A method for efficient utilization of straw resources and improvement of soil carbon pool by interplanting grain and mushroom

Abstract

This invention discloses a method for efficient utilization of straw resources and enhancement of soil carbon pool in grain-mushroom intercropping, comprising the following steps: preparing a layered substrate, the layered substrate comprising 40-45 parts by weight of wheat straw, 35-40 parts by weight of corn straw, 5-8 parts by weight of rice bran, 1-2 parts by weight of gypsum, and 0.5-1 parts by weight of calcium carbonate; subjecting the layered substrate to hydrothermal treatment at 65-70℃ for 90±5 minutes, and then cooling it to 25±2℃; inoculating the cooled substrate with Trichoderma harzianum strain T-22 at an inoculation amount of 10% of the substrate weight. 6 The concentration of CFU / g was measured and cultured at 25±2℃ and 75-85% relative humidity for 24-36 hours. A sequential inoculation system was used, first inoculating oyster mushrooms at 3% of the substrate weight, then inoculating shiitake mushrooms at 2.5% of the substrate weight after 7 days, and finally inoculating king oyster mushrooms at 2% of the substrate weight after another 7 days.

Description

Technical Field

[0001] This invention relates to the field of agricultural resource recycling, and in particular to a method for efficient utilization of straw resources and enhancement of soil carbon pool in grain-mushroom intercropping, belonging to the interdisciplinary field of ecological agriculture, mycology and soil carbon sequestration technology. Background Technology

[0002] With the intensification of global climate change and the deepening of the concept of sustainable development, the resource utilization of agricultural waste and the construction of soil carbon pools have become international research hotspots. China produces approximately 800 million tons of straw resources annually, but traditional utilization methods are inefficient. Common methods such as returning straw to the field or direct burning not only waste the organic carbon resources in the straw but may also lead to environmental pollution problems. At the same time, the organic matter content of farmland soils in my country is generally low, and soil quality is severely degraded, seriously restricting the sustainable development of agriculture.

[0003] Currently, technologies related to straw resource utilization and soil carbon pool enhancement mainly focus on the following aspects:

[0004] Hu Quanyi et al. reported in "Rice-edible mushroom Stropharia rugosoannulatarotation mitigates net global warming potential while enhancing soil fertility and economic benefits" (European Journal of Agronomy, 2024) that a rice-Stropharia rugosoannulata rotation system can increase soil organic carbon by 30%. However, this system uses a rotation rather than intercropping model, so the carbon pool enhancement efficiency is limited. Furthermore, it uses only a single species and fails to fully utilize the differentiated degradation capabilities of different edible fungi on straw components.

[0005] Chinese patent CN105165362A discloses "a method for intercropping corn and king oyster mushroom", which adopts a 4:2 ridge-to-furrow ratio planting pattern. However, this method only involves the combination of a single fungal species and a single crop, and does not consider the construction of soil carbon pool, lacking a systematic resource recycling design.

[0006] The study by Yin et al., “Evaluation of long-term carbon sequestration of biochar insoil with biogeochemical field model” (Science of the Total Environment, 2022: 153576), showed that agricultural waste biochar can sequestrate up to 1.2 tons of carbon per hectare per year. However, the carbon sequestration efficiency relying solely on biochar addition is still limited and has not been effectively combined with edible fungi cultivation systems.

[0007] CN103570453A discloses "a cultivation substrate for enoki mushrooms made from wheat straw and its preparation method", which focuses on the cultivation substrate formula of a single strain, without considering the possibility of sequential inoculation of multiple strains, and does not involve field intercropping patterns and soil carbon pool enhancement.

[0008] The above-mentioned existing technologies have the following defects: (1) They only use a single strain of fungi for cultivation, and fail to make full use of the complementary enzyme systems of different edible fungi; (2) They fail to organically combine edible fungi cultivation with field crop production, resulting in low resource utilization efficiency; (3) They lack a systematic design of the straw-fungus-soil carbon pool, resulting in limited carbon sequestration efficiency; (4) They do not consider the promoting effect of microbial pretreatment on substrate decomposition and mycelial growth; (5) There is insufficient research on the carbon pool enhancement mechanism, making it difficult to achieve targeted regulation.

[0009] Therefore, there is an urgent need to develop a technical system that can comprehensively utilize the differentiated degradation capabilities of various edible fungi, combine them with field grain crop production, and significantly enhance the soil carbon pool, so as to achieve the dual goals of efficient utilization of straw resources and enhanced farmland carbon sequestration. Summary of the Invention

[0010] This invention aims to solve the above-mentioned technical problems and provides a method for efficient utilization of straw resources and improvement of soil carbon pool in grain-mushroom intercropping. By constructing an intercropping system of grain crops and edible fungi with sequential inoculation of multiple strains, combined with the application of biochar-enhanced fertilizer, multiple goals are achieved, including efficient utilization of straw resources, high yield of edible fungi, and significant improvement of soil carbon pool.

[0011] To address the aforementioned technical problems, this invention provides a method for the efficient utilization of straw resources and enhancement of soil carbon pool in grain-mushroom intercropping, comprising the following steps:

[0012] A layered matrix was prepared, comprising 40-45 parts by weight of wheat straw, 35-40 parts by weight of corn straw, 5-8 parts by weight of rice bran, 1-2 parts by weight of gypsum, and 0.5-1 parts by weight of calcium carbonate. The layered matrix was subjected to hydrothermal treatment at 65-70°C for 90±5 minutes, and then cooled to 25±2°C. Trichoderma harzianum strain T-22 was inoculated into the cooled matrix at an inoculation amount of 10% of the matrix weight. 6 The substrate was cultured at CFU / g at 25±2℃ and 75-85% relative humidity for 24-36 hours. A sequential inoculation system was used, first inoculating with oyster mushrooms at 3% of the substrate weight, then inoculating with shiitake mushrooms at 2.5% of the substrate weight after 7 days, and then inoculating with king oyster mushrooms at 2% of the substrate weight after another 7 days. Mushroom beds were prepared between rows of grain crops at a ratio of 2:1:2, where 2 rows of grain crops, 1 row of mushroom bed, and 2 rows of grain crops were planted, with a width of 60-70 cm. After harvesting the mushroom beds, waste mushroom substrate was applied to the soil at a rate of 15-20 tons / hectare, and 2-3 tons / hectare of biochar-fortified fertilizer was added. The soil organic carbon content was monitored and verified to increase by 25-35% within 24 months.

[0013] Preferably, the wheat straw and corn straw have a particle size of 2-5 cm and a moisture content of 14-18%, obtained through mechanical processing; the final moisture content of the layered matrix is ​​65-68%, and the C:N ratio is 28-32:1.

[0014] Preferably, in the hydrothermal treatment step, the ratio of water to substrate is 4:1 (volume ratio), and after treatment, the substrate is naturally cooled at a cooling rate of 1-2℃ / 15 minutes for 4-6 hours.

[0015] Preferably, the Trichoderma harzianum strain T-22 (ATCC 20847) is added in the form of a water-soluble powder. A suspension is prepared by dissolving 5 grams of the product in 10 liters of sterile water and spraying it evenly on the surface of the substrate. 1 liter of suspension is used for every 10 kilograms of substrate.

[0016] Preferably, in the sequential inoculation system: oyster mushroom (Pleurotus ostreatus) is inoculated on day 0, with a culture temperature of 24-26℃ and a relative humidity of 85-90%; shiitake mushroom (Lentinula edodes) is inoculated on day 7, with a culture temperature of 21-24℃ and a relative humidity of 80-85%; king oyster mushroom (Pleurotus eryngii) is inoculated on day 14, with a culture temperature of 18-22℃ and a relative humidity of 80-85%; each spawn is inoculated using a stratified or fixed-point inoculation method, with oyster mushrooms preferentially inoculated in wheat straw areas, shiitake mushrooms preferentially inoculated in nutrient-rich supplementary areas, and king oyster mushrooms preferentially inoculated in corn straw areas.

[0017] Preferably, the grain crop used in the grain-mushroom intercropping is selected from wheat or corn, and the mushroom bed is deployed 30-45 days after the grain crop emerges. The mushroom bed is maintained at a relative humidity of 75-85% using a drip irrigation system with a dripper spacing of 30 cm and a daily water supply of 2-4 liters / square meter.

[0018] Preferably, the biochar-fortified fertilizer is composed of the following components: 55-60 parts by weight of pyrolytic agricultural residue, which is obtained by pyrolysis at 450-500°C for 2 hours; 25-30 parts by weight of a mixture of fresh straw; and 10-15 parts by weight of a mineral supplement, which includes 2-3 parts by weight of dolomite lime, 1-2 parts by weight of phosphate rock, and 0.5-1 parts by weight of potassium sulfate.

[0019] Preferably, the preparation of the pyrolytic agricultural residue includes the following steps: mixing wheat straw and corn straw with a moisture content of 8-12% in a 1:1 ratio and cutting them to a length of 2-5 cm; in a fixed-bed slow pyrolysis reactor, heating from ambient temperature to 250°C at a heating rate of 10°C / min and holding for 30 minutes; then heating from 250°C to 450-500°C at a heating rate of 5°C / min and holding at this temperature for 120±10 minutes; naturally cooling to below 50°C under an inert atmosphere, and then exposing to ambient air in a ventilated area for 24 hours to stabilize the biochar.

[0020] Preferably, the waste mushroom substrate is collected after 2-3 harvests. First, the waste mushroom substrate is broken into 5-10 cm pieces and spread evenly in the field. Then, a shallow disc rake with a depth of 10-15 cm is used to mix the soil. The mixing should be carried out within 7 days after the waste mushroom substrate is removed.

[0021] Preferably, the steps for monitoring and verifying soil organic carbon content include: sampling at soil depths of 0-10 cm, 10-20 cm, and 20-30 cm; collecting 5 composite samples per hectare, each composite sample consisting of 10 individual soil cores; determining the total organic carbon content using the Dumas dry combustion method; performing carbon component analysis using the density separation method to separate light components (<1.8 g / cm³), intermediate components (1.8-2.2 g / cm³), and heavy components (>2.2 g / cm³); determining the δ¹³C value by isotope ratio mass spectrometry to track changes in carbon sources; and achieving a system carbon sequestration rate of 2.1-3.4 tons of CO₂ equivalent per hectare per year.

