A forest-grass complex planting method based on ginkgo biloba root secretion for regulating alfalfa nodule and promoting growth

Abstract

The application provides a kind of based on ginkgo root secretion regulation alfalfa nodulation growth-promoting forest-grass interplanting method.The method makes full use of ginkgo under forest space, by land preparation and fertilization between forest rows, constructs high ridge square, selects high-quality alfalfa seed for strip sowing, combined with reasonable spacing and fixing seedling, cultivation and weeding and other field management measures, realize the normal growth and efficient utilization of alfalfa, provide a scientific and reasonable alfalfa interplanting mode.The application combines soil physical and chemical index determination, systematically evaluates forest soil before and after alfalfa planting, confirms that the method can effectively improve soil physical and chemical properties, promote ginkgo-alfalfa synergistic growth, has significant ecological benefits and application prospect.In addition, the application further verifies that ginkgo root secretion has obvious growth-promoting effect on alfalfa rhizobium, and can improve the expression level of alfalfa nod gene, enhance the number and quality of nodule, so as to improve the production performance of alfalfa and soil improvement effect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically relating to a method for forestry-grassland intercropping that utilizes Ginkgo root secretions to regulate alfalfa nodulation and promote growth. Background Technology

[0002] Ginkgo (Ginkgo biloba) is an important economic and ecological tree species with high medicinal, edible, and ornamental value. Ginkgo cultivation is widespread, especially in the middle and lower reaches of the Yangtze River, East China, and Southwest China, where large-scale cultivation bases exist. With the development of the ginkgo industry, the market applications of its leaves, fruits, and seedlings have gradually expanded, becoming an important part of forestry economic development. However, with the continuous expansion of planting area, the ginkgo industry also faces practical problems such as structural oversupply and inefficient resource allocation. The seedling industry has developed rapidly but suffers from severe homogenization; supply and demand imbalances lead to product stockpiling and price fluctuations, resulting in unstable planting benefits and weakening the planting enthusiasm of forest farmers. At the same time, the land under ginkgo forests generally suffers from low utilization rates and poor added value, failing to fully utilize the composite functions of forest land, becoming a key weakness restricting the sustainable development of the industry.

[0003] Alfalfa (Medicago sativa), a perennial leguminous forage, possesses excellent nitrogen-fixing capacity, ecological adaptability, and feed value. It not only improves soil fertility but also provides high-protein forage resources for livestock, making it one of the high-quality forages currently being vigorously promoted by the state. Under forest conditions, alfalfa's deep root system and symbiotic rhizobia can significantly improve soil structure and fertility, achieving synergistic effects between forestry and grassland. In recent years, understory grass planting technology has received increasing attention, but many technical challenges remain in its implementation, particularly regarding signal recognition, nodulation efficiency, and planting management models within the symbiotic system of leguminous forage and rhizobia. Scientific seed selection, rational layout, and enhancement of nodule formation and nitrogen fixation efficiency are key to achieving efficient forestry-grassland integration. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the prior art, this invention provides a forest-grass intercropping method based on the regulation of alfalfa nodulation and growth promotion using Ginkgo root exudates. This method makes full use of idle land under forest cover, creates economic benefits, effectively improves the growth of alfalfa seedlings and the growth of endophytic bacteria in root nodules, and enhances the nodulation ability and biological nitrogen fixation potential of alfalfa.

[0005] Technical solution: The present invention includes a method for forestry-grassland intercropping that regulates alfalfa nodulation and promotes growth based on ginkgo root exudates, comprising the following steps:

[0006] (1) Land preparation and ridge division: Deeply plow the land between rows of ginkgo trees, and then shallowly cultivate and level it before sowing. Establish high ridges between two rows of ginkgo trees, and dig longitudinal ditches in the field to form quadrats.

[0007] (2) Alfalfa seed treatment: Soak alfalfa seeds in water, take out the plump seeds that sink to the bottom and mix them with fine soil and carbendazim;

[0008] (3) Seed row sowing: Make furrows on the raised beds divided in step (1) according to the row spacing, and sow the alfalfa seeds treated in step (2) evenly into the furrows and cover with soil;

[0009] (4) Thinning and fixing seedlings: Thinning is done twice in the same year, removing weak seedlings and keeping strong ones;

[0010] (5) Cultivation and weeding: Cultivate and weed 1-2 times in the first year and 3-4 times in the second year;

[0011] (6) Harvesting: The first harvest is in the second year, followed by monthly harvests, leaving a stubble after each harvest.

[0012] In step (1), the soil is deeply tilled to a depth of more than 25cm.

[0013] Furthermore, in step (1), the width of the raised beds is 1.2-1.5m, and drainage ditches with a depth of 10-15cm are dug every 4-5m to form a (1.2-1.5)m×(4-5m) quadrat.

