Forest and grass composite planting method for regulating clover nodulation and promoting growth based on gingko root exudates

Abstract

The invention provides a forest and grass composite planting method for regulating clover nodulation and promoting growth based on gingko root exudates. According to the method, the space under the gingko forest is fully utilized, normal growth and efficient utilization of alfalfa are achieved through land preparation and fertilization between forest rows, construction of a high-ridge quadrat, selection of high-quality alfalfa seeds for drilling and combination of reasonable field management measures such as thinning and final singling and intertillage weeding, and a scientific and reasonable alfalfa interplanting mode is provided. According to the method, soil physical and chemical index measurement is combined, the forest land soil before and after alfalfa planting is systematically evaluated, it is proved that the method can effectively improve the soil physical and chemical properties and promote ginkgo-alfalfa synergistic growth, and the method has remarkable ecological benefits and application and popularization prospects. In addition, the invention further verifies that the gingko root exudates have an obvious growth promoting effect on alfalfa rhizobium, and can improve the expression level of the alfalfa nod gene and enhance the number and quality of root nodules, thereby improving the alfalfa production performance and the soil improvement effect.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture, and in particular relates to a forest-grass composite planting method for regulating alfalfa nodulation and growth promotion by utilizing ginkgo root secretions. Background Art

[0002] Ginkgo (Ginkgo biloba) is an important economic and ecological tree species with high medicinal, edible, and ornamental value. Ginkgo is cultivated over a wide area, particularly in the middle and lower reaches of the Yangtze River, East China, and Southwest China, where large-scale cultivation bases are located. With the development of the ginkgo industry, the market application of its leaves, fruits, and seedlings has gradually expanded, becoming an important component of forestry economic development. However, as the cultivated area continues to expand, the ginkgo industry also faces practical problems such as structural surplus and inefficient resource allocation. The seedling industry has developed rapidly but is highly homogenized. The imbalance between supply and demand has led to product backlogs and price fluctuations, unstable planting benefits, and weakened forest farmers' enthusiasm for planting. At the same time, the land under ginkgo forests generally has low utilization rates and poor added benefits, failing to fully utilize the complex functions of forest land, becoming a key shortcoming restricting the sustainable development of the industry.

[0003] Alfalfa (Medicago sativa), a perennial legume forage, has excellent nitrogen fixation ability, ecological adaptability and feeding value. It can not only improve soil fertility, but also provide high-protein forage resources for animal husbandry. It is one of the high-quality forages that the country is currently vigorously promoting. Under forest conditions, the deep root system and symbiotic rhizobia of alfalfa can significantly improve soil structure, increase soil fertility, and achieve synergistic benefits between forest and grass. In recent years, understory grass planting technology has received increasing attention, but there are still many technical difficulties in the specific implementation process, especially in signal recognition, nodulation efficiency, and planting management mode in the symbiotic system of legume forage and rhizobia. How to scientifically select seeds, rationally layout, and improve nodule formation and nitrogen fixation efficiency are the keys to achieving efficient forest-grass integration. Summary of the Invention

[0004] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a forest-grass composite planting method based on the regulation of alfalfa nodulation and growth promotion by ginkgo root secretions, so as to make full use of idle land under the forest, create economic benefits, effectively improve the growth of alfalfa seedlings and the growth of nodule endophytes, and at the same time enhance the nodulation ability and biological nitrogen fixation potential of alfalfa.

[0005] Technical solution: The present invention includes a forest-grass composite planting method based on regulating alfalfa nodulation and growth promotion by ginkgo root secretions, comprising the following steps:

[0006] (1) Land preparation and border division: Deeply plow the land between the rows of ginkgo trees, then shallowly plow and level it before sowing. Build high borders between two rows of ginkgo trees, and dig trenches at intervals in the vertical direction of the field to form sample units.

[0007] (2) Alfalfa seed treatment: Soak the 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 drill sowing: digging furrows on the bed surface divided in step (1) according to the row spacing, sowing the alfalfa seeds treated in step (2) evenly into the furrows and covering with soil;

[0009] (4) Thinning and finalizing seedlings: Thin out seedlings twice a year, removing weak ones and retaining strong ones;

[0010] (5) Intertillage and weeding: Intertillage and weeding should be done 1-2 times in the first year and 3-4 times in the second year;

[0011] (6) Mowing and harvesting: The first mowing is done in the second year, and mowing is done every month thereafter, leaving stubble after each mowing.