[0022] The present invention has the following beneficial effects:

[0023] 1. By using a sequential inoculation system of three edible fungi, the complementary enzyme systems of different edible fungi are fully utilized, improving the conversion efficiency of straw resources and increasing yield by 31-42% compared with single-strain cultivation;

[0024] 2. Pretreatment with Trichoderma harzianum strain T-22 promoted partial degradation of the substrate, creating favorable conditions for subsequent growth of edible fungi and shortening the mycelial growth cycle by 15-25%.

[0025] 3. The grain-mushroom intercropping model achieves efficient use of land resources. At the same time, the interaction between the grain crop roots and mycelium promotes crop growth and increases grain yield by 12-18%.

[0026] 4. The combined application of waste mushroom substrate and biochar-enhanced fertilizer forms a stable soil carbon pool, with a carbon sequestration efficiency 2-5 times higher than that of conventional straw return to the field, and the system carbon sequestration rate reaches 2.1-3.4 tons of CO2 equivalent / hectare / year;

[0027] 5. It has achieved a win-win situation in terms of economic and ecological benefits, with the overall economic benefits of the system increasing by 57-92%, while significantly increasing the soil organic matter content and improving soil structure and fertility. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0029] I. Raw Materials and Reagents

[0030] 1. Wheat straw: Wheat straw within 90 days after harvest, with no obvious mold and a moisture content of <20%.

[0031] 2. Corn stalks: Corn stalks within 90 days of harvest, with no obvious mold and a moisture content of <20%.

[0032] 3. Rice bran: A byproduct of rice milling, containing 12-15% protein, 15-20% lipids, 34-52% carbohydrates, and 7-11% dietary fiber.

[0033] 4. Gypsum: Agricultural grade calcium sulfate dihydrate (CaSO4·2H2O), purity ≥95%, particle size 95% passing through a 150μm sieve.

[0034] 5. Calcium carbonate: Agricultural grade calcium carbonate (CaCO3), purity ≥95%, particle size 95% passing through a 150μm sieve.

[0035] 6. Trichoderma harzianum strain T-22: Trichoderma harzianum strain T-22 (ATCC 20847), trade name RootShield®, manufactured by BioWorks, with a viable spore content ≥2×10⁻⁶. 9 CFU / g.

[0036] 7. Oyster mushroom strain: Pleurotus ostreatus commercial high-yield strain P-80, provided by the China Edible Fungi Research Institute. It is a grain-type strain with white mycelium and vigorous growth.

[0037] 8. Shiitake mushroom spawn: Lentinula edodes commercial wide-temperature strain LE-4, provided by the Institute of Edible Fungi, Chinese Academy of Agricultural Sciences, spawned from sawdust segments, with dense white mycelium and brown mycelial covering.

[0038] 9. King oyster mushroom strain: Pleurotus eryngii commercial strain PE-2, provided by Shanghai Academy of Agricultural Sciences, grain-type strain, with dense white mycelium.

[0039] 10. Dolomite lime: Agricultural grade calcium magnesium carbonate [CaMg(CO3)2], purity ≥90%, particle size 75-150μm.

[0040] 11. Phosphate rock: Agricultural grade calcium phosphate [Ca3(PO4)2], P2O5 content ≥28%, particle size 100-250μm.

[0041] 12. Potassium sulfate: Agricultural grade potassium sulfate [K2SO4, CAS No.], purity ≥95%, K2O content ≥50%.

[0042] II. Equipment and Instruments

[0043] 1. Rotary straw shredder: power 15-25kW, processing capacity 500-1000kg / h, equipped with 5cm screen.

[0044] 2. Fixed-bed slow pyrolysis reactor: 100L volume, maximum temperature up to 800℃, adjustable heating rate, equipped with a temperature monitoring system and inert gas introduction device.

[0045] 3. Hydrothermal treatment device: Stainless steel immersion tank, equipped with a constant temperature heating system, with a temperature control accuracy of ±1℃.

[0046] 4. Low-pressure drip irrigation system: 16mm polyethylene main pipe, 12mm drip tape, integrated dripper, spacing 30cm, flow rate 1-2L / h, working pressure 1.0-1.5bar.

[0047] 5. Shallow disc harrow: Adjustable working depth (5-20cm), working width 2m, suitable for light to moderate farmland operations.

[0048] 6. Soil sampler: Stainless steel soil drill, inner diameter 2.5cm, length 50cm, with depth scale.

[0049] 7. Elemental analyzer: Elementar vario MACRO cube elemental analyzer, used for the determination of C and N content.

[0050] 8. Isotope ratio mass spectrometer: Thermo Fisher Scientific Delta V isotope ratio mass spectrometer, used for δ¹³C value determination.

[0051] Example 1: Grain-Mushroom Intercropping Method Based on Endpoint Values ​​of Wheat Straw Substrate Ratio and Median Values ​​of Corn Straw Substrate Ratio

[0052] This embodiment provides a method for intercropping grains and mushrooms using wheat straw substrate ratio endpoints and corn straw substrate ratio intermediate values, specifically including the following steps:

[0053] (1) Layered substrate preparation: First, fresh wheat straw was collected and cut into 2-3 cm lengths using a rotary straw shredder. The moisture content after screening was 14%. At the same time, corn straw was collected and cut into 3-4 cm lengths, with the moisture content controlled at 16%. The substrate was prepared according to the following ratio: 40 parts by weight of wheat straw, 37.5 parts by weight of corn straw, 6 parts by weight of rice bran, 1.5 parts by weight of gypsum, and 0.8 parts by weight of calcium carbonate. First, wheat straw was laid at the bottom layer, corn straw in the middle layer, and rice bran, gypsum, and calcium carbonate were mixed and sprinkled on the top layer. The mixture was mixed in a horizontal paddle mixer at a speed of 20 RPM for 15 minutes. During the mixing process, water was gradually added until the final moisture content of the substrate reached 65%. At this point, the C:N ratio of the substrate was measured to be 30:1.

[0054] (2) Hydrothermal Treatment: The mixed substrate is placed into a heat-resistant mesh container, ensuring the substrate thickness does not exceed 25 cm. A water bath at 65°C is prepared, with a water-to-substrate volume ratio of 4:1. The substrate is completely immersed in the water, maintaining the water temperature between 65-67°C for 90 minutes. The water temperature is monitored every 15 minutes during treatment to ensure stability. After treatment, the substrate is placed on a clean drainage rack and allowed to cool naturally at an ambient temperature of 22°C at a cooling rate of approximately 1.5°C / 15 minutes for 5 hours, until the final substrate temperature drops to around 25°C.

[0055] (3) Trichoderma harzianum inoculation: Dissolve 5 grams of RootShield® commercial Trichoderma harzianum T-22 strain powder (ATCC20847) in 10 liters of sterile water to prepare a uniform suspension. Use a low-pressure sprayer to evenly spray the suspension onto the substrate surface, ensuring that 1 liter of suspension is used for every 10 kg of substrate, and the inoculation amount reaches 10% of the substrate weight. 6 CFU / g. After spraying, gently agitate the substrate to ensure even distribution of Trichoderma harzianum. Incubate the inoculated substrate at 25°C and 80% relative humidity for 30 hours, during which time a white mycelial network begins to appear on the substrate surface.

[0056] (4) Sequential inoculation system: First, on day 0, inoculate with Pleurotus ostreatus strain P-80 at a rate of 3% of the substrate weight. A layered inoculation method was used, with a layer of Pleurotus ostreatus seed grains evenly sprinkled between substrate layers every 10 cm, focusing on the wheat straw area. The inoculated substrate was cultured for 7 days at 24-25℃ and 85-88% relative humidity. At this time, it was observed that Pleurotus ostreatus mycelium covered approximately 70% of the substrate surface, mainly concentrated in the wheat straw area.

[0057] On day 7, inoculate with Lentinula edodes strain LE-4 at a rate of 2.5% of the substrate weight. Use a spot inoculation method, making small holes 2-3 cm deep every 10 cm along the substrate surface, and filling each hole with 5-8 grams of shiitake mushroom sawdust, focusing on areas rich in rice bran. After inoculation, adjust the ambient temperature to 22℃ and reduce the relative humidity to 82%, and continue cultivation for 7 days.

[0058] On day 14, inoculate with Pleurotus eryngii strain PE-2 at a rate of 2% of the substrate weight. Use the trench inoculation method, creating shallow trenches 1-2 cm deep on the substrate surface. Distribute Pleurotus eryngii inoculum evenly along the trenches, using approximately 20 grams of inoculum per meter of trench, focusing on areas rich in corn stalks. After inoculation, further lower the ambient temperature to 20°C and maintain a relative humidity of 80-82%, continuing cultivation for 14-16 days until the substrate is completely covered by mycelium.

[0059] (5) Field configuration for grain-mushroom intercropping: Select farmland planted with wheat (variety: Jimai 22), and configure the mushroom beds 35 days after wheat emergence. Arrange the wheat in a 2:1:2 ratio (2 rows of wheat: 1 row of mushroom bed: 2 rows of wheat), maintaining a wheat row spacing of 45 cm, a distance of 75 cm between the wheat row and the mushroom bed, and a mushroom bed width of 65 cm. Install a drip irrigation system, with drip tape laid along the center of the mushroom bed, dripper spacing set at 30 cm, and a flow rate of 1.5 L / h. Adjust the irrigation frequency according to the temperature; in spring when the average daily temperature is 18-25℃, irrigate twice a day, 2 L / m² each time, maintaining a relative humidity of about 80% in the mushroom bed.

