Poria cocos strain with high disease resistance and high yield and application thereof

Abstract

The invention discloses a Wolfiporia cocos Wc2408, the preservation number of the Wolfiporia cocos Wc2408 in the China Center for Type Culture Collection is CCTCC NO: M 2026123, and in a plate confrontation experiment, the strain can still keep normal growth and cover a mould colony in the presence of soil competitive fungi represented by trichoderma longibrachiatum; the strain is applied to poria cocos cultivation, compared with a commercial strain, the strain has the capacity of resisting infection of infectious microbes such as trichoderma longibrachiatum and the like, the strain has the high poria cocos fruiting rate and the single plant yield, and the strain with the high space competition capacity, the stress resistance and the high poria cocos yield is provided for poria cocos production. And the method is suitable for large-scale production and market promotion.

Description

Technical Field

[0001] This invention belongs to the field of edible and medicinal fungal strain resources and cultivation technology, specifically involving a strain of Poria cocos (… Wolfiporia cocos Wc2408, and its use in Poria cocos cultivation to enhance resistance to soil-competitive fungi and increase Poria cocos yield, this strain has the ability to inhibit Trichoderma longifolia ( Trichoderma longibrachiatum Trichoderma harzianum ( T. harzianum ), green Trichoderma ( T.viride The activity of soil competitive fungi, represented by ) Background Technology

[0002] Poria cocos Wolfiporia cocos [(Schwein.) Ryvarden et Gilb.] Belonging to the order Polyporaceae, family Polyporaceae, and genus Poria, it is a traditional Chinese medicinal and edible fungus. Its dried sclerotia are used medicinally, possessing diuretic, spleen-strengthening, and calming effects. Modern pharmacological studies have shown that Poria is rich in triterpenoids, polysaccharides, and other bioactive components, exhibiting significant application value in areas such as immune regulation. With increasing market demand and the difficulty in obtaining wild Poria sclerotia resources, the standardized artificial cultivation of Poria is of great significance for ensuring a stable supply of raw materials.

[0003] Currently, the artificial cultivation of Poria cocos mainly uses pine wood as a substrate and is carried out in an open environment. This model simulates the natural habitat to some extent, but due to the presence of a large number of competing bacteria in the cultivation substrate and environment, coupled with the loss of natural antibacterial components in the pine wood during the drying process, it is highly susceptible to bacterial infection during the long growth cycle, which in turn leads to fungal decay, hindered colonization of Poria cocos mycelium, reduced wood conversion, reduced sclerotium yield, and even mold growth.

[0004] Saprophytic fungi, represented by genera such as *Trichoderma* and *Penicillium*, are abundant in soil. Under suitable conditions, they can cause diseases in edible mushroom cultivation, such as mold growth on substrate bags, competitive inhibition of mycelial colonization, and mold growth on primordia and mature fruiting bodies, commonly known as "green mold disease." Common soil-borne *Trichoderma* species include *Trichoderma harzianum* (…). T. harzianum Trichoderma longifolia ( T. longibrachiatum green Trichoderma ( T. viride Infections such as those caused by pathogens can inhibit the growth of target mycelia, leading to reduced yields or even cultivation failure. Current control measures mainly rely on chemical agents or environmental regulation, but chemical control carries the risk of pesticide residues, and breeding superior varieties that combine high yield with broad-spectrum resistance also faces technical bottlenecks. Therefore, screening for and obtaining Poria cocos germplasm resources with natural disease resistance is of significant practical importance for improving the biosafety and yield stability of Poria cocos cultivation. Summary of the Invention

[0005] This invention provides a strain of *Poria cocos* with strong spatial competition ability, stress resistance, and high yield of *Poria cocos*. Wolfiporia cocos Wc2408 was deposited on January 15, 2026 at the China Center for Type Culture Collection (CCTCCNO: M 2026123), located at Wuhan University, Wuhan, China.