[0014] As a preferred option, a raised bed with a width of 1.2m is selected, with drainage ditches enclosing both sides of the raised bed. Horizontal ditches are dug longitudinally in the forest at 4-meter intervals to divide the raised bed surface into standard quadrature units of 1.2m×4m, which facilitates subsequent planting management and data collection.

[0015] In step (2), the mass ratio of seeds: fine soil: carbendazim is 1:1.5-2.5:0.01-0.02.

[0016] As a preferred method, after soaking the plump seeds that have sunk to the bottom for 24 hours, take them out and mix them with 2 kg of fine soil per kg of seeds, and then mix in 10 g of carbendazim for disinfection.

[0017] In step (3), dig furrows 2-3 cm deep with a row spacing of 10-20 cm, use 0.8-1.2 kg of seeds per mu, and cover with 0.2-0.5 cm of soil.

[0018] As a preferred method, the seed usage is 1 kg per mu (approximately 0.067 hectares) with a row spacing of 15 cm.

[0019] In step (4), when the seedlings are 2-3cm tall, the first thinning is carried out, and when the seedlings are 8-12cm tall, the second thinning is carried out.

[0020] Among them, the weeding and cultivation in step (5) are carried out at the time of sowing, when the seedlings are 3-10 cm tall, or when the seedlings are 20-25 cm tall, and deep hoeing is carried out when the seedlings are 20-25 cm tall.

[0021] In step (6), the alfalfa is first harvested when the budding rate reaches 8-15%.

[0022] Furthermore, in step (6), after the first mowing, mowing is performed once every 25-35 days, for a total of 3-5 times.

[0023] As a preferred method, harvesting should begin at the end of May of the following year after sowing.

[0024] In step (6), the stubble height after each cut is 5-7 cm.

[0025] The application of the Ginkgo root exudate described in this invention in promoting the growth of alfalfa seedlings, promoting the growth of endophytic bacteria in alfalfa root nodules, and enhancing the nodulation ability and biological nitrogen fixation potential of alfalfa.

[0026] The collection of Ginkgo root exudates involved selecting robust large Ginkgo trees as test plants. The trees were excavated along the root distribution direction, exposing part of the main and lateral roots. After the roots were exposed, the root area was wrapped in a transparent plastic bag and backfilled with soil to restore the natural growth environment. The plants were left to stand, then removed and thoroughly rinsed with deionized water to remove soil particles adhering to the root surface. The roots were then wrapped in moist, sterile filter paper and kept moist with deionized water. The wrapped plants were placed in centrifuge tubes, the tube openings were sealed with sealing film, and the centrifuge tubes were reburied in the soil at the original excavation location for continued treatment to promote the enrichment and adsorption of root exudates. The plants were then removed, and root segments wrapped in filter paper were cut. Under sterile conditions, the root segments were immersed in deionized water and extracted by shaking. The extract was filtered through filter paper, and the filtrate was collected and concentrated by rotary evaporation. The resulting liquid or dry powder is the Ginkgo root exudate extract.

[0027] This invention develops a forest-grassland intercropping method based on the regulation of alfalfa root nodule formation and growth promotion using Ginkgo root exudates. This method not only helps improve the planting efficiency and nitrogen fixation capacity of alfalfa, but also effectively revitalizes idle resources under Ginkgo forests, enhancing the dual ecological and economic functions of forest land. It has broad application prospects and promotional value.

[0028] This invention fully utilizes the space under ginkgo forests and proposes a scientifically sound alfalfa intercropping model. By preparing and fertilizing the land between ginkgo rows, standard 1.2m × 4m raised plots are constructed. High-quality alfalfa seeds are selected and sown in rows after soaking and disinfection. Combined with reasonable thinning, weeding, and pest and disease control, efficient growth and utilization of alfalfa are achieved. Furthermore, this invention collects ginkgo root exudates using in-situ extraction technology and analyzes their active components using metabolomics. These exudates are then used in alfalfa rhizobia and seedling treatment experiments to verify their biological functions in promoting nodule formation, improving nitrogen fixation capacity, and enhancing alfalfa growth. Experimental results show that ginkgo root exudates have a significant growth-promoting effect on alfalfa rhizobia, increase the expression level of the alfalfa nod gene, and enhance the number and quality of nodules, thereby improving alfalfa production performance and soil improvement effects. In addition, this invention also combines soil physicochemical index measurement to systematically evaluate the pH, total N, P, and K of forest soil before and after alfalfa planting, confirming that the method can effectively improve soil physicochemical properties, promote the synergistic growth of ginkgo and alfalfa, and has significant ecological benefits and prospects for promotion and application.