[0012] Wherein, in step (1), the soil is deep-turned by more than 25 cm.

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

[0014] As a preference, a high ridge with a width of 1.2m is selected, and the two sides of the high ridge are surrounded by drainage ditches. Horizontal ditches are dug at intervals of 4 meters in the longitudinal direction of the forest, dividing the ridge surface into standard sample units of 1.2m×4m to facilitate subsequent planting management and data collection.

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

[0016] Preferably, the plump seeds that have sunk to the bottom are soaked for 24 hours, taken out and mixed with 2 kg of fine soil per kg of seeds, and then 10 g of carbendazim for disinfection.

[0017] Wherein, in step (3), a furrow 2-3 cm deep is opened with a row spacing of 10-20 cm, the seed dosage per mu is 0.8-1.2 kg, and the soil is covered with 0.2-0.5 cm.

[0018] As a preferred method, the row spacing is 15 cm and the seed dosage per mu is 1 kg.

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

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

[0021] Wherein, in step (6), the first mowing is performed when the budding rate of alfalfa reaches 8-15%.

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

[0023] As a best practice, mowing should start at the end of May of the year following sowing.

[0024] Wherein, in step (6), the stubble height after each mowing is 5 to 7 cm.

[0025] The ginkgo root secretions of the present invention are used to promote the growth of alfalfa seedlings, promote the growth of endophytes in alfalfa root nodules, and enhance the nodulation ability and biological nitrogen fixation potential of alfalfa.

[0026] Among them, the ginkgo root secretions were collected by selecting healthy ginkgo trees as test plants, digging along the root distribution direction, exposing part of the main root and lateral roots, and after the roots were exposed, wrapping the root area with a transparent plastic bag and backfilling the soil to restore the natural growth environment. After standing for treatment, the plants were taken out and the soil particles attached to the root surface were thoroughly rinsed with deionized water. The roots were then wrapped with moistened sterile filter paper and kept moist with deionized water. The plants with wrapped roots were placed in a centrifuge tube, the tube mouth was sealed with a sealing film, and the centrifuge tube was buried in the soil at the original excavation position again for continuous treatment to promote the enrichment and adsorption of root secretions. The plants were then taken out, the root segments wrapped in the filter paper were cut, and the root segments were placed in deionized water under sterile conditions for oscillation extraction. After the extract was filtered through the filter paper, the filtrate was collected and concentrated by rotary evaporation. The resulting liquid or dry powder was the ginkgo root secretion extract.

[0027] The present invention develops a forest-grass composite planting method based on the regulation of alfalfa nodule formation and growth promotion by ginkgo root secretions, which not only helps to improve the planting efficiency and nitrogen fixation capacity of alfalfa, but also effectively activates idle resources under the ginkgo forest, enhances the dual ecological and economic functions of the forest, and has broad application prospects and promotion value.

[0028] The method of the present invention fully utilizes the space under the ginkgo forest and proposes a scientific and reasonable alfalfa intercropping model. By preparing the land and applying fertilizers between the rows of the ginkgo forest, a standard 1.2-meter-by-4-meter high-bed plot is constructed. High-quality alfalfa seeds are selected and soaked and disinfected before being sown in rows. Combined with reasonable field management measures such as thinning, weeding, and pest and disease control, efficient growth and utilization of alfalfa are achieved. The present invention further collects ginkgo root secretions through in situ extraction technology and uses metabolomics methods to analyze its active ingredients. The secretions are then used in alfalfa rhizobium and seedling treatment experiment to verify its biological functions in promoting nodule formation, improving nitrogen fixation capacity, and enhancing alfalfa growth. The experimental results show that ginkgo root secretions have a significant growth-promoting effect on alfalfa rhizobium, and can increase the expression level of alfalfa nod gene, enhance the number and quality of nodules, thereby improving alfalfa production performance and soil improvement effects. In addition, the present invention also combines the determination of soil physical and chemical indicators to systematically evaluate the pH, total N, P, and K of forest soil before and after alfalfa planting, confirming that this method can effectively improve soil physical and chemical properties, promote the coordinated growth of ginkgo and alfalfa, and has significant ecological benefits and prospects for promotion and application.

