A wild red mushroom and its understory cultivation method

Abstract

This invention discloses a wild Russula sp. Meizhou Songyuan257 and its understory cultivation method, belonging to the field of microbial technology. The wild Russula sp. Meizhou Songyuan257 of this invention is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024308. This invention is the first to isolate a stable, prolific pure strain of Russula sp. Meizhou Songyuan257 from a red pine forest, solving the problems of lack of pure Russula strains, lack of pure strains, difficulty in preserving pure strains, and the inability of mycelium to stably proliferate under artificial control under the original wild environment in existing technologies. This invention also establishes an understory cultivation method for this wild Russula, which can achieve fruiting in the same year and stable production in the following year using artificial spawn bags, providing technical support for the off-site cultivation and industrial-scale cultivation of wild Russula.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a wild red mushroom and its understory cultivation method. Background Technology

[0002] Russula spp. is a rare wild edible fungus with significant edible and medicinal value, containing ectomycorrhizal components such as polysaccharides, amino acids, and polyphenols. It possesses various biological activities including antioxidant and immunomodulatory effects, making it expensive, with market prices reaching as high as 2000 yuan / kg dry weight, indicating extremely high economic value. However, the current Russula industry suffers from the following shortcomings:

[0003] 1. Scarcity of wild resources: Wild red mushrooms rely on mycorrhizal symbiosis with specific host plants (such as Fagaceae plants), resulting in a low natural fruiting rate. Over-collection leads to a sharp decline in resources.

[0004] 2. Difficulty in isolating pure strains and a lack of strain resources: Russula mycelium is sensitive to culture conditions, and traditional methods are insufficient to isolate pure strains. Existing literature reports new records only at the level of classification and identification, without addressing strain preservation and propagation. Wild Russula sp. in Meizhou relies on ectomycorrhizal symbiosis with the red pine tree, resulting in slow natural reproduction and an annual decline rate of over 30% in wild resources. There are currently no publicly reported pure strains with stable propagation, either domestically or internationally.

[0005] 3. Lack of technology for the propagation and expansion of Russula mycelium: Russula mycelium is sensitive to nutrition and environment and is prone to degeneration in conventional culture media, making it impossible to achieve stable propagation. After three generations of propagation in conventional culture media, its viability drops sharply by 50%, making it impossible to expand production on a large scale and restricting research on artificial cultivation.

[0006] 4. Lack of artificial cultivation system: Wild red mushrooms take 3-5 years to produce fruiting under natural conditions, and artificial cultivation is still a blank. Although some studies have attempted to use fermentation technology to enhance active ingredients, there is a lack of mature forest symbiotic cultivation technology. Existing technologies are only at the theoretical exploration stage, especially lacking key technologies for mycorrhizal establishment, and have not broken through the problem of "strain-mycorrhizal-host" synergy.

[0007] These shortcomings severely limit the development of the red mushroom industry, resulting in persistently high market prices and unstable nutritional value. Therefore, developing a wild red mushroom variety that can be artificially cultivated and stably propagated is crucial for the industrialization of red mushrooms. Summary of the Invention

[0008] The purpose of this invention is to provide a wild Russula var. rubrum and its understory cultivation method to solve the problems existing in the prior art. This invention is the first to isolate a stable, prolific pure strain of Russula var. rubrum from *Pinus koraiensis* forests, solving the problems of lack of pure Russula var. rubrum strains, lack of pure strains for artificial propagation, and difficulty in preserving pure strains in the prior art. This invention also establishes an understory cultivation method for this wild Russula var. rubrum, enabling artificial spawn bags to produce fruiting in the same year and achieve stable production in the following year, providing technical support for the off-site cultivation and industrial-scale cultivation of wild Russula var. rubrum.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] This invention provides a wild Russula sp. Meizhou Songyuan257, deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2024308 and deposit date of January 31, 2024.

[0011] This invention also provides the application of the above-mentioned wild Russula sp. Meizhou Songyuan257 in the artificial cultivation of Russula.