[0006] The present invention relates to a strain of *Poria cocos*, Wc2408, which is a strain of *Poria cocos* resistant to infection by other microorganisms, specifically *Trichoderma*, the genus that causes "green mold" in *Poria cocos*. Trichoderma Soil-competitive fungi, represented by *Trichoderma longifolia* (spp.), including but not limited to *Trichoderma longifolia* (spp.) Trichoderma longibrachiatum Trichoderma harzianum ( T. harzianum ), green Trichoderma ( T.viride Although different Trichoderma species exhibit differences in molecular characteristics, their mechanisms of infecting Poria cocos are essentially similar. They rely on rapid growth and the secretion of cell wall-degrading enzymes to seize ecological niches. When faced with the rapid growth pressure of Trichoderma longiflora, Wc2408 of Poria cocos fungus can effectively inhibit the spread of the pathogen by forming dense antagonistic zones, partially or even completely covering the Trichoderma longiflora colonies, and reducing the spore production of Trichoderma longiflora. Thus, it exhibits a reverse compression effect on the growth space of Trichoderma longiflora.

[0007] Another objective of this invention is to apply the aforementioned Poria cocos strain Wc2408 to the cultivation of Poria cocos and to increase its yield. The cultivation methods include bag cultivation or log cultivation, wherein the logs are preferably pine logs; the cultivation environment can be greenhouse cultivation, forest cultivation, semi-wild cultivation or factory cultivation.

[0008] The cultivation method of Poria cocos sclerotia includes (1) making sclerotium bags; (2) inoculating with Poria cocos fungus Wc2408; (3) mycelial culture; and (4) growth and formation of sclerotia.

[0009] The culture medium formula for the above-mentioned cultivation bags, by weight percentage, is 50-65% pine sawdust, 15-25% hardwood sawdust, 8-12% wheat bran, 8-12% corn flour, 0.5-1.5% sucrose, 0.5-1.5% gypsum, and 0.2-0.6% magnesium sulfate; the moisture content of the culture medium is 55%-60%.

[0010] The culture temperature is 22-28℃, the relative humidity is about 50-60%, and the culture is carried out in the dark.

[0011] The growth and formation method adopts the bag inoculation method, in which the mycelium-covered bag is fixed to one end of a pine wood segment with a moisture content of 45-55%, and directly placed in the cellar for cultivation at 20-28℃ to promote mycelial colonization and formation of granules.

[0012] Through plate confrontation experiments and log cultivation experiments, it was verified that the Poria cocos fungus Wc2408 of this invention can still maintain normal growth and form sclerotia under the condition of contamination by other fungi, showing good resistance to contamination by other fungi. It is suitable for the production of Poria cocos sclerotia, which helps to reduce the risk of soil competitive fungal contamination represented by Trichoderma and improve cultivation stability.

[0013] Advantages and benefits of this invention: 1. The *Poria cocos* strain Wc2408 obtained by screening in this invention exhibits stable genetic traits and significant competitive advantage. Plate confrontation and cultivation experiments confirmed that this strain demonstrates high resistance in the presence of soil-competitive fungi, such as *Trichoderma longifolia*, while maintaining normal growth vigor, covering with fungal colonies, and forming sclerotia. It also possesses excellent commercial traits such as "thick skin and white flesh," firm texture, high *poria cocos* formation rate, and high yield per plant (average 2.5 kg). 2. The optimized cultivation substrate formula of this invention uses pine sawdust, hardwood sawdust, bran and corn flour as the main ingredients. The raw materials are widely available and inexpensive, which not only meets the carbon and nitrogen source requirements, but also effectively reduces the production cost and is conducive to promotion. 3. This invention adopts a comprehensive strategy of "bag inoculation" combined with "resistant strains". By utilizing the natural stress resistance and physical barrier of strain Wc2408, it solves the problem of susceptible contaminants in traditional open cultivation. This method promotes rapid mycelial colonization and can effectively resist soil-competitive fungal infection represented by Trichoderma longifolia without relying on chemical fungicides. It ensures the quality of Chinese medicinal materials from the source, significantly reduces the rate of rotten sticks, and is suitable for standardized production. Attached Figure Description