[0029] The present invention is the first to discover that ginkgo root secretions can promote alfalfa growth and nitrogen fixation. The present invention utilizes ginkgo root secretions to regulate the activity of alfalfa rhizobia and the expression of nodulation genes, thereby enhancing the nodulation and nitrogen fixation capabilities of alfalfa, further enabling efficient composite planting within ginkgo woodlands.

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

[0031] The present invention makes full use of the long-term idle land under the ginkgo forest, and constructs a forest-grass composite system by interplanting alfalfa, which significantly improves the efficiency of land composite utilization, expands the economic function of the forest, and alleviates the efficiency bottleneck caused by single forestry management. Alfalfa has a well-developed root system and strong nitrogen-fixing ability. Symbiosis 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, the present invention proposes and verifies for the first time that ginkgo root secretions can significantly promote the growth of alfalfa rhizobia and the expression of nodulation genes, enhance the nodulation and nitrogen-fixing ability of alfalfa, and thus improve the nutritional quality and yield of alfalfa, and achieve a continuous and stable supply of high-quality forage.

[0032] This invention breaks the traditional idea of ​​forest-grass composite planting that only stays at the spatial superposition utilization. For the first time, ginkgo secretions are collected and studied as biological active factors in the forest-grass composite system, and the research on the biological induction of leguminous forage rhizobia using tree root secretions is combined with its application in actual planting. An integrated and standardized field planting technology is established, and a standardized sample plot (1.2m×4m) planting unit is constructed under the ginkgo forest. Combined with the high ridge + drainage ditch setting, the visualization and precision of alfalfa sowing management are realized. This is a standardized design that the existing forest-grass planting methods do not have.

[0033] The present invention verifies the biological regulation function of ginkgo secretions on alfalfa root nodules and seedlings, experimentally analyzes the functional intercropping mechanism of ginkgo root secretions in regulating alfalfa nodulation formation and growth and development, and combines the expression level of the nod gene (Nucleotide Binding Oligomerization Domain gene) to analyze its mechanism of action on nodulation, constructs an interaction network among "ginkgo-secretions-alfalfa-rhizobia", and effectively improves the nodulation ability and production performance of legume forage in the understory growth environment.

[0034] In addition, the present invention proposes for the first time the use of ginkgo root secretions as a natural plant-derived regulatory factor to effectively improve the expression level of the alfalfa NOD gene and the efficiency of nodule formation. Through systematic seed treatment, sowing pattern and field management technology optimization, combined with the regulatory effect of physiological synergistic factors, the plant height, leaf area and nutritional value of alfalfa all reach the level of first-class high-quality forage. This method is simple to operate, eco-friendly, and low-cost. It can be widely used in various ginkgo planting areas and is particularly suitable for promotion and implementation in existing ginkgo forests, providing new paths and technical support for the integrated development of forest and grass and the transformation of ecological agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a real picture of the field where alfalfa is intercropped under the ginkgo forest;

[0036] Figure 2 This is a graph showing the growth status and agronomic characteristics of alfalfa under the forest;

[0037] Figure 3 Changes in soil physical and chemical properties before and after planting alfalfa under a ginkgo forest;

[0038] Figure 4 In situ collection of root exudates 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 of the isolation and identification of rhizobia;

[0041] Figure 7 This is a diagram showing the effect of ginkgo root secretions on alfalfa root nodule bacteria.

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

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

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

[0045] Ginkgo biloba was a wild plant found in nature, and alfalfa was the cultivated variety 'Huaiyang No. 4', both of which were provided by Yangzhou University.

[0046] Example 1

[0047] Intercropping of alfalfa under ginkgo trees

[0048] (1) Land preparation and bed division: In mid-to-late August, land preparation was carried out between the rows of large ginkgo trees. Weeds were removed and the soil was plowed more than 25 cm deep. The soil was then exposed to the sun for weathering and ripening. Before planting, 20 kg of compound fertilizer was applied per mu in combination with land preparation and incorporated into the soil as base fertilizer. Before sowing, shallow plowing was performed once more. A 1.2-meter-wide raised bed was made between the two rows of ginkgo trees. The raised bed was surrounded by 10-cm-deep drainage ditches on both sides. Horizontal ditches were dug at 4-meter intervals in the longitudinal direction of the field to divide the bed into standard sample units of 1.2 m × 4 m to facilitate subsequent planting management and data collection.

