A compound microbial agent for improving saline-alkali soil, a preparation method and application thereof

Abstract

This invention discloses a composite microbial agent for improving saline-alkali soil, its preparation method, and its application. The composite microbial agent is composed of *Frederick's chamaejasminoides* and *Zoococcus halophilus* with a viable count ratio of (0.2–2):1. Research shows that, under the same total viable count conditions, the composite microbial agent exhibits significantly higher salt tolerance, IAA production, siderophore production capacity, ACC deaminase activity, phosphorus solubility, nitrogen fixation capacity, and extracellular polysaccharide production compared to single-strain agents, demonstrating a significant synergistic effect. The composite agent can also be combined with the salt-tolerant plant *Arundo donax* to construct a synergistic remediation system for saline-alkali land. This system significantly reduces the pH and sodium ion content of saline-alkali soil, improves soil nutrient availability, and significantly promotes *Arundo donax* growth and biomass accumulation, providing an innovative technical solution for rapid, efficient, and sustainable ecological restoration and agricultural utilization of saline-alkali land.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology and saline-alkali land ecological restoration technology, and more specifically, to a composite microbial agent for improving saline-alkali soil, its preparation method and application. Background Technology

[0002] Saline-alkali soil is one of the most significant obstacles to agricultural production globally. It is characterized by high pH (typically >8.0), high soluble salt content, strong ion toxicity, poor soil structure, and low bioavailability of available nutrients (such as phosphorus and iron). Traditional physical and chemical improvement methods (such as topsoil application, gypsum, sulfur, and organic fertilizers) are often costly, have short-lasting effects, easily cause secondary pollution, and are unlikely to fundamentally improve the soil microbial community.

[0003] In recent years, bioremediation technology has attracted much attention due to its environmentally friendly and sustainable characteristics. It mainly includes two aspects: first, phytoremediation using salt-tolerant plants (such as reed, Suaeda salsa, and wolfberry), which gradually improve the soil through root activity, water transpiration, and partial ion absorption during their growth; and second, the application of functional microbial agents, which directly improve the rhizosphere microenvironment by utilizing their salt tolerance, organic acid secretion, nutrient activation, and growth-promoting substances. However, both of these bioremediation approaches face significant bottlenecks in practical applications.

[0004] For microbial remediation, the colonization and survival rate of existing microbial agents under extreme saline-alkali stress is the biggest challenge. Many exotic strains struggle to establish effective populations and maintain their function in harsh saline-alkali soils, leading to inconsistent field results. Furthermore, single-strain or simply combined microbial agents often have limited functionality and cannot synergistically address the complex and intertwined stresses of alkali damage, salt damage, nutrient deficiency, and poor soil structure in saline-alkali soils. For phytoremediation, simply planting salt-tolerant plants results in extremely slow improvement. In severely saline-alkali areas, the survival and growth of seedlings are still severely inhibited, limiting their ecological functions. While plant root exudates can attract some microorganisms, this natural process is highly random and inefficient, insufficient to quickly establish a robust and efficient rhizosphere microbial community.

[0005] Currently, research and practice on artificially and directionally coupling specific functional microorganisms with suitable plants to construct synergistic remediation systems are still in their infancy. The key issue lies in the lack of specialized compound microbial agents capable of forming efficient interactions with target plants and significantly enhancing plant stress tolerance and improving their functions. Existing technologies often involve haphazard combinations of microbial agents and plants, failing to achieve precise matching and bidirectional synergy among the three elements: agent function, plant needs, and soil problems. Summary of the Invention

[0006] The purpose of this invention is to address the problems in existing saline-alkali land bioremediation technologies, such as the single function of microbial agents, low synergistic efficiency with plants, and slow remediation process of plants alone, by providing a composite microbial agent.

[0007] A second objective of this invention is to provide a method for preparing the aforementioned composite microbial agent.

[0008] A third objective of this invention is to provide the application of the aforementioned compound microbial agent.

