Low-carbon cultivation method of poria cocos by using yunnan pine stumps

Abstract

The application relates to the technical field of medicinal fungus cultivation, and discloses a low-carbon cultivation method for Poria cocos by using Yunnan pine felling stakes, which comprises the following steps: drilling holes on the surface of the Yunnan pine felling stakes; preparing and using a biomass guide rope carrying a signal synergistic activation bacterial agent to carry out directional activation inoculation; after in-situ activation culture, inoculating Poria cocos spores to carry out cultivation. The signal synergistic activation bacterial agent is compounded by high-efficiency lignin-degrading bacteria, combined nitrogen-fixing bacteria, lignin degradation product analogues and Poria cocos mycelium metabolic extract. The biomass guide rope guides the activation bacterial agent into the inside of the felling stake. The application also discloses the steps of continuously cultivating fast-growing edible fungi on the felling stake residues after Poria cocos is harvested, through the in-situ microbial activation step, lignin is effectively degraded and biological nitrogen fixation is simultaneously carried out, the carbon-nitrogen ratio of the substrate is improved, the planting period of Poria cocos is shortened, the unit yield and the effective component content are improved, and the cascade utilization of the felling stake biomass is realized.

Description

Technical Field

[0001] This invention relates to the field of medicinal fungi cultivation technology, specifically a low-carbon cultivation method for Poria cocos using Yunnan pine stumps. Background Technology

[0002] Poria cocos is an important medicinal fungus that relies on pine wood as a nutrient substrate for its growth. Traditional Poria cocos cultivation methods consume large amounts of pine logs, putting pressure on forest resources. Therefore, using Yunnan pine stumps left after logging for in-situ cultivation of Poria cocos has become a way to save resources and reduce forest waste.

[0003] However, there are several technical problems with directly using Yunnan pine stumps for Poria cocos cultivation in existing technologies. Yunnan pine has a dense wood structure and high lignin content, resulting in a very slow biodegradation process under natural conditions. This makes it difficult for Poria cocos mycelium to quickly infect and colonize, leading to an excessively long cycle from inoculation to the formation of effective biomass production. Simultaneously, the inherently high carbon-to-nitrogen ratio of the wood itself creates a nutrient environment unfavorable for the efficient growth of Poria cocos mycelium, limiting the biomass conversion efficiency of the substrate and thus affecting the final yield. Existing cultivation methods typically involve only simple physical drilling and inoculation, failing to effectively improve the physicochemical properties of the stump substrate, resulting in unstable cultivation success rates and yields. Furthermore, after the harvest of Poria cocos, a large amount of incompletely decomposed stump residue remains in the forest, failing to maximize its biomass value. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a low-carbon cultivation method for Poria cocos using Yunnan pine stumps. This method solves the problems of slow decomposition of lignocellulose, long mycelial colonization cycle due to substrate carbon-nitrogen imbalance, low biomass conversion rate, and insufficient utilization of stump resources in existing Yunnan pine stump Poria cocos cultivation methods.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a low-carbon cultivation method for Poria cocos using Yunnan pine stumps, comprising: The first aspect of this invention provides a low-carbon cultivation method for Poria cocos using Yunnan pine stumps, comprising the following steps: S1. Pre-treatment of stumps: Drill holes on the surface of the xylem of Yunnan pine stumps. The hole diameter is 1.5-2.0cm, the hole depth is 5-8cm, and the hole spacing is 15-20cm.

[0006] S2. Targeted activation inoculation: Preparation and use of biomass guide lines carrying signal-enhancing activating agents. This step specifically includes: First, a signal-enhancing activating bacterial agent was prepared. This agent was composed of a bacterial suspension of highly efficient lignin-degrading bacteria, a bacterial suspension of syn-nitrogenous bacteria, lignin degradation product analogs, and a metabolic extract of Poria cocos mycelium. The highly efficient lignin-degrading bacteria was *Phanerochaete chrysosporium*, and the syn-nitrogenous bacteria was *Azotobacter brownifolia*, with a bacterial suspension volume ratio of 1:1 to 1:1.5. The lignin degradation product analogs were guaiacol or vanillin, with a final concentration of 0.01%-0.05% (w / v) of the total volume of the agent. The metabolic extract of Poria cocos mycelium was obtained from the sterile supernatant after culturing Poria cocos mycelium in liquid culture medium.