[0049] (2) Alfalfa seed treatment: Select alfalfa seeds with an orange to yellowish brown appearance and no mold, and remove impurities, mold, or insect-damaged seeds; place the seeds in water, stir, and remove impurities floating on the water surface; soak the plump seeds that sink to the bottom for 24 hours, remove them, and mix them with 2 kg of fine soil per kg of seeds, and then mix them with 10 g of carbendazim for disinfection;

[0050] (3) Seed drilling: Sowing is carried out at the end of September. A 2-3 cm deep furrow is opened on the bed surface of step (1) with a row spacing of 15 cm. The seeds treated in step (2) are evenly sown in the furrow at a seed rate of 1 kg per mu, and covered with 0.2-0.5 cm of soil;

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

[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, the second time is when the seedlings are 3 cm tall, combined with thinning and weeding; the third time is when the seedlings are 10 cm tall, combined with thinning and weeding again; the fourth time is when the seedlings are 20-25 cm tall, weeding and deep hoeing;

[0053] (6) Mowing and harvesting: Around the end of May of the following year, the first mowing should be carried out when one-tenth of the alfalfa seedlings in the field are in bloom. Mowing should be carried out once a month thereafter, for a total of 3-5 times. After each mowing, a 5-7 cm stubble should be left to prevent regeneration from being blocked.

[0054] The actual scene of the field where alfalfa is interplanted under the ginkgo forest is as follows Figure 1 shown.

[0055] Example 2

[0056] Determination of agronomic traits of alfalfa

[0057] The agronomic traits of the harvested alfalfa were analyzed, including plant height, leaf area, fresh grass yield, and hay yield. The specific method is: randomly select several plots in the alfalfa field. The size of the plot is generally 1m 2 The number of plots should be sufficient to ensure representative data, but can be adjusted based on actual conditions. The number of plots should be sufficient to ensure representative data, typically no less than five. When selecting plots, avoid the edges of the field and areas of uneven growth. The plots should be selected in areas with uniform plant growth and no obvious missing or broken ridges. Within each plot, all alfalfa plants should be cut.

[0058] Harvest measurements were carried out at the first to fourth mowing stubbles. The fresh alfalfa plant height was measured with a ruler, the fresh alfalfa leaf area was measured using the planimeter method, the fresh alfalfa leaf and petiole weight were measured using the direct weighing method, and the leaf-stem ratio was calculated. The crude protein content was determined by the Kjeldahl method, and the ADF and NDF contents were determined by the Van Soest washing method. The relative feeding value (RFV) was calculated based on the ADF and NDF.

[0059] Determine fresh grass yield: Weigh the fresh alfalfa grass within each plot and record the weight of each plot. Use a high-precision electronic scale for weighing. Add the fresh grass weights of all plots and divide by the number of plots to calculate the average fresh grass weight. Then, convert the fresh grass yield per unit area based on the plot area.

[0060] Hay yield determination: Take a portion of alfalfa sample from each sample plot, weigh its fresh weight (g1), then dry it in a forced air drying oven at 40-60°C, weigh it after drying for 48 hours (g2), and continue drying until the mass is constant (g3). Calculate the dry matter content according to the formula: Dry matter content (dm%) = 100×(g1-g3) / (g1-g0)%, where g0 is the weight of the paper bag of the sample. Calculate the hay yield based on the dry matter content and fresh grass yield of each sample plot. Hay yield = fresh grass yield × dry matter content. Finally, take the average hay yield of all sampling points as the hay yield of the alfalfa field.

[0061] The results of the test are as follows Figure 2 The results showed that alfalfa under the ginkgo forest showed good performance in terms of plant height, leaf area, leaf-to-stem ratio, fresh grass yield, hay yield, crude protein content, ADF, NDF and relative feeding value (RFV). Based on the data of four crops, the plant height ranged from 70.25 to 78.55 cm, and the leaf area was maintained at 1.86 to 2.06 cm. 2 The leaf-to-stem ratio ranged from 1.98 to 2.17, indicating that alfalfa plants under the ginkgo forest grew robustly and had abundant leaves, which was beneficial for improving its feed value. The fresh grass yield and hay yield reached their highest levels in the second crop, at 28,791.67 kg / hm2, respectively. 2 and 4666.67 kg / hm 2 , the crude protein content is between 19.94% and 26.4%, and the nutritional level is relatively high. In addition, the ADF and NDF of each crop are 30.4% to 31.8% and 36.8% to 39.3% respectively, and the relative feeding value (RFV) is 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, the RFV range meets the first-level forage grade standard, indicating that the forest alfalfa has high palatability and digestibility. Therefore, the planting of purple alfalfa under the forest performs well in agronomic traits and feeding quality, meets the standards for high-quality forage production, and reflects the significant advantages of this forest-grass composite planting model in improving forest land utilization efficiency and increasing forage yield and quality, and has good prospects for promotion and application.

