A method for cultivating soybeans in saline-alkali land with resistance based on rhizosphere probiotic regulation

Abstract

This invention relates to the field of agricultural planting technology and discloses a method for cultivating soybeans in saline-alkali land with stress resistance based on rhizosphere probiotic regulation. The method includes: applying component B (1.0-2.0 mM salicylic acid) to the topsoil for pretreatment 7-10 days before sowing; mixing component A (containing Bacillus subtilis and Pseudomonas fluorescens) with component C (organic fertilizer or biochar carrier) at a weight ratio of 1:100-1:200 to prepare a probiotic composite substrate; sowing soybean seeds at a depth of 3-5 cm and applying the probiotic composite substrate 3-5 cm below or to the side of the seeds; and managing field water. This invention induces soybean stress resistance through salicylic acid pretreatment, improves the rhizosphere microenvironment by combining it with the probiotic composite substrate, synergistically enhancing soybean stress resistance, nutrient absorption efficiency, and soil health, thereby promoting soybean growth and yield.

Description

Technical Field

[0001] This invention relates to the field of agricultural planting technology, specifically to a method for cultivating soybeans in saline-alkali land with stress resistance based on the regulation of rhizosphere probiotics. Background Technology

[0002] Saline-alkali land is a significant factor limiting agricultural production, with a vast area globally, severely impacting crop growth and yield. Soybeans, as an important oilseed and protein crop, play a crucial role in global food security due to their planting area and yield. However, in saline-alkali environments, soybeans often experience stunted growth and development, physiological dysfunction, and ultimately, a significant decrease in yield due to stress factors such as high salt and high pH levels.

[0003] For a long time, researchers and producers have tried various methods to address the challenges posed by saline-alkali land to soybean cultivation. These include breeding salt-tolerant soybean varieties and improving their adaptability through genetic modification; as well as adopting chemical improvement measures, such as applying gypsum and organic fertilizers to improve soil physical and chemical properties. At the same time, biological methods have also received attention, such as using single plant growth promoters (PGPR) or biofertilizers to alleviate salt stress and promote soybean growth.

[0004] However, these existing methods generally have limitations in practical applications. Variety breeding cycles are long, and the stress resistance of existing salt-alkali tolerant varieties remains insufficient under extreme saline-alkali conditions. Chemical amendment methods may be costly and have limited effects on improving soil structure, sometimes accompanied by environmental risks. The growth-promoting effects of single microbial agents are greatly affected by the soil environment; their survival, colonization, and function may be inhibited in severely saline-alkali soils, making it difficult to provide continuous and stable stress resistance support. These shortcomings mean that current soybean cultivation in saline-alkali land still lacks a comprehensive, efficient, and sustainable solution, failing to fundamentally and effectively address the multiple impacts of saline-alkali stress on soybean growth and improve its production potential under harsh environments. Therefore, developing a comprehensive cultivation method that can synergistically enhance soybean's own stress resistance, improve the rhizosphere microenvironment, and promote growth is of practical significance. Summary of the Invention

[0005] The technical problem solved by this invention is that during the soybean planting process in saline-alkali land, soybeans often suffer from limited growth and reduced yield due to salt and alkali stress, and existing planting methods are difficult to effectively improve the stress resistance and yield of soybeans.

[0006] To address the above problems, the present invention provides the following technical solution:

[0007] This invention provides a method for stress-resistant soybean cultivation in saline-alkali land based on rhizosphere probiotic regulation, comprising the following steps:

[0008] (1) Soil pretreatment: 7 to 10 days before the scheduled soybean sowing date, a soil pretreatment solution containing component B is applied to the topsoil of the target plot. The concentration of component B in the soil pretreatment solution is 1.0 mM to 2.0 mM, and the topsoil layer of 0-12 cm is moistened. The component B is salicylic acid.

[0009] (2) Preparation of probiotic composite matrix: A component and C component are mixed to prepare a probiotic composite matrix, wherein the weight ratio of A component to C component is 1:100 to 1:200, wherein A component contains Bacillus subtilis and Pseudomonas fluorescens, and C component is selected from organic fertilizer, biochar granules or a combination thereof.

[0010] (3) Sowing and substrate application: Using a sowing machine with independent sowing and fertilization functions, the soybean seed sowing depth is set to 3cm to 5cm, and the probiotic composite substrate prepared in step (2) is applied to the soybean seed 3cm to 5cm below or to the side of the soybean seed.

[0011] (4) Field management: The first irrigation should be carried out within 24 hours after sowing. The amount of water should be enough to moisten the soil layer to 15cm to 20cm. Water management should be carried out according to the soil moisture during the soybean seedling stage.

[0012] By adopting the above technical solution, this invention combines the application of salicylic acid soil pretreatment solution with a specific probiotic composite substrate, achieving stress-resistant cultivation of soybeans in saline-alkali land. Its mechanism of action and beneficial effects are reflected in the following aspects:

[0013] Inducing effect of salicylic acid (component B) in soil pretreatment solution:

[0014] Pretreatment time and concentration: Apply a soil pretreatment solution containing salicylic acid 7 to 10 days before soybean sowing, and control the concentration of salicylic acid in the solution to 1.0 mM to 2.0 mM, so that the salicylic acid can diffuse in the soil and be absorbed and utilized by the roots in the early stage of soybean germination.

