Rapid restoration method for degraded grassland based on microbial agent

Abstract

The invention discloses a rapid restoration method for degraded grassland based on a microbial agent. The microbial agent is formed by compounding 15-30 parts of a nitrogen-fixing microbial agent, 10-20 parts of a phosphate-solubilizing microbial agent, 8-15 parts of a plant growth-promoting microbial agent, 25-40 parts of a humic acid carrier, 3-8 parts of an embedding agent sodium alginate and 5-12 parts of a nutritional agent molasses. Wherein the nitrogen-fixing bacteria are azotobacter chroococcum and alfalfa rhizobium (the viable count is greater than or equal to 5 * 10 < 9 > CFU / g), the phosphate solubilizing bacteria are bacillus megatherium (the viable count is greater than or equal to 1 * 10 < 10 > CFU / g), and the growth-promoting bacteria are bacillus subtilis and pseudomonas fluorescens (the viable count is greater than or equal to 2 * 10 < 10 > CFU / g). The microbial inoculum is subjected to activation, carrier adsorption, sodium alginate embedding granulation and CaCl2 cross-linking processes to be prepared into slow-release particles of 1-3 mm. 200-250 kg of organic fertilizer is applied to each hectare in a mild area, 300-400 kg of organic fertilizer is applied to each hectare in a moderate and severe area, and leymus chinensis and wheatgrass are sown in a mixed mode. After 90 days of application, the vegetation coverage is improved by 40% or above, the soil organic matter is increased by 25-40%, the microbial diversity index is larger than or equal to 3.5, and the comprehensive remediation cost is reduced by 53%.

Description

Technical Field

[0001] This invention belongs to the field of ecological restoration technology for degraded grasslands, specifically relating to a multifunctional composite microbial agent and its preparation method, as well as a rapid grassland restoration method based on graded application according to the degree of degradation. Background Technology

[0002] The degradation of grassland ecosystems is triggering severe ecological chain reactions, manifested in a continuous decline in vegetation cover, accelerated decomposition of topsoil organic matter, a sharp reduction in microbial diversity, and the expansion of desertification. Current mainstream technologies for grassland restoration face three major bottlenecks:

[0003] 1) Limitations of physical measures: Although fencing can reduce human interference, it does not activate soil biological activity, and the natural recovery cycle often exceeds 5 years; loosening the soil and reseeding can improve the seedling rate in the short term, but the vegetation sustainability is poor because the soil nutrient cycle is not rebuilt.

[0004] 2) Side effects of chemical methods: Relying on chemical fertilizers to stimulate pasture growth in the short term can lead to soil compaction, pH imbalance and simplification of microbial communities, which in the long term exacerbates the risk of ecosystem degradation.

[0005] 3) Deficiencies of traditional bioremediation: Single-function bacterial agents (such as nitrogen-fixing bacteria) have a survival rate of less than 30% under drought stress and cannot simultaneously solve multi-dimensional problems such as phosphorus fixation and root development.

[0006] Existing microbial remediation technologies face systemic challenges:

[0007] 1. Functional synergy barrier: Simple mixing of multiple bacterial species can easily lead to niche competition, resulting in reduced colonization efficiency of functional strains (e.g., mismatch in growth rates between nitrogen-fixing bacteria and phosphate-solubilizing bacteria).

[0008] 2. Insufficient environmental tolerance: Conventional carriers (such as peat moss) experience a sharp drop in water retention of more than 50% under strong ultraviolet and drought conditions, and encapsulation materials (such as starch-based materials) are prone to disintegration when exposed to extreme temperatures, causing premature release and inactivation of the microbial community.

[0009] 3. Economic cost constraints: Highly active engineered microbial agents require low-temperature storage and transportation, and are applied frequently in the field (3-4 times per year), resulting in remediation costs exceeding 3,000 yuan / hectare, making it difficult to promote on a large scale.

[0010] The deeper technical contradiction lies in:

[0011] 1) Most compound microbial agents neglect the synergistic design of carrier-microbial community-nutrients, for example:

[0012] The lack of optimization of carrier particle size and pore structure limits the adsorption density of bacterial cells.

[0013] 2) The encapsulation material lacks a temperature-sensitive response mechanism, making it impossible to achieve intelligent slow release under moisture stress;

[0014] 3) Lack of endogenous energy supply layer (such as molasses) causes the proliferation of microbial community to lag behind the repair window period.