[0062] Example 3

[0063] Determination of soil physical and chemical properties

[0064] The changes in soil physicochemical indices such as pH value, total potassium, total nitrogen, and total phosphorus in soils without intercropping alfalfa and with intercropping alfalfa and ginkgo were determined.

[0065] Soil pH determination: Using water as the extractant at a water-to-soil ratio of 2.5:1, immerse the indicator electrode and reference electrode (or pH combination electrode) in the soil suspension to form a galvanic cell. At a certain temperature, the electromotive force (EMF) of the cell is related to the pH value of the suspension. The soil pH value can be determined by measuring the electromotive force of the galvanic cell. Weigh 4g of soil sample into a 50mL beaker, add 10mL of CO2-free distilled water, and vigorously shake with a horizontal oscillator for 2 minutes to fully disperse the soil particles. After standing for 30 minutes, perform the measurement. The measurement should be completed within 1 hour.

[0066] Determination of total nitrogen in soil: Weigh 0.4000g of sample and place it at the bottom of a Kelvin flask. Rinse any sample adhering to the flask walls with a small amount of water. Add 5.0mL of sulfuric acid and 2g of accelerator, shake carefully, and place a small curved-neck funnel at the flask mouth. Heat at low temperature for approximately 10 minutes. Continue heating until the digestion solution and soil particles have completely turned off-white with a slight green tint. Continue digesting for another hour. After digestion, cool the solution and prepare for distillation. Before distillation, thoroughly preheat the nitrogen analyzer with the prepared sodium hydroxide and sulfuric acid standard solutions and mixed indicator. Clean the pipes by air distillation until the reading stabilizes. Calculation: 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 nitrogen, g / mol; and m is the sample weight, g.

[0067] Determination of total soil phosphorus: Accurately weigh 0.2g of sieved, air-dried sample to the nearest 0.0001g and carefully place it in the bottom of a nickel crucible, ensuring that no residue adheres to the sides. Add 5 drops of anhydrous ethanol to moisten the sample, and spread 2g of sodium hydroxide evenly on top. Place the crucible in a high-temperature electric furnace and heat it. When the temperature reaches approximately 400°C, turn off the power and pause for 15 minutes. Then, continue heating to 720°C and hold for 15 minutes. Remove and cool. Add 10mL of water at approximately 80°C and allow to cool. Once the frit has dispersed, transfer the dispersion to a 50mL volumetric flask with deionized water. Rinse the crucible with 10mL of 6 mol / L hydrochloric acid. Rinse the crucible several times with deionized water, transferring all the washes to the volumetric flask. Cool, adjust to volume, mix thoroughly, and allow to settle. This is the soil melt. Simultaneously, prepare a reagent blank solution as described above. Pipette 0, 1, 2, 4, 6, 8, and 10 mL of a 5 mg / L phosphorus standard solution into a 25 mL volumetric flask. Add an equal volume of blank solution and 2-3 drops of dinitrophenol indicator to the sample solution used for the colorimetric assay. Adjust the solution to a slightly yellowish color with 10% sodium carbonate solution or 5% sulfuric acid solution. Add 2.5 mL of molybdenum antimony anti-colorimetric reagent, shake well, and bring to volume with water. Shake well, incubate at a temperature above 15°C for 30 minutes, and then measure the absorbance at 700 nm. Plot a standard curve with absorbance as the y-axis and phosphorus concentration (mg / L) as the x-axis. Pipette the sample solution to be tested into a 25 mL volumetric flask and dilute to approximately 3 / 5 of the total volume with water. Add 2-3 drops of dinitrophenol indicator and adjust the solution to a slightly yellowish color. Add 2.5 mL of molybdenum antimony anti-colorimetric reagent, shake well, and bring to volume with water. Incubate at room temperature above 15°C for 30 minutes. Measure the absorbance at a wavelength of 700 nm, using the blank test solution as the reference solution to adjust to zero. Obtain the corresponding phosphorus content from the standard curve. C is the phosphorus content in the color developing solution obtained from the calibration curve or regression equation, mg / L; m is the mass of the sample weighed, g; V1 is the volume of the sample after molten solution, mL; V2 is the volume of the solution during color development, mL; V3 is the volume taken from the molten sample after molten solution is fixed to volume, mL.

