother culture

[0188] The mother strain of Abies abies ZJLS001 was inoculated onto PDA solid plate medium and cultured in the dark at 25°C for 10 days to expand the culture.

[0189] 2. Preparation of primary seed culture medium

[0190] Weigh 200 parts of potatoes, slice them and boil them for 20 minutes, filter them through 4 layers of gauze, and collect the filtrate in a conical flask; add 20 parts of glucose, 2 parts of corn flour, 3 parts of potassium dihydrogen phosphate, 2 parts of magnesium sulfate, 3 parts of yeast powder, 1 part of peptone, and 0.01 part of VB1 to the filtrate, and sterilize them at 121°C and 0.15 MPa for 30 minutes to obtain a first-level seed culture medium.

[0191] After cooling, the mother culture expansion medium was punched with a 5 mm puncher, and the obtained fungus block was inoculated into the primary seed culture solution, placed in a shaking table at 25° C. and 160 rpm, and cultured for 7 to 10 days to obtain the first-level liquid fungus of Abies abies.

[0192] 3. Preparation of secondary fermentation broth

[0193] Weigh 650 parts of soybeans, add water, and grind using a 100-mesh grinder. Collect the filtrate. Crush the resulting residue again, collect and combine the filtrates, and pour the filtrates into a 100-L fermenter. Then, weigh 2000 parts of glucose, 120 parts of potassium dihydrogen phosphate, 100 parts of magnesium sulfate, 150 parts of yeast powder, and 11 parts of VB into the fermenter. Sterilize at 121°C and 0.15 MPa for 40 minutes to obtain the secondary fermentation broth.

[0194] The primary liquid strain was inoculated into a fermentation tank culture medium loaded with secondary fermentation liquid, and cultured at 25° C. and 0.05 MPa for 7 days to obtain the secondary fermentation liquid strain of Abies abies.

[0195] 2. Factory cultivation

[0196] 1. Cultivated seed preparation

[0197] The cultivation material is composed of 40 parts by weight of sawdust, 22 parts by weight of wheat bran, 20 parts by weight of cottonseed hulls, 8 parts by weight of corn flour, 8 parts by weight of corn cobs, 1 part by weight of lime, and 1 part by weight of sucrose;

[0198] The culture material was mixed and the water content was controlled to 55% and the pH was natural. The culture material was placed in 1100 mL culture bags, with 400 g of material per bag (based on dry material). The culture bags were sterilized at 121°C and 0.15 MPa for 2 h. The specification of the culture bags was 18 × 33 × 0.005 cm.

[0199] 2. Cooling and inoculation

[0200] The sterilized cultivation bottles were moved into the cooling room. After the cultivation bags cooled to room temperature, they were moved into the inoculation room and the secondary fermentation liquid was inoculated onto the surface of the material. The inoculation volume per bottle was 21 mL.

[0201] 3. Mycelium culture

[0202] Move the inoculated cultivation bag into the culture room and culture it in the dark at 24℃ with proper ventilation for 30 days. After the mycelium has grown all over the cultivation bag, after-ripen it for 10 days, scratch the fungus, and then move it out to the culture room.

[0203] 4. Induce budding

[0204] At a temperature of 14-22℃, humidity of 95%, carbon dioxide concentration of 1000ppm, and light intensity of 500lx, ventilation is used to increase the temperature difference to promote primordium differentiation, and rice-grain-shaped primordia can be formed in 5 days.

[0205] 5. Mushroom production management

[0206] After the primordium appeared, the cells were cultured at a temperature of 18°C, a humidity of 95%, a carbon dioxide concentration of 700 ppm, and diffuse light.

[0207] 6. Harvesting

[0208] After 4-6 days, most of the fruiting bodies have grown to 70% maturity and the cap has expanded to 50%, then the first batch of mushrooms can be harvested. After the first batch of mushrooms are harvested, stop humidification, remove the remaining mushroom roots, mushroom feet and dead mushrooms in time, and scratch the mushrooms.

[0209] After 5-7 days of fruiting, the mushrooms were harvested again, following the same management procedures as the first crop. The two crops together produced 350g of fresh mushrooms, with a biological efficiency of 87.5%.