[0012] This invention also provides a method for understory cultivation of the above-mentioned wild red mushroom Russula sp. Meizhou Songyuan 257, comprising the following steps:

[0013] (1) The liquid spawn of the wild Russula sp. Meizhou Songyuan257 was inoculated into the Russula spawn bag substrate and cultured to obtain Russula spawn bags;

[0014] The substrate for the Russula ovata spawn bags includes the following raw materials: broadleaf sawdust, corn cobs, wheat bran, rice husks, calcium carbonate, gypsum, fructose, humus from red pine forests, and water;

[0015] (2) Dig cultivation pits in the red pine forest, and sprinkle the red mushroom bag, red mushroom bag transition substrate, humus red pine leaves and Fusarium liquid in sequence to carry out fruiting culture;

[0016] The transition substrate for the Russula ovata spawn bags includes the following raw materials: Russula ovata tree sawdust, wheat bran, rice husks, Russula ovata forest humus, vermiculite, and soil covering the roots of Russula ovata trees;

[0017] The Fusarium species described is Fusarium sp. 752, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66463 and deposit date of June 4, 2025.

[0018] Furthermore, the Russula ovata var. rubrum substrate comprises the following raw materials by weight percentage:

[0019] The composition consists of: 18wt%-22wt% broadleaf sawdust, 8wt%-12wt% corn cob, 10wt%-20wt% wheat bran, 8wt%-12wt% rice husk, 1wt%-3wt% calcium carbonate, 0.5wt%-2wt% gypsum, 0.5wt%-2wt% fructose, 4wt%-6wt% red pine forest humus, with the remainder being water.

[0020] Furthermore, the transition substrate for the Russula ovata var. rubrum spawn comprises the following raw materials by weight percentage:

[0021] The mixture consists of 30wt%-40wt% red pine tree wood chips, 8wt%-12wt% wheat bran, 8wt%-12wt% rice husks, 10wt%-20wt% red pine forest humus, 2wt%-4wt% vermiculite, and the remainder is soil covering the roots of the red pine trees.

[0022] Furthermore, the mycelial biomass of wild Russula sp. Meizhou Songyuan257 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℃.

[0023] Furthermore, the mass ratio of the Russula ovata var. rubra ...

[0024] Furthermore, the thickness of the humus-rich red cone leaves is 4-6 cm.

[0025] Furthermore, the concentration of the Fusarium bacterial solution is not less than 10. 8 CFU / mL; the amount of Fusarium oxysporum liquid added is 1wt%-3wt% of the transition substrate of the Russula ovata var. rubrum spawn bag.

[0026] Furthermore, the humidity of the mushroom cultivation is 80%-85%, and the temperature is 20-28℃.

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

[0028] This invention is the first to isolate a stable, pure strain of wild Russula sp. Meizhou Songyuan 257 from red pine forests, solving the problems of lack of pure Russula strains, lack of pure strains for artificial propagation, and difficulty in preserving pure strains in the existing technology.

[0029] This invention also establishes a forest understory cultivation technique for the wild Russula sp. Meizhou Songyuan257. Firstly, by optimizing the substrate formula for Russula spawn bags and developing a biomimetic formula using humus from red cone forests, the growth rate and vitality of Russula mycelium are improved, laying the foundation for faster fruiting. Secondly, by determining the transition substrate formula for Russula spawn bags, a triple design of "environmental simulation + symbiotic pre-adaptation + slow nutrient release" is implemented to solve the "acclimatization problem" of Russula from pure culture to the natural environment, significantly improving the success rate of understory planting. This invention also isolates a Fusarium sp. 752 strain that promotes Russula mycorrhizal formation, significantly increasing the fruiting rate and achieving fruiting within 2-3 months after establishment, while simultaneously solving the problem of wild Russula's inability to be cultivated in other locations. The forest understory cultivation method of this invention can achieve fruiting in the same year and stable production in the following year using artificial spawn bags, providing technical support for the off-site cultivation and industrial-scale cultivation of wild Russula. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 Colony morphology of wild Russula sp. Meizhou Songyuan257 on a modified isolation medium slant;

[0032] Figure 2 Image of mycelial morphology of wild Russula sp. Meizhou Songyuan257 in a modified liquid culture medium;