[0015] Plant stress resistance induction: Salicylic acid, as an endogenous signaling molecule in plants, can induce the systemic acquired resistance (SAR) pathway in plants. Before the occurrence of salt and alkali stress, external application of salicylic acid can activate the soybean's defense system in advance, increase the activity of antioxidant enzymes and the synthesis of osmotic regulators, thereby enhancing the soybean's tolerance to salt, alkali, drought and other abiotic stresses.

[0016] Soil microenvironment regulation: Salicylic acid can regulate the structure of soil microbial communities, promote the growth of beneficial microorganisms, and create conditions for the subsequent introduction of probiotics.

[0017] Wetting depth: A wetting depth of 0-12cm ensures that salicylic acid can be evenly distributed in the main active layer of the root system.

[0018] Preferably, the concentration of component B in the soil pretreatment solution is 1.2 mM to 1.8 mM to obtain a better stress resistance induction effect.

[0019] Preferably, the application rate of the soil pretreatment solution is 30L to 40L per acre to ensure uniform treatment.

[0020] Synergistic effect of probiotic complex matrix (components A+C):

[0021] Synergistic effect of specific strains: This invention selects Bacillus subtilis and Pseudomonas fluorescens as component A, both of which are plant growth-promoting rhizosphere bacteria (PGPR).

[0022] Bacillus subtilis: It has the functions of secreting antibacterial substances, producing plant hormones, and solubilizing phosphorus and fixing nitrogen. It can inhibit pathogens, promote plant growth, and improve soil aggregate structure and regulate local salt ion balance by secreting extracellular polysaccharides and enzymes.

[0023] Fluorescent Pseudomonas: It can secrete siderophores, which can improve the absorption efficiency of plants for trace elements such as iron; at the same time, it can produce plant growth hormones such as indoleacetic acid, which can promote root development and increase the root absorption area.

[0024] Supporting and improving role of carrier materials: This invention selects organic fertilizer, biochar granules or a combination thereof as component C.

[0025] Organic fertilizer: increases soil organic matter content, improves soil aggregate structure, enhances water and fertilizer retention capacity, and regulates soil pH.

[0026] Preferably, the organic fertilizer has an organic matter content of not less than 45% and a pH value of 7.0 to 7.5.

[0027] Biochar particles: They have a large specific surface area and porous structure, which can adsorb salt ions in the soil and reduce salt stress; at the same time, they can improve soil aeration and acid-base buffering capacity.

[0028] Preferably, the biochar particles have a particle size of 2 to 4 mm and a pH value of 7.5 to 8.0.

[0029] Protective effect of probiotics and carrier: The carrier material provides attachment sites and living space for probiotics, reducing the mortality rate of probiotics in saline-alkali environments. The weight ratio of component A to component C is controlled between 1:100 and 1:200 to ensure an effective bacterial load.

[0030] Moisture content adjustment: Preferably, the total moisture content of the probiotic composite substrate is adjusted to 35% to 40%, which is beneficial to maintaining the activity of probiotics and promoting the bonding between the substrate and the soil.

[0031] Spatial layout for sowing and substrate application:

[0032] Spatial isolation and early colonization: Soybean seeds are sown at a depth of 3 to 5 cm using seeding machinery with independent seeding and fertilization functions. The probiotic composite substrate is applied 3 to 5 cm below or to the side of the soybean seeds. This spatial distribution allows the probiotic composite substrate to quickly come into contact with the soybean seedlings after seed germination, promoting early colonization of probiotics in the root system and thus providing stress resistance protection for soybean seedlings.

[0033] Preferably, the soybean seeds are sown at a depth of 3.5 cm to 4.5 cm.

[0034] Preferably, the probiotic composite matrix is ​​applied 4 cm directly below the soybean seeds.

[0035] Preferably, the application rate of the probiotic compound substrate is 25 kg to 35 kg per mu.

[0036] Field water management:

[0037] The first irrigation should be carried out within 24 hours after sowing, with the amount of water moistening the soil layer to 15cm to 20cm. This helps promote seed germination, salicylic acid activation, and the revival of beneficial bacteria. During the soybean seedling stage (V1-V3), subsequent water management should be carried out according to soil moisture conditions to ensure water supply for the soybean seedlings.

[0038] In summary, this invention induces stress pre-adaptation in soybeans through salicylic acid pretreatment, while simultaneously improving the rhizosphere microecological environment and enhancing soybean stress resistance and nutrient absorption efficiency by applying a specific composite matrix containing Bacillus subtilis and Pseudomonas fluorescens. These two strategies work synergistically in terms of time, space, and function, thereby increasing the survival rate and yield of soybeans in saline-alkali soils.

[0039] This invention provides a method for cultivating soybeans in saline-alkali land with resistance based on rhizosphere probiotic regulation. It has the following beneficial effects:

[0040] 1. This invention pre-treats the soil with salicylic acid 7 to 10 days before sowing, thereby inducing the stress resistance mechanism of soybeans and enabling them to more effectively activate their defenses when encountering saline-alkali stress. Simultaneously, Bacillus subtilis and Pseudomonas fluorescens in the probiotic composite matrix directly promote soybean root development, improve nutrient absorption, and secrete substances that help alleviate stress. This combined treatment enhances the soybean's tolerance to adverse environments such as salinity, drought, and alkalinity from multiple perspectives.