[0014] Figure 1 The hyphal morphology of Trichoderma longifolia T1 on a PDA plate; Figure 2 The microstructure of Trichoderma longifolia T1 under a scanning electron microscope; Figure 3 Phylogenetic tree of Trichoderma longibranch T1 constructed using the NJ method; Figure 4 Fifteen tested Poria cocos strains (corresponding to numbers Wc2401 to Wc2415 in the figure) were confronted with Trichoderma longifolia T1 on PDA plates. Colony morphology and resistance phenotype were observed on day 6. Figure 5 The images show the colony morphology (a) and hyphae (b) and spores (c) of the highly competitive fungal strain Wc2408 obtained in this invention after 5 days of culture on a PDA plate. Figure 6 Scanning electron microscope image of the mycelium of Poria cocos strain Wc2408; Figure 7Phylogenetic tree of Poria cocos strain Wc2408 constructed based on rDNA-ITS sequence and related type strains; Figure 8 This is a schematic diagram of the on-site cultivation process of Poria cocos logs in Example 5; Figure 9 This is a morphological image of the sclerotium of Poria cocos Wc2408 collected in Example 5. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the reagents and methods used in the embodiments are all conventional reagents and conventional methods. The strains of *Poria cocos* in the examples were sourced as follows: Wc2401 was purchased from Changning County Gumuyuan Chuangzhibo Port Bonsai Horticulture Farm; Wc2402 was purchased from Jialin Agricultural Products Store in Daguokou Township, Chuxiong City; Wc2403 was purchased from Longying Agricultural Products Wholesale Store in Ceheng County; Wc2404 was purchased from Qingyeshengyuan Agricultural Products Store in Chuxiong City; and Wc2405 was purchased from Jinhua Jisen Trading Co., Ltd. Wc2406-Wc24015 were collected from Dali, Cangyuan, and Chuxiong in Yunnan Province and identified as *Poria cocos* (…). Wolfiporia cocos ).

[0016] Example 1: Isolation of invasive fungal strains from Poria cocos planting logs 1. Isolation of invasive soil fungal strains Samples collected: Inoculum bags and bark of diseased pine logs with typical green mold contamination were selected from the Poria cocos cultivation site as isolation sources. Isolation method: In a clean bench, use a sterile inoculation knife to pick up a small amount of representative green mycelium and place it in a centrifuge tube containing sterile water, then suspend it by shaking. Using the dilution plating method, take appropriate amounts of the serially diluted solution and spread it on PDA (potato dextrose agar) plates, then incubate them upside down in a 28°C incubator until visible single colonies appear.

[0017] 2. Cultivation of invasive strains After the colonies have grown on the plates, select dominant colonies with strong growth and distinct morphological characteristics. Transfer fresh hyphae from the edge of these colonies to the center of a new PDA plate and purify them in a 28°C incubator. Repeat this process 2-3 times until a pure culture is obtained. Observe and record the colony morphology, color, growth rate, and hyphal microscopic characteristics of the purified strain on the PDA plates. Figure 1 , 2 ).

[0018] 3. Molecular biological identification of the strain (1) Genomic DNA extraction The purified strain was inoculated into PDB (potato dextrose broth) medium and cultured at 28°C with shaking for 5 days. Mycelia were collected by filtration, and genomic DNA was extracted from the test strain using the Ezup column-based fungal genomic DNA extraction kit from Sangon Biotech (Shanghai) Co., Ltd. The specific procedures were strictly performed according to the kit instructions: an appropriate amount of fresh mycelia were ground and broken up with liquid nitrogen, followed by lysis, column adsorption, and rinsing. Elution buffer was then used to obtain a high-purity fungal genomic DNA template.