[0068] Determination of total potassium in soil: Accurately weigh 0.2g of sieved, air-dried sample to the nearest 0.0001g and carefully place it in the bottom of a nickel crucible, ensuring that no residue adheres to the sides. Add 5 drops of anhydrous ethanol to moisten the sample, and spread 2g of sodium hydroxide evenly on top. Place the crucible in a high-temperature electric furnace and heat it. When the temperature reaches approximately 400°C, turn off the power and pause for 15 minutes. Then, continue heating to 720°C and hold for 15 minutes. Remove and cool. Add 10mL of water at approximately 80°C and allow to cool. Once the frit has dispersed, transfer the dispersion to a 50mL volumetric flask with deionized water. Rinse the crucible with 10mL of 6 mol / L hydrochloric acid. Rinse the crucible several times with deionized water, transferring all the washes to the volumetric flask. Cool, adjust to volume, mix thoroughly, and allow to settle. This is the soil melt. Simultaneously, prepare a reagent blank solution as described above. Accurately pipette 10mL of 1000mg / L potassium standard solution into a 100mL volumetric flask, dilute to volume with deionized water, and mix well. This is the 100mg / L potassium standard solution. Dilute the 100mg / L potassium standard solution with deionized water to a series of standard solutions with concentrations of 0, 6, 12, 18, 24, 30, 36, and 42mg / L. Determine, draw 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 as follows: C is the potassium content of the soil solution to be tested obtained from the calibration curve, mg / L; m is the mass of the sample weighed, g; V1 is the volume of the digestion solution, mL.

[0069] like Figure 3 As shown in the study, soil physical and chemical properties were measured in the bare plot (CK) without intercropping alfalfa and in three plots (S1, S2, and S3) after planting. The results showed that various indicators changed to varying degrees. Soil pH increased in S1, S2, and S3 after planting, with the average pH increasing from 6.73 in the control (CK) to 6.78-6.88. Soil total nitrogen (TN) content increased significantly after planting, with an average of 32 mg / kg in CK and 38 mg / kg, 39 mg / kg, and 38 mg / kg in S1, S2, and S3, respectively. This significant increase suggests that nitrogen fixation and biological activity in the crop rhizosphere may have contributed to the increase in soil nitrogen content. Soil total phosphorus (TP) and total potassium (TK) contents also increased to a certain extent. In summary, planting activities have had a positive impact on soil physical and chemical properties, particularly with significant improvements in soil pH, total nitrogen, total phosphorus, and total potassium.

[0070] Example 4

[0071] Collection of Ginkgo Root Exudates

[0072] The in situ extraction method was used to collect the root secretions of ginkgo, and its composition structure was analyzed by combining metabolomics technology to identify the main active metabolites. Specific implementation method: Select healthy ginkgo trees in the ginkgo forest that are not interplanted with alfalfa as test plants, dig along the root distribution direction, expose part of the main root and lateral roots, and pay attention to avoid mechanical damage to the root system during the operation. After the roots are exposed, use transparent plastic bags to wrap the root area, and backfill the soil to restore the natural growth environment. Let it stand for 72 hours. Figure 4 shown.

[0073] After the static treatment, the plants were removed and soil particles attached to the root surfaces were thoroughly rinsed with deionized water. The roots were then wrapped with moistened sterile filter paper and kept moist with deionized water. The wrapped plants were placed in a 50 ml centrifuge tube, sealed with parafilm, and the tubes were re-buried in the soil at the original excavation site. The treatment continued for 24 hours to promote the enrichment and adsorption of root exudates. The plants were then removed, and the root segments wrapped in the filter paper were cut and placed in an ice box for low-temperature transport back to the laboratory. Under sterile conditions, the root segments were immersed in 100 ml of deionized water and extracted with 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 was the ginkgo root exudate extract and stored at –80°C for subsequent analysis.