[0041] 2. This invention achieves direct improvement of saline-alkali soil by using organic fertilizer or biochar granules as a carrier for probiotics. Organic fertilizer increases soil organic matter, optimizes soil aggregate structure and pH buffering capacity; biochar can adsorb salt ions in the soil, regulate pH value, and improve soil aeration. In addition, the probiotics in the composite matrix can carry out biological nitrogen fixation and phosphorus solubilization activities, converting nutrients fixed in the soil into forms that can be utilized by soybeans, thereby improving soil fertility and the rhizosphere microecological environment.

[0042] 3. This invention, through the pretreatment of soybean seedlings with salicylic acid and the precise application of a probiotic composite substrate, provides soybean seedlings with dual support in terms of stress resistance and growth promotion from the early stages of growth. Salicylic acid enhances the overall adaptability of the plant, while probiotics support crop development by promoting root growth and improving nutrient utilization efficiency. Combined with meticulous management of sowing, substrate application, and irrigation, the maximum effect of beneficial components is ensured. These measures work together to reduce the negative impact of salt-alkali stress on soybean growth, ultimately resulting in healthy soybean plant growth and increased yield. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the preparation examples, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0045] Component A (Probiotic Preparation): Commercially available Bacillus subtilis wettable powder, using the standard strain ATCC6051 as the core strain, with an effective viable count ≥2.0 × 10⁻⁶ in the commercially available preparation. 10 CFU / g. Commercially available wettable powder of Pseudomonas fluorescens, using the standard strain ATCC13525 as the core strain.

[0046] Preparation Examples 1-2:

[0047] Preparation Example 1: Preparation of Soil Pretreatment Solution (Based on Component B)

[0048] This preparation example aims to formulate soil pretreatment solutions of different concentrations from component B (salicylic acid) for use in soil induction treatment before soybean planting.

[0049] Preparation Example 1-1: 1.0 mM Soil Pretreatment Solution

[0050] (1) Weigh out 138.1g of component B (salicylic acid);

[0051] (2) Dissolve the salicylic acid in 1.5L of ethanol and stir until completely dissolved;

[0052] (3) Add 15 mL of nonionic surfactant (Tween-80), mix well, and prepare concentrated mother liquor;

[0053] (4) Add the concentrated mother liquor to the field application tank, add water to make up to 1000L, and stir evenly to obtain a pretreatment solution with a final concentration of 1.0mM.

[0054] Preparation Examples 1-2: 1.5 mM Soil Pretreatment Solution

[0055] (1) Weigh out 207.2g of component B (salicylic acid);

[0056] (2) The dissolution and volume adjustment steps are the same as in Preparation Example 1-1. Finally, water is added to adjust the volume to 1000L to obtain a pretreatment solution with a final concentration of 1.5mM.

[0057] Preparation Examples 1-3: 2.0 mM Soil Pretreatment Solution

[0058] (1) Weigh out 276.2g of component B (salicylic acid);

[0059] (2) The dissolution and volume adjustment steps are the same as in Preparation Example 1-1. Finally, water is added to adjust the volume to 1000L to obtain a pretreatment solution with a final concentration of 2.0mM.

[0060] Preparation Example 2: Preparation of Probiotic Complex Matrix (Component A + Component C)

[0061] This preparation example aims to prepare a probiotic composite matrix with steric diametrical protection function by loading component A (probiotics) onto component C (carrier) through a physical mixing process.

[0062] Preparation Example 2-1: Composite substrate (organic fertilizer carrier) with A:C weight ratio of 1:100

[0063] (1) Prepare component A: Weigh 0.5 kg of Bacillus subtilis powder (ATCC6051) and 0.5 kg of Pseudomonas fluorescens powder (ATCC13525), mix them evenly to obtain 1.0 kg of mixed bacterial powder of component A;

[0064] (2) Prepare component C: Weigh 100 kg of organic fertilizer of the above specifications;

[0065] (3) Premixing: Premix 1.0 kg of component A and 10 kg of component C (organic fertilizer) in a clean container and stir evenly;

[0066] (4) Mixing: Add the premix to the remaining 90kg C component and mix at low speed for 15 minutes using a horizontal mixer;

[0067] (5) Moisture adjustment: During the mixing process, spray an appropriate amount of water to adjust the total moisture content of the substrate to 35%-40% (it can be formed into a ball when squeezed by hand and crumbles when touched).

[0068] Preparation Example 2-2: Composite substrate (organic fertilizer carrier) with A:C weight ratio of 1:150

[0069] (1) Preparation of component A: Same as in preparation example 2-1, prepare 1.0 kg of mixed bacterial powder of component A;

[0070] (2) Prepare component C: Weigh 150 kg of organic fertilizer of the above specifications;

[0071] (3) Mixing and adjustment: The operation process is the same as steps (3)-(5) of preparation example 2-1, and the result is obtained.

