Wild russule and under-forest cultivation method thereof

Abstract

The invention discloses a wild russule and an under-forest cultivation method thereof, and belongs to the technical field of microorganisms. The wild russula sp. Meizhou Songyuan257 disclosed by the invention is preserved in the China Center for Type Culture Collection, and the preservation number of the wild russula sp. Meizhou Songyuan257 is CCTCC (China Center for Type Culture Collection) NO: M 2024308. According to the method, the wild russule pure strain Russula sp.Meizhou Songyuan257 capable of realizing stable passage is separated from the castanopsis hystrix forest for the first time, and the problems that in the prior art, the russule pure strain lacks, the pure strain does not exist, the pure strain is difficult to preserve, and mycelia cannot be separated from the original wild environment and stably proliferate under the manual control condition are solved. The invention further establishes an under-forest cultivation method of the wild russule, the effects of fruiting in the current year and stable yield in the next year of artificial fungus bags can be achieved, and technical support is provided for remote cultivation and industrial cultivation of the wild russule.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a wild red mushroom and a method for cultivating the same under a forest. Background Art

[0002] Russula spp. is a rare wild edible fungus with important ectomycorrhizal properties and medicinal value. It is rich in active ingredients such as polysaccharides, amino acids, and polyphenols, and exhibits multiple biological activities, including antioxidant and immunomodulatory properties. Consequently, it is expensive, with a market price as high as 2,000 yuan per kilogram of dry weight, and its economic value is extremely high. However, the current Russula industry suffers from the following drawbacks:

[0003] 1. Scarcity of wild resources: Wild red mushrooms rely on forming mycorrhizal symbiosis with specific host plants (such as Fagaceae plants), and the natural mushroom production rate is low. Excessive collection has led to a sharp decline in resources.

[0004] 2. Difficulty isolating pure strains and a lack of strain resources: Russula mycelium is sensitive to culture conditions, making it difficult to isolate pure strains using traditional methods. Existing literature reports on newly recorded species have only focused on classification and identification, without addressing strain preservation and propagation. Meizhou's wild Russula sp. relies on ectomycorrhizal symbiosis with Castanopsis rubrum, resulting in slow natural reproduction and an annual decline in wild resources exceeding 30%. No stable pure strains have been publicly reported, either domestically or internationally.

[0005] 3. There is a gap in the technology for the subculture and expansion of Russula mycelium: Russula mycelium is sensitive to nutrition and environment, easily degraded in conventional culture medium, and cannot be stably subcultured. After three generations of subculture in conventional culture medium, its vitality drops sharply by 50%, making it impossible to expand the culture production on a large scale, which restricts the research on artificial cultivation.

[0006] 4. Blank in artificial cultivation system: Under natural conditions, wild red mushrooms take 3-5 years to produce mushrooms, and artificial cultivation is still blank; although some studies have tried to use fermentation technology to improve the active ingredients, there is a lack of mature forest symbiotic cultivation technology. The existing technology is only in the theoretical exploration stage, especially the lack of key technology for establishing mycorrhizae, and has not broken through the "species-mycorrhiza-host" synergistic problem.

[0007] These defects seriously limit the development of the red mushroom industry, resulting in high market prices for red mushrooms and unstable nutritional value of red mushrooms. Therefore, developing a wild red mushroom that can be artificially cultivated and stably passed down from generation to generation is crucial for the industrial development of red mushrooms. Summary of the Invention

[0008] The present invention aims to provide a wild Russula and a method for its understory cultivation, addressing the aforementioned problems of the prior art. The present invention, for the first time, isolates a stable, propagated, pure strain of wild Russula, Russula sp. Meizhou Songyuan 257, from a ruby ​​frutescens forest. This solves the prior art issues of a lack of pure strains of Russula, the inability to artificially propagate pure strains, and the difficulty in preserving pure strains. The present invention also establishes a method for understory cultivation of the wild Russula, enabling the artificial spawning of mushrooms in the same year and stable yields the following year, providing technical support for the off-site and industrialized cultivation of wild Russula.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The present invention provides a wild red mushroom Russula sp. Meizhou Songyuan 257, which is deposited in China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M2024308 and a deposit date of January 31, 2024.