[0007] Secondly, biomass guide ropes are prepared. Cotton or hemp ropes are soaked in a nutrient solution containing 1.0-2.0% corn steep liquor by weight and volume, and after being sterilized by moist heat, the prepared signal-enhancing activating bacterial agent is adsorbed.

[0008] Finally, the biomass guide wire carrying the signal-enhancing activating agent is inserted into the hole described in step S1.

[0009] S3. In-situ Activation Culture: The stumps that have undergone directional activation inoculation are cultured in situ in the forest under natural conditions for 30-60 days. During this stage, highly efficient lignin-degrading bacteria, induced by lignin degradation product analogs, secrete extracellular enzyme systems such as lignin peroxidase to decompose the lignin in the xylem of the stumps. At the same time, syn-nitrogen-fixing bacteria convert atmospheric nitrogen into a bioavailable nitrogen source, reducing the carbon-nitrogen ratio of the substrate.

[0010] S4. Inoculation with Poria cocos inoculum: Inoculate the holes in the stumps that have completed in-situ activation culture with Poria cocos inoculum. Specifically, insert a solid block of Poria cocos inoculum into the hole, ensuring close contact between it and the internal biomass guide cords and the activated xylem.

[0011] S5. Low-carbon management and harvesting in the forest: After inoculating the stumps with Poria cocos spawn, forest management is carried out, and the Poria cocos is harvested after it matures. The low-carbon management in the forest means that no artificial heating, cooling or supplemental lighting is carried out throughout the entire cultivation cycle.

[0012] The second aspect of this invention provides a low-carbon cultivation method for Poria cocos using Yunnan pine stumps. Based on the method described in the first aspect, after harvesting the Poria cocos in step S5, the method further includes step S6: inoculating the stump residue with fast-growing edible fungi for continued cultivation. The fast-growing edible fungi are oyster mushrooms or wood ear mushrooms.

[0013] This invention provides a low-carbon cultivation method for Poria cocos using Yunnan pine stumps. It has the following beneficial effects: 1. This invention pre-activates and cultivates the stumps in situ using an activating agent composed of highly efficient lignin-degrading bacteria and nitrogen-fixing bacteria before inoculating with Poria cocos mycelium. This pre-treatment decomposes the complex lignin structure in the xylem in advance and simultaneously replenishes bioavailable nitrogen sources in situ, effectively improving the physicochemical properties of the culture medium. This pretreatment creates a more easily absorbed and utilized nutrient environment for the subsequent growth of Poria cocos mycelium, thereby shortening the time required for mycelial colonization and growth.

[0014] 2. In the signal-enhancing activating agent used in this invention, lignin degradation product analogs and Poria cocos mycelial metabolic extracts are added as signaling molecules. The former can actively induce the expression of key degradation enzyme systems in the activated bacterial community, while the latter can reduce the antagonistic effects between different microbial populations. Simultaneously, the use of biomass guidelines as carriers and channels for the agent ensures that the functional bacterial community can rapidly establish a dominant population within the stump, improving the stability and directionality of the entire bioactivation process.

[0015] 3. After harvesting Poria cocos, this invention further utilizes the partially degraded stump residue to cultivate fast-growing edible fungi, achieving tiered and cyclical utilization of single forestry waste resources. This method not only improves the comprehensive conversion rate and total economic output of stump biomass, but also allows the final residue to be directly returned to the field as high-quality organic matter, avoiding waste generation and promoting the ecological improvement of forest soil. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0017] Example: Example 1 This embodiment provides a low-carbon cultivation method for Poria cocos using Yunnan pine stumps. The specific steps are as follows: Preparation of signal-enhancing activated bacterial agents After culturing *Phanerochaete chrysosporium* and *Azotobacter chrysophyte* separately, a concentration of 5 × 10⁻⁶ was prepared. 7 Bacterial suspension A with spores / mL and a concentration of 5×10 8 CFU / mL bacterial suspension B; Prepare a 0.75 g / L guaiacol ethanol solution (stock solution C); after culturing Poria cocos mycelium in liquid culture medium at 26°C for 12 days, take sterile metabolic extract solution D; Bacterial suspension A and bacterial suspension B were mixed at a volume ratio of 1:1.25. Then, 1.5 mL of stock solution C and 125 mL of solution D were added to the mixed bacterial suspension at a ratio of 1000 mL to obtain a signal-enhancing activating bacterial agent.