[0019] (2) PCR amplification of ITS sequence The rDNA-ITS region was amplified using the universal fungal primers ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The PCR reaction system (25µL) consisted of 12.5µL of 2× RapidTaq Master Mix, 1µL of upstream primer ITS5 (10µM), 1µL of downstream primer ITS4 (10µM), 2µL of DNA template (approximately 50ng / µL), and nuclease-free water to a final volume of 25µL.

[0020] PCR amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 25 s, 54℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; final extension at 72℃ for 10 min.

[0021] (3) Sequencing and phylogenetic analysis After passing electrophoresis, the amplified products were sent to Beijing Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The sequencing results were assembled and edited using DNAMAN v8.0 software, and the sequences were submitted to the NCBI database for BLAST homology comparison. Reference sequences with high homology were selected, and a phylogenetic tree was constructed using the Neighbor-Joining method (NJ) with MEGA12.0 software. Bootstrap analysis was then performed (1000 replicates).

[0022] 4. Identification After isolation and purification, three dominant infectious fungal strains were obtained, numbered T1, T2, and T3. Morphological observation combined with molecular biological identification results showed that the ITS sequence of strain T1 was similar to that of *Trichoderma longicornis*. Trichoderma longibrachiatum The reference strains clustered into one line and had a high self-spreading support rate, compared with other closely related species (such as...). T. atroviride Clear distinctions (see phylogenetic tree) Figure 3Similarly, strains T2 and T3 were identified. Based on a combination of morphological characteristics and molecular phylogenetic analysis, the three isolated Trichoderma strains were identified as *Trichoderma longichair* (…). T. longibrachiatum Trichoderma harzianum ( T. harzianum ) and green Trichoderma ( T. viride ).

[0023] Example 2: Screening of disease-resistant Poria cocos strains 1. Determining the screening method Characteristics such as mycelial growth rate, spore production, extracellular lignocellulose-degrading enzyme activity, and toxin production capacity are positively correlated with the survival and exclusionary ability of fungi in the environment. The *Trichoderma longicornis* T1 strain possesses strong infectivity, including rapid mycelial growth, high spore production, strong extracellular lignocellulose degradation capacity, and the ability to secrete toxins that inhibit other fungal infections. Therefore, *Trichoderma longicornis* T1 was selected as the target strain for screening resistant *Poria cocos* strains in this experiment.

[0024] After activating 15 strains of Poria cocos, mycelial blocks were punched using a 6mm diameter punch and inoculated onto one end of a PDA plate (approximately 2.0cm from the edge). The plates were then incubated at a constant temperature of 28℃ in the dark with pre-incubation time gradients of 1 day, 2 days, and 3 days. After the preset time was reached, a Trichoderma longifolia T1 mycelial block of the same diameter was inoculated onto the other end of the plate (approximately 2.0cm from the edge) and incubated at 28℃ for 2 days. Observations showed that during the 1-2 day pre-incubation period, the Poria cocos colonies were too small and easily covered by Trichoderma, making it impossible to observe obvious antagonistic characteristics. During the 3-day pre-incubation period, Poria cocos and Trichoderma were in contact in the central area of ​​the plate, with clear antagonistic lines and the most significant difference in resistance phenotypes.

[0025] The results showed that direct confrontation culture on PDA medium more accurately reflected the interaction between Poria cocos and Trichoderma. Therefore, the final screening model parameters were determined as follows: Poria cocos strains were pre-cultured on PDA for 3 days, then inoculated with Trichoderma longifolia for confrontation culture, without the addition of physical barrier media.