[0011] The present invention also provides application of the wild red mushroom Russula sp. Meizhou Songyuan 257 in artificial cultivation of red mushroom.

[0012] The present invention also provides a method for cultivating the wild red mushroom Russula sp. Meizhou Songyuan 257 under a forest, comprising the following steps:

[0013] (1) inoculating the liquid spawn of the wild red mushroom Russula sp. Meizhou Songyuan 257 into the red mushroom spawn bag base material, culturing, and obtaining the red mushroom spawn bag;

[0014] The base material of the red mushroom spawn bag comprises the following raw materials: broadleaf sawdust, corn cobs, bran, rice husks, calcium carbonate, gypsum, fructose, red cone humus soil and water;

[0015] (2) digging a cultivation pit in a forest of red cones, and sequentially spreading the red mushroom spawn, the red mushroom spawn transition base, the humus red cone leaves and the Fusarium fungus solution into the pit to carry out fruiting culture;

[0016] The transition base material for the red mushroom spawn bag comprises the following raw materials: red cone wood chips, bran, rice husks, red cone forest humus, vermiculite and red cone tree root covering soil;

[0017] The Fusarium is Fusarium sp. 752, which is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), the deposit address of which is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the deposit number is GDMCC No: 66463, and the deposit date is June 4, 2025.

[0018] Furthermore, the base material of the red mushroom spawn bag comprises the following raw materials in terms of mass percentage:

[0019] Broadleaf sawdust 18wt%-22wt%, corn cobs 8wt%-12wt%, bran 10wt%-20wt%, rice husks 8wt%-12wt%, calcium carbonate 1wt%-3wt%, gypsum 0.5wt%-2wt%, fructose 0.5wt%-2wt%, red cone humus 4wt%-6wt%, and the balance is water.

[0020] Furthermore, the transition base material for the red mushroom spawn bag comprises the following raw materials in terms of mass percentage:

[0021] 30wt%-40wt% of red cone wood chips, 8wt%-12wt% of bran, 8wt%-12wt% of rice husk, 10wt%-20wt% of red cone forest humus, 2wt%-4wt% of vermiculite, and the remainder is soil covering the roots of the red cone trees.

[0022] Furthermore, the mycelial biomass of wild red mushroom Russula sp. Meizhou Songyuan 257 in the liquid spawn is not less than 4.5 g / L; the inoculation amount is 30%; the culture time is 15-20 days, and the temperature is 28-32° C.

[0023] Furthermore, the mass ratio of the red mushroom spawn bag to the red mushroom spawn bag transition base material is 1:1.

[0024] Furthermore, the thickness of the humified coneflower leaves is 4-6 cm.

[0025] Furthermore, the concentration of the Fusarium spore solution is not less than 10 8 CFU / mL; the addition amount of the Fusarium spore liquid is 1wt%-3wt% of the transition base material of the Russula spore bag.

[0026] Furthermore, the humidity of the mushroom culture is 80%-85%, and the temperature is 20-28°C.

[0027] The present invention discloses the following technical effects:

[0028] The invention separates for the first time a wild pure strain of Russula sp. Meizhou Songyuan 257 that can be stably propagated from a red cone forest, solving the problems in the prior art of lack of pure strains of Russula sp., no pure strains that can be artificially expanded, and difficulty in preserving pure strains.

[0029] The present invention also establishes the understory cultivation technology of the wild red mushroom Russula sp. Meizhou Songyuan 257. First, by optimizing the base material formula of the red mushroom spawn and preparing a biomimetic formula of red cone forest humus soil, the growth rate and vitality of the red mushroom mycelium are improved, laying the foundation for accelerating the fruiting of the red mushroom; further, by determining the transition base material formula of the red mushroom spawn, through the triple design of "environmental simulation + symbiotic pre-adaptation + nutrient slow release", the problem of "water and soil incompatibility" of the red mushroom from pure culture to the natural environment is solved, and the success rate of understory planting is significantly improved; the present invention also isolates a strain of Fusarium sp. 752 that can promote the mycorrhizal generation of red mushrooms, which can significantly increase the fruiting rate of red mushrooms and achieve fruiting 2-3 months after colonization, while solving the problem that wild red mushrooms cannot be cultivated in other places. The understory cultivation method of the present invention can achieve the effect of fruiting in the same year of artificial spawn and stable yield in the next year, providing technical support for the off-site cultivation and industrial cultivation of wild red mushrooms. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 This is a colony morphology of wild red mushroom Russula sp. Meizhou Songyuan 257 on the improved isolation medium slope;