[0009] The fourth objective of this invention is to provide a synergistic remediation system for saline-alkali land.

[0010] The fifth objective of this invention is to provide a method for ecological restoration of saline-alkali land and / or for improving the salt tolerance of plants.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: A compound microbial agent containing *Frederickia oryzae* (Indo-Pacific variella) Vreelandella indica ) and salt-tolerant zoococci ( Planococcus halotolerans The active ingredient is a complex of microorganisms composed of ( ) and ( ); the ratio of viable counts of *Frederickia natans* to halophilic zoococci is (1.5–2.5):1.

[0012] The compound microbial agent of this invention is composed of existing known bacterial strains. Under the same total viable bacterial count, its salt and alkali tolerance, IAA production, siderophore production capacity, ACC deaminase activity, phosphorus solubility, nitrogen fixation capacity, and extracellular polysaccharide production are all significantly higher than those of single bacterial agents. It has comprehensive functions such as acid production to neutralize alkalinity, nutrient activation, growth promotion and improvement of the rhizosphere microenvironment. It can synergistically alleviate salt and alkali stress and has a significant synergistic effect. It is a novel compound microbial agent with multiple growth-promoting and stress-resistance functions.

[0013] Furthermore, the ratio of viable counts of *Frederickia natans* to halophilic zoococci is (1.8–2.2):1; preferably 2:1.

[0014] Furthermore, the compound microbial agent is a solid or liquid formulation; the effective viable count of the solid formulation is not less than 1×10⁻⁶. 8 CFU / g (preferably not less than 1×10⁻⁶) 9 (CFU / g), the effective viable count of liquid preparations is not less than 1×10⁻⁶. 8 CFU / mL (preferably not less than 1×10⁻⁶) 9 (CFU / mL).

[0015] Furthermore, the compound microbial agent also includes a carrier or a protectant; the carrier is selected from at least one of peat, vermiculite, talc, bentonite, and wheat bran; the protectant is selected from at least one of trehalose, skim milk powder, glycerin, and polyvinylpyrrolidone.

[0016] The present invention also provides a method for preparing any of the above-described composite microbial agents, comprising the following steps: S1. Activate and expand the culture of *Frederickia natans* and *Halococcus salina* to obtain bacterial solutions; S2. Mix the two bacterial solutions obtained in step S1 according to the ratio of the corresponding number of viable bacteria to obtain the compound microbial agent.

[0017] Furthermore, the culture medium used in step S1 for the expansion culture is LB medium containing salt and alkali stress.

[0018] Preferably, the culture medium consists of: 5 g / L yeast extract, 30 g / L NaCl, 10 g / L peptone, and pH 8.0.

[0019] The research of this invention shows that the compound microbial agent of this invention has comprehensive functions such as producing acid to neutralize alkalinity, activating nutrients, promoting growth and improving the rhizosphere microenvironment. It can synergistically alleviate salt and alkali stress and has a significant synergistic effect. It can be used for the remediation and improvement of saline-alkali soil and / or to improve the salt and alkali tolerance of plants.

[0020] Therefore, the present invention also provides the application of any of the above-described composite microbial agents in the remediation and / or improvement of saline-alkali soil and the enhancement of plant salt tolerance.

[0021] Furthermore, the research of this invention shows that the above-mentioned compound microbial agent can be used in combination with salt-tolerant plants to synergistically remediate saline-alkali soil.

[0022] Therefore, the present invention also provides the application of any of the above-described composite microbial agents combined with salt-tolerant plants in the construction of a saline-alkali land remediation system.

[0023] This invention also provides a saline-alkali land synergistic remediation system, composed of any of the aforementioned composite microbial agents and salt-tolerant plants. That is, it constructs a "microorganism-salt-tolerant plant" synergistic remediation system.