[0029] This invention is the first to discover that root exudates from Ginkgo biloba can promote alfalfa growth and nitrogen fixation. This invention utilizes Ginkgo biloba root exudates to regulate alfalfa rhizobium activity and nodulation gene expression, thereby enhancing alfalfa's nodulation ability and nitrogen fixation function, and further enabling efficient intercropping in Ginkgo biloba forests.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0031] This invention fully utilizes long-term idle land under ginkgo forests by intercropping alfalfa to construct a forest-grass composite system, significantly improving the efficiency of land use, expanding the economic functions of understory, and alleviating the bottleneck of benefits brought about by single forestry operations. Alfalfa has a well-developed root system and strong nitrogen-fixing capacity. Its symbiotic relationship with rhizobia can effectively improve key indicators such as total nitrogen and organic matter in the soil, improve soil structure and fertility, create a better rhizosphere environment for ginkgo growth, and indirectly promote the healthy growth of ginkgo. At the same time, this invention proposes and verifies for the first time that ginkgo root exudates can significantly promote the growth of alfalfa rhizobia and the expression of nodulation genes, enhance alfalfa nodulation and nitrogen-fixing capacity, thereby improving the nutritional quality and yield of alfalfa and achieving a continuous and stable supply of high-quality forage.

[0032] This invention breaks away from the traditional approach of simply utilizing space in forest-grassland intercropping. For the first time, it uses ginkgo secretions as a bioactive factor for collection and research within a forest-grassland intercropping system. It combines the use of forest tree root secretions for the bio-induction of rhizobia in leguminous forage grasses with practical application in planting, establishing an integrated and standardized field planting technique. Standardized (1.2m×4m) planting units are constructed under ginkgo forests, combined with raised beds and drainage ditches, to achieve visualization and precision in alfalfa sowing management. This is a standardized design not found in existing forest-grassland planting methods.

[0033] This invention verifies the bioregulatory function of ginkgo secretions on alfalfa rhizobia and seedlings. It experimentally analyzes the functional intercropping mechanism by which ginkgo root secretions regulate alfalfa nodulation and growth, and combines the expression level of the nod gene (Nucleotide Binding Oligomerization Domain gene) to analyze its mechanism of nodulation. It constructs an interaction network between "ginkgo-secretions-alfalfa-rhizobia", which effectively improves the nodulation ability and production performance of leguminous forage in the understory environment.

[0034] Furthermore, this invention is the first to propose using Ginkgo root exudates as natural plant-derived regulatory factors to effectively enhance alfalfa nod gene expression levels and root nodule formation efficiency. Through systematic optimization of seed treatment, sowing patterns, and field management techniques, combined with the regulatory effects of physiological efficacious factors, alfalfa plant height, leaf area, and nutritional value all reached the level of first-grade high-quality forage. This method is simple to operate, eco-friendly, and low-cost, and can be widely applied in various Ginkgo planting areas, especially suitable for promotion and implementation in existing Ginkgo forest areas, providing a new path and technical support for the integrated development of forestry and grassland and the transformation of ecological agriculture. Attached Figure Description

[0035] Figure 1 This is a real-life photo of alfalfa intercropping under ginkgo trees in a field.

[0036] Figure 2 This is a diagram showing the growth status and agronomic traits of alfalfa under forest cover.

[0037] Figure 3 Graph showing the changes in soil physicochemical properties before and after planting alfalfa under ginkgo trees;

[0038] Figure 4 In-situ collection image of root exudates from under a ginkgo forest;

[0039] Figure 5 This is a diagram showing the effect of ginkgo root secretions on alfalfa growth.

[0040] Figure 6 This is a diagram showing the isolation and identification of rhizobia;

[0041] Figure 7 This is a diagram showing the effect of Ginkgo root secretions on alfalfa rhizobia.

[0042] Figure 8 This is a graph showing the changes in the expression level of the nod gene in alfalfa root nodules under treatment with ginkgo secretions. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0045] Ginkgo is a common wild-type plant in nature, and alfalfa is a cultivated variety called 'Huaiyang No. 4', both provided by Yangzhou University.

[0046] Example 1

[0047] Intercropping of alfalfa under ginkgo trees

[0048] (1) Land preparation and ridge division: In mid-to-late August, land preparation is carried out between rows of large ginkgo trees. Weeds are removed, and the soil is deeply tilled to a depth of more than 25 cm. The soil is then allowed to dry and weather in the sun. Before planting, 20 kg of compound fertilizer per mu is applied in conjunction with land preparation and incorporated into the soil as base fertilizer. Before sowing, the soil is lightly tilled once more. Ridges with a width of 1.2 meters are made between two rows of ginkgo trees. The ridges are surrounded by drainage ditches with a depth of 10 cm on both sides. Horizontal ditches are dug longitudinally in the field at 4-meter intervals to divide the ridge surface into standard quadrature units of 1.2 meters × 4 meters, which facilitates subsequent planting management and data collection.

[0049] (2) Alfalfa seed treatment: Select alfalfa seeds that are orange to yellowish-brown in appearance and free from mold, and remove impurities, mold, and insect damage. Place the seeds in water, stir, and remove the impurities floating on the surface. Soak the plump seeds that sink to the bottom for 24 hours, then take them out and mix them with 2 kg of fine soil per kg of seeds, and then mix in 10 g of carbendazim for disinfection.