[0018] Preparation of biomass guide cord Cotton ropes with a diameter of 1.2 cm were cut into 12 cm segments and soaked in a nutrient solution containing 1.5% corn steep liquor for 3 hours, followed by moist heat sterilization (121°C, 35 minutes). After cooling, the signal-enhancing activating bacterial agent prepared in step 1 was adsorbed until saturation.

[0019] Cultivation Implementation On the surface of a Yunnan pine log with a diameter of 30cm, several holes with a diameter of 1.8cm, a depth of 6cm, and a spacing of 18cm were drilled. Insert the biomass guide wire prepared in step S2 into the hole and seal it with sterilized humus. Incubate in situ in the forest for 45 days. After activation, solid spawn blocks of Poria cocos were inoculated into the wells for natural forest management. Poria cocos is harvested after 8 months of cultivation. After the Poria cocos is harvested, oyster mushroom spawn is inoculated onto the stump remains for further cultivation.

[0020] Example 2 This embodiment provides a low-carbon cultivation method for Poria cocos using Yunnan pine stumps. The main difference between this method and Embodiment 1 is that the component ratios and process parameters are adjusted to the lower limit of the scope of the claims. The specific steps are as follows: Preparation of signal-enhancing activated bacterial agents The prepared concentration was 1.0 × 10⁻⁶. 7 A suspension of *Phanerochaete chrysospora* with spores / mL and a concentration of 1.0 × 10⁻⁶ spores / mL. 8 CFU / mL brown nitrogen-fixing bacteria suspension B; Prepare a 0.5 g / L vanillin ethanol solution (stock solution C); after culturing Poria cocos mycelium in liquid culture medium at 24°C for 10 days, take sterile metabolic extract solution D; Bacterial suspension A and bacterial suspension B were mixed at a volume ratio of 1:1. Then, 1 mL of stock solution C and 100 mL of solution D were added to each 1000 mL of the mixed bacterial suspension to obtain a signal-enhancing activating bacterial agent.

[0021] Preparation of biomass guide cord Cut 1.0 cm diameter hemp rope into 10 cm segments, soak them in a nutrient solution containing 1.0% corn steep liquor for 2 hours, and then perform moist heat sterilization (121°C, 30 minutes). After cooling, the signal-enhancing activating bacterial agent prepared in step 1 is adsorbed.

[0022] Cultivation Implementation On the surface of a Yunnan pine log with a diameter of 20cm, several holes with a diameter of 1.5cm, a depth of 5cm, and a spacing of 15cm were drilled. Insert the biomass guide wire prepared in step S2 into the hole and seal it with sterilized humus. Incubate in situ in the forest for 30 days. After activation, solid spawn blocks of Poria cocos were inoculated into the wells for natural forest management. Poria cocos is harvested after 10 months of cultivation. After the Poria cocos is harvested, Auricularia auricula-judae spawn is inoculated onto the stumps and planted for further cultivation.

[0023] Example 3 This embodiment provides a low-carbon cultivation method for Poria cocos using Yunnan pine stumps. The main difference between this method and Embodiment 1 is that the component ratio and process parameters are adjusted to the upper limit of the scope of the claims. The specific steps are as follows: Preparation of signal-enhancing activated bacterial agents The prepared concentration was 1.0 × 10⁻⁶. 8 A suspension of *Phanerochaete chrysospora* with spores / mL and a concentration of 1.0 × 10⁻⁶ spores / mL. 9 CFU / mL brown nitrogen-fixing bacteria suspension B; Prepare a 1.0 g / L guaiacol ethanol solution (stock solution C); after culturing Poria cocos mycelium in liquid culture medium at 28°C for 15 days, take sterile metabolic extract solution D; Bacterial suspension A and bacterial suspension B were mixed at a volume ratio of 1:1.5. Then, 2 mL of stock solution C and 150 mL of solution D were added to each 1000 mL of the mixed bacterial suspension to obtain a signal-enhancing activating bacterial agent.

