Functional microbial complex microbial inoculant for ecological improvement of moderate and severe saline-alkali soil and preparation method and application of functional microbial complex microbial inoculant

Abstract

The invention discloses a functional composite microbial inoculant for ecological improvement of moderate and severe saline-alkali soil and a preparation method and application thereof. The composite bacterial agent is prepared by compounding bacterial solutions of paenibacillus polymyxa SMr02, bacillus velezensis DS-R-B-3-5, bacillus laterosporus BHr6-5, lysinibacillus fusiformis SCB19 and pseudomonas japonicus D109 according to the volume ratio of 3: 2: 2: 2: 1. The complex microbial inoculant disclosed by the invention has good saline-alkali tolerance, can obviously improve the stable soil fertility and nutrient content in moderate and severe saline-alkali soil, reduce the soil salinity and alkalinity, relieve saline-alkali stress of salt-tolerant crops and promote growth of the salt-tolerant crops, is simple and convenient to operate, low in cost and environment-friendly, and has a wide application prospect. The method is suitable for ecological improvement of moderate and severe saline-alkali soil in arid areas, irrigation areas in the middle and upstream of the Yellow River and the like, and has good ecological benefits and economic benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of moderate and severe saline-alkali land treatment and microbial technology, and particularly relates to a moderate and severe saline-alkali land ecological improvement functional microbial compound inoculant, a preparation method and application thereof. BACKGROUND

[0002] Soil salinization is a worldwide ecological problem. Saline-alkali soils are widely distributed in the world, and the global area of saline-alkali soils is about 9.55×10 8 hm 2 , mainly in arid regions. China is the third largest country in the world in terms of the distribution of saline-alkali lands, with an area of 3.6×10 7 hm 2 , mainly distributed in the northeast, north and northwest regions. The areas of different regions are different, and the composition and causes of salinity are also different. It is an important exploitable arable land resource. The surface of saline-alkali land accumulates a large amount of salt, resulting in low soil permeability, which is not conducive to plant growth. In arid and semiarid regions, saline-alkali land seriously affects land productivity, thereby restricting the sustainable development of agriculture, and has become one of the major problems of soil ecological environment that needs to be solved urgently. In recent years, soil salinization improvement has been highly valued by the government and relevant scientific research institutions, and a large amount of manpower and financial resources have been invested. Water conservancy engineering, chemical and biological improvement measures have been taken to improve saline-alkali land, and certain results have been achieved. However, water conservancy engineering measures are not suitable for large-scale promotion and use due to high investment. Planting halophytes has a small investment and is sustainable, but it is difficult to promote and apply in the improvement of saline-alkali land in arid regions.

[0003] Soil salinization improvement agent has the characteristics of easy implementation, low cost, high efficiency and wide application range, and has been widely used in many regions. To some extent, it improves the physical and chemical properties of saline-alkali land soil, reduces the salt content of soil, improves the yield and quality of plants in saline-alkali land, and increases the diversity of soil microorganisms. There are many types of soil salinization improvement agents at home and abroad, mainly divided into three categories: chemical improvement agent, organic improvement agent and biological improvement agent. The properties, composition, mechanism and application effects of different types of improvement agents are quite different. Among them, the biological improvement agent mainly using microorganisms has attracted widespread attention. Many different functional microorganisms have been found, such as phosphorus-solubilizing bacteria, potassium-solubilizing bacteria, nitrogen-fixing bacteria and mycorrhizal fungi. By using the life activities of microorganisms to increase the content of nitrogen, available phosphorus and available potassium in soil, to convert some substances in soil that plants cannot directly utilize into nutrients that can be absorbed and utilized by plants, to inhibit plant pathogens by regulating the material level of crops, to improve soil fertility, and to reduce the salt content and pH of saline-alkali land soil, the purpose of improving the soil conditions of saline-alkali land is achieved.

[0004] Currently, existing functional microbial agents for saline-alkali land improvement mainly consist of single strains, which are susceptible to competition from native microorganisms and adverse environmental conditions, resulting in poor survival and colonization rates, thus reducing the improvement effect. Furthermore, salt-tolerant functional microbial agents generally suffer from low effective yields and high production costs, reducing the practicality of microbial improvement of saline-alkali land. Functional microbial compound agents developed based on the theory of "synthetic microbial communities" combine salt-tolerant microbial strains with different ecological functions, fully leveraging their synergistic effects to achieve complementary advantages and a powerful synergy. This enhances the survival and growth-promoting capabilities of various functional bacteria and increases the diversity of beneficial microorganisms in moderately to severely saline-alkali soils. Simultaneously, functional microbial compound agents can activate and improve soil nutrients, reduce soil salinity, and increase the yield of salt-tolerant crops. They also possess advantages such as being environmentally friendly, sustainable, and pollution-free, making them an ideal choice to replace or reduce the use of chemical amendments. Summary of the Invention

[0005] The purpose of this invention is to provide a functional microbial compound agent for improving moderately to severely saline-alkali land, its preparation method and application, to solve the shortcomings of existing microbial agents for improving saline-alkali land, and to achieve ecological improvement of saline-alkali soil and promotion of salt-tolerant crops.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A microbial compound inoculant for ecological improvement of moderately to severely saline-alkali land, which is composed of Bacillus polymyxa (… Paenibacillus polymyxa )SMr02, Bacillus belysinus ( Bacillus velezensis DS-RB-3-5, Bacillus laterosporus ( Brevibacillus laterosporus BHr6-5, Bacillus fusiformis ( Lysinibacillus fusiformis SCB19 and Pseudomonas japonicus ( Pseudomonas japonica The bacterial culture medium of D109 was mixed in a volume ratio of 3:2:2:2:1.