[0050] (3) Seed row sowing: Sow at the end of September. On the raised bed in step (1), make furrows 2-3 cm deep with a row spacing of 15 cm. Sow the seeds treated in step (2) evenly into the furrows according to the seed rate of 1 kg per mu, and cover with 0.2-0.5 cm of soil.

[0051] (4) Thinning and fixing seedlings: When the seedlings are 2-3 cm tall, the first thinning is carried out; when the seedlings are about 10 cm tall, the thinning is carried out again, removing the weak ones and keeping the strong ones.

[0052] (5) Intertillage and weeding: 1-2 times in the first year, 3-4 times in the second year; the first time is at sowing time, the second time is when the seedlings are 3 cm tall, combined with thinning and weeding once; the third time is when the seedlings are 10 cm tall, combined with thinning and weeding once; the fourth time is when the seedlings are 20-25 cm tall, weeding and deep hoeing.

[0053] (6) Harvesting: The first harvest should be carried out around the end of May of the following year when one-tenth of the alfalfa seedlings in the field are flowering. After that, harvest once a month for a total of 3-5 harvests. After each harvest, a 5-7 cm stubble should be left to prevent regrowth from being hindered.

[0054] Actual scene of alfalfa intercropped under ginkgo trees, as shown in the picture. Figure 1 As shown.

[0055] Example 2

[0056] agronomic trait determination of alfalfa

[0057] Agronomic traits of harvested alfalfa, including plant height, leaf area, fresh forage yield, and dry forage yield, were analyzed. The specific method involved randomly selecting several quadrats in the alfalfa field. The size of each quadrat was generally 1 m². 2 However, adjustments can be made based on actual conditions. The number of quadrats should be sufficient to ensure data representativeness, typically no fewer than five. When selecting quadrats, avoid field edges and areas with uneven growth. Quadraats should be chosen in areas with uniform plant growth and no obvious gaps or breaks in the rows. Within each quadrat, cut all alfalfa plants.

[0058] Harvesting measurements were conducted at the first through fourth cuts. The height of fresh alfalfa plants was measured using a ruler, the leaf area was measured using a planimeter, the weight of fresh alfalfa leaves and petioles was measured using direct weighing, and the leaf-to-stem ratio was calculated. The crude protein content was determined using the Kjeldahl method, and the ADF and NDF contents were determined using the Van Soest washing method. The relative feed value (RFV) was calculated based on the ADF and NDF.

[0059] Fresh grass yield determination: The fresh alfalfa grass in each quadrat was weighed and the weight of the fresh grass in each quadrat was recorded. A high-precision electronic scale was used for weighing. The total weight of the fresh grass in all quadrats was summed and divided by the number of quadrats to obtain the average weight of the fresh grass. Then, the fresh grass yield per unit area was calculated based on the area of ​​the quadrats.

[0060] Hay yield determination: A portion of alfalfa samples was taken from each quadrat, and its fresh weight (g1) was measured. The samples were then dried in a forced-air drying oven at 40-60℃ for 48 hours, weighed again (g2), and dried further until a constant weight was achieved (g3). The dry matter content was calculated using the formula: Dry matter content (dm%) = 100 × (g1 - g3) / (g1 - g0)%, where g0 is the weight of the sample in the paper bag. Hay yield was calculated based on the dry matter content and fresh hay yield of each quadrat: Hay yield = Fresh hay yield × Dry matter content. Finally, the average hay yield of all sampling points was taken as the hay yield of the alfalfa field.

[0061] The measurement results are as follows Figure 2 As shown, the results indicate that alfalfa exhibited good performance in terms of plant height, leaf area, leaf-to-stem ratio, fresh forage yield, dry forage yield, crude protein content, ADF, NDF, and relative feed value (RFV) under ginkgo forest conditions. Data from four crop cycles showed that plant height ranged from 70.25 to 78.55 cm, and leaf area remained between 1.86 and 2.06 cm². 2 The leaf-to-stem ratio was between 1.98 and 2.17, indicating that the alfalfa plants under the ginkgo forest were robust and had abundant leaves, which is beneficial for improving their forage value. Fresh grass yield and hay yield reached their highest levels in the second crop, at 28791.67 kg / hm². 2 and 4666.67 kg / hm 2 The crude protein content ranges from 19.94% to 26.4%, indicating a high nutritional level. Furthermore, the ADF and NDF of each crop are 30.4%–31.8% and 36.8%–39.3%, respectively, with a relative feed value (RFV) between 152.96 and 163.28. According to the "First-Crop Hay Quality Grading Standard" (T / CAAA001-2018) issued by the China Animal Husbandry Association, this RFV range meets the first-grade forage standard, indicating that forest-grown alfalfa has high palatability and digestibility. Therefore, forest-grown alfalfa exhibits excellent performance in both agronomical traits and feed quality, meeting the standards for high-quality forage production. This demonstrates the significant advantages of this forest-grass intercropping model in improving forest land utilization efficiency and increasing forage yield and quality, showing promising prospects for widespread application.