[0015] The restoration process is not coupled with the degradation grading, microbial agent adaptation, and native species reconstruction technology chain, resulting in resource waste in mildly degraded areas and insufficient restoration in severely degraded areas.

[0016] Therefore, there is an urgent need for innovative solutions, such as developing multifunctional microbial community compound systems, constructing structured carrier systems, and establishing hierarchical remediation application paradigms. Summary of the Invention

[0017] To address the above problems, this invention discloses a rapid restoration method for degraded grasslands based on microbial agents.

[0018] The objective of this invention is achieved through the following technical solution.

[0019] A microbial inoculant for the restoration of degraded grasslands, comprising, by weight:

[0020] 15-30 parts of nitrogen-fixing bacteria agent;

[0021] 10-20 parts of phosphate-solubilizing bacteria agent;

[0022] 8-15 parts of plant rhizosphere growth-promoting bacteria agent;

[0023] 25-40 parts of humic acid carrier;

[0024] Encapsulation agent: 3-8 parts sodium alginate;

[0025] Nutritional supplement: 5-12 parts molasses;

[0026] The nitrogen-fixing bacterial agent is a compound of *Azotobacter chroococcum* and *Sinorhizobium meliloti*, with a viable count ≥ 5 × 10⁻⁶. 9 CFU / g;

[0027] The phosphate-solubilizing agent is Bacillus megaterium, with a viable count of...

[0028] ≥1×10 10 CFU / g;

[0029] The plant rhizosphere growth-promoting bacterial agent is a compound of Bacillus subtilis and Pseudomonas fluorescens, with a viable count ≥2×10⁻⁶. 10 CFU / g.

[0030] Furthermore, in the aforementioned microbial inoculant, the humic acid carrier is a weathered coal extract with a particle size ≤150 mesh (passable through a 150 mesh sieve), an organic matter content ≥60%, and a pH of 6.5-7.5.

[0031] Furthermore, the aforementioned microbial inoculant, specifically the molasses nutrient, is sugarcane molasses with a total sugar content ≥45% and an ash content ≤15%.

[0032] This invention also discloses a method for preparing the above-mentioned microbial inoculant, comprising the following steps:

[0033] (1) Activation of strains: Inoculate nitrogen-fixing bacteria, phosphate-solubilizing bacteria and plant rhizosphere growth-promoting bacteria into liquid culture medium and culture at 28-32℃ with shaking for 24-48 hours;

[0034] (2) Carrier mixing: Mix the humic acid carrier, molasses nutrient solution and bacterial solution from step (1), and stir at 40-50 r / min for 30 minutes.

[0035] (3) Encapsulation and granulation: Sodium alginate is added as an encapsulating agent, stirred evenly for encapsulation, and then formed into granules with a diameter of 1-3 mm by an extrusion granulator.

[0036] (4) Curing and drying: Crosslink with 2% CaCl2 solution for 10 minutes, and dry in a ventilated environment at 40°C until the moisture content is ≤8%.

[0037] Furthermore, in the above preparation method, the liquid culture medium in step (1) is beef extract peptone culture medium, with a shaking speed of 150-180 r / min.

[0038] Furthermore, in the above preparation method, the extrusion pressure of the extrusion granulator in step (3) is 0.3-0.5 MPa.

[0039] This invention also discloses a method for rapid restoration of degraded grasslands, using any of the above-mentioned microbial agents, comprising the following steps:

[0040] (a) Surface treatment: Shallow tillage of degraded grassland to a depth of 5-10 cm and remove weeds;

[0041] (b) Application of microbial agents: Spread microbial agent granules at a rate of 200-400 kg / hectare;

[0042] (c) Covering with soil and then irrigating: Cover with 1-2cm of soil and then spray irrigation until the soil moisture is 40-60%;

[0043] (d) Reseeding with grass seeds: 7 days later, mix in drought-resistant grass seeds.

[0044] Furthermore, for the above-mentioned restoration methods, in areas with mild degradation (vegetation coverage of 30-50%): the dosage of microbial agent is 200-250 kg / hectare;

[0045] In areas with moderate to severe degradation (vegetation coverage <30%): apply 300-400 kg / hectare of microbial agent and add 500 kg / hectare of well-rotted sheep manure organic fertilizer.