[0032] Figure 2 This is a picture of the mycelial morphology of wild Russula sp. Meizhou Songyuan 257 in the improved liquid propagation medium;

[0033] Figure 3 This is a picture of the mycelial morphology of wild red mushroom Russula sp. Meizhou Songyuan257 in a traditional edible mushroom bag inoculation bottle;

[0034] Figure 4 This is a picture of the mycelial morphology of wild Russula sp. Meizhou Songyuan 257 in the improved Russula spawn inoculation bottle;

[0035] Figure 5 This is a picture of the mycelial morphology of wild red mushroom Russula sp. Meizhou Songyuan257 in a traditional edible mushroom bag;

[0036] Figure 6 This is a picture of the mycelial morphology of wild Russula sp. Meizhou Songyuan 257 in the improved Russula fungus bag;

[0037] Figure 7 This is a picture of the mushroom production situation of the Hongkuikeng experimental group in Songyuan Town, Meixian District, Meizhou City, Guangdong Province;

[0038] Figure 8 This is a picture of the mushroom fruiting situation of the control group in Hongkuikeng, Songyuan Town, Meixian District, Meizhou City, Guangdong Province;

[0039] Figure 9 This is a picture of the mushroom fruiting situation of the control group in Hongkuikeng, Songyuan Town, Meixian District, Meizhou City, Guangdong Province;

[0040] Figure 10 This is a morphological picture of mature fruiting bodies in the Hongkuikeng experimental group in Songyuan Town, Meixian District, Meizhou City, Guangdong Province;

[0041] Figure 11 This is a map of mushroom production in Luofu Town, Xingning City, Meizhou City, Guangdong Province;

[0042] Figure 12 This is a map of the mushroom production in Taoyuan Village, Shixing County, Shaoguan City, Guangdong Province.

[0043] Preservation Instructions

[0044] Wild red mushroom Russula sp. Meizhou Songyuan257 is deposited in China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China. The deposit date is January 31, 2024, and the deposit number is CCTCCNO: M 2024308.

[0045] Fusarium sp. 752 is deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is June 4, 2025, and the deposit number is GDMCC No: 66463. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0052] Example 1 Isolation and Identification of Wild Russula sp. Meizhou Songyuan 257

[0053] 1. Sampling

[0054] Wild Russula fruiting bodies, which coexist with Castanopsis rubripes in the Meizhou mountains, were collected. Their caps were red and their gills were white. The collected fruiting bodies were first soaked in 75% ethanol for 30 seconds and then rinsed five times with sterile water. A 5 mm × 5 mm tissue block was obtained from the junction of the cap and stipe under sterile conditions.

[0055] 2. Isolation and culture

[0056] In order to increase the growth rate of wild Russula, speed up the mycelial germination and accelerate the purification of the strain, the isolation culture medium was improved.

[0057] The traditional isolation medium is PDA isolation medium; the improved isolation medium formula is: 200g / L potato + 20g / L fructose + 10g / L bran + 100mL / L red cone humus extract (red cone humus and water are mixed in a ratio of 1:3, soaked for 12 hours, and then filtered; used to simulate the natural symbiotic environment and improve the mycelium germination rate) + 10mg / L vitamin C + 5g / L calcium carbonate, pH 5.5.