[0062] Example 3

[0063] Soil physicochemical property determination

[0064] The changes in physicochemical indicators such as pH, total potassium, total nitrogen, and total phosphorus in soils of ginkgo forests with and without alfalfa intercropping were measured.

[0065] Soil pH determination: Using water as the extraction solvent, with a water-to-soil ratio of 2.5:1, the indicator electrode and reference electrode (or pH composite electrode) are immersed in the soil suspension to form a galvanic cell. At a certain temperature, its electromotive force is related to the pH value of the suspension. The soil pH value can be obtained by measuring the electromotive force of the galvanic cell. Weigh 4g of soil sample into a 50mL beaker, add 10mL of CO2-removed distilled water, and shake vigorously with a horizontal shaker for 2min to fully disperse the soil particles. After standing for 30min, the measurement is performed, and the measurement should be completed within 1 hour.

[0066] Determination of total nitrogen in soil: Weigh 0.4000 g of sample and place it at the bottom of a Kjeldahl flask. Rinse the sample adhering to the flask wall with a small amount of water. Add 5.0 mL of sulfuric acid and 2 g of accelerator, and shake carefully. Place a small curved-neck funnel at the mouth of the flask. Heat at a low temperature for about 10 minutes, then increase the temperature and continue heating until the digestion solution and soil particles turn completely grayish-white with a slight green tinge. Continue digestion for another hour. After digestion, cool and prepare for distillation. Before distillation, preheat the nitrogen analyzer with the prepared sodium hydroxide, sulfuric acid standard solution, and mixed indicator. Clean the pipeline with an empty distillation apparatus until the reading stabilizes. Calculation of results: Total nitrogen content in soil (g / kg) = c × (V - V0) × 14.01 / m, where c is the concentration of the sulfuric acid standard solution (mol / L), V is the volume of the acid standard solution used to titrate the sample (mL), V0 is the volume of the acid standard solution used to titrate the blank (mL), 14.01 is the molar mass of N (g / mol), and m is the sample weight (g).

[0067] Determination of total phosphorus in soil: Accurately weigh 0.2 g of sieved, air-dried sample (accurate to 0.0001 g) and carefully place it at the bottom of a nickel crucible, ensuring it does not stick to the walls. Add 5 drops of anhydrous ethanol to moisten the sample, and then spread 2 g of sodium hydroxide evenly on the sample. Place the crucible in a high-temperature electric furnace and heat it. When the temperature reaches approximately 400°C, disconnect the power and pause for 15 minutes. Then continue heating to 720°C and maintain for 15 minutes, then remove and cool. Add 10 mL of water at approximately 80°C, allow to cool, and after the molten material disperses, transfer the dispersion to a 50 mL volumetric flask with deionized water. Wash the crucible with 10 mL of 6 mol / L hydrochloric acid. Wash the crucible several times with deionized water, transferring all the washings to the volumetric flask. Cool, dilute to volume, mix well, and allow to settle. This is the soil molten liquid. Simultaneously prepare a reagent blank solution using the same method. Pipette 0, 1, 2, 4, 6, 8, and 10 mL of 5 mg / L phosphorus standard solution into 25 mL volumetric flasks, respectively. Simultaneously add an equal volume of blank solution (equal to the sample solution used in the colorimetric assay) and 2-3 drops of dinitrophenol indicator. Adjust the solution to a slightly yellow hue with 10% sodium carbonate solution or 5% sulfuric acid solution. Add 2.5 mL of molybdenum-antimony anti-colorimetric reagent, shake well, and dilute to volume with water. Shake well and incubate at 15°C or higher for 30 min. Measure the absorbance at 700 nm. Plot a standard curve with absorbance on the ordinate and phosphorus concentration (mg / L) on the abscissa. Pipette the sample solution into a 25 mL volumetric flask and dilute with water to approximately 3 / 5 of the total volume. Add 2-3 drops of dinitrophenol indicator and adjust the solution to a slightly yellow hue. Add 2.5 mL of molybdenum-antimony anti-colorimetric reagent, shake well, and dilute to volume with water. Incubate at room temperature (15°C or higher) for 30 min. The absorbance was measured at a wavelength of 700 nm, with a blank test used as a reference solution for zeroing. The corresponding phosphorus content was then obtained from the standard curve. C—Phosphorus content in the colorimetric solution obtained from the calibration curve or regression equation, mg / L; m—Sample mass, g; V1—Volume after sample melting and final volume adjustment, mL; V2—Volume after colorimetric development and final volume adjustment, mL; V3—Volume aliquoted from the melted sample after final volume adjustment, mL.