[0072] Preparation Example 2-3: Composite matrix (biochar carrier) with A:C weight ratio of 1:200

[0073] (1) Preparation of component A: Same as in preparation example 2-1, prepare 1.0 kg of mixed bacterial powder of component A;

[0074] (2) Prepare component C: Weigh 200 kg of biochar granules of the above specifications;

[0075] (3) Mixing and adjustment: The operation process is the same as steps (3)-(5) of preparation example 2-1, and the result is obtained.

[0076] Examples 1-3:

[0077] Example 1:

[0078] This embodiment provides a method for cultivating soybeans in saline-alkali land with resistance based on rhizosphere probiotic regulation. The specific steps are as follows:

[0079] (1) Soil pretreatment: Eight days before the scheduled soybean sowing date, the 1.5 mM soil pretreatment solution (B component derivative) prepared in Preparation Examples 1-2 was used to spray the surface soil of the experimental field evenly to ensure that the solution moistened the top 0-10 cm soil layer. The application rate was 35 L per mu.

[0080] (2) Preparation of probiotic composite substrate: The probiotic composite substrate (organic fertilizer carrier) with an A:C weight ratio of 1:150 prepared in Preparation Example 2-2 was selected.

[0081] (3) Spatial Isolation Sowing and Application: A no-till precision seeder with an independent fertilizer application box was used. The soybean seed sowing depth was set to 4cm; the probiotic composite substrate prepared in step (2) was loaded into the fertilizer application box, and the fertilizer furrow opener was adjusted so that the substrate was applied 4cm directly below the soybean seeds (i.e., about 8cm deep into the soil), forming a spatial isolation. The application rate of the probiotic composite substrate was 30kg per mu. After sowing, the soil was routinely covered and compacted.

[0082] (4) Field management: Irrigate for the first time within 24 hours after sowing, with the amount of water being enough to moisten the soil layer to a depth of 15-20 cm. Subsequent water management during the soybean seedling stage (V1-V3 stage) should be carried out according to the soil moisture.

[0083] Example 2:

[0084] This embodiment provides a method for cultivating soybeans in saline-alkali land with resistance based on rhizosphere probiotic regulation. The specific steps are as follows:

[0085] (1) Soil pretreatment: Seven days before the scheduled soybean sowing date, the 1.0 mM soil pretreatment solution prepared in Preparation Example 1-1 was used to spray the surface soil of the experimental field evenly to ensure that the solution moistened the top 0-8 cm soil layer. The application rate was 30 L per mu.

[0086] (2) Preparation of probiotic composite substrate: The probiotic composite substrate (organic fertilizer carrier) with an A:C weight ratio of 1:100 prepared in Preparation Example 2-1 was selected.

[0087] (3) Spatial difference sowing and application: No-till precision seeders with independent fertilizer boxes were used. The sowing depth of soybean seeds was set to 3cm; the probiotic composite substrate prepared in step (2) was applied 3cm below and to the side of the soybean seeds. The application rate of the probiotic composite substrate was 20kg per mu. After sowing, the soil was routinely covered and compacted.

[0088] (4) Field management: Irrigate for the first time within 24 hours after sowing, with the amount of water being enough to moisten the soil layer to a depth of 15-20 cm. Subsequent water management during the soybean seedling stage (V1-V3 stage) should be carried out according to the soil moisture.

[0089] Example 3:

[0090] This embodiment provides a method for cultivating soybeans in saline-alkali land with resistance based on rhizosphere probiotic regulation. The specific steps are as follows:

[0091] (1) Soil pretreatment: 10 days before the scheduled soybean sowing date, the 2.0 mM soil pretreatment solution prepared in Preparation Examples 1-3 was used to spray the surface soil of the experimental field evenly to ensure that the solution moistened the top 0-12 cm soil layer. The application rate was 40 L per mu.

[0092] (2) Preparation of probiotic complex matrix: The probiotic complex matrix (biochar carrier) with an A:C weight ratio of 1:200 prepared in Preparation Example 2-3 was selected.

[0093] (3) Spatial difference sowing and application: No-till precision seeders with independent fertilizer boxes were used. The sowing depth of soybean seeds was set to 5cm; the probiotic composite substrate prepared in step (2) was applied 5cm directly below the soybean seeds. The application rate of the probiotic composite substrate was 40kg per mu. After sowing, the soil was covered and compacted as usual.

[0094] (4) Field management: Irrigate for the first time within 24 hours after sowing, with the amount of water being enough to moisten the soil layer to a depth of 15-20 cm. Subsequent water management during the soybean seedling stage (V1-V3 stage) should be carried out according to the soil moisture.

[0095] Comparative Examples 1-6:

[0096] Comparative Example 1:

[0097] Compared with Example 1, this comparative example is a conventional soybean planting method (blank control), the difference being that: the soil pretreatment in step (1) is not performed, and the application of probiotic composite substrate in step (3) is not performed. That is, after conventional tillage, soybean seeds are directly sown and conventional water and fertilizer management is carried out.

[0098] Comparative Example 2:

[0099] Compared with Example 1, the difference is that step (1) is omitted, that is, component B (salicylic acid pretreatment solution) is not used to pretreat the soil. Everything else is the same.