[0024] Furthermore, the salt-tolerant plant is Reed (Phragmites australis). Arundo donax L. When the compound microbial agent of this invention is used in combination with Reed amurensis, a positive cycle of "microorganisms promoting bamboo growth and bamboo assisting microorganisms in multiplying" can be formed, improving the survival rate, early growth and biomass of Reed amurensis, and promoting the improvement of soil physicochemical properties. The synergistic system can reduce the pH of saline-alkali soil and increase the content of available nutrients, and has good ecological and application promotion value.

[0025] Therefore, the present invention also provides the application of the above-mentioned saline-alkali land remediation system in the remediation and / or improvement of saline-alkali soil and the enhancement of plant salt tolerance.

[0026] This invention also provides a method for ecological restoration of saline-alkali land and / or improving plant salt tolerance, comprising applying an effective amount of any of the above-described compound microbial agents to the saline-alkali land. When the compound microbial agent is used for potted or field restoration of saline-alkali soil, it can establish itself for a long time and continuously exert its effects of salt and alkali improvement and growth promotion, significantly improving the survival rate of plants under salt and alkali stress.

[0027] Furthermore, the method involves planting salt-tolerant plants in saline-alkali land and applying an effective amount of any of the aforementioned compound microbial agents to the saline-alkali land; the compound microbial agents can be applied before, during, or after the planting of salt-tolerant plants.

[0028] Preferably, the application rate of the compound microbial agent is 1×10⁻⁶. 10 Up to 1×10 12 CFU / acre; Application method: dilute the liquid formulation and drench the roots, or mix the solid formulation with organic fertilizer and apply in holes or trenches.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a composite microbial agent for improving saline-alkali soil, its preparation method, and its application. The composite microbial agent is composed of *Frederick's var. indicum* and *Zoococcus halophilus* with a viable count ratio of (0.2–2):1. Research shows that, under the same total viable count conditions, the composite microbial agent exhibits significantly higher salt and alkali tolerance, IAA production, siderophore production capacity, ACC deaminase activity, phosphorus solubility, nitrogen fixation capacity, and extracellular polysaccharide production compared to single-strain agents. It possesses comprehensive functions such as acid production to neutralize alkalinity, nutrient activation, growth promotion, and improvement of the rhizosphere microenvironment. It can synergistically alleviate saline-alkali stress, demonstrating a significant synergistic effect, and is a novel composite microbial agent with multiple growth-promoting and stress-resistance functions. The compound microbial agent can also be combined with salt-tolerant plants to construct a synergistic restoration system for saline-alkali land. This synergistic restoration system can significantly reduce the pH value and sodium ion content of saline-alkali soil, improve soil nutrient availability, and significantly promote the growth and biomass accumulation of salt-tolerant plants, providing an innovative technical solution for the rapid, efficient, and sustainable ecological restoration and agricultural utilization of saline-alkali land. Attached Figure Description

[0030] Figure 1 The growth (OD) of strains YL01, PC02 and their compound inoculum (MIX) under different initial pH conditions. 600 ).

[0031] Figure 2 The termination pH changes after the end of each treatment under different initial pH conditions.

[0032] Figure 3 This is a schematic diagram of colony growth on an Ashby nitrogen-free medium plate (verification of nitrogen fixation potential).

[0033] Figure 4 Soil pH changes during a pot experiment involving the combined application of compound microbial agents and reed. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] Test strain: Fritillaria cirrhosa (Indo-Pacific spirochetes) Vreelandella indica The product was purchased from Beijing Bio-Bio Biotechnology Co., Ltd., product number bio-139207, and will be referred to as such in the following examples. YL01 Salt-tolerant zoococci ( Planococcus halotolerance Purchased from the China General Microbiological Culture Collection Center, catalog number CGMCC 1.13628, hereinafter referred to as... PC02 .

[0037] Example 1: Preparation of Compound Microbial Agent (1) Seed liquid preparation: Take separately YL01 and PC02 In LB liquid medium (pH 8.0) containing 3% NaCl, culture at 30°C and 180–200 r / min for 24–48 h until the logarithmic or stationary phase.