[0068] Determination of total potassium in soil: Accurately weigh 0.2 g of sieved, air-dried sample (accurate to 0.0001 g) and carefully place it at the bottom of a nickel crucible, ensuring it does not stick to the walls. Add 5 drops of anhydrous ethanol to moisten the sample, and then spread 2 g of sodium hydroxide evenly on the sample. Place the crucible in a high-temperature electric furnace and heat it. When the temperature reaches approximately 400°C, disconnect the power and pause for 15 minutes. Then continue heating to 720°C and maintain for 15 minutes, then remove and cool. Add 10 mL of water at approximately 80°C, allow to cool, and after the molten material disperses, transfer the dispersion to a 50 mL volumetric flask with deionized water. Wash the crucible with 10 mL of 6 mol / L hydrochloric acid. Wash the crucible several times with deionized water, transferring all the washings to the volumetric flask. Cool, dilute to volume, mix well, and allow to settle. This is the soil molten liquid. Simultaneously prepare a reagent blank solution using the same method. Accurately pipette 10 mL of 1000 mg / L potassium standard solution into a 100 mL volumetric flask, dilute to volume with deionized water, and mix well. This is the 100 mg / L potassium standard solution. Dilute the 100 mg / L potassium standard solution with deionized water to prepare a series of standard solutions with concentrations of 0, 6, 12, 18, 24, 30, 36, and 42 mg / L. Measure the concentrations, plot a standard curve, and calculate the linear regression equation. Calculate the potassium concentration of the test solution from the linear regression equation. The total potassium (K) content of the soil is calculated using the following formula: C—Potassium content in the soil test solution obtained from the calibration curve, mg / L; m—Mass of the sample taken, g; V1—Volume of the digestion solution, mL.

[0069] like Figure 3 As shown, the soil physicochemical properties of the bare area (CK) without alfalfa intercropping and three plots (S1, S2, and S3) after planting were measured. The results showed that all indicators changed to varying degrees. Soil pH increased in S1, S2, and S3 after planting, with the average pH rising from 6.73 in the control (CK) to 6.78-6.88. Soil total nitrogen (TN) content increased significantly after planting. The average TN content in CK was 32 mg / kg, while the TN contents in S1, S2, and S3 reached 38 mg / kg, 39 mg / kg, and 38 mg / kg, respectively, showing a significant increase. This indicates that crop rhizosphere nitrogen fixation and biological activity may have promoted the increase in soil nitrogen content. Soil total phosphorus (TP) and total potassium (TK) contents also increased to some extent. In conclusion, planting activities had a positive impact on soil physicochemical properties, especially with significant improvements in soil pH, total nitrogen, total phosphorus, and total potassium contents.

[0070] Example 4

[0071] Collection of Ginkgo root secretions

[0072] In-situ extraction of Ginkgo root exudates was used, and their composition and structure were analyzed using metabolomics techniques to identify the main active metabolites. Specific implementation method: Healthy large Ginkgo trees in a Ginkgo forest that were not intercropped with alfalfa were selected as test plants. The trees were dug along the root distribution direction, exposing part of the taproot and lateral roots, taking care to avoid mechanical damage to the roots during the operation. After the roots were exposed, the root area was wrapped with a transparent plastic bag and backfilled with soil to restore the natural growth environment. The plants were left to stand for 72 hours. Figure 4 As shown.

[0073] After static treatment, the plants were removed and the soil particles adhering to the root surface were thoroughly rinsed with deionized water. The roots were then wrapped with moist sterile filter paper and kept moist with deionized water. The wrapped plants were placed in 50ml centrifuge tubes, the tubes were sealed with sealing film, and the tubes were reburied in the soil at the original excavation location for 24 hours to promote the enrichment and adsorption of root exudates. The plants were then removed, and root segments wrapped in filter paper were cut and transported back to the laboratory at low temperature in an ice box. Under sterile conditions, the root segments were immersed in 100ml of deionized water and extracted by shaking for 30 minutes. The extract was filtered through filter paper, and the filtrate was collected and concentrated by rotary evaporation. The resulting liquid or dry powder is the Ginkgo root exudate extract, which was finally stored at -80℃ for subsequent analysis.

[0074] Example 5

[0075] The effect of ginkgo root exudates on alfalfa growth

[0076] Under laboratory conditions, one-month-old alfalfa seedlings with uniform growth were selected, and different treatment groups were set up: the ginkgo root exudate prepared in Example 4 was diluted with ultrapure water to OD. 600 =0.0086 and 0.0172, with the water treatment serving as the control group. Each group had 10 plants as replicates. Each treatment applied 500 mL of the diluted solution, irrigated every 4-6 days, for a total of 16 days. After treatment, the plant height was measured using a ruler; leaf area was measured using a leaf area meter (or planimeter method); the total number of root nodules was calculated based on the leaf area (cm²) of the nodule distribution area. 2 Convert to root nodule density per unit area (number of nodules / m²) 2 ).