[0074] Example 5

[0075] Effects of Ginkgo Root Exudates on the Growth of Alfalfa

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

[0077] The results are as follows Figure 5 As shown in the figure, the effects of different concentrations of Ginkgo root exudates on the growth of alfalfa plants are shown. The results show that compared with the control (CK) with water application, the treatment with Ginkgo root exudates has a positive effect on the growth and development of alfalfa, and can significantly increase plant height, leaf area and nodule formation density. Among them, the root exudate concentration is OD 600=0.0172, the growth-promoting effect was the most significant, 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, and YMA Congo red medium was used for the preliminary screening and identification of alfalfa rhizobia by adding 5mL of 1% Congo red solution to YMA. Select the plump, pink typical nodules on the roots of alfalfa in the ginkgo forest, and retain a small amount of attached root segments for operation in a clean bench. First, rinse the soil impurities on the surface of the nodules with sterile water, and then soak them in 70% ethanol solution for 30s and 2% sodium hypochlorite (NaClO) solution for 2min for surface disinfection, and then rinse with sterile water 3 times to remove residual disinfectants. The treated nodules were placed in a sterile mortar, 2mL of sterile water was added to fully grind, and the obtained homogenate was diluted tenfold in a series of gradients. Take 10 -3 , 10 -4 and 10 -5 50 μL of each concentration gradient dilution was evenly spread on a YMA Congo red medium plate, with three replicates for each dilution gradient. The culture medium was placed in a constant temperature incubator at 28°C for 5-7 days, and the colony morphology was observed. Single colonies with a smooth, sticky surface that did not absorb Congo red pigment were selected for streak purification. After obtaining the purified strain, it was stored at 4°C for later use. The purified strain was then amplified by colony PCR and sent to Shanghai Sangon Biotechnology Co., Ltd. for 16S rDNA sequencing. After the sequencing results were returned, they were compared with the NCBI BLAST database for similarity comparison with known rhizobia sequences to confirm the species affiliation and taxonomic status of the isolated strain, and ultimately determine the type of rhizobium.

[0081] like Figure 6 As shown, typical colonies with smooth surfaces and sticky textures were initially screened on YMA medium containing Congo red pigment. These colonies were subsequently transferred to YMA medium without pigment for further purification and culture, and their growth morphology was observed. The colonies were round, with a sticky, raised surface, translucent or opaque, and off-white or milky white in color, consistent with the basic characteristics of rhizobia. The strain was further amplified and sequenced by 16S rDNA fragments. Comparison to 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 and dilute to OD 600 =0.0172 as the treatment group, and a culture medium supplemented with an equal volume of ddH2O as the control group. Each group was inoculated with Sinorhizobium meliloti from Example 6 and cultured at 28°C. Starting from the third day after inoculation, the colony growth was observed daily, 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 exudates (OD 600 =0.0172) dilution to treat meliloti root nodule bacteria. The results showed that ginkgo root secretions had a significant promoting effect on the growth of rhizobia, which was manifested by accelerated colony growth.

[0086] Example 8

[0087] Effects of Ginkgo Root Exudates on Nod Gene Expression

[0088] To further explore the molecular mechanism of Ginkgo root secretions in promoting alfalfa nodulation, five nod genes (Nucleotide Binding Oligomerization Domain genes) closely related to nodulation were selected, including MS.gene023118, MS.gene028334, MS.gene35123, MS.gene28707, and MS.gene35125. 600 =0.0086, 0.0172) expression levels under the treatment of ginkgo root secretions. The method of treating alfalfa with ginkgo root secretions is the same as that in Example 5, specifically: alfalfa seedlings with uniform growth are cultured under greenhouse conditions for one month, and their roots are irrigated with the above-mentioned concentration of ginkgo root secretion solution, irrigated once every 4 days, 500 mL each time, for 16 consecutive days, and the water treatment group was used as a control. After the treatment, the nodule tissue was collected, and the expression levels of the five target nod genes were detected by qRT-PCR to analyze the regulatory effect of ginkgo secretions on the induction of nodulation signaling pathways. The results are shown in Figure 2. Figure 8The results showed that all of the aforementioned NOD genes were significantly upregulated at all ginkgo exudate concentrations compared to the water-treated control group (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 nodule endophytes but also enhance alfalfa's nodulation and biological nitrogen fixation potential by upregulating nodulation gene expression, thereby improving the nutrient status and soil quality of the understory soil. This further validates the feasibility and scientific nature of the proposed forest-grassland hybrid planting system in enhancing ecological benefits.