[0038] (2) Collection of bacterial cells: Centrifuge the culture medium at 8000 r / min for 10 min, discard the supernatant, resuspend in sterile physiological saline and adjust to the same viable cell count level of 1×10⁻⁶. 9 CFU / mL.

[0039] (3) Compound preparation: according to the set ratio of live bacteria. YL01 : PC02 =2:1 mixture.

[0040] (4) Formulation: a) Liquid preparations: Add 0.5% to 2% glycerol or trehalose as a preservative, dispense into sterile containers, and store at 4°C for a short period or at -20°C. b) Solid preparation: Mix the mixed bacterial solution with the carrier (peat / vermiculite / talc, etc.) at a mass ratio of 1: (1-5), add a protective agent (trehalose / skimmed milk powder, etc. 0.5%-5%), dry at low temperature or freeze-dry, then pulverize and sieve to obtain a solid bacterial preparation.

[0041] (5) Quality control: The effective viable count (CFU / g or CFU / mL) shall be tested and shall not be less than 1×10 8 Preferably not less than 1×10 9 .

[0042] Example 2 YL01 , PC02 Salt tolerance test of compound microbial agents Following the method described in Example 1, the activated YL01 , PC02 Single bacteria and a compound bacterial agent (denoted as MIX) of the two mixed at a live bacteria ratio of 2:1 (both live bacteria count 1×10⁻⁶) 9 CFU / mL), and at the same inoculum size, inoculated into LB liquid medium (pH 7.2) containing different concentrations of NaCl (0%, 5%, 10%, 15%, 20%) at 1% inoculum size. Incubated at 30°C with shaking at 180 r / min for 48 hours, OD was measured. 600 The values ​​were used to assess its growth. The results are shown in Table 1. PC02 It can still maintain a high growth rate even under 15% NaCl. YL01 It maintained a certain growth rate even at NaCl concentrations as high as 20%, demonstrating extremely strong salt tolerance. Meanwhile, the MIX group showed varying OD values ​​at different salt concentrations. 600 The values ​​are all no lower than or even slightly higher than the theoretical summation value, indicating that the composite system does not produce antagonism and has the potential to synergistically promote growth under moderate salinity (10%–20%).

[0043] Table 1. Growth of strains at different NaCl concentrations (OD) 600 )

[0044] Example 3 YL01 , PC02 Alkali resistance test of compound microbial agents As in Example 2, YL01 , PC02 Single-strain and compound inoculum MIX were inoculated at the same inoculum amount into LB liquid medium (containing 3% NaCl) with initial pH of 7.0, 8.0, 9.0, 10.0, and 10.5, respectively, and cultured at 30℃ with shaking at 180 r / min for 48 hours. The final OD was then measured. 600 and the culture medium pH was terminated. Results were as follows: Figure 1As shown, YL01 It grows well below pH 10.0, while PC02 maintains vigorous growth at pH 10.5, demonstrating excellent alkali tolerance. In the MIX group, the termination pH was significantly lower than the initial pH and the termination pH of single-cell culture within the pH range of 8.0–10.0. Figure 2 This indicates that the compound microbial agent can more effectively metabolize and produce acid, neutralize the alkaline environment, and has the potential to improve the pH of saline-alkali soil, showing a significant synergistic effect.

[0045] Example 4 YL01 , PC02 And the ability of compound microbial agents to produce IAA (Inoculant Acid) The Salkowski colorimetric method was used to determine the ability of the strain to produce indole-3-acetic acid (IAA). As in Example 2, [the following was performed]. YL01 , PC02 Single-strain and compound inoculum MIX were inoculated at the same inoculum amount into LB liquid medium (containing 3% NaCl, pH 8.5) containing 1 mg / mL L-tryptophan and cultured at 30℃, 180 r / min in the dark for 72 hours. The supernatant was collected by centrifugation, reacted with Salkowski's reagent, and the absorbance was measured at 530 nm. The IAA concentration was calculated using a standard curve. The results are shown in Table 2. YL01 and PC02 Both can secrete IAA. Calculations show that in a compound ratio (... YL01 : PC02 The theoretical arithmetic mean of IAA production at a ratio of 2:1 is 23.2 mg / L. However, experimental results show that the IAA production of the compound bacterial agent (MIX) is as high as 68.5 mg / L, which is 2.95 times the theoretical sum and significantly higher than any single bacterial treatment group. This indicates that the two strains have a significant synergistic effect on the synthesis of auxin-like substances during co-culture.

