Butterfly roll edge mushroom ZJXM002 strain and application thereof

Abstract

This application discloses a *Pleurotus ostreatus* strain ZJXM002 and its applications, relating to the field of microbial cultivation technology. The *Pleurotus ostreatus* strain ZJXM002 has the accession number CGMCC No. 41786; its classification is named... From Lep cremeoinvoluta This strain is suitable for artificial cultivation and can produce fruiting bodies of the Creamy Curly Mushroom ZJXM002 strain with high biological efficiency.

Description

Technical Field

[0001] This application relates to the field of microbial cultivation technology, and in particular to a *Mushroom arborescens* strain ZJXM002 and its application. Background Technology

[0002] Creamy Curled Mushrooms ( Lepista cremeoinvoluta YD Xu, Zhu L. Yang & Z.M. He), belonging to the phylum Basidiomycota, class Agaricomycetes, order Agaricales, family Ciliaceae. Clitocybaceae genus *Mushroom* Lepista This genus of fungi is mainly distributed in temperate regions and has both edible and medicinal value. Creamy-edged shiitake mushroom ( Lepista cremeoinvoluta This is a new species and a newly recorded species from Southwestern China, discovered by Xu Yongdi et al. in Kunming, Yunnan in 2024 (Xu YD, Jiang L, Yang ZL, et al. Lepista cremeoinvoluta and L. multiformis (Clitocybaceae, Agaricales)[J].Mycological Progress, 2024, 23(1).DOI:10.1007 / s11557-024-01986-w.). Since no poisoning reports have been found for this species, this strain is edible. However, there are currently no reports of its cultivation.

[0003] There is limited research on *Shiitake mushroom with butter curled edges*, and no methods for its cultivation have been found. Therefore, research on the selection and breeding of new strains of *Shiitake mushroom with butter curled edges* and its soil-covered cultivation techniques is of great significance.

[0004] The information disclosed in the background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] This application addresses the aforementioned technical problems by providing a *Pleurotus ostreatus* strain ZJXM002 and its application. This strain is suitable for artificial cultivation and can produce fruiting bodies of *Pleurotus ostreatus* strain ZJXM002 through artificial cultivation, with high biological efficiency.

[0006] This application provides a strain of *Pleurotus ostreatus* ZJXM002, with the preservation number CGMCC No. 41786; classified as... Lepista cremeoinvoluta .

[0007] Preferably, no mice fed with the ZJXM002 strain of *Pleurotus ostreatus* in acute oral toxicity tests died; The biological efficiency of the first crop of fruiting bodies of the artificially cultivated mushroom strain ZJXM002 was 45.6-56.3%.

[0008] Another aspect of this application provides the application of the above-mentioned *Pleurotus ostreatus* strain ZJXM002, which is suitable for artificial cultivation.

[0009] Preferably, the artificial cultivation method includes the following steps: 1) Inoculate the mother culture of the cream-edged mushroom strain ZJXM002 into the seed culture medium and place it in a constant temperature and light-protected shaking incubator at 24~26℃ with a rotation speed of 140~155rpm for 15~17 days to obtain the seed culture of strain ZJXM002. 2) After inoculating the seed culture of strain ZJXM002 into the fermentation broth, incubate at a constant temperature of 24~26℃ for 9~11 days to obtain the liquid culture strain; 3) After inoculating the liquid culture spawn onto the cultivation substrate and bagging it, incubate it at 21-23℃ in the dark until the mycelium fills the bag to obtain the culture spawn log; 4) Prepare the seedbed, arrange the spawn logs neatly on the seedbed and cover them with the original soil. Spray water according to the moisture level of the soil covering, and cover with pine needles or straw to keep it moist. Manage the mushrooms until harvest.

[0010] Preferably, the seed culture medium consists of: 17-19 g / L glucose, 5-7 g / L potato flour, 0.5-1.5 g / L potassium dihydrogen phosphate, 1-2 g / L magnesium sulfate, and 5-7 g / L yeast extract. The fermentation broth consists of 7-9 g / L soybean flour, 17-19 g / L glucose, 0.5-1.5 g / L potassium dihydrogen phosphate, 1-2 g / L magnesium sulfate, and 5-7 g / L yeast powder, with a natural pH. The cultivation substrate consists of 70-74% fermented hardwood sawdust, 18-20% wheat bran, 6-8% corn flour, 1% gypsum powder, and 1% white sugar. The moisture content of the cultivation substrate is adjusted to 60-62%.