[0026] 2. Screening of highly resistant Poria cocos strains Fifteen tested Poria cocos strains were inoculated onto one end of a PDA plate and pre-cultured at 28°C for 3 days. Then, Trichoderma longifolia strain T1 was inoculated onto the other end of the plate. Mycelial growth and interaction characteristics at the contact interface were observed at 2, 4, and 6 days post-inoculation. To quantitatively screen for the strain with the best resistance, the following three-level resistance evaluation criteria were established: Level 1 (-): Susceptible, Trichoderma hyphae completely cover the Poria cocos colony across the contact surface, and the Poria cocos hyphae show signs of shrinkage, autolysis or death; Level 2 (+): Moderately resistant. The Poria cocos strain was able to establish an effective defense, forming a clear brown antagonistic line at the contact interface, preventing further physical contact with Trichoderma. Level 3 (++): High resistance. The Poria cocos strain has extremely strong defense and competitive capabilities. Trichoderma hyphae cannot invade the Poria cocos colony at all. Moreover, the Poria cocos hyphae have strong growth potential, maintain their original morphology, and even exert reverse pressure on the growth space of Trichoderma.

[0027] The resistance characteristics of each *Poria cocos* strain at different culture stages are shown in Table 1. In the early stage of culture (day 2), before the two bacteria came into contact, all *Poria cocos* strains grew normally. As culture progressed to days 4-6, significant differentiation in resistance traits emerged among the strains. The colony morphology and resistance characteristics of each strain confronting *Trichoderma longifolia* on day 6 are shown in Table 1. Figure 4 As shown; (1) Some strains, represented by Wc2409, Wc2413 and Wc2415, were breached by Trichoderma hyphae on the 4th day, and by the 6th day the colonies were severely covered or infected, showing primary susceptibility (-).

[0028] (2) Most strains such as Wc2401, Wc2402, and Wc2403 showed secondary neutral resistance (+). Strains such as Wc2401 and Wc2402 were able to form obvious brown antagonistic lines, which limited the physical penetration of Trichoderma.

[0029] (3) Strains Wc2404 and Wc2408 exhibited specific level 3 high resistance (++), as shown by morphological observation (see...). Figure 3 On day 6, Wc2408 did not rely on brown antagonistic lines, but effectively blocked Trichoderma infection by forming a dense, white hyphal physical barrier; its colony edges were neat and its hyphae were dense, showing a significant competitive advantage over Trichoderma longibranchissis, and creating a reverse compression effect on Trichoderma colonies.

[0030] Based on the above rigorous screening results, Wc2408 was selected as the target superior strain for resistance to Trichoderma longifolia.

[0031] Table 1 Results of the confrontation experiment between Poria cocos strain and Trichoderma strain on T1 plate

[0032] Note: Data from day 2 was used to predict the growth potential and subsequent trends of the strains; the final evaluation was based on the state on day 6 (the screening endpoint).

[0033] It should be noted that although this embodiment used *Trichoderma longifolia* (T1) as the target strain to verify the defensive performance of Wc2408, given the high consistency among *Trichoderma longifolia*, *Trichoderma harzianum*, and *Trichoderma viride* in their infection kinetics and niche competition strategies (all being competitive fungi that rely on rapid growth to seize space and secrete extracellular enzymes), and considering that the core resistance mechanism exhibited by Wc2408 lies in constructing a robust, non-specific physical defense barrier through hyphal densification (manifesting as tertiary high resistance), those skilled in the art can reasonably expect that this broad-spectrum physical defense structure formed by Wc2408 will also have significant defensive activity against soil-borne pathogenic fungi such as *Trichoderma harzianum* and *Trichoderma viride*, which have similar hyperparasitic and infection patterns.

[0034] Example 3: Identification of Poria cocos Wc2408 This embodiment identifies the bacteria from three dimensions: macroscopic colony morphology, microscopic microstructure, and molecular phylogeny.

[0035] 1. Observation of colony morphology characteristics The purified Wc2408 strain was inoculated into the center of a PDA plate and incubated at 28°C in the dark. Colony growth characteristics were observed and recorded. The results are as follows: Figure 5 As shown in Figure a, strain Wc2408 exhibits strong growth on PDA medium, with white, dense, and cotton-like hyphae and well-developed aerial hyphae; the colony edges are neat and there is no pigment secretion. It can fully colonize a 90mm diameter plate in 5 days at 28℃.