[0007] The preservation information for the above five strains is as follows: Polymyxin Bacillus ( Paenibacillus polymyxa SMr02, deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 8, 2017, with accession number CGMCC No. 15043; Bacillus belesiensis ( Bacillus velezensis DS-RB-3-5, deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 12, 2023, with accession number CGMCC No. 27087; Lateral spores (Bacillus retroflexus) Brevibacillus laterosporus) BHr6-5, preserved in the China General Microbiological Culture Collection Center on April 12, 2023, with a preservation number of CGMCC No. 27089; Bacillus fusiformis Lysinibacillus fusiformis ) SCB19, preserved in the China General Microbiological Culture Collection Center on April 12, 2023, with a preservation number of CGMCC No. 27091; Pseudomonas japonica Pseudomonas japonica ) D109, preserved in the China General Microbiological Culture Collection Center on April 12, 2023, with a preservation number of CGMCC No. 27093.

[0008] The five functional bacteria used in the present application are all derived from the tissues of plants in the saline-alkali land in the northwest arid region, have good salt-alkali tolerance and ecological adaptability, and have no antagonism among the strains, and can synergistically exert the improvement functions: Bacillus polymyxa SMr02 can exert effects from multiple aspects such as improving soil environment, relieving salt stress, providing nutrition, and inhibiting diseases; Bacillus velezensis DS-R-B-3-5 can directly help salt-tolerant plants resist salt stress, improve rhizosphere microenvironment, activate soil nutrients, and inhibit soil-borne diseases; Bacillus laterosporus BHr6-5 helps plants survive and grow in saline-alkali adversity through strong phosphorus solubilization ability, production of plant hormones and extracellular polymers, and rhizosphere colonization ability; Bacillus fusiformis SCB19 has functions such as relieving salt-alkali stress, promoting the growth of salt-tolerant plants, and efficiently degrading organic matter; and Pseudomonas japonica D109 relieves salt-alkali stress, promotes plant growth, and improves soil microenvironment through active metabolic capacity.

[0009] The viable cell number concentration of the culture solution of each of the five bacteria is ≥1.0×10 9 CFU / mL, and the OD 600 value is 0.8-1.0, so as to ensure that each strain has sufficient activity and quantity in the compound microbial agent, and ensure the stability and repeatability of the improvement effect.

[0010] The present application also provides a preparation method of the above-mentioned ecological improvement functional microbial compound agent for moderate and severe saline-alkali land, comprising the following steps: S1. Single colony culture: the above five kinds of bacteria are inoculated on TSA solid culture medium (culture dish diameter 90 mm, culture medium 15-20 mL) respectively by plate streaking method, and placed in a 25-30°C (preferably 26°C) incubator for dark culture for 5-7 days to obtain five kinds of fresh independent single colonies; wherein, the specific operation of plate streaking method is: different strains of microorganisms are respectively diluted and streaked on the surface of the plate culture medium with an inoculation ring to obtain independently distributed single cells, which grow and reproduce into single colonies after culture; if the single colonies are not reproduced from single cells, repeat the streaking until the pure strain is obtained; the formula of TSA solid culture medium is: tryptone 15.0 g, soybean peptone 5.0 g, NaCl 5.0 g, distilled water 1000 mL, agar 20.0 g, pH 7.3±0.2, 121°C sterilization for 25 min.

[0011] S2. Liquid expansion culture: one strain is picked from each of the five single colonies and inoculated in a 500 mL wide-mouth flask containing 200 mL of TSA liquid culture medium, and three replicates are set for each kind of bacteria; the flask is placed in a 25-30°C (preferably 26°C) constant temperature shaking incubator at 150-250 rpm (preferably 200 rpm) for dark culture for 2-3 days; wherein, the formula of TSA liquid culture medium is: tryptone 15.0 g, soybean peptone 5.0 g, NaCl 5.0 g, distilled water 1000 mL, pH 7.3±0.2, 121°C sterilization for 25 min.

[0012] S3. Bacterial liquid adjustment: after the viable bacterial concentration of each liquid bacterial agent is ≥1.0×10 9 CFU / mL, adjust the OD 600 value of each bacterial suspension to 0.8-1.0 with sterile water to obtain five kinds of bacterial culture liquids; this step ensures the consistency of the number of viable bacteria in each fermentation liquid, and ensures the accuracy and practicability of the compound bacterial agent ratio.