[0046] Table 2. Determination of IAA production capacity of strains

[0047] Example 5 YL01 , PC02 And the ability of compound microbial agents to produce iron carriers The Crazin S (CAS) plate assay was used. The CAS blue assay plate (containing 3% NaCl) was formulated as follows: per liter of culture medium, Crazin S (CAS) 60.5 mg, hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg, FeCl3·6H2O 2.7 mg, piperazine-1,4-diethanesulfonic acid (PIPES) 30.24 g, NaCl 30 g, agar 15 g, pH 6.8. As in Example 2, the... YL01 , PC02 Single-strain and compound inoculum MIX were inoculated onto the above-mentioned plates at the same inoculum size (5 μL) and incubated at 30°C for 3 days. The presence of an orange-yellow halo around the colonies was observed, and the ratio of the halo diameter (D) to the colony diameter (d) was measured and calculated. The results showed that... YL01 and PC02 Both colonies exhibited distinct and large-diameter orange-yellow halos, indicating that both bacteria possessed strong siderophore-producing capabilities. Experimental observations and data measurements revealed that while both single bacteria produced orange-yellow halos, under the condition of inoculation with the same total viable bacterial count, the halo color depth and diffusion diameter at the sampling sites of the compound bacterial agent (MIX) were significantly superior to those of the single bacteria. Specifically, the D / d ratio of the compound bacterial agent MIX reached 3.12 ± 0.15, significantly higher than the arithmetic average level of halos produced by YL01 (1.56 ± 0.08) and PC02 (2.14 ± 0.12). This phenomenon indicates that the compound bacterial agent can more efficiently chelate insoluble iron in saline-alkali soil, significantly enhancing the nutrient activation capacity of the agent under extreme environments, and exhibiting a significant synergistic effect.

[0048] Example 6 YL01 , PC02 Test of the ability of compound microbial agents to produce ACC deaminase Quantitative determination was performed using the α-ketobutyric acid colorimetric method. As in Example 2, [the following text is incomplete and requires further context: "to determine..."] YL01 , PC02 Single-cell and compound inoculum MIX were induced and cultured at the same inoculum amount in DF liquid medium containing 3 mM ACC as the sole nitrogen source (the formulation of DF liquid medium was: 4.0 g KH2PO4, 6.0 g Na2HPO4, 0.2 g MgSO4·7H2O, 2.0 g glucose, 1.0 mg FeSO4·7H2O, 10 μg H3BO3, 10 μg MnSO4·H2O, 70 μg ZnSO4·7H2O, 50 μg CuSO4·5H2O, 10 μg MoO3, pH 7.2 per liter). The medium was autoclaved at 121℃ for 20 min, cooled, and aseptically added with filtered and sterilized ACC solution to a final concentration of 3 mM. Cells were lysed to extract the enzyme solution, which was then reacted with the ACC substrate. The absorbance of the generated α-ketobutyric acid at 540 nm was measured. The results are shown in Table 3. YL01 and PC02 Both possess certain ACC deaminase activity. According to the compounding ratio ( YL01 : PC02 Based on the calculation of α-ketobutyrate = 2:1, its arithmetic mean theoretical activity should be 25.82 μmol mg. -1 protein h -1The experimentally measured enzyme activity of the compound bacterial agent (MIX) was as high as 68.46 μmol α-ketobutyrate mg. -1 protein h -1 This is 2.65 times the theoretical value. This significant increase in activity indicates that the compound microbial agent can more effectively degrade ACC in plants, thereby mitigating ethylene toxicity caused by salt and alkali stress on crop growth, and has a significant synergistic effect.