[0058] After the separated Russula tissue blocks were inoculated into the culture medium, they were cultured in the dark at 20℃±1℃. After the formation of white mycelial colonies, the tip picking method was used for purification. After repeating three times, pure Russula sp. Meizhou Songyuan 257 was obtained. The isolation and culture effects of Russula on the two culture media are shown in Table 1. It can be seen that on the traditional PDA culture medium, the contamination rate was high and the time required for mycelial germination was long; on the improved isolation culture medium, the contamination rate was low and mycelial germination was faster. The Russula sp. Meizhou Songyuan 257 was inoculated onto the slant of the improved isolation culture medium and stored ( Figure 1 ).

[0059] Table 1 Comparative data of isolation and culture effects

[0060]

[0061] 3. Molecular identification and preservation

[0062] Genomic DNA was extracted from the isolated and cultured pure strain Russula sp. Meizhou Songyuan 257, and the ITS sequence was amplified using universal primers ITS1 / ITS4. The resulting amplified product was sequenced. The sequencing result is shown in SEQ ID NO. 1.

[0063] SEQ ID NO.1:

[0064]

[0065] The sequencing results were subjected to Blast analysis on NCBI, and the similarity with GenBank accession number MN317302 reached 99.8%, confirming that it belonged to the genus Russula and was named Russula sp. Meizhou Songyuan257.

[0066] The red mushroom Russula sp. Meizhou Songyuan257 was deposited in the China Center for Type Culture Collection (CCTCC) on January 31, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2024308.

[0067] Example 2 Stable propagation and expansion technology of wild Russula sp. Meizhou Songyuan 257

[0068] 1. Optimization of liquid expansion formula

[0069] Russula mycelium is very sensitive to nutrients and is easily degraded in conventional culture, resulting in loss of nutrients and inability to perform stable passage and fermentation. To address this problem, the present invention improves the liquid culture medium for Russula propagation.

[0070] The traditional liquid propagation culture medium formula (refer to patent technology CN101790937A, screening of Russula strains and preparation method, published on August 4, 2010) is: 20wt% potato, 2wt% glucose, 0.2wt% peptone, 0.05wt% potassium dihydrogen phosphate, 0.05wt% magnesium sulfate, and the balance is water, with a natural pH.

[0071] The improved liquid propagation medium formula is: 30g / L corn flour + 15g / L soybean meal + 15g / L fructose + 5g / L yeast powder + 0.5g / L MgSO4·7H2O + 1.0g / L KH2PO4 + 5g / L Castanopsis erythrorhizon root extract (fresh roots of Castanopsis erythrorhizon were collected, cleaned with deionized water, frozen, ground into powder, soaked in ultrapure water for 12 hours, and the water extract was collected, concentrated by vacuum rotary evaporation at 60°C, and then freeze-dried. Contains symbiotic signal molecules to enhance mycelial activity), pH 5.8.

[0072] Tissue blocks of Russula sp. Meizhou Songyuan 257 were inoculated into shake flasks at a 10% inoculum rate at 180 rpm and 25°C for 7 days. After 7 days, the mycelial biomass and mycelial growth rate in the two culture media were counted. The comparative data on the culture effects are shown in Table 2. It can be seen that using the traditional liquid propagation medium, the growth rate of Russula sp. Meizhou Songyuan 257 was slow, and the accumulation of mycelial biomass was small. The improved liquid propagation medium increased the mycelial growth rate by about 100%, with mycelial biomass reaching as high as 4.8 g / L ( Figure 2 ).

[0073] Table 2 Comparative data of culture effects

[0074]

[0075] 2. Establishment and evaluation of stable cell culture methods

[0076] Russula mycelium is also very sensitive to the conditions of liquid deep culture and transfer to solid culture medium. In order to solve this problem, the present invention improves the subculture method.

[0077] The traditional subculture method is as follows: inoculate the slant mother culture into 100 mL of traditional liquid expansion culture medium, with an inoculum volume of 10% of the culture medium volume and an expansion ratio of 1:10 (for example, 100 mL of bacterial liquid is inoculated into 1000 mL of new culture medium). The secondary culture medium is then gradually expanded to 200 L of liquid culture medium according to the same inoculation ratio.