[0033] Figure 3 Image of mycelial morphology of wild Russula sp. Meizhou Songyuan 257 in a traditional edible mushroom spawn bag in an inoculation bottle;

[0034] Figure 4 Image of mycelial morphology of wild Russula sp. Meizhou Songyuan 257 in an inoculation bottle of a modified Russula spawn bag;

[0035] Figure 5 Image of mycelial morphology of wild Russula sp. Meizhou Songyuan 257 in traditional edible mushroom substrate bags;

[0036] Figure 6 Image of mycelial morphology of wild Russula sp. Meizhou Songyuan 257 in a modified Russula spawn bag;

[0037] Figure 7 This is a diagram showing the mushroom production in the Hongkuikeng experimental group in Songyuan Town, Meixian District, Meizhou City, Guangdong Province.

[0038] Figure 8 This is a graph showing the mushroom production in the control group at Hongkuikeng, Songyuan Town, Meixian District, Meizhou City, Guangdong Province.

[0039] Figure 9 This is a graph showing the mushroom production in the control group at Hongkuikeng, Songyuan Town, Meixian District, Meizhou City, Guangdong Province.

[0040] Figure 10 A morphological diagram of mature fruiting bodies from the Hongkuikeng experimental group in Songyuan Town, Meixian District, Meizhou City, Guangdong Province;

[0041] Figure 11 A map showing the mushroom production situation in Luofu Town, Xingning City, Meizhou City, Guangdong Province;

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

[0043] Preservation Instructions

[0044] Wild Russula sp. Meizhou Songyuan257 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on January 31, 2024, with accession number CCTCCNO: M 2024308.

[0045] Fusarium sp. 752 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on June 4, 2025, with accession number GDMCC No: 66463. Detailed Implementation

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0049] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were 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 red mushroom fruiting bodies that coexist with red pine trees in the mountainous areas of Meizhou were collected. The caps were red and the gills were white. The collected fruiting bodies were first soaked in 75% ethanol for 30 seconds, then rinsed 5 times with sterile water. Under sterile conditions, tissue blocks (5mm×5mm) were taken from the junction of the cap and the stipe.

[0055] 2. Isolation and Culture

[0056] To improve the growth rate of wild red mushrooms, accelerate mycelial germination, and expedite strain purification, the isolation culture medium was modified.

[0057] The traditional isolation medium is PDA isolation medium; the modified isolation medium formula is: 200g / L potato + 20g / L fructose + 10g / L wheat bran + 100mL / L red pine forest humus extract (obtained by mixing red pine forest humus and water at a ratio of 1:3, soaking for 12 hours and then filtering; used to simulate the natural symbiotic environment and improve the mycelial germination rate) + 10mg / L vitamin C + 5g / L calcium carbonate, pH 5.5.

[0058] After the isolated Russula sp. tissue blocks were inoculated onto the culture medium and incubated in the dark at 20℃±1℃, the mycelial colonies of white mycelium were observed to form. Purification was then performed using the tip-picking method, repeated three times, to obtain pure Russula sp. MeizhouSongyuan257. 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 medium, the contamination rate was high, and the mycelial germination time was longer; while on the improved isolation medium, the contamination rate was low, and mycelial germination was faster. The Russula sp. MeizhouSongyuan257 strain was inoculated onto slant agar plates of the improved isolation medium for preservation. Figure 1 ).

[0059] Table 1 Comparison of isolation and culture effects

[0060]

[0061] 3. Molecular identification and preservation

[0062] Genomic DNA was extracted from the pure strain *Russula sp.* Meizhou Songyuan257 obtained by isolation and culture. The ITS sequence was amplified using universal primers ITS1 / ITS4, and the amplified products were sequenced. The sequencing results are shown in SEQ ID NO. 1.

[0063] SEQ ID NO.1:

[0064]

[0065] The sequencing results were BLASTed on NCBI and showed a 99.8% similarity to GenBank accession number MN317302, confirming it as belonging to the genus Russula, named Russula sp. Meizhou Songyuan257.