[0011] Preferably, the inoculation amount of the mother culture in the seed culture medium is at least 5 pieces; The inoculum of strain ZJXM002 seed culture should be at least 8% of the mass fraction of the fermentation medium. The inoculation amount of liquid culture strain in the cultivation medium is: at least 20 mL in each bag of cultivation medium, and each cultivation bag is filled with 380~450 g of dry cultivation medium.

[0012] Preferably, the preparation of the seedbed involves spreading 25-30 kg of quicklime per mu, turning the soil, rotary tilling, and sun-drying for 3-5 days. Then, the seedbeds are prepared for use. The seedbeds are 80-85 cm wide, and the furrows between the seedbeds are 25-30 cm wide and 10-15 cm deep.

[0013] Preferably, the water spraying operation is as follows: if the soil to be covered is moist, no watering is required after covering; if the soil to be covered is dry, water is sprayed to retain moisture after covering.

[0014] Preferably, mushroom cultivation management includes: During the fruiting period, maintain the temperature inside the greenhouse between 22 and 26℃, keep the moisture content of the cultivation substrate and soil between 55 and 85%, and the relative humidity between 75 and 85%. During the fruiting body growth period, ensure sufficient diffused light and avoid direct sunlight. After the mycelium in the seedbed begins to twist and crawl out of the soil surface, use drip irrigation or sprinkler irrigation to thoroughly water the soil at least once to stimulate primordia formation. After the primordia appear, keep the soil moist and harvest after 8-10 days.

[0015] Another aspect of this application provides an application of the above-mentioned *Mushroom Flos ...

[0016] The beneficial effects that this application can produce include: 1) The butter-curling mushroom strain ZJXM002 provided in this application and its application: This application successfully obtained a butter-curling mushroom strain ZJXM002 by tissue isolation method, and conducted molecular identification to determine its classification and nomenclature.

[0017] 2) The *Pleurotus ostreatus* strain ZJXM002 provided in this application and its application. This strain can be artificially cultivated to obtain fruiting bodies using the method provided in this application. This strain is an edible fungus variety, which helps to enrich market supply and meet consumer demand. Related toxicological experiments were conducted on the fruiting bodies of *Pleurotus ostreatus* ZJXM002, and the results showed that the fruiting bodies of this strain are non-toxic and meet the safety standards for edible fungi.

[0018] 3) The Creamy-edged Mushroom ZJXM002 strain provided in this application and its application: When artificially cultivated using the method provided in this application, the resulting fruiting bodies have a large yield, strong resistance to diseases and pests, and fast growth rate. The biological efficiency of one crop is 45.6-56.3%, which has economic value.

[0019] Preservation Information: The ZJXM002 strain of *Millettia speciosa*, deposited on February 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Postcode: 100101; Preservation Number: CGMCC No. 41786; Classified as *Millettia speciosa*. Lepista cremeoinvoluta . Attached Figure Description

[0020] Figure 1 Photographs of wild buttercup mushroom fruiting bodies collected in the field environment in Example 1 of this application; Figure 2 The image shows a slant mother culture of *Mushroom shiitake* ZJXM002 obtained in Example 1 of this application, with multiple test tubes of the slant mother culture of *Mushroom shiitake* ZJXM002 shown in the image. Figure 3 The phylogenetic tree of *Pleurotus ostreatus* ZJXM002 constructed based on the ITS+nLSU rDNA sequence in Example 2 provided in this application; Figure 4 The images provided in Example 3 of this application show the liquid culture strain of *Mushroom floribunda* ZJXM002, a, which is the liquid culture fermentation broth; and b, a magnified view of a portion of the fermentation broth. Figure 5 A photograph of a cultivation bag containing *Mushroom floribunda* ZJXM002 mycelium grown on the substrate obtained in Example 3 of this application; the substrate surface inside the cultivation bag is covered with white mycelium. Figure 6 The images provided in Example 3 of this application show the mycelial growth stages of the *Mushroom Flos 'Cream-Rolling' ZJXM002 strain after soil covering cultivation. Image a shows mycelium climbing the soil; image b shows primordia that have already grown; images c-h show cultivation stages from primordia growth to harvest; and images g-h show cultivation stages at harvest. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0022] Unless otherwise specified, all materials and instruments used in the following embodiments were obtained through commercial channels; and all detection methods used are existing methods unless otherwise specified.