[0210] Using this cultivation method, the biological efficiency of "Zhongjun Abies No. 1" is 87.5%. The first harvest can be harvested 5 to 7 days after the bag is opened.

[0211] The control strain was cultivated using the above operation, and the time interval between the first and second crops of the control strain was 9 to 11 days. During this stage, the growth rate of Abies abies ZJLS001 was faster than that of the control strain.

[0212] During the cultivation of Abies cylindrica ZJLS001, the number of mushroom bags that failed to yield due to contamination by foreign fungi was 0.

[0213] Example 5 Antagonism experiment between Abies abies ZJLS001 and control strains

[0214] The mother culture block of the Abies serrata ZJLS001 strain obtained in Example 2 and the original culture block of the control strain were inoculated into the same 90 mm PDA solid culture medium (the same as in Example 2) using a puncher. The inoculation spacing between the two blocks on the culture medium was 1 to 2 cm. They were dark cultured at 25°C for 10 days and taken out as the antagonism test results.

[0215] The results are as follows Figure 3 As shown in b, at the junction of the opposing culture colonies, the antagonistic line of the two strains is obvious, and the colony morphology of the two strains is completely different, indicating that Abies fasciata ZJLS001 and the control strain are not the same species.

[0216] Example 6 Edibility Test of Abies abies ZJLS001

[0217] The fruiting bodies of Abies abies ZJLS001 cultivated in Example 2 were selected, washed, and steamed for 40 minutes, seasoned, and distributed to 10 tasters for tasting, and their reactions were recorded. Each taster tried to consume 100 g of Abies abies.

[0218] After tasting, the testers reported no discomfort. The fruiting body exudes a rich mushroom aroma and has a crisp, tender, and refreshing texture. The stem is crisp, while the cap is smooth and delicate, making it less likely to get stuck in your teeth. Its high hardness and crispness require minimal energy to chew.

[0219] Since most of the genus Agaricus are medicinal or edible fungi, Agaricus fir ZJLS001 belongs to this genus, and the above experiments show that this strain meets the edible requirements.

[0220] Example 7

[0221] The difference from Example 2 is that the first-level seed culture liquid was inoculated with the mother strain of Abies serrata ZJLS001 and then cultured at 28°C for 7 days to obtain the first-level liquid strain of Abies serrata. The second-level fermentation culture was carried out at 28°C and 0.07 MPa with aeration for 6 days to obtain the second-level fermentation liquid strain of Abies serrata.

[0222] The culture medium has a water content of 60%; the culture medium ingredients are: 60 parts by weight of sawdust, 15 parts by weight of wheat bran, 10 parts by weight of cottonseed hulls, 10 parts by weight of soybean flour, 3 parts by weight of lime, and 2 parts by weight of sucrose; the inoculum volume of each bottle of fermentation liquid in the culture medium is 25 mL;

[0223] The mycelial culture conditions were dark culture at 20°C with adequate ventilation for 13 days.

[0224] The carbon dioxide concentration during mushroom production management is 800ppm.

[0225] Example 8

[0226] The difference from Example 3 is: Fruiting management: when the stem grows to 3 cm, open the bag and culture at a temperature of 20° C., a humidity of 95%, a carbon dioxide concentration of 500 ppm, and scattered light conditions.

[0227] Example 9

[0228] The difference from Example 4 is that the cultivation material is composed of 45 parts by weight of sawdust, 25 parts by weight of wheat bran, 15 parts by weight of cottonseed hulls, 13 parts by weight of corn cobs, 1 part by weight of lime, and 1 part by weight of sucrose.

[0229] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fir tree parasol ZJLS001, characterized in that The classification and nomenclature of Pholiota abietis is CGMCC NO.41797; During the cultivation process, the proportion of cultivation bags of Abies serrata ZJLS001 that did not germinate due to contamination by other bacteria was 0; 5 to 9 days after scratching, light yellow, rice-grain-shaped primordia were formed, with an average number of buds of 28 and an average diameter of 1 to 2 mm. Two crops of Abies polyphylla ZJLS001 produced 290-350g of fresh mushrooms, with a biological efficiency of 82-87.5%.