[0024] Preparation of biomass guide cord Cotton ropes with a diameter of 1.5 cm were cut into 15 cm segments and soaked in a nutrient solution containing 2.0% corn steep liquor for 4 hours, followed by moist heat sterilization (121°C, 45 minutes). After cooling, the signal-enhancing activating bacterial agent prepared in step 1 was adsorbed.

[0025] Cultivation Implementation On the surface of a Yunnan pine log with a diameter of 40cm, several holes with a diameter of 2.0cm, a depth of 8cm, and a spacing of 20cm were drilled. Insert the biomass guide wire prepared in step S2 into the hole and seal it with sterilized humus. Incubate in situ in the forest for 60 days. After activation, solid spawn blocks of Poria cocos were inoculated into the wells for natural forest management. Poria cocos is harvested after 6 months of cultivation. After the Poria cocos is harvested, oyster mushroom spawn is inoculated onto the stump remains for further cultivation.

[0026] Comparative example: Comparative Example 1 Compared to Example 1, the difference is that the directional activation inoculation and in-situ activation culture steps are omitted. Instead, after the stump pretreatment, solid Poria cocos inoculum blocks are directly inoculated into the holes. All other aspects are the same.

[0027] Comparative Example 2 Compared with Example 1, the difference is that in step S1, when preparing the activated bacterial agent, guaiacol ethanol solution (stock solution C) and Poria cocos mycelium metabolic extract solution (solution D) are not added; that is, only a mixture of Phanerochaete chrysospora suspension A and Azotobacter brownifolia suspension B is used to adsorb the biomass guide rope. All other aspects are the same.

[0028] Comparative Example 3 Compared to Example 1, the difference is that biomass guide wires are not used. The directional activation operation in step S3 is changed to mixing the signal-enhancing activating agent prepared in step S1 with sterilized humus at a volume ratio of 1:10, and then filling the holes with this mixture. Everything else is the same.

[0029] Comparative Example 4 Compared with Example 1, the difference is that in step S1, only *Phanerochaete chrysospora* suspension A is used when preparing the activated bacterial agent, and *Azotobacter brownifolia* suspension B is not added. All other aspects are the same.

[0030] Test Example 1: Determination of Poria cocos planting cycle and unit yield Experimental steps Ten stumps from each of the prepared Examples 1, 2, 3, Comparative Examples 1, 2, 3 and 4 were taken as test samples, numbered and grouped.

[0031] The timeline begins from the day the Poria cocos spawn is inoculated onto each group of stumps. The base of each stump and the surrounding soil are checked daily at fixed times to observe and record the formation of Poria cocos primordia. When a spherical Poria cocos primordia with a diameter greater than or equal to 1 cm is first observed, the total number of days from the inoculation date to that date is recorded; this number of days is the establishment period for that sample.

[0032] After the cultivation cycle of each group of stumps was completed, each sample stump was excavated, and all the Poria cocos fruiting bodies growing from the stump were collected. Using an electronic balance with an accuracy of 0.01 kg, the total fresh weight of Poria cocos corresponding to each sample stump was weighed and recorded as the yield per unit stump of fresh weight.

[0033] Calculate the arithmetic mean of the planting period and the yield per unit fresh weight of stump for each of the 10 samples in each group.

[0034] Experimental data Table 1 Comparison of Poria cocos planting cycle and unit yield in each embodiment and comparative example. III. Results Analysis Experimental data show that, compared to Comparative Example 1, Examples 1, 2, and 3 significantly reduced the Poria cocos planting cycle and significantly increased the fresh weight yield per unit stump. This is because Examples 1, 2, and 3 used an activating agent containing highly efficient lignin-degrading bacteria and nitrogen-fixing bacteria for in-situ activation culture of the stumps. This step, through microbial action, decomposes the complex lignin structure in the stump xylem into more readily available carbon sources and simultaneously converts atmospheric nitrogen into bioavailable nitrogen, effectively adjusting the carbon-nitrogen ratio of the substrate. In contrast, Comparative Example 1 did not undergo this activation step; the Poria cocos mycelium directly faced undegraded lignocellulose, resulting in slow nutrient acquisition and limiting its planting and growth processes.