[0060] (6) Application of Waste Mushroom Substrate and Biochar: After three harvests (approximately 90-100 days), waste mushroom substrate was collected, and the yield was measured to be 3.1 tons fresh weight / hectare. The waste mushroom substrate was broken into 5-8 cm blocks and evenly spread in the field at a rate of 17 tons / hectare. At the same time, 2.5 tons / hectare of biochar-fortified fertilizer was applied. The biochar-fortified fertilizer consisted of the following components: 58 parts by weight of pyrolyzed agricultural residue (obtained by pyrolysis at 475°C for 2 hours), 27 parts by weight of fresh straw mixture, and 15 parts by weight of mineral supplement (including 2.5 parts by weight of dolomite lime, 1.5 parts by weight of phosphate rock, and 0.8 parts by weight of potassium sulfate). The waste mushroom substrate and biochar-fortified fertilizer were mixed with the topsoil using a shallow disc rake with a set depth of 12 cm. The mixing operation was completed on the 5th day after the waste mushroom substrate was collected.

[0061] (7) Soil carbon pool monitoring: Stratified sampling was conducted at three soil depths: 0-10 cm, 10-20 cm, and 20-30 cm. Five sampling points were randomly placed per hectare, and ten soil cores were collected from each sampling point to form a composite sample. The total organic carbon content of the soil was determined using the Dumas dry combustion method, carbon component analysis was performed using the density separation method, and the δ¹³C value was determined by isotope ratio mass spectrometry. The monitoring results showed that within 24 months after treatment, the organic carbon content of the 0-30 cm soil layer increased by an average of 28.5%, and the carbon sequestration rate reached 2.4 tons of CO2 equivalent / hectare / year.

[0062] Example 2: Grain-Mushroom Intercropping Method Based on the Median Value of Wheat Straw Substrate Ratio and the End Value of Corn Straw Substrate Ratio

[0063] This embodiment provides a method for intercropping grains and mushrooms using wheat straw substrate ratios at intermediate and corn straw substrate ratios, specifically including the following steps:

[0064] (1) Layered substrate preparation: Wheat straw after harvest was collected and processed to a length of 4-5 cm using a rotary shredder, with a moisture content of 16%; corn straw after harvest was also collected and cut to a length of 2-3 cm, with a moisture content of 15%. The substrate was prepared according to the following ratio: 42.5 parts by weight of wheat straw, 35 parts by weight of corn straw, 7 parts by weight of rice bran, 1.2 parts by weight of gypsum, and 0.6 parts by weight of calcium carbonate. Wheat straw was placed at the bottom layer, corn straw in the middle layer, and the rice bran, gypsum, and calcium carbonate were mixed and placed at the top layer. The mixture was then mixed in a horizontal paddle mixer at 22 RPM for 18 minutes, gradually adding water until the final moisture content of the substrate reached 67%. The C:N ratio of the substrate was measured to be 29:1.

[0065] (2) Hydrothermal Treatment: The mixed substrate was placed into a heat-resistant mesh container, ensuring the substrate thickness did not exceed 28 cm. A water bath at 68°C was prepared, with a water-to-substrate volume ratio of 4:1. The substrate was completely immersed in the water, maintaining the water temperature between 67-70°C for 85 minutes. The water temperature was monitored every 20 minutes during the treatment. After treatment, the substrate was placed on a clean drainage rack and allowed to cool naturally at an ambient temperature of 24°C. The cooling rate was approximately 1.8°C / 15 minutes, and the cooling time was 4.5 hours, until the final substrate temperature dropped to around 25°C.

[0066] (3) Trichoderma harzianum inoculation: Dissolve 5 grams of RootShield® commercial Trichoderma harzianum T-22 strain powder (ATCC20847) in 10 liters of sterile water to prepare a uniform suspension. Use a low-pressure sprayer to evenly spray the suspension onto the substrate surface, ensuring that 1 liter of suspension is used for every 10 kg of substrate, and the inoculation amount reaches 10% of the substrate weight. 6 CFU / g. After spraying, gently agitate the substrate to ensure even distribution of Trichoderma harzianum. Incubate the inoculated substrate at 25°C and 78% relative humidity for 32 hours, during which time a white mycelial network is observed on the substrate surface.

[0067] (4) Sequential inoculation system: First, on day 0, inoculate with Pleurotus ostreatus strain P-80 at a rate of 3% of the substrate weight. A layered inoculation method was used, with a layer of Pleurotus ostreatus wheat spawn evenly sprinkled between substrate layers every 12 cm in thickness, focusing on inoculating the wheat straw areas. The inoculated substrate was cultured for 7 days at 25-26℃ and 88-90% relative humidity. At this time, it was observed that Pleurotus ostreatus mycelium covered approximately 75% of the substrate surface.

[0068] On day 7, inoculate with Lentinula edodes strain LE-4 at a rate of 2.5% of the substrate weight. Use a fixed-point inoculation method, making small holes 2-3 cm deep every 12 cm along the substrate surface. Fill each hole with 6-9 grams of shiitake mushroom sawdust, focusing on areas rich in rice bran. After inoculation, adjust the ambient temperature to 21-22℃ and reduce the relative humidity to 80-83%, then continue cultivation for another 7 days.

[0069] On day 14, inoculate with Pleurotus eryngii strain PE-2 at a rate of 2% of the substrate weight. Use the trench inoculation method, creating shallow trenches 1.5-2 cm deep on the substrate surface. Distribute Pleurotus eryngii inoculum evenly along the trenches, using approximately 22 grams of inoculum per meter of trench, focusing on areas rich in corn stalks. After inoculation, further lower the ambient temperature to 18-19℃ and maintain the relative humidity at 82-84%, continuing cultivation for 16-18 days until the substrate is completely covered by mycelium.

[0070] (5) Field configuration for grain-mushroom intercropping: Select farmland planted with corn (variety: Zhengdan 958), and configure the mushroom beds 40 days after corn emergence. Arrange the corn in a 2:1:2 ratio (2 rows of corn: 1 row of mushroom bed: 2 rows of corn), maintaining a corn row spacing of 50 cm, a distance of 80 cm between the corn row and the mushroom bed, and a mushroom bed width of 60 cm. Install a drip irrigation system, with drip tape laid along the center of the mushroom bed, dripper spacing set at 30 cm, and a flow rate of 1.8 L / h. Adjust the irrigation frequency according to the temperature; in summer, when the average daily temperature is >25℃, irrigate 3 times a day, 3 L / m² each time, maintaining the relative humidity of the mushroom bed at around 75-80%.

[0071] (6) Application of Waste Mushroom Substrate and Biochar: After two harvests (approximately 70-80 days), waste mushroom substrate was collected, and the yield was measured to be 2.8 tons fresh weight / hectare. The waste mushroom substrate was broken into 7-10 cm blocks and evenly spread in the field at a rate of 16 tons / hectare. At the same time, 2.2 tons / hectare of biochar-fortified fertilizer was applied. The biochar-fortified fertilizer consisted of the following components: 55 parts by weight of pyrolyzed agricultural residue (obtained by pyrolysis at 450°C for 2 hours), 30 parts by weight of fresh straw mixture, and 15 parts by weight of mineral supplement (including 2 parts by weight of dolomite lime, 1 part by weight of phosphate rock, and 0.5 parts by weight of potassium sulfate). The waste mushroom substrate and biochar-fortified fertilizer were mixed with the topsoil using a shallow disc rake with a set depth of 10 cm. The mixing operation was completed on the 3rd day after the waste mushroom substrate was collected.

[0072] (7) Soil carbon pool monitoring: Stratified sampling was conducted at three soil depths: 0-10 cm, 10-20 cm, and 20-30 cm. Five sampling points were randomly placed per hectare, and ten soil cores were collected from each sampling point to form a composite sample. The total organic carbon content of the soil was determined using the Dumas dry combustion method, carbon component analysis was performed using the density separation method, and the δ¹³C value was determined by isotope ratio mass spectrometry. The monitoring results showed that within 24 months after treatment, the organic carbon content of the 0-30 cm soil layer increased by an average of 26.8%, and the carbon sequestration rate reached 2.2 tons of CO2 equivalent / hectare / year.

[0073] Example 3: Grain-Mushroom Intercropping Method Based on Endpoint Values ​​of Wheat Straw Substrate Ratio and Corn Straw Substrate Ratio

[0074] This embodiment provides a method for intercropping grains and mushrooms using wheat straw substrate ratio endpoints and corn straw substrate ratio endpoints, specifically including the following steps:

[0075] (1) Layered substrate preparation: Wheat straw after harvest was collected and processed to a length of 2-3 cm using a rotary shredder, with a moisture content of 18%; corn straw after harvest was also collected and cut to a length of 2-3 cm, with a moisture content of 18%. A mixture was prepared using 45 parts by weight of wheat straw, 40 parts by weight of corn straw, 8 parts by weight of rice bran, 2 parts by weight of gypsum, and 1 part by weight of calcium carbonate. Wheat straw was placed at the bottom layer, corn straw in the middle layer, and the rice bran, gypsum, and calcium carbonate were mixed and placed at the top layer. The mixture was then mixed in a horizontal paddle mixer at 25 RPM for 20 minutes, gradually adding water until the final moisture content of the substrate reached 68%. The C:N ratio of the substrate was measured to be 28:1.

[0076] (2) Hydrothermal Treatment: The mixed substrate is placed into a heat-resistant mesh container, ensuring the substrate thickness does not exceed 30 cm. A water bath at 70°C is prepared, with a water-to-substrate volume ratio of 4:1. The substrate is completely immersed in the water, maintaining the water temperature between 68-70°C for 95 minutes. The water temperature is monitored every 15 minutes during the treatment. After treatment, the substrate is placed on a clean drainage rack and allowed to cool naturally at an ambient temperature of 20°C at a cooling rate of approximately 1°C / 15 minutes for 6 hours, until the final substrate temperature drops to around 25°C.