[0046] Furthermore, in the above-mentioned restoration method, the drought-resistant grass species mentioned in step (d) are Leymus chinensis and Agropyron cristatum, with a sowing density of 15-20 kg / ha and a mixed planting ratio of 1:1.

[0047] This invention also discloses the application of the above-mentioned microbial agents in the restoration of degraded grasslands.

[0048] After 90 days of restoration, vegetation coverage increased by more than 40%;

[0049] Soil organic matter content increased by 25-40%, and the Shannon diversity index of microorganisms was ≥3.5.

[0050] Compared with existing technologies, the present invention has the following advantages and beneficial effects:

[0051] This invention overcomes the bottlenecks of traditional grassland restoration, such as fragmented microbial community functions, insufficient environmental tolerance, and low restoration efficiency, by innovatively constructing a "multifunctional microbial community-structured carrier-tiered restoration" technical system. Specifically, it manifests as follows:

[0052] 1. Synergistic microbial community design: nitrogen-fixing bacteria (Azotobacter chrysotile and Rhizobium alfalfa), phosphate-solubilizing bacteria (Bacillus megaterium), and rhizosphere growth-promoting bacteria (Bacillus subtilis and Pseudomonas fluorescens) form a functionally complementary microecological cycle chain, which simultaneously activates soil nitrogen and phosphorus transformation and plant stress resistance mechanisms, significantly improving nutrient utilization efficiency;

[0053] 2. Intelligent sustained-release carrier system: Humic acid carrier (high organic matter, near-neutral pH) provides a stable microbial habitat, coupled with sodium alginate encapsulation layer (Ca... 2+ Cross-linked networks enable water stress-responsive slow release, allowing the bacterial agent to maintain high activity for a long time under drought and strong ultraviolet conditions;

[0054] 3. Precise and appropriate restoration: Based on the graded application of microbial agents according to the degree of grassland degradation (vegetation coverage), and in conjunction with the reconstruction of native grass species, vegetation coverage can be increased by more than 40% within 90 days, and the biomass restoration efficiency in moderate to severe areas reaches 320g / m². 2 ;

[0055] 4. Sustainable application and economic efficiency: By utilizing weathered coal humic acid and molasses industrial by-products to reduce raw material costs, and combined with low-energy granulation technology, the overall restoration cost is reduced by 53% compared to commercially available microbial agents, and the investment payback period is shortened to 1.2 years, providing a scalable solution for large-scale grassland ecological management. Attached Figure Description

[0056] Figure 1 For the environmental tolerance test of the microbial agent (15-day survival rate);

[0057] Figure 2 For the environmental tolerance test of the microbial agent (30-day survival rate);

[0058] Figure 3 For the environmental tolerance test of the microbial agent (45-day survival rate);

[0059] Figure 4 The increase in available nitrogen in the comparison of soil nutrient activation capacity;

[0060] Figure 5 This is a comparison of microbial biomass carbon (mg / kg) in the comparison of soil nutrient activation capacity. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0062] Example 1

[0063] 1. Microbial inoculant formula (parts by weight):

[0064]

[0065] 2. Preparation method:

[0066] Strain activation: Each bacterial agent was inoculated into beef extract peptone medium and cultured at 30°C and 170 r / min for 36 hours with shaking.

[0067] Carrier mixing: Mix humic acid carrier, molasses and bacterial solution, and stir at 45 r / min for 30 minutes;

[0068] Encapsulation and granulation: Sodium alginate is added for encapsulation, and the mixture is granulated into 2mm diameter particles using an extrusion granulator (pressure 0.4MPa);

[0069] Curing and drying: Crosslink with 2% CaCl2 solution for 10 minutes (20℃), then air dry at 40℃ until the moisture content is 7%.

[0070] 3. Restoration methods (slightly degraded areas, vegetation coverage 45%):

[0071] (a) Shallow tillage to 8cm to remove weeds;

[0072] (b) Apply the microbial agent at a rate of 220 kg / ha by broadcasting;

[0073] (c) Cover with 1.5cm of soil and then spray water until the soil moisture reaches 50%;

[0074] (d) Seven days later, mix sheepgrass: icegrass (1:1) at a seeding rate of 18 kg / ha.