[0023] Example 1: Obtaining the ZJXM002 strain of *Mushroom floribunda*. (1) In September 2023 in Dali Bai Autonomous Prefecture, Yunnan Province, such as Figure 1Fruiting bodies of the *Pleurotus ostreatus* strain ZJXM002 were collected under the conditions shown and placed in a clean bench. (2) Using a sterile scalpel, cut the connection between the stipe and cap of the fruiting body into several small pieces (0.5cm*0.5cm). Pick one piece and place it in a test tube slant for culture. Make 5 replicates. Incubate the test tube slant at 25℃ in the dark for 6 days. Then pick an uncontaminated piece (0.5cm*0.5cm) and place it in a test tube slant. Incubate the test tube slant at 25℃ in the dark for 6 days. After 3 purifications, a pure culture of the strain was obtained. The culture medium used for the test tube slant was sterilized modified PDA medium. The modified PDA medium consisted of 200 g / L potato, 20 g / L glucose, 20 g / L agar, 1 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, and 6 g yeast extract, with the pH set naturally.

[0024] (3) The obtained pure culture was inoculated into slant test tubes containing modified PDA medium and cultured at 25°C in the dark for 12 days to obtain the following results. Figure 2 The slant culture of the butter-curled mushroom ZJXM002 shown was used to obtain strain "ZJXM002".

[0025] Example 2 Molecular identification of the *Pleurotus ostreatus* strain ZJXM002. The following steps were followed to obtain the ITS sequences of the mycelium and fruiting body of *Mushroom with Curly Edges* ZJXM002, and then to identify and preserve the strain: (1) DNA extraction The ZJXM002 strain obtained in Example 1 was transferred to a modified PDA medium and cultured at 25°C for 14 days before mycelia were collected. Genomic DNA was extracted from the mycelia and fruiting bodies using the TSP101-200 kit, and the purity and concentration of the obtained DNA were determined by agarose gel electrophoresis and biospectrophotometry.

[0026] (2) ITS amplification and detection PCR amplification was performed using ITS (ITS4, ITS5) and nrLSU (LROR, LR5) as primers. The 25 μL amplification system consisted of: 20 μL of Gold Mix (green, TSE101), 0.5 μmol / L forward and reverse primers, and 50 ng of genomic template DNA. The PCR program was: 94℃ for 2 minutes; 94℃ for 15 seconds, 60℃ for 30 seconds, 72℃ for 60 seconds, 30 cycles; 72℃ for 10 minutes. After PCR, 5 μL of 6× loading buffer was added and mixed thoroughly, followed by 1.0% agarose gel electrophoresis for detection. After passing the detection, sequencing was performed to obtain the ITS and nrLSU sequences.

[0027] (3) Sequence comparison and phylogenetic analysis The obtained ITS and nrLSU sequences were compared for homology in the NCBI database using BLAST software, and the sequence showed over 99% identity with the corresponding sequences of various *Mammillaria rubra* strains already existing in the database. The obtained *Mammillaria rubra* strain ZJXM002 was deposited on February 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 41786.