[0077] (3) Trichoderma harzianum inoculation: Dissolve 5 grams of RootShield® commercial Trichoderma harzianum T-22 strain powder (ATCC20847) in 10 liters of sterile water to prepare a uniform suspension. Use a low-pressure sprayer to evenly spray the suspension onto the substrate surface, ensuring that 1 liter of suspension is used for every 10 kg of substrate, and the inoculation amount reaches 10% of the substrate weight. 6CFU / g. After spraying, gently agitate the substrate to ensure even distribution of Trichoderma harzianum. Incubate the inoculated substrate at 25°C and 85% relative humidity for 36 hours, during which a distinct white mycelial network is observed on the substrate surface.

[0078] (4) Sequential inoculation system: On day 0, Pleurotus ostreatus strain P-80 was inoculated at a rate of 3% of the substrate weight. A layered inoculation method was used, with a layer of Pleurotus ostreatus wheat seed evenly sprinkled between substrate layers every 10 cm, focusing on inoculating the wheat straw area. The inoculated substrate was cultured for 7 days at 26℃ and 90% relative humidity, at which point it was observed that Pleurotus ostreatus mycelium covered approximately 80% of the substrate surface.

[0079] On day 7, *Lentinula edodes* strain LE-4 was inoculated at a rate of 2.5% of the substrate weight. A fixed-point inoculation method was used, with small holes 3 cm deep made every 10 cm on the substrate surface. Each hole was filled with 10 grams of *Lentinula edodes* sawdust, focusing on areas rich in rice bran. After inoculation, the ambient temperature was adjusted to 24°C, and the relative humidity was reduced to 85%, and cultivation continued for another 7 days.

[0080] On day 14, *Pleurotus eryngii* strain PE-2 was inoculated at 2% of the substrate weight. A trench inoculation method was used, with shallow trenches 2 cm deep created on the substrate surface. *Pleurotus eryngii* seed grains were evenly distributed along the trenches, using approximately 25 grams of inoculum per meter of trench, focusing on areas rich in corn stalks. After inoculation, the ambient temperature was further lowered to 22°C, and the relative humidity was maintained at 85%. Culture continued for 14 days until the substrate was completely covered by mycelium.

[0081] (5) Field configuration for grain-mushroom intercropping: Select farmland planted with wheat (variety: Jimai 22), and configure the mushroom beds 30 days after wheat emergence. Arrange the wheat in a 2:1:2 ratio (2 rows of wheat: 1 row of mushroom bed: 2 rows of wheat), maintaining a wheat row spacing of 40 cm, a distance of 70 cm between the wheat row and the mushroom bed, and a mushroom bed width of 70 cm. Install a drip irrigation system, with drip tape laid along the center of the mushroom bed, dripper spacing set at 30 cm, and a flow rate of 2 L / h. Adjust the irrigation frequency according to the temperature; in spring when the average daily temperature is 15-20℃, irrigate once a day at a rate of 4 L / m², maintaining a relative humidity of approximately 85% in the mushroom bed.

[0082] (6) Application of Waste Mushroom Substrate and Biochar: After three harvests (approximately 90-100 days), waste mushroom substrate was collected, and the yield was measured to be 3.4 tons fresh weight / hectare. The waste mushroom substrate was broken into 5-7 cm blocks and evenly spread in the field at a rate of 20 tons / hectare. At the same time, 3 tons / hectare of biochar-fortified fertilizer was applied. The biochar-fortified fertilizer consisted of the following components: 60 parts by weight of pyrolyzed agricultural residue (obtained by pyrolysis at 500℃ for 2 hours), 25 parts by weight of fresh straw mixture, and 15 parts by weight of mineral supplement (including 3 parts by weight of dolomite lime, 2 parts by weight of phosphate rock, and 1 part by weight of potassium sulfate). The waste mushroom substrate and biochar-fortified fertilizer were mixed with the topsoil using a shallow disc rake with a set depth of 15 cm. The mixing operation was completed on the second day after the waste mushroom substrate was collected.

[0083] (7) Soil carbon pool monitoring: Stratified sampling was conducted at three soil depths: 0-10 cm, 10-20 cm, and 20-30 cm. Five sampling points were randomly placed per hectare, and ten soil cores were collected from each sampling point to form a composite sample. The total organic carbon content of the soil was determined using the Dumas dry combustion method, carbon component analysis was performed using the density separation method, and the δ¹³C value was determined by isotope ratio mass spectrometry. Monitoring results showed that within 24 months after treatment, the organic carbon content in the 0-30 cm soil layer increased by an average of 35%, and the carbon sequestration rate reached 3.4 tons of CO2 equivalent / hectare / year.

[0084] Example 4: Grain-Mushroom Intercropping Method Based on Endpoint Values ​​of Wheat Straw Substrate Ratio and Corn Straw Substrate Ratio

[0085] This embodiment provides a method for intercropping grains and mushrooms using wheat straw substrate ratio endpoints and corn straw substrate ratio endpoints, specifically including the following steps:

[0086] (1) Layered substrate preparation: Wheat straw after harvest was collected and processed to a length of 4-5 cm using a rotary shredder, with a moisture content of 14%; corn straw after harvest was also collected and cut to a length of 4-5 cm, with a moisture content of 14%. The substrate was prepared according to the following ratio: 40 parts by weight of wheat straw, 40 parts by weight of corn straw, 5 parts by weight of rice bran, 1 part by weight of gypsum, and 0.5 parts by weight of calcium carbonate. Wheat straw was placed at the bottom layer, corn straw in the middle layer, and the rice bran, gypsum, and calcium carbonate were mixed and placed at the top layer. The mixture was then mixed in a horizontal paddle mixer at 20 RPM for 15 minutes, gradually adding water until the final moisture content of the substrate reached 65%. The C:N ratio of the substrate was measured to be 32:1.

[0087] (2) Hydrothermal Treatment: The mixed substrate is placed into a heat-resistant mesh container, ensuring the substrate thickness does not exceed 25 cm. A water bath at 65°C is prepared, with a water-to-substrate volume ratio of 4:1. The substrate is completely immersed in the water, maintaining the water temperature between 65-67°C for 90 minutes. The water temperature is monitored every 15 minutes during the treatment. After treatment, the substrate is placed on a clean drainage rack and allowed to cool naturally at an ambient temperature of 25°C at a cooling rate of approximately 2°C / 15 minutes for 4 hours, until the substrate temperature finally drops to around 25°C.

[0088] (3) Trichoderma harzianum inoculation: Dissolve 5 grams of RootShield® commercial Trichoderma harzianum T-22 strain powder (ATCC20847) in 10 liters of sterile water to prepare a uniform suspension. Use a low-pressure sprayer to evenly spray the suspension onto the substrate surface, ensuring that 1 liter of suspension is used for every 10 kg of substrate, and the inoculation amount reaches 10% of the substrate weight. 6 CFU / g. After spraying, gently turn the substrate to ensure even distribution of Trichoderma harzianum. Incubate the inoculated substrate at 25°C and 75% relative humidity for 24 hours, during which time a white mycelial network begins to appear on the substrate surface.

[0089] (4) Sequential inoculation system: On day 0, Pleurotus ostreatus strain P-80 was inoculated at a rate of 3% of the substrate weight. A layered inoculation method was used, with a layer of Pleurotus ostreatus wheat seed evenly sprinkled between substrate layers every 15 cm in thickness, focusing on inoculating the wheat straw area. The inoculated substrate was cultured for 7 days at 24℃ and 85% relative humidity, at which point it was observed that Pleurotus ostreatus mycelium covered approximately 65% ​​of the substrate surface.

[0090] On day 7, inoculate with Lentinula edodes strain LE-4 at a rate of 2.5% of the substrate weight. Use a fixed-point inoculation method, making 2 cm deep holes every 15 cm along the substrate surface and filling each hole with 5 g of shiitake mushroom sawdust, focusing on areas rich in rice bran. After inoculation, adjust the ambient temperature to 21°C and reduce the relative humidity to 80%, and continue cultivation for 7 days.

[0091] On day 14, *Pleurotus eryngii* strain PE-2 was inoculated at 2% of the substrate weight. A trench inoculation method was used, with shallow trenches 1 cm deep created on the substrate surface. *Pleurotus eryngii* seed grains were evenly distributed along the trenches, using approximately 20 grams of inoculum per meter of trench, focusing on areas rich in corn stalks. After inoculation, the ambient temperature was further lowered to 18°C, and the relative humidity was maintained at 80%. Culture continued for 21 days until the substrate was completely covered by mycelium.

[0092] (5) Field configuration for grain-mushroom intercropping: Select farmland planted with corn (variety: Zhengdan 958), and configure the mushroom beds 45 days after corn emergence. Arrange the corn beds in a 2:1:2 ratio (2 rows of corn: 1 row of mushroom bed: 2 rows of corn), maintaining a corn row spacing of 50 cm, a distance of 70 cm between the corn row and the mushroom bed, and a mushroom bed width of 70 cm. Install a drip irrigation system, with drip tape laid along the center of the mushroom bed, dripper spacing set at 30 cm, and a flow rate of 1.5 L / h. Adjust the irrigation frequency according to the temperature; in summer, when the average daily temperature is >28℃, irrigate 3 times a day, 3 L / m² each time, maintaining the relative humidity of the mushroom bed at around 75%.

[0093] (6) Application of Waste Mushroom Substrate and Biochar: After two harvests (approximately 70-80 days), waste mushroom substrate was collected, and the yield was measured to be 2.6 tons fresh weight / hectare. The waste mushroom substrate was broken into 8-10 cm blocks and evenly spread in the field at a rate of 15 tons / hectare. At the same time, 2 tons / hectare of biochar-fortified fertilizer was applied. The biochar-fortified fertilizer consisted of the following components: 55 parts by weight of pyrolyzed agricultural residue (obtained by pyrolysis at 450°C for 2 hours), 30 parts by weight of fresh straw mixture, and 15 parts by weight of mineral supplement (including 2 parts by weight of dolomite lime, 1 part by weight of phosphate rock, and 0.5 parts by weight of potassium sulfate). The waste mushroom substrate and biochar-fortified fertilizer were mixed with the topsoil using a shallow disc rake with a set depth of 10 cm. The mixing operation was completed on the 7th day after the waste mushroom substrate was collected.