[0075] Example 2

[0076] 1. Microbial inoculant formula (parts by weight):

[0077]

[0078]

[0079] 2. Preparation method: Same as Example 1, with the following adjustments:

[0080] The extrusion granulation pressure is 0.45 MPa, and the particle diameter is 2.5 mm.

[0081] The moisture content after drying is 6%.

[0082] 3. Restoration methods (moderately degraded areas, vegetation coverage 25%):

[0083] Inoculant application rate: 350 kg / ha;

[0084] Apply an additional 500 kg / ha of well-rotted sheep manure organic fertilizer;

[0085] The rest is the same as in Example 1.

[0086] Example 3

[0087] Microbial inoculant formulation (parts by weight):

[0088] Nitrogen-fixing bacteria (Azotobacter chrysoprase: Alfalfa rhizobia = 2:1, viable count 9 × 10⁻⁶) 9 28 samples (CFU / g)

[0089] Phosphate-solubilizing bacteria (Bacillus megaterium, viable count 1.8 × 10⁻⁶) 10 18 servings of CFU / g

[0090] Plant rhizosphere growth-promoting bacteria (Bacillus subtilis: Pseudomonas fluorescens = 1:2, viable count 3.5 × 10⁻⁶) 10 14 copies of CFU / g

[0091] 38 portions of humic acid carrier

[0092] 7 parts sodium alginate

[0093] 11 portions of sugarcane molasses

[0094] Preparation method: Same as Example 1, with the following adjustments:

[0095] The extrusion granulation pressure is 0.35 MPa, and the particle diameter is 1.8 mm.

[0096] The moisture content after drying is 5%.

[0097] Restoration methods (severely degraded areas, vegetation coverage 15%):

[0098] Inoculant application rate: 380 kg / ha;

[0099] Apply an additional 500 kg / ha of well-rotted sheep manure organic fertilizer;

[0100] The amount of grass seed to be reseeded is 20 kg / hectare.

[0101] Comparative Example 1

[0102] Sodium alginate encapsulation agent missing:

[0103] Formulation: Same as Example 2, but sodium alginate encapsulating agent is removed;

[0104] Preparation: Step (3) Direct granulation (without encapsulation and crosslinking);

[0105] Repair method: Same as in Example 2.

[0106] Comparative Example 2

[0107] Lacking humic acid carrier:

[0108] Formula: Same as Example 2, except that the humic acid carrier is replaced with an equal amount of ordinary peat moss (40% organic matter, pH 5.8);

[0109] Preparation: Same as in Example 2;

[0110] Repair method: Same as in Example 2.

[0111] Comparative Example 3

[0112] Single-strain alternatives to compound bacterial agents:

[0113] formula:

[0114] Only Bacillus megaterium (1.5 × 10¹ viable cells) was retained. 0 25 portions of CFU / g;

[0115] The remaining components are the same as in Example 2;

[0116] Preparation and repair methods: Same as in Example 2.

[0117] Comparative Example 4

[0118] Ungraded repair (uniform dosage):

[0119] Inoculant formulation: Same as in Example 2;

[0120] Repair method:

[0121] The uniform application rate for moderately to severely degraded areas is 250 kg / ha (without additional organic fertilizer).

[0122] The rest is the same as in Example 2.

[0123] Comparative Example 5

[0124] Simplified preparation process:

[0125] Formula: Same as Example 2;

[0126] preparation:

[0127] Delete the CaCl2 crosslinking step;

[0128] Dry directly at 60℃ until the moisture content is 8%;

[0129] Repair method: Same as in Example 2.

[0130] Test Example 1

[0131] Environmental tolerance test of microbial agents

[0132] Objective: To verify the protective effect of sodium alginate encapsulation system on bacterial communities under drought-strong ultraviolet stress.

[0133] method:

[0134] Accelerated coercive treatment:

[0135] Take the bacterial agent granules from each example / comparative example and place them in an artificial climate chamber (temperature 40℃±2, UV intensity 0.8W / m²). 2 (humidity 15%).

[0136] Processing time: 0, 15, 30, 45 days.

[0137] Survival rate detection:

[0138] After grinding, the particles were diluted and spread onto beef extract peptone plates. The plates were incubated at 30°C for 48 hours, and the viable bacteria count (CFU / g) was calculated. The survival rate was calculated as (CFU after stress / initial CFU) × 100%.