[0035] The results of Examples 1, 2, and 3, compared with those of Comparative Examples 2 and 3, show that the signaling molecule and biomass guide wire have a clear technical role in the cultivation process. Comparative Example 2, which did not use a signaling molecule, had inferior colonization cycle and yield data compared to the Example group, indicating that the introduction of lignin degradation product analogs and *Poria cocos* mycelial metabolic extracts played roles in inducing the expression of degradation enzyme systems and reducing interspecies antagonism among microorganisms, respectively. Comparative Example 3, which did not use a biomass guide wire, had inferior colonization cycle data compared to the Example group, indicating that this guide wire structure provided a physical basis for the rapid colonization of functional microbial communities and the establishment of dominant populations within the stump, and provided a pre-defined channel for subsequent *Poria cocos* mycelial infection.

[0036] Comparing the data from Examples 1, 2, and 3 with Comparative Example 4 reveals that the presence of nitrogen-fixing bacteria in the activating agent is a necessary condition for obtaining high yields. Comparative Example 4, even with the inclusion of lignin-degrading bacteria, still showed a significantly lower yield per unit stump fresh weight compared to the Example group, indicating that effective carbon source release alone, without sufficient nitrogen source matching, cannot support efficient biomass accumulation in Poria cocos mycelium. This invention, through the synergistic effect of two functional bacterial strains, simultaneously solves the technical problems of carbon source utilization and nitrogen source supply, forming the basis for obtaining yield data.

[0037] Test Example 2: Determination of the content of key active ingredients in Poria cocos Experimental steps From the cultivation results of Examples 1, 2, 3, Comparative Examples 1, 2, 3, and 4, 500g of Poria cocos fruiting bodies were randomly selected from each group. The samples were washed, sliced, and dried in a constant temperature forced-air drying oven at 60℃ until constant weight. The dried samples were then pulverized using a pulverizer and passed through an 80-mesh sieve to produce uniform Poria cocos powder, which was then packaged and sealed for later use.

[0038] Accurately weigh 1.0 g of each group of Poria cocos powder samples, place them in a flask, add 100 mL of distilled water, and extract in a 90℃ water bath for 2 hours. Repeat twice. Combine the two extracts, centrifuge, and collect the supernatant.

[0039] Add 4 times the volume of anhydrous ethanol to the supernatant and allow it to stand at 4°C for 12 hours to precipitate. Collect the precipitate by centrifugation; this is the crude polysaccharide.

[0040] A standard curve was prepared using the phenol-sulfuric acid method with glucose as the standard. An appropriate amount of sample solution was taken, and phenol solution and concentrated sulfuric acid were added. After the reaction, the absorbance was measured at a wavelength of 490 nm, and the polysaccharide content was calculated based on the standard curve.

[0041] Accurately weigh 1.0 g of each group of Poria cocos powder samples, place them in an Erlenmeyer flask, add 50 mL of ethanol, and extract by ultrasonication at 60 °C for 40 minutes. Filter and collect the filtrate.

[0042] A standard curve was prepared using the vanillin-perchloric acid method with oleanolic acid as the standard. An appropriate amount of sample filtrate was taken, evaporated to dryness, and then a colorimetric reagent (vanillin-perchloric acid solution) was added. The mixture was reacted in a 70°C water bath for 20 minutes, cooled, diluted, and the absorbance was measured at 548 nm. The total triterpenoid content was calculated based on the standard curve.

[0043] Each sample was tested three times, and the arithmetic mean of the contents of Poria cocos polysaccharides and total triterpenes was calculated. The results were expressed as a percentage of dry weight (%).

[0044] Experimental data Table 2 Comparison of the content of effective components of Poria cocos in each example and the comparative example. III. Results Analysis Data shows that the Poria cocos samples obtained through the methods of Examples 1, 2, and 3 had higher contents of Poria cocos polysaccharides and total triterpenes than all comparative groups. Comparative Example 1, which directly inoculated Poria cocos, had the lowest content of effective components due to the low nutrient utilization efficiency of the unbiodegraded stump substrate, which limited the synthesis of secondary metabolites by the Poria cocos mycelium. The Example groups, through the in-situ microbial activation step, provided a more sufficient and readily available carbon and nitrogen source for the metabolic activities of the Poria cocos mycelium, which is the material basis for their higher content of effective components.