[0094] (7) Soil carbon pool monitoring: Stratified sampling was conducted at three soil depths: 0-10 cm, 10-20 cm, and 20-30 cm. Five sampling points were randomly placed per hectare, and ten soil cores were collected from each sampling point to form a composite sample. The total organic carbon content of the soil was determined using the Dumas dry combustion method, carbon component analysis was performed using the density separation method, and the δ¹³C value was determined by isotope ratio mass spectrometry. Monitoring results showed that within 24 months after treatment, the organic carbon content in the 0-30 cm soil layer increased by an average of 25%, and the carbon sequestration rate reached 2.1 tons of CO2 equivalent / hectare / year.

[0095] Example 5: Biochar-fortified fertilizer with endpoint values ​​for pyrolysis agricultural residues and mineral supplements

[0096] This embodiment provides a biochar-fortified fertilizer with endpoint values ​​for pyrolysis agricultural residues and mineral supplements, specifically including the following steps:

[0097] (1) Preparation of pyrolyzed agricultural residues: Wheat straw and corn straw with a moisture content of 8% were collected, mixed in a 1:1 ratio, and mechanically cut to a length of 2 cm. The treated mixture was loaded into a fixed-bed slow pyrolysis reactor, and the temperature was increased from the ambient temperature of 25°C to 250°C at a rate of 10°C / min, and held for 30 minutes to fully release the moisture; then the temperature was increased from 250°C to 500°C at a rate of 5°C / min, and held at this temperature for 130 minutes. Subsequently, it was naturally cooled to 45°C under nitrogen protection, and then exposed to ambient air in a ventilated area for 24 hours to stabilize the biochar. The prepared biochar had a pH of 9.5, a specific surface area of ​​230 m² / g, and a fixed carbon content of 75%.

[0098] (2) Preparation of biochar-fortified fertilizer: The following mixture was prepared: 60 parts by weight of the above-mentioned pyrolytic agricultural residues, 25 parts by weight of a fresh straw mixture (wheat straw and corn straw mixed in a 1:1 ratio, cut to 1 cm length), and 15 parts by weight of a mineral supplement. The mineral supplement included 3 parts by weight of dolomite lime, 2 parts by weight of phosphate rock, and 1 part by weight of potassium sulfate. First, the mineral supplement components were mixed evenly, then thoroughly mixed with the biochar, and water was added to a moisture content of 30%. Finally, the fresh straw mixture was added, and the mixture was stirred in a rotary mixer at 15 RPM for 20 minutes. After mixing, the material was piled up under aerobic conditions for 72 hours of maturation, with the maturation temperature maintained at 30°C. The pile was turned over every 24 hours to ensure uniformity.

[0099] (3) Application of biochar-fortified fertilizer: The prepared biochar-fortified fertilizer is evenly spread in the field at a rate of 3 tons / hectare. Then, a shallow disc harrow with a depth of 15 cm is used to mix the fertilizer with the topsoil to ensure that the biochar-fortified fertilizer is fully mixed with the topsoil. The application time is during the soil preparation period after crop harvest or at the same time as waste mushroom substrate.

[0100] (4) Effect evaluation: The impact of biochar-fortified fertilizer on the soil carbon pool was evaluated through soil sampling and analysis. The changes in carbon composition were assessed using density separation method and thermogravimetric analysis. The results showed that the proportion of stable carbon components (half-life > 100 years) in the total organic carbon in the treated soil increased from 35% in the control area to 65%, indicating that biochar-fortified fertilizer significantly improved the stability and persistence of soil carbon.

[0101] Example 6: Biochar-fortified fertilizer with endpoint values ​​for pyrolysis agricultural residues and mineral supplements

[0102] This embodiment provides a biochar-fortified fertilizer with endpoint values ​​for pyrolysis agricultural residues and mineral supplements, specifically including the following steps:

[0103] (1) Preparation of pyrolyzed agricultural residues: Wheat straw and corn straw with a moisture content of 12% were collected, mixed in a 1:1 ratio, and mechanically cut to a length of 5 cm. The treated mixture was loaded into a fixed-bed slow pyrolysis reactor, and the temperature was increased from ambient temperature (22°C) to 250°C at a rate of 10°C / min and held for 30 minutes; then the temperature was increased from 250°C to 450°C at a rate of 5°C / min and held at that temperature for 110 minutes. Subsequently, the mixture was naturally cooled to 50°C under nitrogen protection, and then exposed to ambient air in a ventilated area for 24 hours to stabilize the biochar. The prepared biochar had a pH of 8.8, a specific surface area of ​​200 m² / g, and a fixed carbon content of 70%.

[0104] (2) Preparation of biochar-fortified fertilizer: The following mixture was prepared: 55 parts by weight of the above-mentioned pyrolytic agricultural residues, 30 parts by weight of a fresh straw mixture (wheat straw and corn straw mixed in a 1:1 ratio, cut to 2 cm length), and 15 parts by weight of a mineral supplement. The mineral supplement included 2 parts by weight of dolomite lime, 1 part by weight of phosphate rock, and 0.5 parts by weight of potassium sulfate. First, the mineral supplement components were mixed evenly, then thoroughly mixed with the biochar, and water was added to a moisture content of 35%. Finally, the fresh straw mixture was added, and the mixture was stirred in a rotary mixer at 18 RPM for 15 minutes. After mixing, the material was piled up under aerobic conditions for 48 hours of maturation, with the maturation temperature maintained at 25°C. The pile was turned over every 24 hours to ensure uniformity.

[0105] (3) Application of biochar-fortified fertilizer: The prepared biochar-fortified fertilizer is evenly spread in the field at a rate of 2 tons / hectare. Then, a shallow disc rake with a depth of 10 cm is used to mix the fertilizer with the topsoil to ensure that the biochar-fortified fertilizer is fully mixed with the topsoil. The application time is during the soil preparation period after crop harvest or at the same time as waste mushroom substrate.

[0106] (4) Effect evaluation: The impact of biochar-fortified fertilizer on the soil carbon pool was evaluated through soil sampling and analysis. The changes in carbon composition were assessed using density separation method and thermogravimetric analysis. The results showed that the proportion of stable carbon components (half-life > 100 years) in the total organic carbon in the treated soil increased from 32% in the control area to 58%, indicating that biochar-fortified fertilizer significantly improved the stability and persistence of soil carbon.

[0107] Example 7: Grain-Mushroom Intercropping Method Based on Optimized Combination of Intermediate Value Parameters

[0108] This embodiment provides a method for grain-mushroom intercropping with optimized combinations of intermediate value parameters, specifically including the following steps:

[0109] (1) Layered matrix preparation: Wheat straw after harvest was collected and processed to a length of 3-4 cm using a rotary shredder, with a moisture content of 16%; corn straw after harvest was also collected and cut to a length of 3-4 cm, with a moisture content of 16%. The matrix was prepared according to the following ratio: 42.5 parts by weight of wheat straw, 37.5 parts by weight of corn straw, 6.5 parts by weight of rice bran, 1.5 parts by weight of gypsum, and 0.75 parts by weight of calcium carbonate. Wheat straw was placed at the bottom layer, corn straw in the middle layer, and the rice bran, gypsum, and calcium carbonate were mixed and placed at the top layer. The mixture was then mixed in a horizontal paddle mixer at 22 RPM for 18 minutes, gradually adding water until the final moisture content of the matrix reached 66.5%. The C:N ratio of the matrix was measured to be 30:1.

[0110] (2) Hydrothermal Treatment: The mixed substrate is placed into a heat-resistant mesh container, ensuring the substrate thickness does not exceed 27 cm. A water bath at 67°C is prepared, with a water-to-substrate volume ratio of 4:1. The substrate is completely immersed in the water, maintaining the water temperature between 67-68°C for 90 minutes. The water temperature is monitored every 15 minutes during the treatment. After treatment, the substrate is placed on a clean drainage rack and allowed to cool naturally at an ambient temperature of 23°C at a cooling rate of approximately 1.5°C / 15 minutes for 5 hours, until the final substrate temperature drops to around 25°C.

[0111] (3) Trichoderma harzianum inoculation: Dissolve 5 grams of RootShield® commercial Trichoderma harzianum T-22 strain powder (ATCC20847) in 10 liters of sterile water to prepare a uniform suspension. Use a low-pressure sprayer to evenly spray the suspension onto the substrate surface, ensuring that 1 liter of suspension is used for every 10 kg of substrate, and the inoculation amount reaches 10% of the substrate weight. 6 CFU / g. After spraying, gently agitate the substrate to ensure even distribution of Trichoderma harzianum. Incubate the inoculated substrate at 25°C and 80% relative humidity for 30 hours, during which a distinct white mycelial network is observed on the substrate surface.

[0112] (4) Sequential inoculation system: On day 0, Pleurotus ostreatus strain P-80 was inoculated at a rate of 3% of the substrate weight. A layered inoculation method was used, with a layer of Pleurotus ostreatus wheat seed evenly sprinkled between substrate layers every 12 cm in thickness, focusing on inoculating the wheat straw area. The inoculated substrate was cultured for 7 days at 25℃ and 87% relative humidity, at which point it was observed that Pleurotus ostreatus mycelium covered approximately 75% of the substrate surface.

[0113] On day 7, *Lentinula edodes* strain LE-4 was inoculated at a rate of 2.5% of the substrate weight. A fixed-point inoculation method was used, with small holes 2.5 cm deep made every 12 cm along the substrate surface. Each hole was filled with 7 grams of *Lentinula edodes* sawdust, focusing on areas rich in rice bran. After inoculation, the ambient temperature was adjusted to 22°C, and the relative humidity was reduced to 82%, and cultivation continued for another 7 days.

[0114] On day 14, *Pleurotus eryngii* strain PE-2 was inoculated at 2% of the substrate weight. A trench inoculation method was used, with shallow trenches 1.5 cm deep created on the substrate surface. *Pleurotus eryngii* seed culture was evenly distributed along the trenches, using approximately 22 grams of inoculum per meter of trench, focusing on areas rich in corn stalks. After inoculation, the ambient temperature was further lowered to 20°C, and the relative humidity was maintained at 82%. Culture continued for 17 days until the substrate was completely covered by mycelium.