[0013] S4. Compound ratio: mix the five kinds of bacterial culture liquids in a volume ratio of 3:2:2:2:1 to obtain the compound bacterial agent; this ratio is the optimal ratio obtained through indoor hole plate experiment and pot experiment, which can maximize the synergistic effect between each strain. This ratio can ensure the harmonious symbiosis of each strain in the rhizosphere, no mutual antagonism, and can maximize the quorum sensing and functional complementation, and realize the ecological improvement effect of “1+1>2”.

[0014] The application provides the application of the above-mentioned functional microbial compound bacterial agent in improving the soil of moderate to severe saline-alkali land and / or promoting the growth of salt-tolerant plants.

[0015] The application is to apply the functional microorganism composite microbial agent to saline-alkali soil or salt-tolerant plant roots. The application mode is: after the functional microorganism composite microbial agent stock solution is diluted 50-150 times (preferably 100 times), it is applied to the roots of salt-tolerant plants, and the recommended dosage is 150 ml of diluted bacterial solution per plant. In field application, the application amount of the functional microorganism composite microbial agent is 5.0-15.0 kg of stock solution per mu, which can be adjusted according to the degree of soil salinity and the growth stage of plants. The moderate to severe saline-alkali soil is suitable for the saline-alkali soil in the arid region, especially the moderate to severe saline-alkali soil in the irrigation area of the middle and upper reaches of the Yellow River. The saline-alkali soil in the irrigation area of the middle and upper reaches of the Yellow River: the saline-alkali soil in the irrigation area of the middle and upper reaches of the Yellow River, which has high salt content in soil parent material, unbalanced mineral nutrients, lack of organic matter and other nutrients, poor soil structure, and difficulty in growing crops or low yield. The salt-tolerant plants include but are not limited to corn, sunflower and the like.

[0016] Compared with the prior art, the present application has the following advantages: 1. Strong synergy and strong ecological adaptability: the five functional bacteria used are all derived from plants in saline-alkali soil, can quickly adapt to the climate and soil environment of saline-alkali soil, have good ecological adaptability, and have no antagonistic effect between strains. Through scientific compounding, synergistic effect is achieved, and the strains can quickly colonize in the rhizosphere soil and roots of plants, effectively alleviate the salt stress of plants, promote the growth of plants, improve the microbial community structure of saline-alkali soil, increase the soil nutrient content, and reduce the soil salinity, so as to realize the rapid improvement of moderate to severe saline-alkali soil.

[0017] 2. Significant improvement effect and comprehensive function: the composite microbial agent can improve the saline-alkali soil through multiple ways, which can not only reduce the soil salt content (such as electrical conductivity, water-soluble salt content, Na + , etc.) and pH value, but also activate soil nutrients, increase the content of soil organic matter, total nitrogen, available phosphorus, available potassium and other nutrients, alleviate the salt stress of salt-tolerant plants, promote the growth of plants, and significantly improve the crop yield. Field experiments show that after applying 15 kg / mu of the composite microbial agent, the dry weight yield of corn is increased by 31.55% compared with the control, and the dry weight yield of sunflower is increased by 126.70% compared with the control.

[0018] 2. Flexible application and wide adaptability: the ratio and usage amount of the composite microbial agent can be adjusted according to the climate, soil and growth period of crops, which is suitable for different types of moderate to severe saline-alkali soil, especially suitable for the saline-alkali soil in the arid region and the irrigation area of the middle and upper reaches of the Yellow River; the product is a liquid microbial agent, which can be directly diluted and applied, and is convenient to use and easy to popularize.

[0019] 4. Environment-friendly and outstanding economic benefits: the microbial agent is a biological preparation, which is environment-friendly, sustainable and pollution-free, and can replace or reduce the use of chemical improvement agents; it can significantly improve the yield of salt-tolerant crops, reduce the cost of saline-alkali soil improvement, and has good ecological and economic benefits. Attached Figure Description

[0020] Figure 1 This is the experimental design diagram for a field plot in moderately to severely saline-alkali land in Jingtai County, according to the present invention.

[0021] Figure 2 This is a diagram showing the application of the functional microbial compound inoculant for moderately to severely saline-alkali land in Jingtai County according to the present invention.

[0022] Figure 3 This invention relates to the yield of corn (A) and sunflower (B) in moderately to severely saline-alkali land in Jingtai County.

[0023] Figure 4 This invention relates to the rhizosphere soil salinity index of maize (A) and sunflower (B) in moderately to severely saline-alkali land in Jingtai County.

[0024] Figure 5 This invention relates to the rhizosphere acid ion index of corn (A) and sunflower (B) in moderately to severely saline-alkali land in Jingtai County.

[0025] Figure 6 This invention relates to the cation content of salt in the roots of corn (A) and sunflower (B) in moderately to severely saline-alkali land in Jingtai County.