[0028] The ITS sequence of strain ZJXM002 is shown in SEQ NO.1: cctgcggaag gatcattatt gaataaactt ggtcgggttg ctgctggctc cttggagcat 60 gtgcacacct gccaccattt ttaccacctg tgcactcttt gtagatctgg aattatctct 120 cgaggcaact cggtctgagg gatgcttcgc gcaagcttag cttctcttgc attccaggtc 180 tatgtttaat tatatacccc atatcatagt caagaatgtc attaatgggc cttgtgcctt 240 taaattaata caactttcaa caacggatct cttggttctc gcatcgatga agaacgcagc 300 gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat catcgaatct ttgaacgcac 360 cttgcgctcc ttggtattcc gaggagcatg cctgtttgag tgtcattaaa ttctcaacct 420 tttcagcttg tacaagttga atttggcttg gatgtggggg ctgcgggctt ctcagaagtc 480 ggctcctctt aaatgcatta gcggaacctt tgtggaccag ctctggtgtg ataattatct 540 acgccactgt tgtgaagcag cttacatgg ggttcagctt ctaaccgtcc attgacttgg 600 acaaatttg acattttgac ctcaaatcag gtaggattac ccgctgaact taagcatatc 660 665 years ZJXM002 Register No.LSU Registered SEQ NO.2 Registered: gagaaagaac tacaaggatt cccctagtac ctgcgagtga agcgggaaaa gctcaaattt 60 taaatctggc agtctttggc tgtccgagtt gtaatctaga gaagtgttat ccgcgctgga 120 ccgtgtacaa gtctcctgga acggagcgtc atagagggtg agaatcccgt ctttgacacg 180 gactaccagg gctttgtgat gcgctctcaa agagtcgagt tgtttgggaa tgcagctcta 240 aatgggtggt aaattccatc taaagctaaa tattggcgag agaccgatag cgaacaagta 300 ccgtgaggga aagatgaaaa gaactttgga aagagagtta aacagtacgt gaaattgttg 360 aaagggaaac gcttgaagtc agtcgcgttg gctggggatc aaccttgctc ttttgcttgg 420 tgtacttccc agttgatggg tcagcatcaa ttttgaccgg tggataaagg ccaagggaat 480 gtggcatctt cggatgtgtt atagcccatg gttgcataca tcggttggga ttgaggaact 540 cagcacgccg caaggccggg tctttgacca cgtacgtgct taggatgctg gcataatggc 600 tttaatcgac ccgtcttgaa acacggacca aggagtctaa catgcccgcg agtgtttggg 660 tggaaaaccc ggacgcgtaa tgaaagtgaa agttgagatc cctgtcgtgg ggagcatcga 720 cgcccggacc agaccttttg tgacggttcc gcggtggagc gtgtatgttg ggacccgaaa 780 gatggtgaac tatgcctgaa tagggtgaag ccagaggaaa ctctggtgga ggctcgtagc 840 gattctgacg tgcaaatcga tcgtcgaatt t 871 At the same time, download the genus *Mushroom* (*Mushroom*) from the database. Lepista ), genus *Vessel-cup* ( Singerocybe ) and Umbelliferae ( Clitocybe ITS and nrLSU sequences of the species, phylogenetic tree based on ITS-nrLSU sequences ( Figure 3 The results showed that strain ZJXM002 clustered with other samples of *Mammillaria rubra* with a branching support rate of 100%. Therefore, strain ZJXM002 was identified as *Mammillaria rubra*. Lepista cremeoinvoluta ) strain.

[0029] Example 3: Artificial cultivation of the butter-curling mushroom strain ZJXM002 1) Weigh 18g glucose, 6g potato flour, 1g potassium dihydrogen phosphate, 1.5g magnesium sulfate, and 6g yeast powder, then dilute with water to 1 liter to obtain seed culture medium. Fill each bottle with the liquid to 2 / 3 of the container volume. Sterilize the liquid culture medium at 121℃ for 30 minutes, then cool and set aside. Inoculate 5 circular pieces (R=0.5cm) of the mother culture mycelium of strain ZJXM002 into the seed culture medium and incubate at 25℃ in a constant temperature, dark, shaking incubator at 150 rpm for 16 days to obtain the seed culture of strain ZJXM002. Figure 4 In Figures a and b, a large amount of mycelium can be seen at the bottom, indicating that this method can produce a seed liquid with a large number of mycelial clusters, which is beneficial for subsequent fermentation and cultivation. 2) Prepare a fermentation broth using 8 g / L soybean flour, 18 g / L glucose, 1 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, 6 g / L yeast powder, and natural pH. Add the fermentation broth to a fermenter and sterilize at 121℃ for 60 min. Slowly cool the broth while maintaining the pressure inside the broth at no less than 0.05 MPa. After the liquid has cooled, inoculate the seed culture into the fermentation broth at 8% of the fermentation medium mass. Incubate at a constant temperature of 25℃. During the incubation process, maintain the pressure inside the broth at 0.04 MPa and the aeration rate at 5 L / min. Incubate for 10 days to obtain the liquid culture strain ZJXM002 of *Millettia speciosa*.