[0115] (5) Field configuration for grain-mushroom intercropping: Select farmland planted with wheat (variety: Jimai 22), and configure the mushroom beds 38 days after wheat emergence. Arrange the wheat in a 2:1:2 ratio (2 rows of wheat: 1 row of mushroom bed: 2 rows of wheat), maintaining a wheat row spacing of 45 cm, a distance of 75 cm between the wheat row and the mushroom bed, and a mushroom bed width of 65 cm. Install a drip irrigation system, with drip tape laid along the center of the mushroom bed, dripper spacing set at 30 cm, and a flow rate of 1.7 L / h. Adjust the irrigation frequency according to the temperature; in spring when the average daily temperature is 18-22℃, irrigate twice a day at a rate of 2.5 L / m² each time, maintaining a relative humidity of approximately 80% in the mushroom bed.

[0116] (6) Application of Waste Mushroom Substrate and Biochar: After three harvests (approximately 85-95 days), waste mushroom substrate was collected, and the yield was measured to be 3.2 tons fresh weight / hectare. The waste mushroom substrate was broken into 6-8 cm blocks and evenly spread in the field at a rate of 17.5 tons / hectare. At the same time, 2.5 tons / hectare of biochar-fortified fertilizer was applied. The biochar-fortified fertilizer consisted of the following components: 57.5 parts by weight of pyrolyzed agricultural residue (obtained by pyrolysis at 475°C for 2 hours), 27.5 parts by weight of fresh straw mixture, and 15 parts by weight of mineral supplement (including 2.5 parts by weight of dolomite lime, 1.5 parts by weight of phosphate rock, and 0.75 parts by weight of potassium sulfate). The waste mushroom substrate and biochar-fortified fertilizer were mixed with the topsoil using a shallow disc rake with a set depth of 12 cm. The mixing operation was completed on the 4th day after the waste mushroom substrate was collected.

[0117] (7) Soil carbon pool monitoring: Stratified sampling was conducted at three soil depths: 0-10 cm, 10-20 cm, and 20-30 cm. Five sampling points were randomly placed per hectare, and ten soil cores were collected from each sampling point to form a composite sample. The total organic carbon content of the soil was determined using the Dumas dry combustion method, carbon component analysis was performed using the density separation method, and the δ¹³C value was determined by isotope ratio mass spectrometry. Monitoring results showed that within 24 months after treatment, the organic carbon content in the 0-30 cm soil layer increased by an average of 30%, and the carbon sequestration rate reached 2.8 tons of CO2 equivalent / hectare / year.

[0118] Example 8: Biochar-fortified fertilizer with extreme pyrolysis temperatures

[0119] This embodiment provides a biochar-fortified fertilizer with a pyrolysis temperature endpoint, specifically including the following steps:

[0120] (1) Preparation of pyrolytic agricultural residues: Wheat straw and corn straw with a moisture content of 10% were collected, mixed in a 1:1 ratio, and mechanically cut to a length of 3.5 cm. The treated mixture was loaded into a fixed-bed slow pyrolysis reactor, and the temperature was increased from ambient temperature (23°C) to 250°C at a rate of 10°C / min and held for 30 minutes; then the temperature was increased from 250°C to 475°C at a rate of 5°C / min and held at that temperature for 120 minutes. Subsequently, it was naturally cooled to 48°C under nitrogen protection, and then exposed to ambient air in a ventilated area for 24 hours to stabilize the biochar. The prepared biochar had a pH of 9.2, a specific surface area of ​​215 m² / g, and a fixed carbon content of 73%.

[0121] (2) Preparation of biochar-fortified fertilizer: The following mixture was prepared: 57.5 parts by weight of the above-mentioned pyrolytic agricultural residues, 27.5 parts by weight of a fresh straw mixture (wheat straw and corn straw mixed in a 1:1 ratio, cut to 1.5 cm length), and 15 parts by weight of a mineral supplement, comprising 2.5 parts by weight of dolomite lime, 1.5 parts by weight of phosphate rock, and 0.75 parts by weight of potassium sulfate. First, the mineral supplement components were mixed evenly, then thoroughly mixed with the biochar, and water was added to a moisture content of 32.5%. Finally, the fresh straw mixture was added, and the mixture was stirred in a rotary mixer at 17 RPM for 17 minutes. After mixing, the material was piled up under aerobic conditions for 60 hours of maturation, with the maturation temperature maintained at 28°C. The pile was turned over every 24 hours to ensure uniformity.

[0122] (3) Application of biochar-fortified fertilizer: The prepared biochar-fortified fertilizer is evenly spread in the field at a rate of 2.5 tons / hectare. Then, a shallow disc harrow with a depth of 12.5 cm is used to mix the fertilizer with the topsoil to ensure that the biochar-fortified fertilizer is fully mixed. The application time is during the soil preparation period after crop harvest or at the same time as waste mushroom substrate.

[0123] (4) Effect evaluation: The impact of biochar-fortified fertilizer on the soil carbon pool was evaluated through soil sampling and analysis. The changes in carbon composition were assessed using density separation method and thermogravimetric analysis. The results showed that the proportion of stable carbon components (half-life > 100 years) in the total organic carbon in the treated soil increased from 34% in the control area to 62%, indicating that biochar-fortified fertilizer significantly improved the stability and persistence of soil carbon.

[0124] Comparative Example 1: Single-strain cultivation in a non-sequential inoculation system

[0125] This comparative example used the same substrate formulation as Example 1 (40 parts by weight of wheat straw, 37.5 parts by weight of corn straw, 6 parts by weight of rice bran, 1.5 parts by weight of gypsum, and 0.8 parts by weight of calcium carbonate), but was inoculated with only a single strain of Pleurotus ostreatus, strain P-80, at an inoculation amount of 3% of the substrate weight. Other conditions were the same as in Example 1, including substrate preparation, hydrothermal treatment, Trichoderma harzianum pretreatment, field preparation, and waste substrate treatment.

[0126] The results showed that the yield of oyster mushrooms cultivated with a single strain was 2.2 tons of fresh weight per hectare, which was 29.0% lower than the 3.1 tons of fresh weight per hectare in Example 1. Meanwhile, the soil organic carbon content increased by only 15.3% over 24 months, and the carbon sequestration rate was 1.3 tons of CO2 equivalent per hectare per year, significantly lower than the 2.4 tons of CO2 equivalent per hectare per year in Example 1. This indicates that the sequential inoculation system can significantly improve the utilization efficiency of straw resources and the soil carbon sequestration capacity, mainly due to the complementarity of different edible fungi enzyme systems and the staged degradation of the substrate.

[0127] Comparative Example 2: Grain-Mushroom Intercropping Method Without Trichoderma harzianum Pretreatment

[0128] This comparative example used the same substrate formulation and sequential inoculation system as Example 3, but omitted the pretreatment step of Trichoderma harzianum strain T-22. Other conditions were the same as in Example 3.

[0129] The results showed that without Trichoderma harzianum pretreatment, the total yield of the three edible fungi was 2.8 tons fresh weight / ha, 17.6% lower than the 3.4 tons fresh weight / ha in Example 3. Simultaneously, mycelial growth was significantly slower, with the mycelial coverage of *Pleurotus ostreatus* reaching only 65% ​​on day 7 (compared to 80% in Example 3), and the overall cultivation period was extended by approximately 15%. Soil organic carbon content increased by 28.4% over 24 months, with a carbon sequestration rate of 2.7 tons CO2 equivalent / ha / year, lower than the 3.4 tons CO2 equivalent / ha / year in Example 3. This indicates that Trichoderma harzianum pretreatment creates favorable conditions for subsequent edible fungi growth by prematurely degrading some complex substrates and accelerates carbon conversion and stabilization in the soil.

[0130] Comparative Example 3: Grain-Mushroom Intercropping Method without Biochar-Fortified Fertilizer

[0131] This comparative example used the same substrate formulation, Trichoderma harzianum pretreatment, and sequential inoculation system as Example 7, but no biochar-fortified fertilizer was added when applying the waste mushroom substrate; instead, the waste mushroom substrate was applied to the soil at a rate of 17.5 tons / hectare. Other conditions were the same as in Example 7.

[0132] The results showed that, without biochar-fortified fertilizer, the yield of edible fungi was similar to that of Example 7, at 3.1 tons fresh weight / ha. However, the soil organic carbon content increased by only 18.2% over 24 months, significantly lower than the 30% increase in Example 7. The carbon sequestration rate was 1.5 tons CO2 equivalent / ha / year, far lower than the 2.8 tons CO2 equivalent / ha / year in Example 7. Meanwhile, density separation analysis showed that the light component (<1.8 g / cm³) accounted for 45% of the total organic carbon, higher than the 28% in Example 7, indicating that without biochar-fortified fertilizer, the newly added organic carbon was mainly in easily decomposable forms with poor stability. This demonstrates that biochar-fortified fertilizer plays an irreplaceable role in improving the stability and persistence of the soil carbon pool.

[0133] Comparative Example 4: Traditional Straw Returning Methods

[0134] This comparative example uses the traditional method of directly returning straw to the field. Wheat and corn straw are mixed in a 1:1 ratio, chopped to 5-10 cm, and applied evenly to the soil surface at a rate of 40 tons / hectare. Then, shallow disc harrows are used for tilling and mixing.

[0135] The results showed that traditional straw return methods increased soil organic carbon content by 10.5% within 24 months, with a carbon sequestration rate of only 0.7 tons of CO2 equivalent / hectare / year, far lower than the levels in Examples 1-8. Simultaneously, the high emissions of methane and nitrous oxide generated during straw decomposition partially offset the carbon sequestration benefits. Furthermore, the slow decomposition of untreated straw affected the growth of subsequent crops, leading to a slight decrease in grain yield (approximately 3%). This indicates that the grain-mushroom intercropping method of this invention, combined with biochar-fortified fertilizer, has significant advantages in straw resource utilization efficiency and soil carbon pool enhancement.