[0036] 2. Observation using laser scanning confocal microscopy To further investigate the cell wall integrity and microstructural characteristics of Wc2408, fresh mycelial and spore samples cultured for 5 days were taken, stained, and observed under a laser scanning confocal microscope.

[0037] The results are as follows Figure 5 b、 Figure 5 As shown in Figure c, under a specific excitation wavelength, both the mycelium and spores of Wc2408 exhibit bright blue fluorescence. Under a microscope, the mycelium shows clear septa and branching structures with uniform diameter; the spores are elliptical or oval, plump, with smooth edges and uniform fluorescence signal distribution. This cell wall integrity and density are consistent with its strong physical barrier capability exhibited in macroscopic plate confrontation.

[0038] 3. Microscopic morphological observation using scanning electron microscopy (SEM) Wc2408 mycelial samples cultured for 5 days were fixed with 2.5% glutaraldehyde, dehydrated by ethanol gradient, critical point drying, and sputter-coated with gold, and then observed under a scanning electron microscope; the results are as follows. Figure 6As shown, the hyphae of Wc2408 are plump, relatively uniform in thickness, and approximately 3.5-4.8 μm in diameter. The surface of the hyphae is relatively smooth. The hyphae intertwine to form a dense network structure, which may be the structural basis for its formation of a physical barrier and effective resistance to Trichoderma infection.

[0039] 4. Molecular biological identification and phylogenetic analysis Genomic DNA was extracted from strain Wc2408 using the Ezup column-based fungal genomic DNA extraction kit from Sangon Biotech (Shanghai) Co., Ltd. The rDNA-ITS region was amplified by PCR using universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). Sequencing of the amplified products revealed that the full-length rDNA-ITS region of strain Wc2408 was 1627 bp. This sequence was submitted to the NCBI GenBank database for BLAST alignment analysis. The results showed that the ITS sequence of strain Wc2408 was similar to that of several other strains. Wolfiporia cocos The homology of the strains was over 99%; a phylogenetic tree was constructed using MEGA 12.0 software with the neighbor-joining method (bootstrap set to 1000 replicates), and the results are as follows. Figure 7 As shown, strain Wc2408 and Wolfiporia cocos The reference sequences clustered tightly into one line with a bootstrap value as high as 99%, which is significantly different from other polyporaceae fungi.

[0040] In summary, based on morphological characteristics and molecular biological evidence, the highly resistant selection strain Wc2408 was identified as a fungus belonging to the genus *Poria* of the family Polyporaceae. Wolfiporia cocos (Schwein.) Ryvarden et Gilb.

[0041] Example 4: Preparation of Poria cocos strain Wc2408 This embodiment aims to disclose a method for preparing production primary strains using the highly resistant and superior strain Wc2408 obtained through screening. The method mainly includes two steps: mother strain propagation and primary strain preparation. Here, the "mother culture" (also known as the primary culture) refers to the initial inoculum obtained by inoculating the screened and purified Wc2408 mycelium onto slant or plate culture media and then culturing and propagating it. It is mainly used for preserving the strain genotype and propagating the next level of strain. The "original culture" (also known as the secondary culture) refers to the mycelium obtained by transferring the mother culture mycelium to a solid culture medium such as sawdust and then culturing it. It serves as the direct inoculum source for subsequent production cultivation (tertiary culture). The specific preparation steps are as follows: 1. Preparation of mother culture (1) Preparation of mother culture medium (PDA): Potato glucose agar (PDA) medium was used, with the following formula: 200g peeled potatoes, 20g glucose, 15-20g agar powder, and distilled water to a final volume of 1000 mL.

[0042] Preparation method: Peel and cut potatoes into chunks, boil for 20-30 minutes, filter with gauze to collect the filtrate, add glucose and agar powder to dissolve, and add water to make up to 1000 mL. Dispense into test tubes (to make slant agar) or Erlenmeyer flasks, seal and autoclave at 121℃ and 0.105MPa for 30 minutes.