2. A cultivation method of Abies polyphylla ZJLS001 according to claim 1, characterized in that: The following steps are involved: The mother seed of Abies serrata ZJLS001 was expanded and inoculated into the primary seed culture solution, and primary seed culture and secondary fermentation culture were performed to obtain the secondary fermentation solution of Abies serrata. The secondary fermentation liquid of Abies aegypti was inoculated on the sterilized cultivation material, and Abies aegypti ZJLS001 was obtained through mycelium culture, bud induction, mushroom fruiting management and harvesting.

3. The cultivation method of Abies polyphylla ZJLS001 according to claim 1, characterized in that: The first-level seed liquid culture medium used for first-level seed culture is prepared as follows: potato slices are boiled, filtered, and the filtrate is collected; glucose, corn starch, potassium dihydrogen phosphate, magnesium sulfate, yeast powder, peptone, and VB are added to the filtrate, and the mixture is sterilized to obtain the first-level seed liquid culture medium; The secondary fermentation liquid used in the secondary fermentation culture includes: soybean powder, corn powder, glucose, potassium dihydrogen phosphate, magnesium sulfate, yeast powder, VB; Preferably, the secondary fermentation broth used in the secondary fermentation culture is prepared by: grinding soybeans with water, collecting the filtrate, crushing the filter residue again, collecting and combining the filtrates; taking glucose, potassium dihydrogen phosphate, magnesium sulfate, yeast powder, VB and the filtrate and sterilizing them to obtain the secondary fermentation broth.

4. The cultivation method of Abies polyphylla ZJLS001 according to claim 3, characterized in that: The first-level seed culture is to inoculate the mother strain of Abies serrata ZJLS001 into the first-level seed liquid culture medium, and then culture in a shake flask at 25-28°C for 5-10 days to obtain the first-level liquid strain; The secondary fermentation culture is as follows: the primary liquid strain is inoculated into the secondary fermentation liquid, and cultured under ventilation conditions of 25-28° C. and 0.05-0.07 MPa for 6-8 days to obtain the secondary fermentation liquid strain of Abies abies.

5. The cultivation method of Abies polyphylla ZJLS001 according to claim 2, characterized in that: The cultivation material is composed of 40-55 parts by weight of sawdust, 15-22 parts by weight of wheat bran, 16-20 parts by weight of cottonseed hulls, 8 parts by weight of corn flour, 8 parts by weight of corn cobs, 1 part by weight of lime and 1 part by weight of sucrose.

6. The cultivation method of Abies polyphylla ZJLS001 according to claim 2, characterized in that: Inoculation amount: inoculate at least 15 mL of Abies abies secondary fermentation liquid per 300 g of culture medium; Preferably, the inoculation amount is: 15 to 21 mL of the secondary fermentation liquid of Abies abies per 300 g of the culture medium.

7. The cultivation method of Abies polyphylla ZJLS001 according to claim 2, characterized in that: The mycelium culture is to inoculate the cultivation material with the secondary fermentation liquid of Abies aegyptifolia in the dark at 22-25°C, culture it with ventilation for 30 days, after-ripen it for 10 days, and induce budding after scratching the fungus.

8. The cultivation method of Abies polyphylla ZJLS001 according to claim 1, characterized in that: The bud-inducing operation is as follows: bud-inducing is carried out in an environment of 12-22℃, humidity 80-95%, carbon dioxide concentration 1000-1100ppm, and light intensity 400-500lx. Ventilation increases the temperature difference to promote primordium differentiation. After rice-grain-shaped primordium is formed in 5-9 days, mushroom fruiting management can be carried out.

9. The cultivation method of Abies polyphylla ZJLS001 according to claim 8, characterized in that: The mushroom management is to cultivate the mushrooms at a temperature of 18°C, a humidity of 95%, a carbon dioxide concentration of 700 ppm, and scattered light conditions after the primordium appears, and harvest them when most of the fruiting bodies are 70% mature and the cap expansion reaches 50%.

10. The cultivation method of Abies polyphylla ZJLS001 according to claim 2, characterized in that: After harvesting the first batch of mushrooms, stop humidification, remove the remaining mushroom roots, mushroom feet and dead mushrooms, scratch the fungi, and repeat the mushroom fruiting management for 5 to 8 days before harvesting the second batch of mushrooms.

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