[0030] 3) According to the mass percentage, mix 70% fermented hardwood sawdust, 20% wheat bran, 8% corn flour, 1% gypsum powder, and 1% white sugar to obtain the cultivation substrate; adjust the moisture content of the obtained cultivation substrate to 60%. Fill cultivation bags with the cultivation substrate, seal them, and sterilize at 121℃ for 2.5 hours. After cooling to below 25℃, use an inoculation gun to take 20mL of liquid culture of *Millettia speciosa* strain ZJXM002 and inoculate it into cultivation bags (18 cm × 33 cm × 0.005 cm thickness) containing the cultivation substrate. Each bag should contain 380-450g of dry cultivation substrate. Incubate at 22℃ in the dark for 55 days until the mycelium fully fills the bag. Figure 5 As shown, the preparation of the cultivar is now complete.

[0031] After fermentation, the sawdust is adjusted to a moisture content of 60%–65% by adding water, ensuring the material is uniform and free of dry lumps. It is then piled into a long heap 1.5–2 meters wide at the base and 1–1.5 meters high, with ventilation holes to ensure oxygen supply. The core of fermentation management is "temperature monitoring and turning": the core temperature of the heap must be rapidly raised to a suitable range of 60–70°C and maintained for 24–48 hours to kill miscellaneous bacteria and insect eggs and initiate the degradation of lignocellulose. Afterward, the heap is turned for the first time to achieve material exchange, oxygen replenishment, and heat dissipation. Subsequently, depending on the temperature recovery, the heap is usually turned 2–3 more times, with the entire fermentation cycle lasting 15–25 days.

[0032] 4) Creamy-edged shiitake mushrooms are a medium-high temperature variety. They should be sown and cultivated in mid-April when the temperature inside the greenhouse is maintained at 20-25℃. 5) Before sowing, spread 30 kg of quicklime per mu, turn the soil, rotary till, and expose it to the sun for 5 days. Then, make beds for use. The beds should be 85 cm wide, and the furrows between the beds should be 30 cm wide and 10 cm deep.

[0033] 6) Cultivation using mushroom bags with soil covering: Cut open the outer bag of the cultivation bag filled with mycelium, remove the spawn, and arrange them neatly on the prepared beds in step 6). Cover with the original soil, generally 1cm ± 0.5cm thick. If the covering soil is moist, do not water after covering; if the covering soil is dry, spray water to retain moisture after covering, and finally cover with pine needles, straw, etc. to retain moisture.

[0034] 7) Mushroom Management During the fruiting period, maintain the temperature inside the greenhouse between 22~26℃, keep the moisture content of the cultivation substrate and soil between 55~60%, and the relative humidity of the air at 80%. During the fruiting body growth period, ensure sufficient diffused light and avoid direct sunlight. Wait until the mycelium in the seedbed begins to twist and crawl out of the soil surface (e.g., ...). Figure 6 After step a), thoroughly water the mushroom once using drip irrigation or sprinkler irrigation to stimulate primordia formation. Simultaneously, carefully observe the growth of the *Mammillaria rubra*, promptly identifying and treating any diseased or pest-infested strains. Primordia will appear 2-3 days after covering with soil. Keep the soil moist; harvesting can begin 10 days later. Begin daily manual inspections after primordia stimulation, taking photos when significant changes are observed. The resulting photos should be as follows: Figure 6 As shown in b~h, Figure 6 The photos in g~h show the cultivation site at the time of harvest, indicating that this strain is suitable for artificial cultivation and fruiting bodies can be obtained.