[0077] The results are as follows Figure 5 As shown, the effects of different concentrations of Ginkgo root exudates on alfalfa plant growth are illustrated. The results indicate that, compared to the control (CK) treated with water, Ginkgo root exudate treatment had a positive effect on alfalfa growth and development, significantly increasing plant height, leaf area, and root nodule density. The optimal concentration of root exudate was OD0.05. 600The growth-promoting effect was most significant when the coefficient of growth ratio was 0.0172, indicating that alfalfa achieved the optimal growth response and symbiotic nodulation effect under this condition.

[0078] Example 6

[0079] Isolation and identification of rhizobia

[0080] YMA (Yeast Mannitol Agar) medium was used as the basic growth medium for rhizobia. YMA Congo Red medium, obtained by adding 5 mL of 1% Congo Red solution to YMA, was used for preliminary screening and identification of alfalfa rhizobia. Typical, plump, pinkish root nodules from alfalfa roots in Ginkgo forests were selected, with a small number of attached root segments retained. The process was performed in a clean bench. First, the surface of the root nodules was rinsed with sterile water to remove soil impurities. Then, they were sequentially immersed in 70% ethanol solution for 30 seconds, followed by immersion in 2% sodium hypochlorite (NaClO) solution for 2 minutes for surface disinfection. Finally, they were rinsed three times with sterile water to remove residual disinfectant. The treated root nodules were placed in a sterile mortar, and 2 mL of sterile water was added for thorough grinding. The resulting homogenate was serially diluted tenfold. 10... -3 10 -4 and 10 -5 50 μL of each concentration gradient dilution was evenly spread onto YMA Congo Red agar plates, with three replicates for each dilution gradient. The medium was incubated at 28°C for 5–7 days, and colony morphology was observed. Single colonies with smooth surfaces, stickiness, and no absorption of Congo Red pigment were selected for streak purification. The purified strains were stored at 4°C for later use. Subsequently, colony PCR amplification was performed on the purified strains, and they were sent to Shanghai Sangon Biotech Co., Ltd. for 16S rDNA sequencing. After the sequencing results were returned, they were compared with known rhizobium sequences using the NCBI BLAST database to confirm the species and taxonomic position of the isolated strains, thus ultimately identifying the rhizobium species.

[0081] like Figure 6 As shown, typical colonies with smooth surfaces and viscous textures were initially screened on YMA medium containing Congo red pigment. These colonies were then transferred to pigment-free YMA medium for further purification and culture, and their colony growth morphology was observed. The colonies were round, with a viscous, raised surface, translucent or opaque, and grayish-white or milky-white, consistent with the basic characteristics of rhizobia. Further 16S rDNA fragment amplification and sequencing of this strain, along with comparison with the NCBI database and phylogenetic tree analysis, ultimately identified the strain as *Sinorhizobium meliloti*.

[0082] Example 7

[0083] Effects of Ginkgo root exudates on rhizobia

[0084] Add to YMA medium diluted to OD 600 A root exudate concentration of 0.0172 was used as the treatment group, while a culture medium supplemented with an equal volume of ddH2O served as the control group. Each group was inoculated with *Sinorhizobium meliloti* from *Alfalfa* (Example 6) and cultured at 28°C. Colony growth was observed daily starting from day 3 after inoculation, and changes in plaque area were recorded to evaluate the promoting effect of root exudates and their representative components on rhizobium growth.

[0085] The results are as follows Figure 7 As shown, the optimal concentration of root exudate (OD) selected in Example 5 was used. 600 Treatment of alfalfa rhizobia with a dilution of 0.0172) revealed that Ginkgo root exudates significantly promoted the growth of rhizobia, resulting in accelerated colony growth.

[0086] Example 8

[0087] Effects of Ginkgo root exudates on nod gene expression

[0088] To further explore the molecular mechanism by which Ginkgo root exudates promote alfalfa nodule formation, five nod genes (Nucleotide Binding Oligomerization Domain genes) closely related to nodule formation were selected. The five key genes were: MS.gene023118, MS.gene028334, MS.gene35123, MS.gene28707, and MS.gene35125. Their concentrations (OD) at different levels were measured. 600 =0.0086, 0.0172) Expression levels under Ginkgo root exudate treatment. The method of treating alfalfa with Ginkgo root exudate was the same as in Example 5, specifically: alfalfa seedlings with uniform growth were cultured in a greenhouse for one month, and their roots were irrigated with the above-mentioned concentration of Ginkgo root exudate solution, once every 4 days, 500 mL each time, for 16 consecutive days, with the water treatment group as the control. After treatment, root nodule tissue was collected, and the expression levels of 5 target nod genes were detected by qRT-PCR to analyze the regulatory effect of Ginkgo exudate on the nodulation signaling pathway. The results are as follows: Figure 8The results showed that, compared with the control group treated with water, all the above-mentioned nod genes were significantly upregulated at all treatment concentrations of Ginkgo root exudates (p<0.05), indicating that Ginkgo root exudates can effectively induce the expression of key genes in the nod signaling pathway. Ginkgo root exudates not only significantly promote the growth of endophytic bacteria in root nodules, but also enhance the nodulation ability and biological nitrogen fixation potential of alfalfa by upregulating the expression of nodulation genes, thereby improving the nutrient status of understory soil and enhancing soil quality. This further verifies the feasibility and scientific validity of the forest-grassland intercropping system of this invention in improving ecological benefits.