[0066] This Russula sp. Meizhou Songyuan257 was deposited at the China Center for Type Culture Collection (CCTCC) on January 31, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2024308.

[0067] Example 2: Stable subculturing and propagation techniques of wild red mushroom Russula sp. Meizhou Songyuan257

[0068] 1. Optimization of liquid culture formulation

[0069] Russula mycelium is highly sensitive to nutrients, and the strain is prone to degeneration during conventional cultivation, resulting in nutrient loss and making stable subculturing and fermentation impossible. To address this issue, this invention improves the liquid culture medium for Russula.

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

[0071] The improved liquid culture medium formula is as follows: 30 g / L corn flour + 15 g / L soybean meal powder + 15 g / L fructose + 5 g / L yeast powder + 0.5 g / L MgSO4·7H2O + 1.0 g / L KH2PO4 + 5 g / L Conifera spp. root extract (collected from fresh Conifera spp. roots, cleaned with deionized water, frozen, ground into powder, soaked in ultrapure water for 12 hours, the aqueous extract collected, concentrated by vacuum rotary evaporation at 60℃, and then freeze-dried; contains symbiotic signaling 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, and cultured at 180 rpm and 25℃ for 7 days. After 7 days, mycelial biomass and growth rate were measured in both culture media. The comparison data of the culture effects are shown in Table 2. It can be seen that using the traditional liquid expansion medium, the growth rate of *Russula sp. Meizhou Songyuan 257* was slow, and the accumulation of mycelial biomass was low; the improved liquid expansion medium could increase the mycelial growth rate of *Russula sp. Meizhou Songyuan 257* by about 100%, and the mycelial biomass reached as high as 4.8 g / L. Figure 2 ).

[0073] Table 2 Comparison of Cultivation Effects

[0074]

[0075] 2. Establishment and evaluation of stable propagation methods

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

[0077] The traditional subculture method is as follows: Inoculate the slant mother culture into 100mL of traditional liquid expansion medium, the inoculation amount is 10% of the medium volume, and the expansion ratio is 1:10 (e.g., 100mL of bacterial culture is inoculated into 1000mL of new medium). The secondary culture is then expanded step by step to 200L of liquid medium according to the same inoculation ratio.

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

[0079] The slant culture of *Russula sp. Meizhou Songyuan 257* was first transferred to agar plates. The plate culture tissue blocks (mother culture) were then inoculated at a rate of 10% into a modified liquid expansion medium in 100 mL / 250 mL shake flasks. The flasks were shaken at 180 rpm and the temperature was 25℃ for 7 days to obtain primary mycelial liquid seed culture of *Russula*. The primary mycelial liquid seed culture was then transferred at a rate of 15% to a secondary liquid expansion medium in 500 mL / L shake flasks. Under the same conditions, the culture was incubated for 7 days to obtain secondary mycelial culture broth of *Russula*. This secondary mycelial culture broth can then be directly transferred at a rate of 15% to solid culture media in mushroom bags or bottles.

[0080] The mycelium was passaged under the same conditions as in submerged liquid culture. After 10 passages, the mycelial growth rate was measured. The results showed that after 10 passages, the mycelial growth rate remained at 0.3 cm / day, compared to 0.32 cm / day for the mother culture, representing a decrease of <7%.

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

[0082] (1) Genomic DNA was extracted from Russula rubra mycelia using a fungal DNA extraction kit. After determining the concentration of the extracted DNA, it was diluted to approximately 20 ng / μL for later use. Ensure DNA purity A. 260 / A 280 The value should be around 1.8 to ensure the accuracy of subsequent experiments.

[0083] (2) qPCR was performed using telomere primer pairs (T-reaction primers) and single-copy gene 36b4 primer pairs (S-reaction primers). 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 primer:

[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 consisted of: 10 μL of 2×PCR Master Mix (containing hot-start Taq DNA polymerase, dNTPs, and the Master Mix compatible with StarLighter hot-start Taq DNA polymerase); 0.5 μL each of the forward and reverse T-reaction primers (10 μM); 0.5 μL each of the forward and reverse S-reaction primers (10 μM); 3 μL of template DNA; and sterile deionized water to a final volume of 20 μL.