[0043] (2) Inoculation and culture: After the culture medium has cooled and solidified, under aseptic conditions, the tip hyphae of the highly resistant Poria cocos strain Wc2408, screened and identified in Example 3, are picked and inoculated into the center of the mother culture medium. The medium is then placed in a 28°C incubator and cultured in the dark for 5-7 days. Once the dense, white hyphae have covered the surface of the culture medium, the Wc2408 mother culture is obtained.

[0044] 2. Preparation of the original strain (1) Original culture medium formula Weigh the following raw materials by weight percentage (wt%): 60% pine sawdust, 19% hardwood sawdust, 10% wheat bran, 9.6% corn flour, 0.5% sucrose, 0.5% gypsum, and 0.4% magnesium sulfate.

[0045] (2) Preparation of culture medium After mixing the above dry materials evenly, add an appropriate amount of water and stir to adjust the moisture content of the culture medium to 55%-60%. Pack the mixed material into polyethylene inoculum bags, compact the material surface, and seal the bags with rings.

[0046] (3) Sterilization Place the filled mushroom bags in an autoclave and sterilize them at 121℃ and 0.105MPa for 2.5-3 hours to ensure that all bacteria and insect eggs are completely killed. After sterilization, remove the mushroom bags and place them in a clean and dry place to cool naturally to room temperature.

[0047] (4) Inoculation and culture In a sterile inoculation box or laminar flow hood, evenly cut the Wc2408 mother culture prepared in step 1 into pieces and inoculate them into the center of the cooled primary culture bags. Place the inoculated bags in a culture room at 22-28℃ in the dark, ensuring good ventilation and dryness during the incubation period, and regularly check and remove contaminated bags. After approximately 25-35 days, when the white mycelium has completely covered the bags and is dense and white, the physiologically mature, highly resistant Poria cocos strain Wc2408 primary culture has been obtained.

[0048] Example 5: Cultivation of Poria cocos Wc2408 on pine logs like Figure 8 As shown, this embodiment uses the traditional pine log cultivation method and selects strains Wc2401 and Wc2405 as controls to evaluate the disease resistance, sclerotium yield, and other cultivation indicators of the highly resistant strain Wc2408 obtained by plate culture under the complex environment of actual field.

[0049] In early spring, select healthy pine logs as the cultivation substrate. The pine logs should be at least 9cm in diameter and about 50-70cm in length. After removing part of the outer bark, allow them to air dry naturally until the moisture content of the logs is reduced to a suitable range before use.

[0050] After the temperature stabilizes and rises, choose a period of consecutive sunny days to dig cultivation pits along the slope in the pre-prepared poria cocos field. The cultivation pits should be approximately 0.8-1.2m long, with the width and depth controlled within the range of 40-60cm. A certain distance should be maintained between adjacent cultivation pits, and drainage ditches should be set up according to the terrain to avoid water accumulation affecting mycelial growth.

[0051] The prepared pine logs were placed in the cultivation pit as a culture medium. To ensure the comparability of the experimental results, the same amount of material was used in each treatment group: three pine logs of basically the same size (10cm in diameter and 65cm in length) were placed in parallel in each pit, and the total fresh weight of the pine logs in each pit was controlled at 7kg. Then, the cultivation bags (14×28cm in size, with a wet weight of about 650g of inoculum per bag) that had been covered with Poria cocos mycelium were directly inoculated onto one end of the pine log, so that the mycelium was in full contact with the cut surface of the log. After inoculation, no intermediate culture treatment was carried out. Instead, the inoculated pine logs were placed directly in the cultivation pit for dark environment culture.

[0052] After inoculation, cover the surface of the pine log with an appropriate amount of soil, with the soil thickness controlled between 4-6 cm, to maintain suitable temperature and humidity conditions. During the cultivation process, maintain a relatively stable environment inside the cultivation cellar and regularly observe the mycelial expansion and sclerotium formation; when cracks appear on the surface of the soil covering, promptly replenish the soil to prevent the sclerotia from being exposed and affecting normal development.