[0078] The improved stable passage method (three-stage expansion + gradient transfer) is as follows:

[0079] The slant culture of Russula sp. Meizhou Songyuan 257 was first transferred to a plate culture, and the plate culture tissue block (mother culture) was inoculated into a 100 mL / 250 mL shake flask modified liquid propagation culture medium at a 10% inoculation rate. The shake flask speed was 180 rpm, the temperature was 25°C, and the culture was carried out for 7 days to obtain the first-level mycelium liquid seeds of Russula. The first-level mycelium liquid seeds of Russula were transferred to the second-level liquid propagation culture medium of 500 mL / L shake flask at a 15% inoculation rate and cultured for 7 days under the same culture conditions to obtain the second-level mycelium culture liquid of Russula. Subsequently, the second-level mycelium culture liquid of Russula can be directly transferred to the solid culture medium in the fungus bag or fungus bottle at a 15% inoculation rate.

[0080] The mycelial growth rate was measured after 10 generations of subculture according to the conditions of submerged liquid culture. The results showed that the mycelial growth rate remained at 0.3 cm / day after 10 generations, compared with the mycelial growth rate of 0.32 cm / day of the mother culture, with a decay rate of <7%.

[0081] To determine the stability of Russula sp. Meizhou Songyuan 257 during the passage process, qPCR technology was used to detect changes in telomere length. The specific process is as follows:

[0082] (1) Use a fungal DNA extraction kit to extract genomic DNA from Russula mycelium. After measuring the concentration of the extracted DNA, dilute it to a concentration of about 20 ng / μL for later use. Ensure the DNA purity is A 260 / A 280 Around 1.8 to ensure the accuracy of subsequent experiments.

[0083] (2) qPCR was performed using a telomere primer pair (T reaction primer) and a single copy gene 36b4 primer pair (S reaction primer). The primer sequences are as follows:

[0084] T reaction primers:

[0085] Forward primer: 5′-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3′ (SEQ ID NO. 2);

[0086] Reverse primer: 5′-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCT-3′ (SEQ ID NO. 3).

[0087] S reaction primers:

[0088] Forward primer: 5′-CAGCAAGTGGGAAGGTGTAATCC-3′ (SEQ ID NO. 4);

[0089] Reverse primer: 5′-CCCATTCTATCATCAACGGGTACAA-3′ (SEQ ID NO. 5).

[0090] The PCR reaction system is as follows: 10 μL of 2×PCR Master Mix (including hot-start Taq DNA polymerase, dNTPs, and Master Mix for StarLighter hot-start Taq DNA polymerase); 0.5 μL each of forward and reverse primers (10 μM) for the T reaction; 0.5 μL each of forward and reverse primers (10 μM) for the S reaction; 3 μL of template DNA; and sterile deionized water to make up to 20 μL.

[0091] The PCR reaction procedure is as follows: a. PCR activation reaction: Place the PCR reaction tube in a fluorescent quantitative PCR instrument and heat at 95°C for 10 minutes to activate the Hot StarTaq DNA polymerase in the system.

[0092] b. Cycling reaction: A two-step cycling method is used for amplification:

[0093] T reaction: denaturation at 95°C for 15 seconds, annealing / extension mix at 54°C for 2 minutes, for a total of 18 cycles. This process primarily amplifies telomere sequences.

[0094] The S reaction was performed by denaturation at 95°C for 15 seconds and annealing / extension at 58°C for 1 minute for 30 cycles. This process primarily amplified the single-copy 36b4 gene sequence.

[0095] (3) After the reaction is completed, the software supporting the fluorescence quantitative PCR instrument is used to determine the Ct values ​​of the T reaction and the S reaction respectively - Ct (telomeres) and Ct (36b4). According to the principle that the PCR reaction product increases exponentially by 2, the T / S ratio is close to the formula (2 Ct（telomeres） / 2 Ct（36b4） )-1=2 -ΔCt If the telomere lengths of different passages of Russula samples are compared, 2 -（ΔCt1-ΔCt2） =2 -ΔΔCt At the same time, the data reading is optimized by setting the baseline and threshold. For example, the internal reference (36b4) baseline is set to 6-15 cycles, and the telomere baseline is set to 3-15 cycles. The internal reference threshold can be set to a Ct value of 21±1, and the telomere threshold is set to a Ct value of 13±1.