[0100] Comparative Example 3:

[0101] Compared with Example 1, the difference is that steps (1) and (3) are combined. Specifically, instead of pre-treating the soil, on the day of sowing, the B component (salicylic acid) solution prepared in Example 1-2 is sprayed into the sowing furrow, followed by sowing and application of the A+C substrate. Everything else is the same.

[0102] Comparative Example 4:

[0103] Compared with Example 1, the difference is that the pretreatment in step (1) is omitted; instead, an equal amount of component B (salicylic acid) solution is applied as a foliar fertilizer / root irrigation agent during the field management stage (soybean V1 seedling stage). The remaining steps are the same as in Example 1.

[0104] Comparative Example 5:

[0105] Compared with Example 1, the difference lies in the application location in step (3). Specifically, the A+C probiotic composite substrate is mixed evenly with soybean seeds before sowing, and then sown into the same soil depth using a single feed box of a seeder (i.e., there is no spatial separation between the seeds and the substrate). Everything else is the same.

[0106] Comparative Example 6:

[0107] Compared with Example 1, the difference lies in the change of steps (2) and (3). Specifically, instead of preparing the A+C composite matrix, component A (probiotic powder) is directly mixed with soybean seeds at the conventional seed mixing ratio (powder:seed = 1:50) and then sown. Ordinary organic fertilizer without bacteria is applied as base fertilizer at the time of sowing. All other steps are the same.

[0108] Test Examples 1-4:

[0109] Test Example 1: Time Inhibition Window Test of Component B on Indigenous Microorganisms

[0110] This test case aims to verify the short-term inhibitory effect of component B (salicylic acid) on the original soil microbial community and to determine the time window of this inhibitory effect, so as to support the feasibility of the time difference pretreatment mechanism of the present invention.

[0111] Experimental steps:

[0112] Air-dried and sieved test soil was placed into 20 sterile pots, 5 kg per pot. Before filling, the soil moisture content was adjusted to 60% of field capacity and mixed evenly. The surface of the soil in 15 pots was uniformly sprayed with a 1.5 mM salicylic acid pretreatment solution prepared in Examples 1-2, at a rate equivalent to 25 L / acre per pot. The remaining 5 pots served as a control group, sprayed with an equal volume of sterile water. On days 0 (before spraying), 3, 7, 10, and 14 after treatment, 3 pots were randomly selected from both the salicylic acid treatment group and the control group. A 10 g soil sample was collected from the 0-10 cm soil layer of each pot using a sterile sampler, immediately placed in a sterile self-sealing bag, and labeled. The control group was sampled only on days 0 and 7. Weigh 10.0 g of each soil sample, add 90 mL of sterile physiological saline, and shake on a shaker for 30 minutes. Then, perform a serial dilution of the soil suspension (10⁻⁶). -1 Up to 10 -6Take 100 μL of suspensions at different dilutions and spread them onto the corresponding culture media: nutrient agar was used for bacterial counting, and potato dextrose agar was used for fungal counting. Three replicates were set up for each dilution. Bacterial culture dishes were incubated at 30°C for 48 hours, and fungal culture dishes were incubated at 28°C for 72 hours. The number of colonies (CFU) in each culture dish was counted, and the total number of bacteria and fungi per gram of dry soil was calculated based on the dilution factor and soil dry weight.

[0113] Experimental data:

[0114] Table 1: Changes in total soil bacteria and fungi counts over time

[0115]

[0116] Note: "-" indicates that data was not collected at that time point.

[0117] Conclusion: The experimental data in Table 1 show that the total number of bacteria and fungi in the salicylic acid-treated group decreased from day 0 to day 7 after treatment. Compared with day 0 before treatment, the total number of bacteria in the soil decreased to 30.9% and the total number of fungi decreased to 30.5% on day 7. On days 10 and 14, the soil microbial counts began to recover, but their values ​​were still lower than the initial levels and the levels in the control group at the same time point. The soil microbial counts in the control group remained relatively stable during the experiment.

[0118] These results indicate that applying salicylic acid to saline-alkali soils can, in stages, inhibit the growth of native soil microorganisms. This inhibitory effect reaches its lowest point 7 days after application, followed by a gradual increase in microbial community count as the salicylic acid degrades in the soil. This phased decrease in microbial numbers provides a less competitive environment for subsequently introduced probiotics. During this stage, externally applied probiotics can germinate, proliferate, and colonize the plant rhizosphere more effectively, thereby improving their survival rate under saline-alkali stress. This test case validates the feasibility of time-difference pretreatment in regulating the soil microbial environment and reducing competition in the early stages of probiotic colonization.

[0119] Test Example 2: Effects of A+C composite substrate application method on probiotic rhizosphere colonization and early soybean growth

[0120] This test case aims to evaluate the effects of different application methods of the A+C composite matrix on the colonization of probiotics in the soybean rhizosphere and the early growth of soybean, in order to support the feasibility of the spatial difference protection mechanism of the present invention.