[0026] Figure 7 This invention relates to the salt cation content of aboveground corn (A) and sunflower (B) plants in moderately to severely saline-alkali land in Jingtai County. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. In the following embodiments, unless otherwise specified, the methods used are conventional methods, and the reagents and materials used are commercially available.

[0028] Example 1: Preparation of Functional Microbial Compound Agent Polymyxin Bacillus ( Paenibacillus polymyxa SMr02, deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 8, 2017, with accession number CGMCC No. 15043; Bacillus belyssus ( Bacillus velezensis DS-RB-3-5, deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 12, 2023, with accession number CGMCC No. 27087; Bacillus laterosporus ( Brevibacillus laterosporus BHr6-5, deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 12, 2023, with accession number CGMCC No. 27089; *Bacillus fusiformis* ( Lysinibacillus fusiformis) SCB19, preserved in the China General Microbiological Culture Collection Center on April 12, 2023, with the preservation number CGMCC No. 27091; Pseudomonas japonica (Pseudomonas japonica) Pseudomonas japonica ) D109, preserved in the China General Microbiological Culture Collection Center on April 12, 2023, with the preservation number CGMCC No. 27093.

[0029] The five strains of the present application are microbial bacterial strains isolated and purified from plants in the saline-alkali land in the northwest arid region, and are screened through the indexes of salt-alkali tolerance, salt-tolerant plant growth promotion, soil nutrient activation promotion in saline-alkali land, and salinity reduction. The specific functions of each strain are as follows: Bacillus polymyxa SMr02 (Bacillus polymyxa SMr02) Paenibacillus polymyxa has the functions of improving soil environment, relieving salt stress, providing nutrition, and inhibiting diseases.

[0030] Bacillus velezensis DS-R-B-3-5 (Bacillus velezensis DS-R-B-3-5) Bacillus velezensis has the functions of helping salt-tolerant plants resist salt stress, improving rhizosphere microenvironment, activating soil nutrients, and inhibiting soil-borne diseases.

[0031] Bacillus laterosporus BHr6-5 (Bacillus laterosporus BHr6-5) Brevibacillus laterosporus has the functions of phosphorus solubilization, plant hormone and extracellular polymer production, and helping plants survive and grow in saline-alkali adversity.

[0032] Bacillus fusiformis SCB19 (Bacillus fusiformis SCB19) Lysinibacillus fusiformis has the functions of relieving salt-alkali stress, promoting the growth of salt-tolerant plants, and efficiently degrading organic matter.

[0033] Pseudomonas japonica D109 (Pseudomonas japonica D109) Pseudomonas japonica has the functions of active metabolism, relieving salt-alkali stress, promoting plant growth, and improving soil microenvironment.

[0034] The preparation method of the medium and heavy saline-alkali land ecological improvement functional microbial compound inoculant is as follows: S1. Single colony culture: Paenibacillus polymyxa SMr02, B. velezensis DS-R-B-3-5, B. laterosporus BHr6-5, Lysinibacillus fusiformis SCB19 and Pseudomonas japonica D109 were respectively inoculated on TSA solid medium (diameter of culture dish 90 mm, 18 mL medium per dish) by plate streaking method; the formula of TSA solid medium was: tryptone 15.0 g, soybean peptone 5.0 g, NaCl 5.0 g, distilled water 1000 mL, agar 20.0 g, pH 7.3, sterilized at 121 ℃ for 25 min; the inoculated plates were placed in a 26 ℃ incubator for dark culture for 6 d, and five kinds of fresh independent single colonies were obtained.

[0035] S2. Liquid expansion culture: one strain was picked from each of the five single colonies and inoculated in a 500 mL wide-mouth flask containing 200 mL of TSA liquid medium, with three replicates for each bacterium; the formula of TSA liquid medium was: tryptone 15.0 g, soybean peptone 5.0 g, NaCl 5.0 g, distilled water 1000 mL, pH 7.3, sterilized at 121 ℃ for 25 min; the flasks were placed in a 26 ℃ constant temperature shaking incubator at 200 rpm for dark culture for 2.5 d.

[0036] S3. Bacterial liquid adjustment: the viable cell concentration of the liquid bacterial agent in each flask was detected, all ≥1.0×10 9 CFU / mL, the OD 600 value of each bacterial suspension was adjusted to 0.9 with sterile water, and five kinds of bacterial culture solutions were obtained.

[0037] S4. Compound proportioning: five kinds of bacterial culture solutions were respectively measured according to the volume ratio of 3:2:2:2:1, and mixed uniformly to obtain the ecological improvement functional microbial compound inoculant for moderate and severe saline-alkali land.

[0038] Example 2 Field plot experiment of application of functional microbial compound inoculant A field plot experiment of the functional microbial compound inoculant prepared in Example 1 was carried out in a moderate and severe saline-alkali land (soil salt content 22.46 g / kg, pH 8.72, soil type silty loam and sandy clay loam, light texture, poor structure, weak water and fertilizer retention capacity, and salt accumulation in the soil surface) in Jingtai County, Gansu Province, to verify its effectiveness.