[0035] Post-harvest statistics showed that the biological efficiency of strain ZJXM002 was 49.6%. Mycelial growth rate was 2.9–3.2 mm / d, with an average of 3.05 mm / d; the time to fully colonize the bag was 45–65 days, with an average of 55 days; the bag contamination rate was 2–3%; fresh mushroom yield (g / bag): 452–498 g / bag; the mycelial growth was vigorous and robust, but the growth rate was moderate. The weight of a single fruiting body ranged from 13.19 to 26.57 g.

[0036] This indicates that the strain grows well under artificial cultivation, with high yield, fast growth rate, strong resistance, and large single fruiting body weight.

[0037] Example 4 The difference from Example 3 is as follows: The cultivation substrate consists of 74% fermented hardwood sawdust, 18% wheat bran, 6% corn flour, 1% gypsum powder, and 1% white sugar, with a moisture content of 62%. The inoculation amount of the spawn is 25 mL / bag. The surface width of the seed coat is 80 cm, and the furrows between the seed coats are 25 cm wide and 15 cm deep.

[0038] Mushroom management: Increase air humidity to 85%. Once the mycelium in the seedbed begins to twist and emerge from the soil surface, thoroughly water twice using drip irrigation or sprinkler irrigation to stimulate primordium formation. Creamy-edged mushroom primordia will appear 20 days after covering with soil. Keep the soil moist, and harvesting can begin after 8 days.

[0039] According to statistics, the biological efficiency of ZJXM002 in one crop was 52%.

[0040] Example 5 Acute oral toxicity test of the fruiting body of *Mushroom with Creamy Edges* ZJXM002 Twenty healthy mice (half male and half female, weighing 18-22g) were selected for the experiment. The fruiting bodies of *Pleurotus ostreatus* ZJXM002 were dried and thoroughly ground to prepare the test sample. 12.5360g of the test sample was weighed and placed in a beaker. A small amount of sterile water for injection was added to mix the sample, and the mixture was poured into a 50mL volumetric flask. The beaker was rinsed several times with a small amount of sterile water for injection, and the rinse water was added to the volumetric flask. Sterile water for injection was added to bring the volume to the mark to obtain the gavage-treated sample. The gavage-treated sample was thoroughly shaken, placed into sample tubes, and labeled for use. Samples were prepared and used immediately.

[0041] Animals were acclimatized in a barrier-controlled animal facility for 4 days prior to the experiment. The maximum limit method was used, with a gavage dose of 10028.8 mg / g (two doses per day, combined as one dose). Animals were fasted for 4 hours before administration.

[0042] After the experiment began, the animals were administered the poison twice daily via oral gavage, with an interval of 4 hours between each administration. After the second administration, the animals were kept fasted for 1 hour. Symptoms of poisoning and behavioral changes were observed daily, and the animals were weighed weekly. The poisoning process and the incidence and mortality of the animals during the observation period were observed and recorded. The observation period was 14 days. After the observation period, the surviving animals were euthanized and grossly dissected. The results are shown in Table 1.

[0043] Table 1 Results of Acute Oral Toxicity Test

[0044] The above test results were obtained according to the method of "National Food Safety Standard Acute Oral Toxicity Test" (GB 15193.3-2014). As shown in Table 1 and according to the standard of GB 15193.3-2014, the fruiting body of the butter-curled mushroom ZJXM002 belongs to the practically non-toxic category.

[0045] Example 6: Preparation of Cream-Crusted Mushroom ZJXM002 Quick-Frozen Dumplings The fruiting bodies of the strain obtained in Example 3 were washed, chopped, and mixed with minced pork. The mixture was then wrapped in dumpling wrappers and quick-frozen to obtain quick-frozen dumplings.

[0046] Example 7: Preparation of Cream-Curved Shiitake Mushroom Powder ZJXM002 After centrifuging to collect mycelia from the liquid culture strain obtained in Example 3, the mycelia were washed and impurities were removed to obtain mycelial mud. A protective agent (glycerol at 10% of the weight of the mycelial mud) was added, and the mixture was resuspended and then freeze-dried under vacuum (vacuum degree <10 Pa, cold trap -55~-65℃, drying for 30h). The resulting mycelial mud was then pulverized and sieved to obtain mycelial powder.