[0096] Testing showed that the traditional subculture method shortened telomeres by 65 bp per generation, reaching a shortening of 650 bp after 10 generations. The stable subculture method established in this example shortened telomeres by 15 bp per generation, reaching a shortening of 225 bp after 15 generations, significantly less than the traditional method. Further testing of the oxidative stress indicator (MDA content) in Russula mycelium after subculture revealed that, while the MDA content in Russula mycelium reached 3.8 nmol / mg after 10 generations using the traditional subculture method, the MDA content in Russula mycelium after 15 generations using the stable subculture method established in this example was only 1.2 nmol / mg, a 68% reduction compared to the traditional method.

[0097] Mycelia of Russula after 10 generations using the stable subculture method and the traditional subculture method were placed in 20% glycerol and frozen at -80°C. After 12 months of storage, they were revived. The results showed that the mycelia using the stable subculture method had a resuscitation rate of 85%, while the mycelia using the traditional subculture method could not be revived. This shows that the stable subculture method of the present invention can effectively maintain the cell viability of wild Russula, providing the necessary nutritional foundation for the preservation of Russula strains, thereby facilitating the conservation of Russula seeds.

[0098] Example 3: Understory cultivation technology of wild Russula sp. Meizhou Songyuan 257

[0099] 1. Optimization of the base material of red mushroom bags

[0100] The existing wild red mushroom cultivation under the forest has the problems of difficult and late fruiting. In order to promote the fruiting of wild red mushrooms as soon as possible during under-forest cultivation, the present invention optimizes the formula of the mushroom bag base material for under-forest cultivation.

[0101] The formula of traditional edible fungus bag base material is: taking 100 grams of mushroom bag base material as an example: 80wt% wheat bran + 4.5wt% soybean meal + 0.5wt% potassium carbonate, and the rest is water.

[0102] The improved Russula spawn base material formula is as follows: For 100 grams of base material, the main ingredients are: 20wt% broadleaf sawdust, 10wt% corncobs, 15wt% bran, and 10wt% rice husks; supplementary ingredients are: 2wt% calcium carbonate, 1wt% gypsum, 1wt% fructose, and 5wt% humus soil from a red cone forest (from the topsoil beneath the red cone forest); the balance is water. The physical and chemical parameters of this base material are measured to be: pH 5.0-5.5, and C / N ratio 25-30:1.

[0103] The two fungus bag base materials were placed in a 12cm*24cm fungus bag or an 800mL inoculation bottle, and 50mL of the liquid fungus obtained in Example 2 "1. Optimization of liquid propagation formula" (mycelial biomass ≥ 4.5g / L) was added. The fungus bag was placed in a constant temperature incubator at 30℃ for 7 days. The culture results of the traditional edible fungus bag are shown in Figure 3 and Figure 5 The culture results of the improved red mushroom bag are shown in Figure 4 and Figure 6 It can be seen that when the traditional edible fungus bag substrate was used to cultivate Russula sp. Meizhou Songyuan257, no mycelium grew within 7 days; when the improved edible fungus bag substrate was used to cultivate Russula sp. Meizhou Songyuan257, the mycelium grew rapidly within 7 days and basically covered the entire fungus bag.

[0104] After inoculating the modified Russula spawn base with liquid spawn, the modified Russula spawn was placed in a constant-temperature incubator at 20, 25, 30, and 35°C for 7 days. Mycelial growth rates at different temperatures were measured. The results are shown in Table 3. Russula mycelium grew fastest at 30°C.

[0105] Table 3 Comparison of mycelial growth rates at different temperatures

[0106]

[0107] Use the improved red mushroom spawn bag base and inoculate the red mushroom Russula sp. Meizhou Songyuan 257 liquid spawn at an inoculation rate of 30% (v / w, 30 mL of spawn liquid added to 100 g of red mushroom spawn bag base). Cultivate for 15-20 days. After the mycelium has covered the spawn bag base, the red mushroom spawn bags are obtained and used for under-forest cultivation.