[0049] Table 3. Determination of ACC deaminase activity in strains

[0050] Example 7 YL01 , PC02 Phosphate-solubilizing ability test of compound microbial agents The PKO liquid culture method was used. As in Example 2, [the following was done / contained / etc.]. YL01 , PC02 Single-strain and compound inoculum MIX were inoculated at the same inoculum amount into inorganic phosphorus liquid medium with calcium phosphate as the sole phosphorus source (the PKO liquid medium formula used was: 10.0 g glucose, 5.0 g Ca3(PO4)2, 0.5 g (NH4)2SO4, 0.2 g NaCl, 0.1 g MgSO4·7H2O, 0.2 g KCl, 0.5 g yeast extract, 0.002 g FeSO4·7H2O, 0.002 g MnSO4·H2O, pH 7.0 per liter), and cultured at 30℃ and 180 r / min for 7 days. After centrifugation, the available phosphorus content in the supernatant was determined using the molybdenum antimony colorimetric method. A control medium without inoculation was also included. The results are shown in Table 4. Both single strains were able to dissolve inorganic phosphorus. Based on the performance of the single strains, the theoretical expected value of phosphorus dissolution in the compound system was only 24.5 mg / L. However, the measured phosphorus solubility of the compound bacterial agent (MIX) reached 65.6 mg / L, which is 2.68 times the theoretical expected value. This fully demonstrates that the two strains complement and enhance each other in terms of metabolic acid production and the types of organic acids, exhibiting a synergistic phosphorus solubility effect.

[0051] Table 4. Determination of Phosphate Solubility of Strains

[0052] Example 8 YL01 , PC02 Nitrogen fixation capacity test of compound microbial agents The nitrogen-fixing potential of the strain was verified using the Ashby agar plate growth method. The Ashby agar medium formulation was: mannitol 10.0 g, dipotassium hydrogen phosphate 0.2 g, magnesium sulfate 0.2 g, sodium chloride 0.2 g, calcium sulfate 0.1 g, calcium carbonate 5.0 g, agar powder 15.0 g, distilled water 1000 mL, pH 7.0–7.2. This medium contains no combined nitrogen source; the microorganisms capable of growth must have the ability to utilize atmospheric nitrogen (N2). As in Example 2, the activated... YL01 , PC02 Single-strain and compound bacterial agent MIX were prepared into bacterial suspensions of the same concentration (OD600≈1.0) using sterile physiological saline. Using the dilution-spreading method, each bacterial suspension and its 10-fold dilution were spread onto Assoubai nitrogen-free agar plates. Simultaneously, plates inoculated with an equal volume of sterile physiological saline were set up as negative controls. All plates were placed in a 30℃ incubator and inverted for 7–14 days, with colony growth observed and recorded periodically.

[0053] After 7 days of culture, the results are as follows Figure 3 No colonies grew on the negative control plate. YL01 On both plates treated with the compound microbial agent (MIX), distinct and uniformly distributed single colonies were observed, indicating that... YL01 The microorganisms and their associated complexes can grow independently without any exogenous nitrogen, demonstrating significant nitrogen fixation potential. The colony morphology is consistent with that on nitrogen-containing media, proving that the nitrogen required for their growth originates from the biological fixation of atmospheric nitrogen. PC02 After 14 days of cultivation, single-strain treatments showed only a few weak colonies on plates, indicating weak or absent nitrogen-fixing ability. The compound microbial agent (MIX) exhibited significantly better colony counts and growth vigor than [previous treatments]. YL01 Single-strain treatment suggests that in a co-culture system, PC02 It may indirectly support or enhance growth by providing other growth factors or improving the microenvironment. YL01 The nitrogen fixation metabolism activity further demonstrates the synergistic effect among strains.