[0091] The PCR reaction procedure is as follows: a. PCR activation reaction: Place the PCR reaction tube in a real-time PCR instrument at 95°C for 10 min to activate the Hot StarTaq DNA polymerase in the system.

[0092] b. Cyclic reaction: Amplification is performed using a two-step cyclic method:

[0093] T reaction: denaturation at 95℃ for 15 s, followed by annealing / extension mixing at 54℃ for 2 min, for a total of 18 cycles. This process primarily amplifies telomere sequences.

[0094] S reaction: denaturation at 95℃ for 15 s, followed by annealing / extension mixing at 58℃ for 1 min, for a total of 30 cycles. This process mainly amplifies the single-copy gene 36b4 sequence.

[0095] (3) After the reaction, the Ct values ​​of the T reaction and S reaction were determined using the software of the real-time PCR instrument—Ct(telomeres) and Ct(36b4), respectively. Based on the principle that PCR products increase exponentially by 2, the T / S ratio is close to the formula (2...). Ct（telomeres） / 2 Ct（36b4） )-1=2 -ΔCt To compare the differences in telomere length among different generations of *Russula ovata* samples, use 2... -（ΔCt1-ΔCt2） =2 -ΔΔCt The calculation is performed using this method. At the same time, the data reading is optimized by setting the baseline and threshold. For example, the baseline of the intrinsic parameter (36b4) is set to 6-15 cycles, and the telomere baseline is set to 3-15 cycles; the intrinsic parameter threshold can be set to Ct value 21±1, and the telomere threshold can be set to Ct value 13±1.

[0096] Testing revealed that traditional subculturing methods shorten telomeres by 65 bp per generation, reaching 650 bp after 10 generations. The stable subculturing method established in this embodiment shortens telomeres by 15 bp per generation, reaching 225 bp after 15 generations, significantly less than the traditional method. Further analysis of oxidative stress indicators (MDA content) in the subcultured Russula mycelium showed that the MDA content in Russula mycelium after 10 generations using the traditional method reached 3.8 nmol / mg, while the stable subculturing method established in this embodiment resulted in an MDA content of only 1.2 nmol / mg after 15 generations, a 68% reduction compared to the traditional method.

[0097] Russula mycelia after 10 generations using both the stable subculturing method and the traditional subculturing method were placed in 20% glycerol and frozen at -80°C. After 12 months of storage, they were revived. The results showed that the revival rate of Russula mycelia obtained using the stable subculturing method reached 85%, while the mycelia obtained using the traditional subculturing method could not be revived. This indicates that the stable subculturing method of the present invention can effectively maintain the cell viability of wild Russula, providing the necessary nutritional basis for the preservation of Russula strains.

[0098] Example 3: Understory cultivation techniques for wild Russula sp. Meizhou Songyuan 257

[0099] 1. Optimization of substrate for Russula ovata var. rubrum spawn bags

[0100] Existing methods for cultivating wild red mushrooms under forest cover suffer from difficulties in fruiting and delayed fruiting. To promote faster fruiting of wild red mushrooms under forest cover, this invention optimizes the substrate formula for mushroom bags used in under-forest cultivation.

[0101] The traditional formula for edible mushroom substrate is as follows: taking 100 grams of substrate as an example: 80 wt% wheat bran + 4.5 wt% soybean meal + 0.5 wt% potassium carbonate, with the remainder being water.

[0102] The improved formula for the substrate of *Russula ovata* spawn bags is as follows: Taking 100 grams of substrate as an example: Main ingredients: 20wt% broadleaf sawdust + 10wt% corn cob + 15wt% wheat bran + 10wt% rice husk; Auxiliary ingredients: 2wt% calcium carbonate + 1wt% gypsum + 1wt% fructose + 5wt% *Russula ovata* humus (taken from the topsoil under the *Russula ovata* forest); the remainder is water. The physicochemical parameters of this substrate are: pH 5.0-5.5, C / N ratio 25-30:1.