[0121] Experimental steps:

[0122] Prepare saline-alkali soil with a salt content of 0.3% and fill 40 non-porous pots with 3 kg of soil per pot. Set up four treatment groups with 10 replicates per group: blank control group, soybean seeds were directly sown without adding A+C composite substrate; conventional seed dressing group, A component was mixed with soybean seeds at a weight ratio of 1:50 before sowing; seed-fertilizer mixed application group, the probiotic composite substrate of Preparation Example 2-2 was pre-mixed evenly with soybean seeds and then sown together; spatial difference application group, the steps of Example 1 (3) were performed, soybean seeds were sown at a depth of 4 cm, and the probiotic composite substrate of Preparation Example 2-2 was applied 4 cm directly below the soybean seeds. No salicylic acid pretreatment was performed before sowing in any treatment group. 3 soybean seeds were sown in each pot, and after emergence, the seedlings were thinned to 2 plants per pot. The potted plants were placed in an artificial climate chamber with a day / night temperature of 25℃ / 20℃, 14 hours of light / 10 hours of darkness, and a light intensity of 300 μmol / m². -2 s -1 Observe seedling emergence daily and replenish water according to soil moisture to maintain soil moisture content at 60% of field capacity. 21 days after soybean emergence, randomly select 5 pots from each treatment group, remove the plants, and collect soil samples from the area where roots are tightly attached as rhizosphere soil samples. Weigh 10.0g of rhizosphere soil sample, add 90mL of sterile physiological saline, and shake thoroughly to dilute. Spread suspensions of different dilutions onto probiotic selective culture medium, incubate, count colonies, and calculate the CFU of probiotics per gram of dry soil. Simultaneously, measure the plant height and fresh weight of each soybean seedling, and dry them in a 70℃ oven to constant weight, measuring the dry weight.

[0123] Experimental data:

[0124] Table 2: Effects of different application methods on early growth of soybean and colonization of probiotics

[0125]

[0126] Conclusion: According to the experimental data in Table 2, compared with the blank control group, all treatment groups that applied probiotics improved the emergence rate, plant height and biomass of soybeans to varying degrees, and increased the number of probiotic colonies in the rhizosphere.

[0127] Among different application methods, the conventional seed dressing group showed lower growth enhancement and probiotic colonization rates compared to other application groups, with a rhizosphere probiotic count of 1.76 × 10⁻⁶. 5 CFU / g dry soil. The number of probiotic colonies and plant growth indicators in the seed-fertilizer mixture group were better than those in the conventional seed dressing group, with the rhizosphere probiotic count reaching 3.11 × 10⁻⁶. 5 CFU / g dry soil. The spatial difference application group showed better performance in all indicators than other treatment groups, with a rhizosphere probiotic count reaching 6.45 × 10⁻⁶. 5 CFU / g dry soil.

[0128] This result confirms that spatial differential application can effectively promote the colonization of probiotics in the soybean rhizosphere. By applying the probiotic composite substrate at a certain distance below the seed, the substrate provides a relatively stable microenvironment for the probiotics. Simultaneously, this spatial arrangement guides soybean roots to grow towards the probiotic-rich area, promoting early contact and interaction between roots and probiotics, thus ensuring effective colonization and proliferation of the probiotics. Successful colonization of the probiotics allows them to exert their functions of biological nitrogen fixation, phosphorus and potassium solubilization, and secretion of plant growth regulators, ultimately enhancing the early growth performance of soybeans under saline-alkali stress. This test case supports the role of spatial differential protection strategies in improving probiotic colonization efficiency and enhancing soybean stress resistance.

[0129] Test Example 3: The comprehensive impact of the planting method of the present invention on soybean yield and rhizosphere microecology

[0130] This test case aims to comprehensively evaluate the combined effects of the soybean saline-alkali land stress-resistant planting method based on rhizosphere probiotic regulation of the present invention on soybean growth, yield and rhizosphere microecology through field plot trials, and compare it with multiple comparative cases.

[0131] Experimental steps:

[0132] A representative saline-alkali land plot (soil pH 8.5-9.0, electrical conductivity EC value 2-4 dS / m) was selected, and multiple experimental plots were set up, each plot measuring 10m x 5m, with protective rows between plots. A total of 6 treatment groups were set up, with 3 replicates per group. The treatment group design is as follows:

[0133] Example 1 group: Follow all the steps of Example 1.

[0134] Comparative Example 1: No salicylic acid pretreatment was performed, no probiotic compound substrate was applied, and only conventional sowing and management were carried out.

[0135] Comparative Example 2: No salicylic acid pretreatment was performed, but soybean seeds and probiotic composite matrix were applied according to steps (2) and (3) of Example 1.

[0136] Comparative Example 3: The application of salicylic acid pretreatment and probiotic compound substrate was combined on the day of sowing, that is, salicylic acid solution was sprayed and probiotic compound substrate was applied simultaneously in the sowing furrow, and then sowing was carried out immediately.

[0137] Comparative Example 5: No salicylic acid pretreatment was performed. The probiotic composite substrate was mixed with soybean seeds and sown together without spatial isolation.

[0138] Comparative Example 6: No salicylic acid pretreatment was performed. Component A (probiotic powder) was directly mixed with soybean seeds before sowing, and component C substrate was not used. Soybean seeding rate and field management (irrigation, weeding, etc.) were kept consistent across all treatment groups. The following data were collected during the mid-season (initial flowering stage) and harvest period:

[0139] At the initial flowering stage: 20 soybean plants were randomly selected from each plot, and plant height, stem diameter, above-ground fresh weight, and dry weight were measured. At the same time, rhizosphere soil samples were collected, and real-time quantitative PCR was used to quantitatively analyze the gene copy number of the target probiotics (Bacillus subtilis and Pseudomonas fluorescens) in the rhizosphere to reflect their colonization quantity.