[0054] This embodiment uses the classic nitrogen-free culture method of Assumption. YL01 The biological nitrogen fixation ability of the strain has been preliminarily demonstrated, and this ability is comparable to... PC02 The compound formulation further enhances its effectiveness. This provides crucial theoretical and experimental evidence that this compound microbial agent can provide nitrogen nutrition for plants in saline-alkali land and reduce dependence on chemical nitrogen fertilizers.

[0055] Example 9 YL01 , PC02 EPS production capacity test of compound microbial inoculants Extracellular polysaccharides (EPS) were extracted and quantified using ethanol precipitation. As in Example 2, [the following text is incomplete and requires further context: "to extract and quantify the extracellular polysaccharides (EPS) using ethanol precipitation."] YL01 , PC02 Single-strain and compound inoculum MIX were inoculated into LB liquid medium (containing 3% NaCl) at the same inoculum amount and cultured to the stationary phase. The supernatant was collected by centrifugation, and three volumes of pre-cooled anhydrous ethanol were added. The mixture was incubated overnight at 4°C to precipitate EPS. The precipitate was dried and weighed. Results showed that... PC02 The EPS production capacity (specifically, the output was 280.5 ± 8.5 mg / L) was significantly higher than that of other products. YL01 (120.2±5.2 mg / L). The EPS yield of the mixed culture (MIX) reached 350.8±12.4 mg / L, which was significantly higher than the arithmetic mean of the yields of the two single strains (200.35 mg / L), indicating that the combination of the two strains may have promoted the biosynthesis of EPS. EPS helps to improve soil aggregate structure and water retention.

[0056] Example 10: Pot test of the effect of compound microbial agent application To verify the effects of applying compound microbial agents alone and in combination with Reed (Phragmites australis) Arundo donax L. The effects of combined application are shown in Table 5 below: Table 5 Treatment Groups

[0057] Each treatment group was configured with 3 replicates. The soil used in the experiment was taken from a typical coastal saline-alkali land (initial pH 8.0). The compound microbial agent was prepared according to the method in Example 2. All treatment groups were applied once via root drenching when transplanting *Arundo donax* seedlings to ensure a consistent total viable count of 2 × 10⁶ bacteria per pot. 9 CFU. Potted plants are placed in a greenhouse and managed under standard conditions, with a cultivation cycle of 90 days.

[0058] Plant growth index determination: After cultivation, all Arundo donax plants were harvested (no plant data were available for the CK and MIX groups). The following indices were measured and calculated: Plant height: The vertical height from the base of the stem to the tip of the highest leaf.

[0059] Root length: Measure the length of the longest root after washing.

[0060] Biomass: The plant was divided into aboveground parts and root system, and the fresh weight of each was measured. Then, the plant was blanched at 105℃ for 30 minutes, and then dried at 65℃ to constant weight, and the dry weight was measured.

[0061] The results are shown in Table 6. Compared with the single-planting of Arundo donax (AD group), the combined application of compound microbial agent (AD+MIX group) significantly promoted the growth of Arundo donax. The plant height, root length, aboveground dry weight, and root dry weight of Arundo donax in the AD+MIX group increased by 42.1%, 35.7%, 86.5%, and 74.2%, respectively, compared with the AD group.p <0.01). In the MIX group, which only applied the inoculant but did not plant any plants, a small amount of microbial film formed on the surface of the soil in the pots, but no plant indicators were observed.

[0062] Table 6 Effects of different treatments on growth indicators of Arundo donax

[0063] Soil ion content determination: Na in soil + K + Ca 2+ and Mg 2+ The concentrations of Na, K, Ca, and Mg in the digestion solution were determined by inductively coupled plasma optical emission spectrometry (ICP-OES). Soil samples were dried to constant weight at 60°C, finely ground, and approximately 25 mg of sample was digested using a microwave digestion system with 4 mL of concentrated HNO3 and 1 mL of H2O2 until the solution was clear. After cooling, the digest was filtered through a 0.45 μm membrane and diluted to 25 mL with 4% (v / v) HNO3, then stored at 4°C until analysis. The elemental concentrations of Na, K, Ca, and Mg in the digest were determined by ICP-OES according to the previously described procedure, as shown in Table 7.