[0039] 1. Experimental design: Three concentration gradient composite microbial agent treatment groups were set: T1 (5.0 kg of stock solution per mu), T2 (10.0 kg of stock solution per mu), and T3 (15.0 kg of stock solution per mu), with a blank (CK, spraying dilution water without microbial agent) as the control. The field plots were arranged in a randomized block design, with a total of 5 sample areas, each measuring 4 m x 15 m. Each treatment was set up with 3 replicates, and 1 m of isolation row was left between each sample area (see Figure 1 ).

[0040] 2. Planting and application: On April 28, 2025, 2.0 tons of bio-organic fertilizer were evenly applied to the moderately and severely saline-alkali soil, and a rotary cultivator was used for shallow plowing to a depth of about 15 cm. Strong mulch film with a width of 100 cm and a thickness of more than 0.01 mm was used for mulching. The film was pulled tight and laid flat, tightly against the ground, and the edges of the film were pressed firmly with soil. After mulching, a dibbler was used to punch holes in the film, with a depth of 3-5 cm. 1-2 seeds were planted in each hole, and after planting, the seeds were lightly pressed into contact with the soil, then the planting holes were sealed with fine wet soil to form small soil mounds, preventing the loss of heat and moisture in the film and preventing the film from being torn by strong winds. The variety of corn ( Zea mays L.) was Xianyu 335, and the variety of oil sunflower ( Helianthus annuus L.) was T562. The row spacing of corn was 40-50 cm, the plant spacing was 25-30 cm, and there were about 3500 plants per mu. The row spacing of oil sunflower was about 50 cm, the plant spacing was 30-40 cm, and there were about 3000 plants per mu.

[0041] Application time: In early June 2025, when the corn and sunflower in the saline-alkali soil had grown 5 pairs of true leaves, the first root application of functional microbial composite was carried out. In late August 2025, when the corn and sunflower in the saline-alkali soil entered the present budding period, the second root application of functional microbial composite was carried out. In mid-September 2025, when the corn and sunflower in the saline-alkali soil entered the filling stage, the third root application of functional microbial composite was carried out.

[0042] Application method: Choose overcast days or before 11 am in the morning and after 4 pm in the afternoon to apply fertilizer, avoiding high temperature, strong light, and windy weather. Dilute the functional microbial composite 100 times, and use the near-root hole application method to make the functional microbial composite directly reach the root area of corn and sunflower, improving the utilization rate. About 5 cm from the base of the seedling stem, use a thin stick to insert a hole about 15 cm deep at an angle downward (about 45°), then use a water bottle to inject 150 ml of microbial liquid into the hole, and immediately cover the soil to prevent water evaporation and the influence of ultraviolet rays on living bacteria ( Figure 2 ). The blank control (CK) was applied with 150 ml of dilution water without microbial agent per plant, and the application method was consistent with that of the functional microbial composite.

[0043] Experimental site: Caowotan Town, Jingtai County, Baiyin City, Gansu Province (elevation 1524.2 meters, north latitude 37°15'58'', east longitude 104°07'49'').

[0044] Test sample site: moderate to severe saline-alkali soil.

[0045] 3. Index detection and results: The functional microbial compound inoculant was subjected to field plot control experiment, and on June 27, 2025, August 18, 2025, and October 11, 2025, three 1m x 1m fixed quadrats were set up in each sample area in the field plot, and the plant height and ground diameter of corn and sunflower in the fixed quadrat were recorded. The vertical height from the ground root neck to the highest natural stretching point of the plant was measured with a tape measure as the plant height (unit: cm), and the jaws of a vernier caliper were gently clamped around the stem near the ground to ensure perpendicularity without excessive compression of the stem. The stem diameter was measured to measure the lodging resistance and robustness of the plant (unit: mm). On October 11, 2025, the corn and sunflower seeds of the control and each treatment group were harvested, dried in an oven at 65°C for 72h, and then weighed with an electronic balance (accurate to 0.01) to convert the dry weight yield per mu.

[0046] On June 27, 2025, the crop survival rate was 42.96% for corn and 44.46% for sunflower, the plant height and ground diameter detection results are shown in Table 1, and the yield results are shown in Figure 3 .

[0047] The experimental results show that: applying different concentrations of functional microbial compound inoculant can increase the plant height and ground diameter of corn and sunflower in saline-alkali soil, and with the increase of the application concentration, the improvement effect also increases accordingly, showing obvious concentration dependence. In addition, the improvement effect of early application is generally higher than that of later application. Under the concentration of 15 kg / mu of functional microbial compound inoculant, the three times of fertilization increased the corn plant height by 77.69%, 23.51%, and 17.54%, respectively, and the ground diameter by 21.35%, 30.34%, and 29.20%, respectively; the sunflower plant height by 58.80%, 21.28%, and 18.75%, respectively, and the ground diameter by 239.37%, 15.76%, and 13.87%, respectively (Table 1). In terms of yield, the dry weight yield of corn was 302.38-397.38 kg / mu, and the yield of 15 kg / mu of functional microbial compound inoculant was the highest, increasing by 31.55% compared with the control; the dry weight yield of sunflower was 97.78-221.67 kg / mu, and the yield of 15 kg / mu of functional microbial compound inoculant was the highest, increasing by 126.70% compared with the control Figure 3 .