[0140] Harvest period: Randomly select 20 soybean plants from each plot and measure the number of pods per plant, the number of seeds per pod, and the weight of 100 seeds. Calculate the actual soybean yield for each plot.

[0141] Experimental data:

[0142] Table 3: Effects of different treatment groups on soybean growth, yield, and probiotic colonization

[0143]

[0144] Note: ND indicates not detected.

[0145] Conclusion: This test case comprehensively evaluated the combined effects of the planting method of the present invention, which combines time difference pretreatment and spatial difference protection, on soybean growth, yield formation and rhizosphere probiotic colonization through field plot experiments.

[0146] Table 3 shows that Example 1 group demonstrated advantages in all evaluation indicators, including plant height at initial flowering stage, aboveground dry weight, target probiotic colonization, number of pods per plant, 100-seed weight, and final yield, significantly outperforming all comparative groups. The soybean yield of Example 1 group reached 235.1 kg / mu, 2.29 times that of Comparative Group 1 (blank control). Furthermore, Example 1 group exhibited the highest rhizosphere target probiotic colonization, reaching 7.3 × 10⁻⁶. 7 Gene copy number / g dry soil indicates that this method effectively promotes the establishment and proliferation of probiotics in the rhizosphere of saline-alkali soil.

[0147] Detailed analysis of the comparative group data:

[0148] The control group (blank control) performed the worst, with all its indicators at the lowest level, demonstrating the inhibitory effect of saline-alkali land on soybean growth.

[0149] Although the indicators of Comparative Example 2 (without salicylic acid pretreatment) were better than those of Comparative Example 1, they were significantly lower than those of Example 1. This indicates that although a probiotic complex substrate was applied, the lack of salicylic acid pretreatment to inhibit indigenous microorganisms led to competitive pressure on the introduced probiotics in the early stages of colonization, preventing them from fully exerting their growth-promoting effects.

[0150] The results of Comparative Group 3 (where salicylic acid and probiotics were applied simultaneously) were unsatisfactory, with significantly lower probiotic colonization and growth rates compared to Example 1. This confirms the potential inhibitory effect of salicylic acid on probiotics and underscores the necessity of a 7-10 day interval in the time-difference pretreatment to ensure that the concentration of salicylic acid decreases to a level that does not affect the probiotics after treatment.

[0151] The probiotic colonization rate and yield in Comparative Group 5 (probiotic composite substrate mixed with seeds, without spatial separation) were also lower than those in Example 1. This indicates that the lack of spatial isolation, direct contact between probiotics and seeds, or the lack of substrate guidance for the root system are all detrimental to the effective colonization of probiotics and their promotion of soybean growth.

[0152] The comparative group 6 (seed dressing only, without component C substrate) showed lower performance across all indicators. This highlights the importance of the component C composite substrate as a carrier for probiotics and a protective layer for the microenvironment. Under saline-alkali stress, probiotics exposed to direct contact with the soil have limited survival rates and colonization abilities.

[0153] In summary, this invention eliminates or reduces competition from native microorganisms through time-difference pretreatment, creating a favorable ecological niche for the colonization of exogenous probiotics. Simultaneously, by combining spatial differential protection with a composite matrix, it provides probiotics with a sanctuary and nutrient source in a saline-alkali environment, and guides effective interaction between crop roots and probiotics. The synergistic effect of these two innovative strategies improves the colonization efficiency and functional expression of probiotics in the rhizosphere of soybeans in saline-alkali land, ultimately enhancing soybean's stress resistance and increasing yield. The field trial results of this test case provide practical evidence for the application of this invention in soybean cultivation in saline-alkali land.

[0154] Test Example 4: Effects of Salicylic Acid Application Methods on Soybean Physiological Stress Resistance Indicators and Yield

[0155] This test case aims to compare the effects of the application method of salicylic acid as a soil pretreatment agent in this invention with the conventional application method of salicylic acid as a plant growth regulator on the physiological stress resistance index and yield of soybeans under salt-alkali stress.

[0156] Experimental steps:

[0157] The experimental site and plot setup were the same as in Test Example 3. Three treatment groups were set up, with three replicates in each group. Example 1 group followed all the steps of Example 1, i.e., salicylic acid soil pretreatment was performed 7 days before sowing. Comparative Example 2 group did not undergo salicylic acid pretreatment, but applied soybean seeds and probiotic composite substrate according to steps (2) and (3) of Example 1. Comparative Example 4 group did not undergo salicylic acid pretreatment, but applied soybean seeds and probiotic composite substrate according to steps (2) and (3) of Example 1; at the soybean V2 seedling stage (when the second trifoliate leaf unfolded), an equal amount of salicylic acid (prepared as a 1.5 mM solution) was applied as a foliar spray, the same amount as in Example 1 group. The soybean sowing rate and field management were consistent across all treatment groups. During the full bloom of soybeans, plants with uniform growth were randomly selected from each plot, and the 3rd-4th fully unfolded functional leaves at the top were taken for physiological index measurement. At harvest, the actual yield of each plot was measured. Physiological parameters were determined as follows: proline content was determined using the acidic ninhydrin colorimetric method; malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) method; and superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium (NBT) photochemical reduction method. Yield was determined in the same manner as in test example 3.