[0089] In summary, through multi-level and multi-dimensional controlled experiments, this invention utilizes Ginkgo root exudates to regulate alfalfa nodulation and nitrogen fixation processes. Combined with a scientifically standardized land preparation and forest management system, it provides a good growth foundation and drainage / aeration environment for alfalfa. The invention demonstrates significant advantages in soil improvement, alfalfa quality enhancement, and nodule formation regulation, fully showcasing the practicality and innovation of this intercropping method. Setting up raised beds and standardized quadrats under the Ginkgo forest effectively improves forest management efficiency and establishes a standardized model that is easy to promote.

Claims

1. A method for forestry-grassland intercropping based on the regulation of alfalfa nodulation and growth promotion using Ginkgo root exudates, characterized in that, Includes the following steps: (1) Land preparation and ridge division: Deeply plow the land between rows of ginkgo trees, and then shallowly cultivate and level it before sowing. Establish high ridges between two rows of ginkgo trees, and dig longitudinal ditches in the field to form quadrats. (2) Alfalfa seed treatment: Soak alfalfa seeds in water, take out the plump seeds that sink to the bottom and mix them with fine soil and carbendazim; (3) Seed row sowing: Make furrows on the raised beds divided in step (1) according to the row spacing, and sow the alfalfa seeds treated in step (2) evenly into the furrows and cover with soil; (4) Thinning and fixing seedlings: Thinning is done twice in the same year, removing weak seedlings and keeping strong ones; (5) Cultivation and weeding: Cultivate and weed 1-2 times in the first year and 3-4 times in the second year; (6) Harvesting: The first harvest is in the second year, followed by monthly harvests, leaving a stubble after each harvest.

2. The intercropping method according to claim 1, characterized in that, In step (1), the soil is deeply tilled to a depth of more than 25cm.

3. The intercropping method according to claim 1, characterized in that, In step (1), the width of the raised beds is 1.2-1.5m, and drainage ditches with a depth of 10-15cm are dug every 4-5m to form a (1.2-1.5)m×(4-5m) quadrat.

4. The intercropping method according to claim 1, characterized in that, In step (2), the mass ratio of seeds: fine soil: carbendazim is 1:1.5-2.5:0.01-0.

02.

5. The intercropping method according to claim 1, characterized in that, In step (3), dig furrows 2-3 cm deep with a row spacing of 10-20 cm, use 0.8-1.2 kg of seeds per mu, and cover with 0.2-0.5 cm of soil.

6. The intercropping method according to claim 1, characterized in that, In step (4), when the seedlings are 2-3cm tall, the first thinning is carried out, and when the seedlings are 8-12cm tall, the second thinning is carried out.

7. The intercropping method according to claim 1, characterized in that, Step (5) Weeding and hoeing are carried out at the time of sowing, when the seedlings are 3-10 cm tall, or when the seedlings are 20-25 cm tall, and deep hoeing is carried out when the seedlings are 20-25 cm tall.

8. The intercropping method according to claim 1, characterized in that, In step (6), the alfalfa is first cut when the budding rate reaches 8-15%. After the first cut, it is cut once every 25-35 days for a total of 3-5 times. The stubble height is 5-7cm after each cut.

9. Application of Ginkgo root exudates in promoting alfalfa seedling growth, promoting the growth of endophytic bacteria in alfalfa root nodules, enhancing alfalfa nodulation ability and biological nitrogen fixation potential.

10. The application according to claim 9, characterized in that, Ginkgo root exudates were collected by selecting robust ginkgo trees as test plants. The trees were excavated along the root distribution direction, exposing part of the main and lateral roots. After the roots were exposed, the root area was wrapped with a transparent plastic bag and backfilled with soil to restore the natural growing environment. The plants were left to stand, then removed and thoroughly rinsed with deionized water to remove soil particles adhering to the root surface. The roots were then wrapped with moist sterile filter paper and kept moist with deionized water. The wrapped plants were placed in centrifuge tubes, the tube openings were sealed with sealing film, and the centrifuge tubes were reburied in the soil at the original excavation location for continued treatment to promote the enrichment and adsorption of root exudates. The plants were then removed, and root segments wrapped in filter paper were cut. Under sterile conditions, the root segments were immersed in deionized water and extracted by shaking. The extract was filtered through filter paper, and the filtrate was collected and concentrated by rotary evaporation. The resulting liquid or dry powder is the ginkgo root exudate extract.

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