[0048] Effects of functional microbial complex inoculant on soil salt, acid radical ion and physicochemical properties in moderate and severe saline-alkali land This example takes the field plot of functional microbial complex inoculant application in moderate and severe saline-alkali land in Jingtai County as the research object, and detects the indexes of soil salt, acid radical ion, physicochemical properties, etc. to evaluate the improvement effect of a functional microbial complex inoculant for moderate and severe saline-alkali land on the degree of soil salinity and nutrient content in moderate and severe saline-alkali land.

[0049] Sampling time: October 11, 2025.

[0050] Specific implementation method: In the field plot of corn and sunflower treated with control and different concentrations of functional microbial complex inoculant, 10 plants were sampled in each plot. The control and each treatment obtained 30 corn and 30 sunflower plants with soil, respectively. The peripheral soil of the root system was shaken off gently, and the rhizosphere soil attached to the root surface was brushed off gently with a brush. After sieving, the residual roots mixed in the soil were removed and placed in a dry and cool indoor environment. After drying, it was sieved through a 60-mesh sieve.

[0051] Rhizosphere soil salt index: electrical conductivity (EC), water-soluble salt (TDS), Na + , K + , Mg 2+ and Ca 2+ . Refer to the method in "Soil Agricultural Chemical Analysis" (Bao Shidan, third edition). The electrode method was used to determine the soil electrical conductivity (EC), and the water-soluble salt (TDS) was extracted according to the water and saline-alkali soil ratio of 5:1. The weight of the extracted solution after evaporation was the content of soil water-soluble salt. Mg 2+ and Ca 2+ were determined by EDTA titration, and Na + and K + were determined by flame photometry. The results are shown in Figure 4 .

[0052] Rhizosphere soil acid radical ion index: Cl - , SO4 2- and HCO3 - . Refer to the method in "Soil Agricultural Chemical Analysis" (Bao Shidan, third edition). Cl - was determined by silver nitrate titration, SO4 2- was determined by EDTA indirect complexation titration, and HCO3 - was determined by double-indicator neutralization titration. The results are shown in Figure 5 .

[0053] Rhizosphere soil physicochemical properties: pH, organic matter (SOM), organic carbon (SOC), total nitrogen (TN), available nitrogen (AN), total phosphorus (TP), available phosphorus (AP), total potassium (TK), and available potassium (AK). Rhizosphere soil pH was determined using a pH meter with a 1:2.5 water-soil mixture suspension. Organic matter (SOM) was determined using the potassium dichromate-sulfuric acid oxidation method. Organic carbon (SOC) was determined using an elemental analyzer. Total nitrogen (TN) and total phosphorus (TP) were determined using sulfuric acid-catalyst digestion combined with the Kjeldahl method. Available nitrogen (AN) was determined using the ferrous sulfate-alkaline diffusion method. Available phosphorus (AP) was determined using sodium bicarbonate / ammonium fluoride hydrochloric acid extraction and the molybdenum-antimony colorimetric method. Total potassium (TK) was determined using the NaOH alkaline fusion-flame photometric method. Available potassium (AK) was determined using ammonium acetate solution extraction and the flame photometric method. The results are shown in Table 2.

[0054] Application results: (1) The results of the detection of soil salinity index in the rhizosphere of maize and sunflower showed that the application of 5 kg / mu of functional microbial compound inoculant at T1 reduced soil K + and Mg 2+ The content had no significant effect on total soluble salts (TDS); both the T2 treatment (10 kg / mu) and the T3 treatment (15 kg / mu) reduced the electrical conductivity (EC) and sodium content in the rhizosphere soil of maize. + K + Mg 2+ and the total soluble salt content, increasing Ca 2+ content( Figure 4 -A). Treatment with all three concentrations of the functional microbial compound inoculant reduced the rhizosphere soil electrical conductivity and total soluble salt content in sunflowers; treatment T1 reduced Ca... 2+ K + and Mg 2+ The content of Ca; T2 treatment increases the content of Ca. 2+ The content of Na in the soil was reduced. + Content; T3 treatment reduced soil Na content. + and K + content( Figure 4 -B). Overall, the application of functional microbial compound inoculants can effectively reduce total soil salinity and sodium. + Content, lowering soil pH.

[0055] (2) The results of the detection of acid ion index in the rhizosphere soil of maize and sunflower showed that: T1 treatment increased SO4 in the rhizosphere of maize. 2- The content decreased HCO3. - The content of Cl - The content was not significantly affected; the T2 treatment increased the rhizosphere HCO3 in maize. - The content of SO4 was reduced. 2-and Cl - content; T3 treatment increased the HCO3 - content of the corn rhizosphere soil, and decreased the SO4 2- and Cl - content Figure 5 -A). The detection of sunflower rhizosphere soil showed that T1 treatment decreased the HCO3 - and Cl - content, T2 treatment decreased the HCO3 - and Cl - content, but increased the SO4 2- content, T3 treatment decreased the HCO3 - content of the sunflower rhizosphere soil, and increased the SO4 2- and Cl - content Figure 5 -B).