[0053] The traditional pine log cultivation method described above provides favorable conditions for the growth of Poria cocos mycelium in a dark and relatively constant environment, promoting the expansion of mycelium into the wood log and the gradual formation of sclerotia.

[0054] In the traditional pine log cultivation process described above, once the Poria cocos mycelium has fully expanded within the pine log, further inoculation treatment can be carried out to promote sclerotium formation and increase Poria cocos yield. Specifically, after burying the inoculated pine log in a cultivation cellar for 20-30 days, the covering soil layer is dug up from one side of the cultivation cellar, and the uninoculated end of the pine log is observed. If white mycelium has grown in this area and has a distinct Poria cocos odor, it indicates that the Poria cocos mycelium has colonized and expanded within the pine log.

[0055] Based on this, first- or second-generation sclerotia obtained from the same Poria cocos strain were selected as the inoculum material. The sclerotia should be mature, plump, with a diameter of 4-8 cm, a thin outer skin, a light red color, and a white internal tissue. They should be rich in pulp and not easily spoiled. After harvesting, the sclerotia can be stored at approximately 4°C for a short period of time.

[0056] During the inoculation process, the outer skin of the sclerotium is removed. At the uninoculated end of the pine log, the peeled sclerotium is inserted into a natural or artificially created crack on the surface of the log, ensuring full contact between the sclerotium and the mycelium inside the log. The log is then covered with soil again and cultured continuously to complete the inoculation process. Figure 9 ).

[0057] Table 2. Sclerotium results

[0058] Note: Sclerotium formation rate: refers to the percentage of logs with obvious sclerotia (diameter > 5cm) formed at the end of cultivation, out of the total number of effectively inoculated logs.

[0059] Firstly, regarding yield composition, although the average weight of sclerotia per cellar of Wc2408 (2.50 kg) was not statistically significantly different from that of the high-yielding commercial control strain Wc2401 (2.55 kg), Wc2408 achieved a high agglomeration rate of 90.6%, significantly better than the 84.6% of the control strain Wc2401, thanks to its extremely low contamination rate (<2%). This result indicates that, under the same cultivation scale, Wc2408 can substantially increase the total effective yield by reducing wood decay caused by contamination and wood scrapping due to contamination competition.

[0060] Secondly, regarding commercial characteristics, the Wc2408 sclerotium exhibits a unique "thick skin" trait (skin thickness 3.20 mm). This thickened epidermal structure is highly consistent with the dense hyphal barrier characteristics observed in the plate antagonism experiment, constituting an effective physical defense structure protecting the internal flesh from soil pathogens (such as Trichoderma). It is noteworthy that despite the slightly thicker epidermis, its internal flesh is white, firm, and has a moisture content of only 40.25% (significantly lower than the control), demonstrating extremely high dry matter accumulation and dry weight, fully meeting the quality requirements of high-quality Poria cocos: "firm sclerotium and sufficient powder."

Claims

1. A type of Poria cocos fungus ( Wolfiporia cocos Wc2408, its accession number at the China Center for Type Culture Collection is CCTCC NO: M 2026123.

2. The Poria cocos strain Wc2408 according to claim 1, characterized in that: It has the ability to inhibit Trichoderma longifolia ( Trichoderma longibrachiatum Trichoderma harzianum ( T. harzianum ), green Trichoderma ( T.viride The activity of soil competitive fungi, represented by ) 3. The application of the Poria cocos strain Wc2408 as described in claim 1 or 2 in the cultivation of Poria cocos and the improvement of Poria cocos yield.

4. The application according to claim 3, characterized in that: The culture medium formula for Poria cocos cultivation bags by weight percentage is 50-65% pine sawdust, 15-25% hardwood sawdust, 8-12% wheat bran, 8-12% corn flour, 0.5-1.5% sucrose, 0.5-1.5% gypsum, and 0.2-0.6% magnesium sulfate; the moisture content of the culture medium is 55%-60%.

Images