[0089] In summary, through multi-level and multi-dimensional controlled experiments, this method utilizes ginkgo root secretions to regulate alfalfa nodulation and nitrogen fixation, and in conjunction with scientific and standardized land preparation and forest management systems, provides a good growth foundation and drainage and aeration environment for alfalfa. This method demonstrates significant advantages in soil improvement, alfalfa quality enhancement, and nodule regulation, fully demonstrating the practicality and innovation of this composite planting method. The establishment of raised bed land preparation and standardized plotting under the ginkgo forest effectively improves forest management efficiency and establishes a standard model that is easy to promote.

Claims

1. A forest-grass composite planting method based on regulating alfalfa nodulation and growth promotion by ginkgo root secretions, characterized in that: The following steps are involved: (1) Land preparation and border division: Deeply plow the land between the rows of ginkgo trees, then shallowly plow and level it before sowing. Build high borders between two rows of ginkgo trees, and dig trenches at intervals in the vertical direction of the field to form sample units. (2) Alfalfa seed treatment: Soak the alfalfa seeds in water, take out the plump seeds that sink to the bottom and mix them with fine soil and carbendazim; (3) seed drill sowing: digging furrows on the bed surface divided in step (1) according to the row spacing, sowing the alfalfa seeds treated in step (2) evenly into the furrows and covering with soil; (4) Thinning and finalizing seedlings: Thin out seedlings twice a year, removing weak ones and retaining strong ones; (5) Intertillage and weeding: Intertillage and weeding should be done 1-2 times in the first year and 3-4 times in the second year; (6) Mowing and harvesting: The first mowing is done in the second year, and mowing is done every month thereafter, leaving stubble after each mowing.

2. The composite planting method according to claim 1, characterized in that: In step (1), the soil is deep-turned to a depth of more than 25 cm.

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

4. The composite planting 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 composite planting method according to claim 1, characterized in that: In step (3), dig 2-3 cm deep furrows 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 composite planting method according to claim 1, characterized in that: In step (4), when the seedlings are 2-3 cm tall, the first thinning is performed, and when the plant height is 8-12 cm, the second thinning is performed.

7. The composite planting method according to claim 1, characterized in that: Step (5) intertillage and weeding are carried out at the time of sowing, when thinning out the seedlings when the seedlings are 3-10 cm high, or when the seedlings are 20-25 cm high, and deep hoeing is performed when the seedlings are 20-25 cm high.

8. The composite planting method according to claim 1, characterized in that: In step (6), the first mowing is carried out when the budding rate of alfalfa reaches 8-15%, and the mowing is carried out once every 25-35 days after the first mowing, for a total of 3 to 5 times; the stubble height after each mowing is 5 to 7 cm.

9. Application of Ginkgo root secretions in promoting the growth of alfalfa seedlings, promoting the growth of endophytes in alfalfa root nodules, and enhancing the nodulation ability and biological nitrogen fixation potential of alfalfa.

10. The use according to claim 9, characterized in that Ginkgo root secretions were collected by selecting healthy ginkgo trees as test plants, digging along the root distribution direction to expose part of the main root and lateral roots, and after the roots were exposed, wrapping the root area with a transparent plastic bag and backfilling the soil to restore the natural growth environment. After standing for treatment, the plants were taken out and the soil particles attached to the root surface were thoroughly rinsed with deionized water. The roots were then wrapped with moistened sterile filter paper and kept moist with deionized water. The plants with wrapped roots were placed in a centrifuge tube, the tube mouth was sealed with sealing film, and the centrifuge tube was buried in the soil at the original excavation location again for continuous treatment to promote the enrichment and adsorption of root secretions. The plants were then taken out, the root segments wrapped in the filter paper were cut, and the root segments were placed in deionized water under sterile conditions for oscillation extraction. After the extract was filtered through the filter paper, the filtrate was collected and concentrated by rotary evaporation. The resulting liquid or dry powder was the ginkgo root secretion extract.

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