[0056] (3) The detection results of the physicochemical property indexes of the corn and sunflower rhizosphere soil showed that the functional microbial compound agent treatment of the three concentrations could reduce the pH of the corn rhizosphere soil, and increase the contents of total organic carbon, organic matter, total nitrogen, total phosphorus, and alkali-hydrolyzable nitrogen. At the same time, T2 and T3 significantly increased the contents of available phosphorus and available potassium, and T3 had the most obvious effect on the improvement of the corn rhizosphere soil nutrients (Table 2). The detection of the physicochemical properties of the sunflower rhizosphere soil found that T3 reduced the pH of the sunflower rhizosphere soil, T2 and T3 treatment increased the contents of total organic carbon and organic matter of the rhizosphere soil, and increased the contents of total nitrogen, total phosphorus, alkali-hydrolyzable nitrogen, available phosphorus, and available potassium (Table 2). In summary, the application of the functional microbial compound agent improved the internal stability of the soil, activated the soil nutrients, improved the ecological environment of the saline-alkali soil, and was beneficial to the seedling and growth of salt-tolerant plants.

[0057] Example 4 Influence of Functional Microbial Compound Agent on Salt Cation Indexes of Root System and Aboveground Plant of Salt-tolerant Crops in Medium and Severe Saline-alkali Soil In this example, the root system and aboveground plant of corn and sunflower in the field plot of the functional microbial compound agent applied in the medium and severe saline-alkali soil in Jingtai County were taken as the research objects, the salt cation indexes were detected, and the influence of a functional microbial compound agent for ecological improvement of medium and severe saline-alkali soil on the absorption of salt cation by salt-tolerant crops was evaluated.

[0058] Sampling time: October 11, 2025.

[0059] Specific implementation method: in the control and different concentration of functional microbial compound microbial agent treatment of corn and sunflower field plot sample, 10 plants of crops were taken in each sample, and 30 corn and 30 sunflower plants were obtained in the control and each treatment respectively. 10 root segments of 8-10 cm were randomly cut from the root of each corn in the control and treatment, mixed into one root sample, and 4 corn and sunflower root samples were obtained respectively; similarly, 3 small sections of 10-15 cm in length were randomly cut from different heights of the aboveground plants of each corn and sunflower, mixed into one aboveground plant sample, and 4 corn and sunflower aboveground plant samples were obtained respectively. The root and aboveground plant samples were placed in an oven at 65°C for 72 h, dried, crushed and sieved through a 60 mesh sieve.

[0060] Root and aboveground plant salt cation index: Na + , K + , Mg 2+ and Ca 2+ . The detection method is consistent with that of salt cation in rhizosphere soil.

[0061] Application results: (1) The detection results of salt cation index of corn and sunflower root showed that: T2 application of 10 kg / acre and T3 application of 15 kg / acre of functional microbial compound microbial agent promoted the absorption of salt cation in corn and sunflower root in Jingtai County medium and heavy saline-alkali land, T1 application of 5 kg / acre of functional microbial compound microbial agent reduced the Mg 2+ content of corn plant by 2.33%, and also reduced the Na + content of sunflower plant by 7.60%, Ca 2+ 12.44%, Mg 2+ 8.07% ( Figure 6 ). T3 treatment had the most obvious effect on promoting the absorption of salt cation in corn and sunflower root, the Na + content of corn root was 35.41% higher than that of the control, and the Na + content was 18.96% higher; T3 treatment had more obvious effect on promoting the absorption of salt cation in sunflower root than in corn, the Na + , Ca 2+ , Mg 2+ and K + contents were 52.04%, 27.33%, 53.05% and 60.57% higher than those of the control, respectively ( Figure 6 ). Overall, the application of functional microbial compound microbial agent can activate the salt cations in the soil, so that they can be absorbed and utilized by salt-tolerant plants, thereby effectively reducing the salt index in the soil, and the higher the concentration of functional microbial compound microbial agent applied, the more beneficial it is for the plant root to absorb salt cations.

[0062] (2) The detection results of salt cation indexes of corn and sunflower plants show that the Mg 2+ content of corn plants in T1 treatment is slightly lower than that of the control, the Ca 2+ content of corn plants in three concentration treatments is lower than that of the control, indicating that Na + in the soil is replaced by Ca 2+ to form calcium salt that is insoluble in water, so the Ca 2+ content in corn plants is reduced. T2 and T3 treatments increase the contents of other three cations, and the increase in T3 treatment is the most obvious, the Na + , Mg 2+ and K + contents are 226.80%, 9.72% and 24.19% higher than those of the control, respectively. Figure 7 The application of functional microbial composite inoculant with different concentrations increases the salt cation content in sunflower plants, the Na + , Ca 2+ and Mg 2+ contents in T2 treatment are 61.22%, 26.34% and 38.29% higher than those of the control, respectively, and the K + content in T3 treatment is 9.93% higher than that of the control. Figure 7 Overall, the application of functional microbial composite inoculant activates the salt cations in the soil, which are absorbed by the salt-tolerant plant roots, thereby effectively reducing the salt index in the soil, and the salt cations absorbed by the roots can be transported from the roots to the aboveground plants.