[0045] Comparing the results of Examples 1, 2, and 3 with Comparative Examples 2 and 3, the roles of signaling molecules and biomass guide lines in increasing the content of effective components can be determined. Comparative Example 2 lacked the induction of signaling molecules, resulting in insufficient initiation efficiency and directionality of lignin degradation by the activated bacterial community. Comparative Example 3 lacked biomass guide lines, affecting the distribution and colonization rate of the functional bacterial community within the stump. Both situations led to insufficient biotransformation of the substrate, thus affecting the nutrient supply for the synthesis of secondary metabolites by the Poria cocos mycelium, ultimately resulting in lower levels of Poria cocos polysaccharides and total triterpenes compared to the Example groups.

[0046] A comparison of the results of Examples 1, 2, and 3 with Comparative Example 4 reveals the role of synergistic nitrogen-fixing bacteria in the synthesis of active ingredients. Comparative Example 4 only contained lignin-degrading bacteria, which, although able to decompose cellulose to provide a carbon source, lacked a simultaneous biological nitrogen fixation process, leading to an imbalance in the matrix carbon-nitrogen ratio. The synthesis of Poria cocos polysaccharides and total triterpenes requires the participation of multiple enzymes, and enzyme synthesis requires a sufficient nitrogen source. Nitrogen deficiency limits the overall metabolic level within the Poria cocos mycelium, thus restricting its ability to synthesize secondary metabolites. Therefore, the synergistic effect of the two functional bacterial species in the activating agent is a necessary condition for achieving a higher content of active ingredients in the fruiting body of Poria cocos.

[0047] Test Example 3: Determination of cascade utilization output Experimental steps The stumps remaining after the harvesting of Poria cocos in each group of Examples 1, 2, 3, Comparative Examples 1, 2, 3 and 4 were used as the experimental subjects of this test, and the original grouping and numbering were maintained.

[0048] For all groups of stump remains, the same batch of oyster mushroom solid spawn was used for inoculation. The inoculation method involved breaking the spawn block into small pieces, stuffing them into the holes and crevices formed by harvesting or decomposition, and covering them with moist soil.

[0049] After inoculation, all stump remains were cultivated in the same natural forest environment without any artificial intervention.

[0050] Harvesting begins from the date of inoculation with oyster mushroom spawn and continues until the fruiting bodies mature. At harvest, all fruiting bodies growing from a single stump are collected, and their total fresh weight is weighed using an electronic balance and recorded as the fresh weight yield of fast-growing edible fungi (kg / piece).

[0051] Calculate the arithmetic mean of the fresh weight yield of fast-growing edible fungi in each group.

[0052] Experimental data Table 3 Comparison of cascade utilization output between each embodiment and the comparative example III. Results Analysis Experimental data showed that the stump residues from Examples 1, 2, and 3 yielded significantly higher yields than all comparative groups when used for subsequent cultivation of fast-growing edible fungi. This indicates that the in-situ microbial activation step employed during the first stage of Poria cocos cultivation effectively degraded the lignocellulose in the stumps, transforming the stump residues into a culture medium rich in usable nutrients. Comparative Example 1, lacking any activation treatment, had stumps that remained dense after Poria cocos harvesting, exhibiting extremely low degradation and failing to provide sufficient nutrients for subsequent Pleurotus ostreatus mycelial growth, resulting in the lowest yield.

[0053] Comparing the data from Examples 1, 2, and 3 with Comparative Examples 2 and 3, the impact of the integrity of the in-situ activation process on the subsequent cascade utilization effect can be determined. Comparative Example 2 lacked the induction of signaling molecules, and Comparative Example 3 lacked the directional transport of biomass guidewires. Both of these resulted in lower efficiency and thoroughness of the first-stage biodegradation compared to the Example groups. Therefore, the total amount of nutrients available for oyster mushroom utilization in their stump residues was lower, ultimately leading to a lower oyster mushroom yield than the Example groups. This demonstrates the contribution of signal enhancement and directional transport technologies to improving the overall conversion rate of biomass from stumps.