[0103] Both types of substrate were placed into 12cm*24cm mushroom bags or 800mL inoculation bottles, and 50mL of the liquid inoculum (mycelial biomass ≥ 4.5g / L) obtained in Example 2, "1. Optimization of Liquid Expansion Formula" was added. The mushroom bags were placed in a 30℃ constant temperature incubator for 7 days. The cultivation results of traditional edible mushroom bags are shown in [the table below]. Figure 3 and Figure 5 The cultivation results of the improved Russula ovata var. rubrum spawn bags are shown in [the table]. Figure 4 and Figure 6 It is evident that when using traditional edible mushroom substrate to cultivate Russula sp. Meizhou Songyuan257, no mycelial growth was observed within 7 days; however, when using the improved Russula sp. Meizhou Songyuan257 substrate, mycelial growth was rapid within 7 days, and the substrate essentially covered the entire substrate.

[0104] After inoculating the improved Russula rubra spawn substrate with liquid spawn, the substrate was placed in constant temperature incubators at 20, 25, 30, and 35℃ for 7 days, and the mycelial growth rate at different temperatures was measured. The results are shown in Table 3. It can be seen that Russula rubra mycelial growth is fastest at 30℃.

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

[0106]

[0107] Using the improved Russula sp. Meizhou Songyuan257 liquid spawn, inoculate at a rate of 30% (v / w, 30 mL of mycelium solution added to 100 g of Russula sp. substrate) and culture for 15-20 days. Once the mycelium has fully colonized the substrate, the Russula sp. substrate is obtained and used for understory cultivation.

[0108] 2. Screening of symbiotic strains

[0109] Wild red succulents rely on mycorrhizal symbiosis with specific host plants to produce fruiting bodies. This characteristic limits the cultivation areas of wild red succulents, hindering their ex-situ cultivation and industrial-scale production. To address this issue, a symbiotic strain was isolated and screened from the rhizosphere soil of *Castanopsis fargesii*. The genome of this strain was extracted, and its 18S rDNA sequence was amplified using NS1 / NS8 primers. The amplified product was then sequenced. The sequencing results are shown in SEQ ID NO. 6.

[0110] SEQ ID NO.6:

[0111]

[0112] The sequencing results were BLASTed on NCBI and showed the highest homology with Fusarium venenatum, confirming it as Fusarium sp., and named Fusarium sp. 752.

[0113] The strain Fusarium sp. 752 was deposited on June 4, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66463.

[0114] Fusarium sp. 752 was inoculated into PDB medium (Potato Dextrose Broth) and cultured at 30°C with shaking for 2 days until fermentation reached OD. 600 =1.2 (logarithmic growth phase), ensuring a viable count ≥10. 8 CFU / mL.

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

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

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

[0118] (2) Formula for transitional substrate for Russula rubra spawn bags (used for the transition from spawn expansion to understory planting): 35wt% Russula rubra wood chips, 10wt% wheat bran, 10wt% rice husks, 15wt% Russula rubra forest humus, 3wt% vermiculite (2-4mm), with the remainder being soil covering the roots of the host Russula rubra tree. This transitional substrate, through a triple design of "environmental simulation + symbiotic pre-adaptation + slow nutrient release," solves the problem of Russula rubra's "acclimatization" when transitioning from pure culture to the natural environment, and helps improve the success rate of understory planting.

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

[0120] Following this understory cultivation method, the following fruiting experiment was conducted:

[0121] 1) A fruiting experiment was conducted in Hongkuikeng, Songyuan Town, Meixian District, Meizhou City, Guangdong Province. The experimental group followed the above-mentioned understory cultivation method, while the control group did not receive Fusarium sp. 752 mycelial solution. Other operations were the same as the above-mentioned understory cultivation method. The results showed that mycorrhizal formation was observed 10 days after planting in May 2024, and the first fruiting occurred in August 2024. Figure 7 However, no mushrooms were produced in the control group. Figure 8 and Figure 9 The experimental group matured in June 2025, yielding multiple mature fruiting bodies. Figure 10 (6-8cm in diameter, with an annual yield of approximately 350g (wet weight) per package).