[0139] The results are shown in Table 1 and... Figure 1-3 .

[0140] Table 1: Environmental Tolerance Test of Microbial Agent

[0141] Group 15-day survival rate 30-day survival rate 45-day survival rate Example 1 92％ 85％ 78％ Example 2 90％ 82％ 75％ Comparative Example 1 48％ 22％ <5％ Comparative Example 5 65％ 38％ 15％

[0142] The following conclusions can be drawn from the data in Table 1: the survival rate of the encapsulated crosslinking system (example) is more than 15 times that of Comparative Example 1 (45 days), proving that sodium alginate-Ca 2+ The network effectively blocks ultraviolet rays and moisture loss.

[0143] Test Example 2

[0144] Comparison of soil nutrient activation capacity

[0145] Objective: To evaluate the synergistic effect of compound microbial agents on the improvement of degraded soil.

[0146] method:

[0147] Microcosm Experiment:

[0148] Soil samples were collected from degraded grassland (0.8% organic matter, 3.2 mg / kg available phosphorus).

[0149] Add bacterial inoculant (1g / kg soil) and incubate at 25℃ for 30 days.

[0150] Testing indicators:

[0151] Fast-acting nitrogen: Indophenol blue colorimetric method.

[0152] Quick-acting phosphorus: molybdenum antimony resists spectrophotometry.

[0153] Microbial biomass carbon: chloroform fumigation-K2SO4 extraction method.

[0154] The results are shown in Table 2 and Figure 4 and Figure 5 .

[0155] Table 2: Comparison of Soil Nutrient Activation Capacity

[0156] Group Increase in available nitrogen Increase in available phosphorus Microbial biomass carbon (mg / kg) Example 2 +186％* +240％* 352±21 Comparative Example 2 +92％ +105％ 198±17 Comparative Example 3 +67％ +310％ 185±15

[0157] (*P<0.01 vs. comparative example; comparative example 3 showed a high increase in phosphorus but weak nitrogen activation, indicating functional imbalance)

[0158] Table 2 shows that the combination of nitrogen-fixing bacteria and phosphate-solubilizing bacteria achieves simultaneous activation of nitrogen and phosphorus (the nitrogen-phosphorus amplification ratio in Example 2 is >0.77), while the single-strain system is unbalanced (the nitrogen-phosphorus ratio in Comparative Example 3 is only 0.22).

[0159] Test Example 3

[0160] Dynamic monitoring of vegetation restoration

[0161] Objective: To quantify the restoration efficiency of graded restoration strategies on degraded grasslands.

[0162] method:

[0163] Field test area:

[0164] Three hectares each were set up for mild (42% coverage) and severe (18% coverage) degradation plots.

[0165] The repair shall be carried out according to the method of claims 7-9 (3 replicates per group).

[0166] Monitoring technology:

[0167] Vegetation cover: UAV multispectral imagery (once a month, NDVI calculated using ENVI 5.3).

[0168] Biomass: quadrat harvesting method (0.5 × 0.5 m2).

[0169] Data summary (90 days), results are shown in Table 3.

[0170] Table 3. Dynamic monitoring results of vegetation restoration

[0171] Degradation level Processing group Increased coverage Aboveground biomass (g / m2) Mild Example 1 +48％ 286±24 Mild Comparative Example 4 +29％ 178±19 Severe Example 3 +75％ 320±28 Severe Comparative Example 4 +41％ 205±22

[0172] As can be seen from the results in Table 3, the biomass production of graded restoration (Example 3) is more than 60% higher than that of Comparative Example 4, proving the necessity of graded adaptation for degradation.

[0173] Test Example 4

[0174] Verification of the rhizosphere colonization ability of functional bacteria

[0175] Objective: To evaluate the survival and proliferation capacity of functional bacteria in the rhizosphere of grassland plants.

[0176] method:

[0177] Potted plant simulation system:

[0178] Device: PVC pipe (30cm in diameter, 50cm in height) filled with degraded grassland soil (0.9% organic matter).

[0179] Planting: 10 seedlings of Leymus chinensis per pot, apply microbial agent at a rate of 200 kg / ha.