[0136] Comparative Example 5: Single-bed cultivation of grain-free mushrooms

[0137] This comparative example used the same substrate formula, Trichoderma harzianum pretreatment, and sequential inoculation system as Example 2, but instead of the field grain-mushroom intercropping model, edible fungi cultivation was carried out in a separate mushroom house. Other conditions were the same as in Example 2.

[0138] The results showed that the total yield of edible fungi cultivated in a single mushroom bed was 3.5 tons of fresh weight per hectare, slightly higher than the 2.8 tons of fresh weight per hectare in Example 2. However, considering that grain crops were also planted in Example 2, the overall land use efficiency was significantly lower. After waste mushroom substrate was applied to the soil, the soil organic carbon content increased by 21.3% within 24 months, and the carbon sequestration rate was 1.8 tons of CO2 equivalent per hectare per year, lower than the 2.2 tons of CO2 equivalent per hectare per year in Example 2. This indicates that the grain-mushroom intercropping model promotes the conversion of organic matter into stable soil carbon through the interaction between crop roots and mycelium, while improving land use efficiency.

[0139] To evaluate the effectiveness of the grain-mushroom intercropping method of the present invention, systematic tests were conducted on each embodiment and comparative example, mainly including indicators such as changes in edible fungus yield, grain crop yield, soil organic carbon content, carbon composition distribution, and greenhouse gas emissions. The test results are shown in Tables 1-3.

[0140] Table 1. Effects of edible fungi yield on grain crop yield increase

[0141] Sample number Oyster mushroom yield (tons of fresh weight / hectare) Shiitake mushroom yield (tons of fresh weight / hectare) King oyster mushroom yield (tons of fresh weight / hectare) Total yield (tons fresh weight / hectare) Grain crop yield increase rate (%) Example 1 1.4 0.9 0.8 3.1 14.2 Example 2 1.2 0.8 0.8 2.8 12.5 Example 3 1.5 1 0.9 3.4 17.8 Example 4 1.1 0.7 0.8 2.6 12.1 Example 7 1.4 0.9 0.9 3.2 15.3 Comparative Example 1 2.2 0 0 2.2 8.5 Comparative Example 2 1.2 0.8 0.8 2.8 14.1 Comparative Example 3 1.4 0.8 0.9 3.1 15 Comparative Example 4 0 0 0 0 -3.2 Comparative Example 5 1.6 1 0.9 3.5 0

[0142] Table 2. Changes in soil organic carbon content and carbon sequestration effect

[0143] Sample number Initial SOC content (g / kg) SOC content (g / kg) after 24 months SOC increase rate (%) <![CDATA[Carbon sequestration rate (tons CO2e / ha / year)]]> Example 1 12.8 16.4 28.5 2.4 Example 2 13.2 16.7 26.8 2.2 Example 3 11.5 15.5 35 3.4 Example 4 12 15 25 2.1 Example 7 12.5 16.3 30 2.8 Comparative Example 1 12.8 14.7 15.3 1.3 Comparative Example 2 11.5 14.8 28.4 2.7 Comparative Example 3 12.5 14.8 18.2 1.5 Comparative Example 4 12.3 13.6 10.5 0.7 Comparative Example 5 13 15.8 21.3 1.8

[0144] Table 3. Soil carbon composition distribution and greenhouse gas emissions

[0145] Sample number Light component carbon (%) intermediate component carbon (%) Recombinant carbon (%) Stable carbon ratio (%) <![CDATA[Methane emissions (kg CH4 / ha / year)]]> <![CDATA[Nitrous oxide emissions (kg N2O / ha / year)]]> Example 1 30 22 48 58 -1.2 0.8 Example 2 32 21 47 56 -1 0.7 Example 3 25 25 50 65 -1.5 0.6 Example 4 35 20 45 55 -0.8 0.9 Example 7 28 23 49 62 -1.3 0.7 Comparative Example 1 40 20 40 45 -0.5 1.2 Comparative Example 2 32 23 45 52 -0.9 0.8 Comparative Example 3 45 20 35 42 -0.7 1 Comparative Example 4 55 25 20 30 2.5 1.8 Comparative Example 5 38 22 40 48 -0.6 1.1

[0146] Note: Light component carbon (<1.8 g / cm³), intermediate component carbon (1.8-2.2 g / cm³), and heavy component carbon (>2.2 g / cm³) represent soil organic carbon components with different levels of stability; stable carbon ratio refers to the percentage of carbon components with a half-life >50 years in the total organic carbon; negative methane emissions indicate net absorption.

[0147] As can be seen from the test results in Tables 1-3, the grain-mushroom intercropping method of the present invention has the following significant effects:

[0148] 1. Edible fungi yield: The sequential inoculation system (Examples 1-8) increased the total yield by 18-55% compared with single-strain cultivation (Comparative Example 1), demonstrating the advantages of multi-strain complementary utilization of straw resources. Trichoderma harzianum pretreatment (Example 3 vs. Comparative Example 2) increased the total yield of edible fungi by 17.6%, verifying the growth-promoting effect of microbial pretreatment.

[0149] 2. Increased grain crop yield: The grain-mushroom intercropping system increased grain crop yield by an average of 12-18%, which is significantly better than traditional straw return to the field (-3.2%) and single mushroom bed cultivation (0%), indicating the promoting effect of mycorrhizal interaction on crop growth.

[0150] 3. Soil carbon pool enhancement: The method of this invention increases soil organic carbon by 25-35%, and the carbon sequestration rate is 2.1-3.4 tons of CO2 equivalent / hectare / year, which is much higher than the 10.5% and 0.7 tons of CO2 equivalent / hectare / year of traditional straw return to the field. In particular, the example with added biochar-fortified fertilizer significantly improved carbon sequestration efficiency compared with the control example 3 without added biochar (30% vs 18.2%).

[0151] 4. Carbon component stability: The method of this invention achieves a stable carbon content (half-life > 50 years) of 55-65%, while traditional straw return to the field only reaches 30%. This indicates that the present invention significantly improves the stability and persistence of soil carbon through microbial conversion and biochar enhancement.

[0152] 5. Greenhouse gas emission reduction: The method of this invention not only increases the soil carbon pool, but also shows net methane uptake (-0.8 to -1.5 kg CH4 / ha / year) and low nitrous oxide emissions (0.6-0.9 kg N2O / ha / year), which are significantly better than traditional straw return to the field (2.5 and 1.8 kg / ha / year), further improving the climate change mitigation benefits.

[0153] Based on the test results of all embodiments, Example 3 performed best. It used a substrate formulation of 45 parts by weight of wheat straw and 40 parts by weight of corn straw, combined with 3 tons / hectare of biochar-fortified fertilizer (containing 60 parts by weight of pyrolyzed agricultural residues). The carbon sequestration rate reached 3.4 tons of CO2 equivalent / hectare / year, and the stable carbon ratio reached 65%. This indicates that a high proportion of wheat and corn straw formulation, combined with a high dose of biochar-fortified fertilizer, can maximize the improvement of the soil carbon pool.

[0154] The method for efficient utilization of straw resources and enhancement of soil carbon pool in grain-mushroom intercropping of the present invention involves multiple complex biological and chemical processes. Based on experimental results, its mechanism of action can be analyzed from the following aspects:

[0155] 1. Multi-species enzyme complementarity mechanism: This invention employs a sequential inoculation system of oyster mushrooms, shiitake mushrooms, and king oyster mushrooms, fully utilizing the enzyme complementarity of different edible fungi. Oyster mushrooms (Pleurotus ostreatus) produce highly active laccase (150-300 U / g matrix) and manganese peroxidase (50-100 U / g matrix), preferentially degrading lignin components; shiitake mushrooms (Lentinula edodes) produce highly active manganese peroxidase (120-250 U / g matrix) and moderately active laccase (80-150 U / g matrix), enabling balanced degradation of various components; king oyster mushrooms (Pleurotus eryngii) produce highly active multifunctional peroxidase (100-200 U / g matrix), capable of degrading more stubborn substrates.

[0156] This complementary enzyme system ensures the full degradation of lignin, cellulose, and hemicellulose in straw, improving resource utilization efficiency. Experiments have shown that the sequential inoculation system increases the total yield by 18-55% compared to single-strain cultivation (Comparative Example 1), confirming this mechanism.

[0157] 2. Mechanism of growth promotion by pretreatment of Trichoderma harzianum: Pretreatment of Trichoderma harzianum strain T-22 promotes the growth of edible fungi in the following ways: On the one hand, the cellulases (β-1,4-endoglucanase, cellulolytic enzyme) and hemicellulases (endo-1,4-β-xylanase, β-xylosidase) produced by the strain partially degrade complex substrates, providing more readily available nutrients for subsequent edible fungi; on the other hand, the volatile organic compounds (such as 6-pentyl-α-pyranone) and bioactive substances produced by the strain stimulate the growth and branching of mycelia of edible fungi.

[0158] Experiments showed that pretreatment with Trichoderma harzianum (Example 3 vs. Comparative Example 2) increased mycelial coverage rate by approximately 23% (80% vs. 65% coverage in 7 days) and increased total yield by 17.6%, verifying this growth-promoting mechanism.

[0159] 3. Mechanism of Grain-Mushroom Interaction for Growth Promotion: In grain-mushroom intercropping systems, a mutually beneficial relationship similar to mycorrhizae is formed between the roots of grain crops and the mycelium. Edible fungus mycelium helps crop roots obtain insoluble nutrients such as phosphorus and potassium from the soil, while secreting plant hormones (such as auxins and gibberellins) to promote root development; in turn, the crop provides readily available carbon sources for the mycelium through root exudates.

[0160] Furthermore, the well-developed mycelial network improves soil structure and enhances soil drought resistance and water retention capacity. This mutually beneficial relationship results in an average increase of 12-18% in grain crop yields through grain-mushroom intercropping systems, while traditional straw return to the field leads to a 3.2% decrease in yield due to the temporary fixation of nutrients.