[0047] Example 8 The difference from Example 3 is that the seed culture of strain ZJXM002 was cultured in a constant temperature and dark shaker at 26°C for 15 days at a speed of 140 rpm. The culture conditions for the liquid culture strain were constant temperature incubation at 24℃ for 9 days; The culture conditions after the liquid culture strain was inoculated onto the substrate were 21°C and cultured in the dark. The seed culture medium consisted of 17 g / L glucose, 5 g / L potato starch, 0.5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, and 5 g / L yeast extract. The fermentation broth consisted of 7 g / L soybean flour, 17 g / L glucose, 0.5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, and 5 g / L yeast powder, with a natural pH. The cultivation substrate consists of 74% fermented hardwood sawdust, 18% wheat bran, 6% corn flour, 1% gypsum powder, and 1% white sugar, with the moisture content adjusted to 60%. Prepare the seedbeds by spreading 25 kg of quicklime per mu, turning the soil, rotary tilling, and sun-drying for 3 days. Then, make raised beds for later use. The beds should be 80 cm wide, and the furrows between the beds should be 25 cm wide and 10 cm deep. During the fruiting period, maintain the temperature inside the greenhouse at around 22℃, keep the moisture content of the cultivation substrate and soil between 65% and 75%, and the relative humidity of the air at 75%. During the fruiting body growth period, ensure sufficient diffused light and avoid direct sunlight. After the mycelium in the seedbed begins to twist and crawl out of the soil surface, use drip irrigation or sprinkler irrigation to thoroughly water the soil at least once to stimulate primordia formation. After the primordia appear, keep the soil moist and harvest after 9 days.

[0048] Example 9 The difference from Example 3 is that the seed culture of strain ZJXM002 was cultured in a constant temperature and dark shaker at 24°C for 17 days at a speed of 155 rpm. The culture conditions for the liquid culture strain were constant temperature incubation at 26℃ for 11 days; The culture conditions after the liquid culture strain was inoculated onto the substrate were 23°C and cultured in the dark. The seed culture medium consisted of 19 g / L glucose, 7 g / L potato starch, 1.5 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate, and 7 g / L yeast extract. The fermentation broth consisted of 9 g / L soybean flour, 19 g / L glucose, 1.5 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate, and 7 g / L yeast powder, with a natural pH. The cultivation substrate consists of 70% fermented hardwood sawdust, 20% wheat bran, 8% corn flour, 1% gypsum powder, and 1% white sugar, with the moisture content adjusted to 62%. Prepare the seedbeds by spreading 30 kg of quicklime per mu, turning the soil, rotary tilling, and sun-drying for 5 days. Then, make raised beds for later use. The beds should be 85 cm wide, and the furrows between the beds should be 30 cm wide and 15 cm deep. During the fruiting period, maintain the temperature inside the greenhouse at around 26℃, keep the moisture content of the cultivation substrate and soil between 60% and 85%, and the relative humidity of the air at 85%. During the fruiting body growth period, ensure sufficient diffused light and avoid direct sunlight. After the mycelium in the seedbed begins to twist and crawl out of the soil surface, use drip irrigation or sprinkler irrigation to thoroughly water the soil at least once to stimulate primordia formation. After the primordia appear, keep the soil moist and harvest after 8 days.

[0049] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A *Flammulina velutipes* strain ZJXM002, characterized in that, The preservation number of the *Pleurotus ostreatus* strain ZJXM002 is CGMCC No. 41786; its classification name is... Lepista cremeoinvoluta .

2. The *Pleurotus ostreatus* strain ZJXM002 according to claim 1, characterized in that, No mice fed with the ZJXM002 strain of *Mushroom floribunda* in acute oral toxicity tests died; The biological efficiency of the first crop of fruiting bodies of the artificially cultivated mushroom strain ZJXM002 was 45.6-56.3%.