[0180] Colonization detection (routine culture method):

[0181] Time points: 15 days, 30 days, and 60 days after application.

[0182] Sampling: Take rhizosphere soil (within 2 mm of the root surface), add 10 g of sample to 90 mL of sterile water and shake to dilute.

[0183] Selective culture medium count:

[0184] Nitrogen-fixing bacteria: Assumption nitrogen-free medium (cultured at 30℃ for 5 days).

[0185] Phosphate-solubilizing bacteria: Monkina organic phosphorus medium (clear zone method).

[0186] Bacillus subtilis: beef extract peptone + penicillin (5U / mL).

[0187] Calculate the colony density:

[0188] Results expressed as: CFU / g rhizosphere soil (mean of three replicates ± SD).

[0189] The results are shown in Table 4.

[0190] Table 4. Results of verification of the rhizosphere colonization ability of functional bacteria

[0191]

[0192] Table 4 leads to the following conclusions: The colonization density of the encapsulated bacterial agent (Example 2) after 60 days was more than 400 times that of Comparative Example 1. All functional bacteria in the compound bacterial agent colonized stably (Comparative Example 3 only detected phosphate-solubilizing bacteria).

[0193] Test Example 5

[0194] Comprehensive evaluation of soil biological activity

[0195] Objective: To verify the effectiveness of soil microecological restoration using basic indicators.

[0196] method:

[0197] Sample processing:

[0198] After 90 days of remediation, take soil samples from 0-20cm and sieve them through a 2mm sieve.

[0199] Routine testing items (national standard methods):

[0200] Total microbial count: dilution plate count (beef extract peptone medium, incubated at 30°C for 3 days).

[0201] Soil respiration intensity: alkaline absorption method (NaOH titration, unit: mg CO2 / kg·d).

[0202] Functional enzyme activity:

[0203] Dehydrogenase: TTC reduction method (λ=485nm).

[0204] Urease: Sodium phenolate colorimetric method.

[0205] Data comparison: 3 replicate plots in each group.

[0206] The results are shown in Table 5.

[0207] Table 5: Comprehensive Evaluation Results of Soil Biological Activity

[0208]

[0209] (*P<0.01 vs before repair; dehydrogenases reflect microbial oxidative activity, and ureases indicate nitrogen cycling capacity)

[0210] in conclusion:

[0211] In Example 2, the total amount of microorganisms increased by 550%, and the dehydrogenase activity reached 2.8 times that before the repair, proving that the microbial agent effectively activated the biological function of the soil.

[0212] Test Example 6

[0213] Economic analysis

[0214] Objective: To verify the cost advantages of this invention in large-scale application.

[0215] method:

[0216] Cost accounting (unit: yuan / hectare):

[0217] Production cost of microbial agents: microbial culture + carrier + encapsulation material + energy consumption.

[0218] Total cost of restoration: fungicide + labor + machinery + grass seed.

[0219] Benefit assessment:

[0220] Increased grass production = (Post-restoration yield - Local average yield) × Forage price.

[0221] Investment recovery period = total cost / annual increase in production benefits.

[0222] The results are shown in Table 6.

[0223] Table 6 Results of Economic Analysis

[0224] Technical solution Repair costs Annual grass yield (kg / ha) Payback period (years) Example 2 2850 4200 1.2 Traditional fertilizers 3200 3800 2.5 Commercially available bacterial agents 6100 3500 >5

[0225] Conclusion: This invention, through localization of the carrier (humic acid) and low-consumption process, reduces costs by 53% and shortens the payback period by 76% compared to commercially available microbial agents.

[0226] The advantages of this invention can be summarized from the above test examples as follows:

[0227] 1. Overcoming the bottleneck of synergistic function of gut microbiota

[0228] By scientifically combining nitrogen-fixing bacteria, phosphorus-solubilizing bacteria, and growth-promoting bacteria (Test Example 2), simultaneous activation of nitrogen and phosphorus (available nitrogen +186%, available phosphorus +240%) and synergistic root growth promotion are achieved, overcoming the problem of functional imbalance in single-species systems (comparative Example 3 has a nitrogen-phosphorus activation ratio of only 0.22).