[0064] The results are shown in Table 7. Compared with planting Reed alone (AD group), the combined application of compound microbial agent (AD+MIX group) significantly improved the ionic environment of saline-alkali soil. The soil Na+ of the AD+MIX group... + The content was significantly reduced by 36.9% compared to the AD group, demonstrating extremely strong desalination capacity; while the soil nutrient cation K + Ca 2+ and Mg 2+ The levels of these substances were significantly higher than those in the AD group by 93.7%, 39.4%, and 34.7%, respectively (p<0.01).

[0065] Table 7 Effects of different treatments on soil ion content

[0066] The compound microbial agent (MIX) of this invention has the ability to improve the ionic environment of saline-alkali soil and can significantly reduce the sodium ion concentration in the soil. + ) content and increase potassium ion (K) + At the same level, the combined application of the two (AD+MIX) showed a true synergistic effect: at the plant level, the biomass and root development of the AD group were significantly higher than those of the AD group. Furthermore, under the condition of consistent total viable bacterial count, the combined application of AD and MIX demonstrated a genuine synergistic effect: at the plant level, the biomass and root development of the AD group were significantly higher than those of the MIX group. YL01 and PC02The arithmetic mean of individual bacteria was used; at the soil level, the combined treatment was also more effective than the arithmetic mean of individual bacteria in removing sodium ions and maintaining and improving nutrients such as potassium. This fully demonstrates that constructing a "functional microorganism-suitable plant" synergistic system is an effective strategy for achieving rapid, efficient, and sustainable ecological restoration and productivity improvement in saline-alkali land.

Claims

1. A compound microbial agent, characterized in that, Contains Fritillaria cirrhosa (Indo-Pacific Frylandia diffusa) Vreelandella indica ) and salt-tolerant zoococci ( Planococcus halotolerans The active ingredient is a complex of microorganisms composed of ( ) and ( ); the ratio of viable counts of *Frederickia natans* to halophilic zoococci is (1.5–2.5):

1.

2. The composite microbial agent according to claim 1, characterized in that, The ratio of viable counts of *Frederickia natans* to *Zoococcus salina* is (1.8–2.2):1; preferably 2:

1.

3. The compound microbial agent according to claim 1, characterized in that, The total effective viable bacteria count of the compound microbial agent is not less than 1×10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL; preferably not less than 1×10⁻⁶ 9 CFU / g or 1×10 9 CFU / mL.

4. The method for preparing the composite microbial agent according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Activate and expand the culture of *Frederickia natans* and *Halococcus salina* to obtain bacterial solutions; S2. Mix the two bacterial solutions obtained in step S1 according to the ratio of the corresponding number of viable bacteria to obtain the compound microbial agent.

5. The application of the composite microbial agent according to any one of claims 1 to 3 in the remediation and / or improvement of saline-alkali soil and the enhancement of plant salt tolerance.

6. The application of the composite microbial agent described in any one of claims 1 to 3 in combination with salt-tolerant plants in the construction of a synergistic remediation system for saline-alkali land.

7. A synergistic remediation system for saline-alkali land, characterized in that, It is composed of the composite microbial agent described in any one of claims 1 to 3 and salt-tolerant plants.

8. The application of the saline-alkali land remediation system of claim 8 in the remediation and improvement of saline-alkali soil and / or the enhancement of plant salt tolerance.

9. A method for ecological restoration of saline-alkali land and / or improving plant salt tolerance, characterized in that, For applying an effective amount of the compound microbial agent according to any one of claims 1 to 3 to saline-alkali land.

10. The method according to claim 9, characterized in that, To cultivate salt-tolerant plants in saline-alkali land, and to apply an effective amount of the compound microbial agent described in any one of claims 1 to 3 to the saline-alkali land.

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