[0122] 2) A fruiting experiment was conducted in Luofu Town, Xingning City, Meizhou City, Guangdong Province, following the aforementioned understory cultivation method. Results showed that plants planted in May 2024 produced their first fruiting in August 2024. Figure 11 There had been no previous reports of wild red mushrooms appearing in the town.

[0123] 3) A fruiting experiment was conducted in Taoyuan Village, Shixing County, Shaoguan City, Guangdong Province, following the aforementioned understory cultivation method. Results showed that plants planted in May 2024 produced their first fruiting fruit in August 2024. Figure 12 ).

[0124] In summary, this invention collected, isolated, and purified a wild Russula strain from the mountainous area of ​​Meizhou. Molecular identification confirmed it to be of the Russula genus, and it was named Russula sp. Meizhou Songyuan257. It is deposited at the China Center for Microbial Culture Collection (CCTCC) with accession number CCTCC NO: M 2024308.

[0125] This invention optimizes the liquid culture medium formula and determines a stable subculturing method for wild Russula sp. Meizhou Songyuan 257. Even after 10-15 generations of continuous cultivation, it maintains a high mycelial growth rate and stable physiological functions, providing technical support for the development of liquid fermentation technology for wild Russula. This invention also optimizes the substrate for Russula sp. Meizhou Songyuan 257 substrate to promote mycelial growth, laying the foundation for understory cultivation technology of Russula. This invention isolates a symbiotic strain, Fusarium sp. 752, from the rhizosphere soil of Pinus massoniana, which promotes fruiting of wild Russula sp. Meizhou Songyuan 257, and uses this strain to establish understory cultivation technology for wild Russula sp. Meizhou Songyuan 257, providing technical support for the off-site cultivation and industrial-scale cultivation of wild Russula.

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

Claims

1. A method for understory cultivation of wild Russula sp. Meizhou Songyuan 257, characterized in that, Includes the following steps: (1) The liquid spawn of the wild Russula sp. Meizhou Songyuan257 was inoculated into the Russula spawn bag substrate and cultured to obtain Russula spawn bags; The red mushroom spawn substrate comprises the following raw materials by weight percentage: 18wt%-22wt% broadleaf sawdust, 8wt%-12wt% corn cob, 10wt%-20wt% wheat bran, 8wt%-12wt% rice husk, 1wt%-3wt% calcium carbonate, 0.5wt%-2wt% gypsum, 0.5wt%-2wt% fructose, 4wt%-6wt% red pine forest humus, with the balance being water; (2) Dig cultivation pits in the red pine forest, and sprinkle the red mushroom bag, red mushroom bag transition substrate, humus red pine leaves and Fusarium liquid in sequence to carry out fruiting culture; The transition substrate for the Russula ovata var. rubra spawn bags comprises the following raw materials by weight percentage: The mixture consists of 30wt%-40wt% red pine tree wood chips, 8wt%-12wt% wheat bran, 8wt%-12wt% rice husks, 10wt%-20wt% red pine forest humus, 2wt%-4wt% vermiculite, and the remainder is soil used to cover the roots of the red pine trees. The wild Russula sp. Meizhou Songyuan257 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2024308 and deposit date of January 31, 2024. The Fusarium species mentioned is Fusarium sp. 752, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66463 and deposit date of June 4, 2025.

2. The forest understory cultivation method according to claim 1, characterized in that, The mycelial biomass of the wild Russula sp. Meizhou Songyuan257 in the liquid culture 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℃.

3. The forest understory cultivation method according to claim 1, characterized in that, The mass ratio of the Russula ovata spawn bag to the Russula ovata spawn bag transition substrate is 1:

1.

4. The forest understory cultivation method according to claim 1, characterized in that, The thickness of the humus-rich red cone leaves is 4-6 cm.

5. The forest understory cultivation method according to claim 1, characterized in that, The concentration of the Fusarium spore suspension is not less than 10%. 8 CFU / mL; the amount of Fusarium oxysporum liquid added is 1wt%-3wt% of the transition substrate of the Russula ovata var. rubrum spawn bag.

6. The forest understory cultivation method according to claim 1, characterized in that, The humidity for fruiting cultivation is 80%-85%, and the temperature is 20-28℃.

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