[0108] 2. Screening of symbiotic strains

[0109] Wild Russula mushrooms rely on forming a mycorrhizal symbiosis with specific host plants to produce fruiting bodies. This trait limits their cultivation areas, hindering their off-site cultivation and industrialized cultivation. To address this issue, a symbiotic strain was isolated from the rhizosphere soil of Castanopsis rubripes. The genome of this strain was extracted, and the 18S rDNA sequence was amplified using NS1 / NS8 primers. The resulting amplification product was sequenced. The sequencing result is shown in SEQ ID NO. 6.

[0110] SEQ ID NO.6:

[0111]

[0112] The sequencing results were blasted on NCBI and had the highest homology with Fusarium venenatum, confirming it to be Fusarium sp. and named Fusarium sp. 752.

[0113] The strain Fusarium sp. 752 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on June 4, 2025. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 66463.

[0114] The strain Fusarium sp. 752 was inoculated into PDB medium (Potato Dextrose Broth) and cultured at 30℃ for 2 days until the OD 600 =1.2 (logarithmic growth phase), ensure viable cell count ≥10 8 CFU / mL.

[0115] 3. Establishment and verification of understory cultivation methods

[0116] The steps of understory cultivation are as follows:

[0117] (1) Select 3-5 year old red cone trees as hosts, with a plant spacing of 2m × 3m.

[0118] (2) Formula of transitional substrate for red mushroom spawn (used in the transition stage from spawn expansion to forest planting): 35wt% red cone wood chips, 10wt% bran, 10wt% rice husk, 15wt% red cone forest humus, 3wt% vermiculite (2-4mm), and the remainder as soil covering the roots of the host red cone tree. This transitional substrate solves the problem of red mushrooms not adapting to the natural environment when moving from pure culture to the natural environment, and helps to improve the success rate of forest planting.

[0119] (3) Planting operation of spawn bags: dig a 30cm deep pit, put in the red mushroom spawn bags, and sprinkle in the red mushroom spawn bag transition base material. The mass ratio of the red mushroom spawn bag transition base material to the red mushroom spawn bag is 1:1; then cover with 5cm of humus red cone leaves, sprinkle with 2wt% of the transition base material of Fusarium sp.752 spawn solution, and control the humidity under the forest to 80-85% and the temperature to 20-28℃.

[0120] According to the understory cultivation method, the following fruiting test was carried out:

[0121] 1) A fruiting experiment was conducted in Hongkuikeng, Songyuan Town, Meixian District, Meizhou City, Guangdong Province. The experimental group followed the above-mentioned forest cultivation method, while the control group did not apply the Fusarium sp. 752 solution. Other operations were the same as the above-mentioned forest cultivation method. The results showed that mycorrhiza formation was observed 10 days after the experimental group was planted in May 2024, and the first fruiting occurred in August 2024 ( Figure 7 ), but the control group did not produce mushrooms ( Figure 8 and Figure 9 The experimental group matured in June 2025, and harvested multiple mature fruiting bodies ( Figure 10 ), diameter 6-8cm, annual output of a single package is about 350g (wet weight).

[0122] 2) A fruiting experiment was conducted in Luofu Town, Xingning City, Meizhou City, Guangdong Province, according to the above-mentioned forest cultivation method. The results showed that the first fruiting occurred in August 2024 after planting in May 2024. Figure 11 There had been no reports of wild red mushrooms appearing in the town before.

[0123] 3) A fruiting experiment was conducted in Taoyuan Village, Shixing County, Shaoguan City, Guangdong Province, using the above-mentioned understory cultivation method. The results showed that the first fruiting occurred in August 2024 after planting in May 2024. Figure 12 ).

[0124] In summary, the present invention collected, isolated and purified a wild Russula from the mountainous area of ​​Meizhou. Molecular identification confirmed that it was of the genus Russula and named Russula sp. Meizhou Songyuan 257. It was deposited in the China Culture Collection Center of Microorganisms (CCTCC) with the deposit number CCTCC NO: M 2024308.