[0161] 4. Carbon stabilization mechanism: The carbon stabilization in this invention is mainly achieved through the following pathways:

[0162] (1) Fungal transformation pathway: Edible fungi convert carbonaceous materials in straw into more stable fungal chitin and melanin. Chitin (β-(1→4)-N-acetylglucosamine polymer) has a half-life of 5-15 years in soil, while melanin (a complex heterocyclic aromatic polymer) has an even longer half-life, reaching 10-50 years or more.

[0163] (2) Physical protection by biochar: Biochar has a highly developed microporous structure (0.1-10 μm), which can physically encapsulate organic carbon molecules, preventing them from being attacked by microbial enzymes. At the same time, the aromatic structure on the surface of biochar can form π-π interactions with organic matter, further enhancing the protective effect.

[0164] (3) Mineral-organic complex: The mineral supplements (dolomite limestone, phosphate rock) in biochar-fortified fertilizers provide polyvalent cations such as Ca²⁺ and Mg²⁺. These cations can bridge the negatively charged organic matter and mineral surfaces to form a stable mineral-organic complex.

[0165] (4) Carbonate precipitation: Fungal-mediated calcium carbonate precipitation process fixes CO2 into stable carbonate minerals (CaCO3), which can be preserved in the soil for hundreds of years.

[0166] These pathways work together to achieve a stable carbon content (half-life > 50 years) of 55-65% formed by the method of this invention, which is much higher than the 30% of traditional straw return to the field, explaining its excellent carbon sequestration effect.

[0167] 5. Greenhouse gas emission reduction mechanism: The method of this invention exhibits net methane absorption and low nitrous oxide emissions, the mechanism of which is as follows:

[0168] (1) Methane oxidation: The porous structure of biochar provides an ideal habitat for methane-oxidizing bacteria. At the same time, the trace elements it contains, such as copper, can act as cofactors for methane monooxygenase, promoting methane oxidation.

[0169] (2) Nitrogen conversion regulation: Biochar slows down the nitrogen conversion rate by adsorbing NH4⁺ and NO3⁻, and its alkaline properties inhibit the key enzymes of nitrification, thereby reducing the production of N2O.

[0170] (3) Electron donor-acceptor balance: The stable organic carbon in the waste mushroom substrate acts as an electron donor and forms a good balance with the electron acceptors in the soil (such as NO3⁻ and Fe³⁺), which reduces the production of methane and nitrous oxide under anaerobic conditions.

[0171] The combined effect of these mechanisms enables the method of this invention to increase the soil carbon pool while also achieving net reduction in greenhouse gas emissions, further enhancing the benefits of climate change mitigation.

[0172] This invention provides a method for the efficient utilization of straw resources and the enhancement of soil carbon pool in grain-mushroom intercropping. Through technical measures such as constructing a multi-species sequential inoculation system, Trichoderma harzianum pretreatment, grain-mushroom intercropping mode, and biochar-enhanced fertilizer application, the efficient utilization of straw resources and the significant enhancement of soil carbon pool are achieved.

[0173] Experimental results show that the method of the present invention has the following advantages over traditional straw return to the field: (1) the yield of edible fungi reaches 2.6-3.4 tons of fresh weight / hectare; (2) the yield of grain crops increases by 12-18%; (3) the soil organic carbon increase rate reaches 25-35%; (4) the carbon sequestration rate reaches 2.1-3.4 tons of CO2 equivalent / hectare / year; (5) the proportion of stable carbon formed reaches 55-65%; (6) net methane absorption and nitrous oxide emission reduction are achieved.

[0174] These effects are attributed to the synergistic effects of mechanisms such as multi-strain enzyme complementarity, Trichoderma harzianum pretreatment promoting growth, grain-mushroom interaction promoting growth, multi-pathway carbon stabilization, and greenhouse gas emission reduction. Among them, the formulation and process parameters used in Example 3 (45 parts by weight of wheat straw, 40 parts by weight of corn straw, and high-dose biochar-fortified fertilizer) showed the best performance, with a carbon sequestration rate of 3.4 tons of CO2 equivalent / hectare / year and a carbon stabilization ratio of 65%.

[0175] The method of this invention not only solves the problem of resource utilization of agricultural waste, but also achieves the dual environmental benefits of improving soil quality and mitigating climate change. At the same time, it brings significant economic benefits through increased production of edible fungi and grains, providing new ideas and technologies for sustainable agricultural development.

[0176] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for efficient utilization of straw resources and enhancement of soil carbon pool in grain-mushroom intercropping, characterized in that, Includes the following steps: Prepare a layered matrix comprising 40-45 parts by weight of wheat straw, 35-40 parts by weight of corn straw, 5-8 parts by weight of rice bran, 1-2 parts by weight of gypsum and 0.5-1 parts by weight of calcium carbonate. Place wheat straw at the bottom layer, corn straw in the middle layer, and mix rice bran, gypsum and calcium carbonate and sprinkle it on the top layer. The layered matrix was subjected to hydrothermal treatment at 65-70°C for 90±5 minutes, and then cooled to 25±2°C. Trichoderma harzianum strain T-22 was inoculated into the cooled substrate at an inoculum size of 10% of the substrate weight. 6 CFU / g, and cultured at 25±2℃ and 75-85% relative humidity for 24-36 hours; A sequential inoculation system was used. First, oyster mushrooms were inoculated at a rate of 3% of the substrate weight. Seven days later, shiitake mushrooms were inoculated at a rate of 2.5% of the substrate weight. Seven days after that, king oyster mushrooms were inoculated at a rate of 2% of the substrate weight. Mushroom beds are arranged between rows of grain crops in a ratio of 2:1:2, where the ratio refers to 2 rows of grain crops, 1 row of mushroom bed, and 2 rows of grain crops, and the width of the mushroom bed is 60-70 cm. After harvesting the mushroom beds, apply 15-20 tons / hectare of waste mushroom substrate to the soil and add 2-3 tons / hectare of biochar-enhanced fertilizer. Monitoring and verification showed that soil organic carbon content increased by 25-35% within 24 months; The sequential inoculation system: Oyster mushroom (Pleurotus ostreatus) was inoculated on day 0, and cultured at a temperature of 24-26℃ and a relative humidity of 85-90%. Shiitake mushrooms (Lentinula edodes) were inoculated on day 7, and cultured at a temperature of 21-24℃ and a relative humidity of 80-85%. King oyster mushroom (Pleurotus eryngii) was inoculated on day 14, and cultured at a temperature of 18-22℃ and a relative humidity of 80-85%. Each strain was inoculated using a stratified inoculation method. Oyster mushrooms were preferentially inoculated in wheat straw areas, shiitake mushrooms were preferentially inoculated in supplementary areas rich in rice bran, and king oyster mushrooms were preferentially inoculated in corn straw areas. The biochar-fortified fertilizer is composed of the following components: 55-60 parts by weight of pyrolytic agricultural residues, wherein the pyrolytic agricultural residues are obtained by pyrolysis at 450-500°C for 2 hours; 25-30 parts by weight of fresh straw mixture; 10-15 parts by weight of a mineral supplement, wherein the mineral supplement comprises 2-3 parts by weight of dolomite lime, 1-2 parts by weight of phosphate rock and 0.5-1 parts by weight of potassium sulfate; The preparation of the pyrolytic agricultural residue includes the following steps: Mix wheat straw and corn straw with a moisture content of 8-12% in a 1:1 ratio and cut them into lengths of 2-5 cm. In a fixed-bed slow pyrolysis reactor, the temperature was increased from ambient temperature to 250°C at a rate of 10°C / min and held for 30 minutes. Then, increase the temperature from 250℃ to 450-500℃ at a rate of 5℃ / min, and maintain this temperature for 120±10 minutes. The biochar was naturally cooled to below 50°C in an inert atmosphere and then exposed to ambient air in a ventilated area for 24 hours to stabilize it.

2. The method according to claim 1, characterized in that, The wheat straw and corn straw have a particle size of 2-5 cm and a moisture content of 14-18%, obtained through mechanical processing; the final moisture content of the layered matrix is ​​65-68%, and the C:N ratio is 28-32:

1.

3. The method according to claim 1, characterized in that, In the hydrothermal treatment step, the volume ratio of water to substrate is 4:

1. After treatment, the substrate is naturally cooled at a cooling rate of 1-2℃ / 15 minutes for 4-6 hours.

4. The method according to claim 1, characterized in that, The Trichoderma harzianum strain T-22 (ATCC 20847) was added in the form of a water-soluble powder. A suspension was prepared by dissolving 5 grams of the product in 10 liters of sterile water and spraying it evenly on the surface of the substrate. 1 liter of suspension was used for every 10 kilograms of substrate.

5. The method according to claim 1, characterized in that, The grain crop used in the grain-mushroom intercropping is selected from wheat or corn. The mushroom bed is deployed 30-45 days after the grain crop emerges. The mushroom bed uses a drip irrigation system to maintain a relative humidity of 75-85%. The drip irrigation system has a drip head spacing of 30 cm and a daily water supply of 2-4 liters / square meter.

6. The method according to claim 1, characterized in that, The waste mushroom substrate is collected after 2-3 harvests. First, the waste mushroom substrate is broken into 5-10 cm pieces and spread evenly in the field. Then, a shallow disc rake with a depth of 10-15 cm is used to mix the soil. Mixing should be carried out within 7 days after the waste mushroom substrate is removed.

7. The method according to claim 1, characterized in that, The steps for monitoring and verifying soil organic carbon content include: Soil samples were taken at depths of 0-10 cm, 10-20 cm, and 20-30 cm. Five composite samples were collected per hectare, and each composite sample consisted of 10 individual soil cores; Total organic carbon content was determined using the Dumas dry combustion method; Carbon composition analysis was performed using density separation method, separating light components (<1.8 g / cm³), intermediate components (1.8–2.2 g / cm³), and heavy components (>2.2 g / cm³). The δ¹³C value was determined by isotope ratio mass spectrometry to track changes in the carbon source. The system's carbon sequestration rate reaches 2.1-3.4 tons of CO2 equivalent per hectare per year.