[0158] Experimental data:

[0159] Table 4: Effects of different salicylic acid application methods on soybean physiological indicators and yield

[0160]

[0161] Conclusion: This test case evaluated the effects of salicylic acid as a soil pretreatment agent versus conventional application as a plant growth regulator on the physiological stress resistance indicators and yield of soybean under salt-alkali stress.

[0162] The experimental data in Table 4 show that, compared with Comparative Example 2 (without salicylic acid application), the soybean leaves of Example 1 and Comparative Example 4 exhibited increased proline content and SOD activity, decreased malondialdehyde (MDA) content, and ultimately higher yields. This indicates that the application of salicylic acid enhances the salt-alkali stress tolerance of soybeans.

[0163] Comparing the data from Example 1 and Comparative Example 4, the improvement in physiological indicators was more significant in Example 1, with the highest proline content and SOD activity, and the lowest MDA content. This indicates that the plant had the least degree of cell membrane lipid peroxidation, the strongest osmotic regulation and reactive oxygen species scavenging ability, and the best physiological state. This physiological advantage was ultimately reflected in yield, with the yield of Example 1 (235.1 kg / mu) significantly higher than that of Comparative Example 4 (181.3 kg / mu).

[0164] The above results reveal the difference in the mechanism of action between the two salicylic acid application methods. In the comparative group 4, foliar spraying was used, and its mechanism of action involved direct absorption by the plant, inducing systemic resistance. This is a passive response measure when the plant is already under stress, and its effectiveness is limited by the plant's physiological state and absorption efficiency at that time.

[0165] This invention (Example 1) pre-treats the soil microecology by applying salicylic acid, which functions to regulate the soil micro-ecology. Through time-difference pretreatment, salicylic acid improves the survival environment of rhizosphere microorganisms, reduces competition from native microorganisms, and thus ensures efficient colonization of subsequently applied probiotics. These probiotics continuously improve the rhizosphere microenvironment, provide nutrients, and secrete hormones throughout the soybean's growth cycle, helping soybeans resist salt and alkali stress. Therefore, the method of this invention constructs a favorable rhizosphere micro-ecosystem, placing soybeans in a better growth environment throughout their entire life cycle. The enhancement of their stress resistance is systematic and long-lasting, superior to single-stage plant resistance induction measures in the later stages.

Claims

1. A soybean planting method for regulating stress resistance in saline-alkali soil based on rhizosphere probiotics, characterized in that, The method comprises the following steps: Step 1: soil pretreatment: 7-10 days before the scheduled sowing date of soybeans, a soil pretreatment liquid containing a B component is applied to the surface soil of the target plot, the concentration of the B component in the soil pretreatment liquid is 1.0-2.0 mM, and the surface layer of 0-12 cm is moistened, wherein the B component is salicylic acid; In step 1, the concentration of the B component in the soil pretreatment liquid is 1.2-1.8 mM; In step 1, the application amount of the soil pretreatment liquid per mu is 30-40 L; Step 2: preparation of probiotic compound matrix: mixing A component and C component to prepare probiotic compound matrix, wherein the weight ratio of A component to C component is 1:100-1:200, the A component contains Bacillus subtilis and Pseudomonas fluorescens, and the C component is selected from organic fertilizer, biochar particles or a combination thereof; Step 3: sowing and matrix application: using sowing machinery with independent sowing and fertilization functions, the sowing depth of soybean seeds is set to 3-5 cm, and the probiotic compound matrix prepared in step 2 is applied to the position 3-5 cm below or beside the soybean seeds; Step 4: field management: first irrigation within 24 hours after sowing, the irrigation amount is appropriate to wet the soil layer of 15-20 cm, and the water management is carried out according to the soil moisture content during the seedling stage of soybeans.

2. The soybean planting method based on rhizosphere probiotics regulation for salt-alkali soil resistance according to claim 1, characterized in that, The organic matter content of the organic fertilizer is not less than 45%, and the pH value is 7.0-7.5; the particle size of the biochar particles is 2-4 mm, and the pH value is 7.5-8.

0.

3. The soybean planting method based on rhizosphere probiotics regulation for salt-alkali soil resistance according to claim 1, characterized in that, In step 2, the total water content of the probiotic compound matrix is adjusted to 35%-40%.

4. The soybean planting method based on rhizosphere probiotics regulation for salt-alkali soil resistance according to claim 1, characterized in that, In step 3, the sowing depth of the soybean seeds is 3.5-4.5 cm.

5. The soybean planting method based on rhizosphere probiotics regulation according to claim 1, characterized in that, In step 3, the probiotic compound matrix is applied to the position 4 cm below the soybean seeds.

6. The soybean planting method based on rhizosphere probiotics regulation according to claim 1, characterized in that, In step 3, the application amount of the probiotic compound matrix per mu is 25-35 kg.

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