[0063] The above only describes the embodiments of the present application, and does not limit the inventive range of the present application, and any equivalent structure or equivalent process transformation using the content of the present application, or direct or indirect application in other related technical fields, are also included in the inventive protection range of the present application.

Claims

1. A microbial compound inoculant for ecological improvement of moderately to severely saline-alkali land, characterized in that, Bacillus polymyxa ( Paenibacillus polymyxa )SMr02, Bacillus belysinus ( Bacillus velezensis DS-RB-3-5, Bacillus laterosporus ( Brevibacillus laterosporus BHr6-5, Bacillus fusiformis ( Lysinibacillus fusiformis SCB19 and Pseudomonas japonicus ( Pseudomonas japonica The bacterial culture medium of D109 is mixed; wherein, the preservation number of the polymyxa Bacillus SMr02 is CGMCC No.15043; the preservation number of the Bacillus belyssus DS-RB-3-5 is CGMCC No.27087; the preservation number of the Bacillus laterosporus BHr6-5 is CGMCC No.27089; the preservation number of the Bacillus fusiformis lysine Bacillus SCB19 is CGMCC No.27091; and the preservation number of the Pseudomonas japonicus D109 is CGMCC No.27093.

2. The functional microbial compound inoculant according to claim 1, characterized in that, The volumetric mixing ratio of the bacterial culture medium is 3:2:2:2:1 for Bacillus polymyxa SMr02, Bacillus belyssus DS-RB-3-5, Bacillus laterosporus BHr6-5, Bacillus fusiformis SCB19, and Pseudomonas japonicus D109.

3. The functional microbial compound inoculant according to claim 1, characterized in that, The viable cell counts of the bacterial cultures of *Bacillus polymyxa* SMR02, *Bacillus belyceae* DS-RB-3-5, *Bacillus laterosporus* BHr6-5, *Bacillus fusiformis* SCB19, and *Pseudomonas japonicus* D109 were all ≥1.0 × 10⁻⁶. 9 CFU / mL.

4. A method for preparing a microbial compound inoculant for ecological improvement of moderately to severely saline-alkali land as described in claim 1, characterized in that, Includes the following steps: S1. Single colony culture: Bacillus polymyxa SMR02, Bacillus belyeis DS-RB-3-5, Bacillus laterosporus BHr6-5, Bacillus fusiformis SCB19, and Pseudomonas japonicus D109 were inoculated onto TSA solid medium and incubated in the dark at 25-30℃ for 5-7 days to obtain single colonies, resulting in five fresh, independent single colonies. S2. Liquid expansion culture: Pick strains from each single colony and inoculate them into containers containing TSA liquid medium. Incubate in the dark with shaking at 25-30℃ and 150-250 rpm for 2-3 days. S3. Bacterial suspension adjustment: The viable cell count concentration of each liquid bacterial agent should be ≥1.0 × 10⁻⁶. 9 After adjusting the CFU / mL concentration, the OD of each bacterial suspension was adjusted with sterile water. 600 The concentration was adjusted to 0.8~1.0 to obtain culture media for five types of bacteria; S4. Compound formulation: Mix the culture media of the five bacteria in a volume ratio of 3:2:2:2:1 to obtain the compound bacterial agent.

5. The preparation method according to claim 4, characterized in that, The TSA solid culture medium formula is as follows: 15.0 g tryptone, 5.0 g soybean peptone, 5.0 g NaCl, 1000 mL distilled water, 20.0 g agar, pH 7.3±0.2, sterilized at 121℃ for 25 min.

6. The preparation method according to claim 4, characterized in that, The TSA liquid culture medium formula is as follows: 15.0 g tryptone, 5.0 g soybean peptone, 5.0 g NaCl, 1000 mL distilled water, pH 7.3±0.2, sterilized at 121℃ for 25 min.

7. The application of the functional microbial compound inoculant according to any one of claims 1-3 in improving moderately to severely saline-alkali soil and / or promoting the growth of salt-tolerant plants.

8. The application according to claim 7, characterized in that, The application involves applying functional microbial compound agents to saline-alkali soil or the roots of salt-tolerant plants.

9. The application according to claim 8, characterized in that, The application involves diluting the functional microbial compound inoculant stock solution 50-150 times and then watering it onto the roots of salt-tolerant plants.

10. The application according to claim 8 or 9, characterized in that, The moderately to severely saline-alkali land refers to saline-alkali land in arid areas; the application rate of the functional microbial compound inoculant is 5.0-15.0 kg of stock solution per mu.

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