[0054] The differences in data between Examples 1, 2, and 3 and Comparative Example 4 reveal the importance of the substrate carbon-nitrogen ratio for tiered utilization. The activating agent in Comparative Example 4 lacked syn-nitrogen-fixing bacteria, resulting in a nitrogen-limited state during the first stage of biodegradation and incomplete decomposition of lignocellulose. This left the stump residue with an excessively high carbon-nitrogen ratio, unsuitable for the rapid growth of fast-growing edible fungi like oyster mushrooms. The Example groups, through in-situ nitrogen fixation by syn-nitrogen-fixing bacteria, effectively reduced the substrate's carbon-nitrogen ratio. Their stump residue provided a more nutritionally balanced substrate for subsequent oyster mushroom cultivation, which is the foundation for achieving higher yields through tiered utilization.

[0055] The method in this embodiment can be used to execute the above method embodiments, and its principle and technical effect are similar, so it will not be described again here.

[0056] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A low-carbon cultivation method for Poria cocos using Yunnan pine stumps, characterized in that, Includes the following steps: S1. Pre-treatment of stumps: Drilling holes in the xylem surface of Yunnan pine stumps; S2. Targeted activation inoculation: Prepare a biomass guide cord carrying a signal-enhancing activating agent and insert the biomass guide cord into the hole. The signal-enhancing activating agent is a compound of highly efficient lignin-degrading bacteria, nitrogen-fixing bacteria, lignin degradation product analogs, and extracts of Poria cocos mycelium metabolism, wherein the volume ratio of highly efficient lignin-degrading bacteria to nitrogen-fixing bacteria is 1:1 to 1:1.

5. S3. In-situ activation culture: The inoculated stumps are cultured in the in-situ natural environment of the forest for 30-60 days; S4. Inoculation with Poria cocos strain: Inoculate the poria cocos strain into the holes of the stumps that have completed in-situ activation culture; S5. Low-carbon management and harvesting in the forest: Low-carbon management of stumps after inoculation with Poria cocos spawn is carried out in the forest, and the Poria cocos is harvested after it matures. The highly efficient lignin-degrading bacteria is *Proteus xanthosporium*, and the combined nitrogen-fixing bacteria is *Azotobacter browniformis*. The lignin degradation product analogue is guaiacol or vanillin, and its addition amount in the signal-enhancing activated microbial agent is 0.01%-0.05% (w / v) of the total volume of the microbial agent. The preparation method of the biomass guide cord includes: After soaking and sterilizing cotton or hemp rope in a nutrient solution containing 1.0-2.0% corn steep liquor by weight, the rope is then adsorbed with the signal-enhancing activating agent.

2. The method for low-carbon cultivation of Poria cocos using Yunnan pine stumps according to claim 1, characterized in that, The metabolite extract of Poria cocos mycelium is an extract of the sterile supernatant after liquid fermentation culture of Poria cocos mycelium.

3. The method for low-carbon cultivation of Poria cocos using Yunnan pine stumps according to claim 1, characterized in that, In step S1, the diameter of the drilled hole is 1.5-2.0 cm, the hole depth is 5-8 cm, and the hole spacing is 15-20 cm.

4. The method for low-carbon cultivation of Poria cocos using Yunnan pine stumps according to claim 1, characterized in that, In step S4, the method of inoculating with Poria cocos is to insert a solid Poria cocos culture block into the hole so that it is in close contact with the biomass guide cord.

5. The method for low-carbon cultivation of Poria cocos using Yunnan pine stumps according to claim 1, characterized in that, The aforementioned low-carbon management in the forest means that no artificial heating, cooling, or supplemental lighting is carried out throughout the entire cultivation cycle.

6. The method for low-carbon cultivation of Poria cocos using Yunnan pine stumps according to claim 1, characterized in that, After harvesting Poria cocos in step S5, step S6 is also included: Fast-growing edible fungi were inoculated onto the stumps for further cultivation.

7. A low-carbon cultivation method for Poria cocos using Yunnan pine stumps according to claim 6, characterized in that, The fast-growing edible fungus is either oyster mushroom or wood ear fungus.

Images