3. The application of the *Pleurotus ostreatus* strain ZJXM002 as described in claim 1 or 2, characterized in that, The ZJXM002 strain of *Mushroom floribunda* is suitable for artificial cultivation.

4. The application of the *Pleurotus ostreatus* strain ZJXM002 according to claim 3, characterized in that, Artificial cultivation methods include the following steps: 1) Inoculate the mother culture of the cream-edged mushroom strain ZJXM002 into the seed culture medium and place it in a constant temperature and light-protected shaking incubator at 24~26℃ with a rotation speed of 140~155rpm for 15~17 days to obtain the seed culture of strain ZJXM002. 2) After inoculating the seed culture of strain ZJXM002 into the fermentation broth, incubate at a constant temperature of 24~26℃ for 9~11 days to obtain the liquid culture strain; 3) After inoculating the liquid culture spawn onto the cultivation substrate and bagging it, incubate it at 21-23℃ in the dark until the mycelium fills the bag to obtain the culture spawn log; 4) Prepare the seedbed, arrange the spawn logs neatly on the seedbed and cover them with the original soil. Spray water according to the moisture level of the soil covering, and cover with pine needles or straw to keep it moist. Manage the mushrooms until harvest.

5. The application of the *Pleurotus ostreatus* strain ZJXM002 according to claim 4, characterized in that, The seed culture medium consists of: 17-19 g / L glucose, 5-7 g / L potato flour, 0.5-1.5 g / L potassium dihydrogen phosphate, 1-2 g / L magnesium sulfate, and 5-7 g / L yeast extract. The fermentation broth consists of 7-9 g / L soybean flour, 17-19 g / L glucose, 0.5-1.5 g / L potassium dihydrogen phosphate, 1-2 g / L magnesium sulfate, and 5-7 g / L yeast powder, with a natural pH. The cultivation substrate consists of 70-74% fermented hardwood sawdust, 18-20% wheat bran, 6-8% corn flour, 1% gypsum powder, and 1% white sugar. The moisture content of the cultivation substrate is adjusted to 60-62%.

6. The application of the *Pleurotus ostreatus* strain ZJXM002 according to claim 4, characterized in that, The mother culture should be inoculated into at least 5 mycelial blocks in the seed culture medium; The inoculum of strain ZJXM002 seed culture should be at least 8% of the mass fraction of the fermentation medium. The inoculation amount of liquid culture strain in the cultivation medium is: at least 20 mL in each bag of cultivation medium, and each cultivation bag is filled with 380~450 g of dry cultivation medium.

7. The application of the *Pleurotus ostreatus* strain ZJXM002 according to claim 4, characterized in that, To prepare the seedbed, spread 25-30 kg of quicklime per mu, turn the soil, rotary till, and expose it to the sun for 3-5 days. Then, open the seedbeds for later use. The seedbeds should be 80-85 cm wide, and the furrows between the seedbeds should be 25-30 cm wide and 10-15 cm deep.

8. The application of the *Pleurotus ostreatus* strain ZJXM002 according to claim 4, characterized in that, The watering procedure is as follows: if the soil to be covered is moist, no watering is required after covering; if the soil to be covered is dry, water should be sprayed after covering to retain moisture.

9. The application of the *Pleurotus ostreatus* strain ZJXM002 according to claim 4, characterized in that, Mushroom management includes: During the fruiting period, maintain the temperature inside the greenhouse between 22 and 26℃, keep the moisture content of the cultivation substrate and soil between 55 and 85%, and the relative humidity between 75 and 85%. During the fruiting body growth period, ensure sufficient diffused light and avoid direct sunlight. After the mycelium in the seedbed begins to twist and crawl out of the soil surface, use drip irrigation or sprinkler irrigation to thoroughly water the soil at least once to stimulate primordia formation. After the primordia appear, keep the soil moist and harvest after 8-10 days.

10. The application of the *Pleurotus ostreatus* strain ZJXM002 as described in claim 1 or 2, characterized in that, The ZJXM002 strain of Creamy Curling Mushroom can be used to make frozen foods, mushroom powder, umami extracts, mushroom crisps, vegetarian meatballs, vegetarian meat patties, hot pot base, sauces, and soup mixes.

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