[0125] The present invention optimizes the liquid propagation culture medium formula to determine a stable propagation method for wild Russula sp. Meizhou Songyuan 257. After 10-15 generations of continuous culture, the mycelial growth rate and stable physiological functions can still be maintained at a high level, providing technical support for the development of liquid fermentation technology for wild Russula sp. Meizhou Songyuan 257. The present invention also optimizes the base material of the fungus bag for wild Russula sp. Meizhou Songyuan 257 to promote mycelial growth, laying the foundation for the understory cultivation technology of Russula sp. 752. The present invention isolates a symbiotic strain, Fusarium sp. 752, from the rhizosphere soil of Castanopsis rubrum that can promote the fruiting of wild Russula sp. Meizhou Songyuan 257, and uses this strain to establish an understory cultivation technology for wild Russula sp. Meizhou Songyuan 257, providing technical support for the off-site cultivation and industrialized cultivation of wild Russula sp.

[0126] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A wild red mushroom Russula sp. Meizhou Songyuan 257, characterized in that: It was deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2024308 and the deposit date was January 31, 2024.

2. Use of the wild Russula sp. Meizhou Songyuan 257 according to claim 1 in artificial cultivation of Russula.

3. A method for cultivating the wild red mushroom Russula sp. Meizhou Songyuan 257 under forests according to claim 1, characterized in that: The steps include: (1) inoculating the liquid spawn of the wild red mushroom Russula sp. Meizhou Songyuan 257 into the red mushroom spawn bag base material, culturing, and obtaining the red mushroom spawn bag; The base material of the red mushroom spawn bag comprises the following raw materials: broadleaf sawdust, corn cobs, bran, rice husks, calcium carbonate, gypsum, fructose, red cone humus soil and water; (2) digging a cultivation pit in a forest of red cones, and sequentially spreading the red mushroom spawn, the red mushroom spawn transition base, the humus red cone leaves and the Fusarium fungus solution into the pit to carry out fruiting culture; The transition base material for the red mushroom spawn bag comprises the following raw materials: red cone wood chips, bran, rice husks, red cone forest humus, vermiculite and red cone tree root covering soil; The Fusarium is Fusarium sp. 752, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 66463 and a deposit date of June 4, 2025.

4. The understory cultivation method according to claim 3, characterized in that: The base material of the red mushroom spawn bag includes the following raw materials in terms of mass percentage: Broadleaf sawdust 18wt%-22wt%, corn cobs 8wt%-12wt%, bran 10wt%-20wt%, rice husks 8wt%-12wt%, calcium carbonate 1wt%-3wt%, gypsum 0.5wt%-2wt%, fructose 0.5wt%-2wt%, red cone humus 4wt%-6wt%, and the balance is water.

5. The understory cultivation method according to claim 3, characterized in that: The transition base material of the red mushroom spawn bag includes the following raw materials in percentage by mass: 30wt%-40wt% of red cone wood chips, 8wt%-12wt% of bran, 8wt%-12wt% of rice husk, 10wt%-20wt% of red cone forest humus, 2wt%-4wt% of vermiculite, and the remainder is soil covering the roots of the red cone trees.

6. The understory cultivation method according to claim 3, characterized in that: The mycelial biomass of wild red mushroom Russula sp. Meizhou Songyuan 257 in the liquid spawn is not less than 4.5 g / L; the inoculation amount is 30%; the culture time is 15-20 days, and the temperature is 28-32° C.

7. The understory cultivation method according to claim 3, characterized in that: The mass ratio of the red mushroom spawn bag to the red mushroom spawn bag transition base material is 1:

1.

8. The understory cultivation method according to claim 3, characterized in that: The thickness of the humus Hystrix cycad leaves is 4-6 cm.

9. The understory cultivation method according to claim 3, characterized in that: The concentration of the Fusarium spore solution is not less than 10 8 CFU / mL; the addition amount of the Fusarium spore liquid is 1wt%-3wt% of the transition base material of the Russula spore bag.

10. The understory cultivation method according to claim 3, characterized in that: The humidity of the mushroom cultivation is 80%-85%, and the temperature is 20-28°C.

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