[0229] 2. Innovative carrier-based sustained-release protection mechanism

[0230] The humic acid carrier (organic matter ≥60%, pH 6.5-7.5) provides a water-retaining and fertilizer-preserving microenvironment. Sodium alginate encapsulation (degree of substitution 0.8-1.2) forms a temperature-sensitive slow-release network through CaCl2 cross-linking, which enables the bacterial agent to survive for 45 days under drought-strong ultraviolet stress with a survival rate of >75% (Test Example 1), which is 15 times higher than that of the unencapsulated bacterial agent (Comparative Example 1).

[0231] 3. Significantly improves the efficiency of ecological restoration

[0232] The graded remediation strategy (right 8) adapts the dosage of microbial agent to different degrees of degradation. After 90 days, the vegetation coverage increased by 48-75% (test example 3), the aboveground biomass (320g / m2) increased by 56% compared with the uniform dosage (comparative example 4), and the soil dehydrogenase activity increased to 2.8 times that before remediation (test example 5).

[0233] 4. Advantages of low-cost, large-scale application

[0234] By utilizing weathered coal to extract humic acid and sugarcane molasses to reduce raw material costs, and combined with extrusion granulation technology, the production cost of microbial agents is ≤2850 yuan / hectare, with an investment payback period of 1.2 years (Test Example 6), which is 53% lower than the cost of commercially available microbial agents.

[0235] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, any changes and modifications made to the embodiments described herein based on the innovative concept of the present invention, or equivalent structural or procedural transformations made using the content of the present invention specification, that directly or indirectly apply the above technical solutions to other related technical fields, are all included within the scope of protection of the present invention patent.

Claims

1. A microbial inoculant for the restoration of degraded grasslands, characterized in that, Included by weight: 15-30 parts of nitrogen-fixing bacteria agent; 10-20 parts of phosphate-solubilizing bacteria agent; 8-15 parts of plant rhizosphere growth-promoting bacteria agent; 25-40 parts of humic acid carrier; Encapsulation agent: 3-8 parts sodium alginate; Nutritional supplement: 5-12 parts molasses; The nitrogen-fixing bacterial agent is a compound of *Azotobacter chroococcum* and *Sinorhizobium meliloti*, with a viable count ≥ 5 × 10⁻⁶. 9 CFU / g; The phosphate-solubilizing agent is Bacillus megaterium, with a viable count of... ≥1×10 10 CFU / g; The plant rhizosphere growth-promoting bacterial agent is a compound of Bacillus subtilis and Pseudomonas fluorescens, with a viable count ≥2×10⁻⁶. 10 CFU / g.

2. The microbial agent according to claim 1, characterized in that, The humic acid carrier is a weathered coal extract with a particle size ≤150 mesh, an organic matter content ≥60%, and a pH of 6.5-7.

5.

3. The microbial agent according to claim 1, characterized in that, The molasses is sugarcane molasses with a total sugar content of ≥45% and an ash content of ≤15%.

4. The method for preparing the microbial inoculant according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Activation of strains: Inoculate nitrogen-fixing bacteria, phosphate-solubilizing bacteria and plant rhizosphere growth-promoting bacteria into liquid culture medium and culture at 28-32℃ with shaking for 24-48 hours; (2) Carrier mixing: Mix the humic acid carrier, molasses nutrient solution and bacterial solution from step (1), and stir at 40-50 r / min for 30 minutes. (3) Encapsulation and granulation: Sodium alginate is added as an encapsulating agent for encapsulation, and the particles are formed into granules with a diameter of 1-3 mm by an extrusion granulator. (4) Curing and drying: Crosslink with 2% CaCl2 solution for 10 minutes, and dry in a ventilated environment at 40°C until the moisture content is ≤8%.

5. The preparation method according to claim 4, characterized in that, The liquid culture medium mentioned in step (1) is beef extract peptone medium, with a shaking speed of 150-180 r / min.

6. The preparation method according to claim 4, characterized in that, The extrusion pressure of the extrusion granulator in step (3) is 0.3-0.5 MPa.

7. A method for rapid restoration of degraded grasslands, characterized in that, The use of the microbial agent according to any one of claims 1-3 includes the following steps: (a) Surface treatment: Shallow tillage of degraded grassland to a depth of 5-10 cm and remove weeds; (b) Application of microbial agents: Spread microbial agent granules at a rate of 200-400 kg / hectare; (c) Covering with soil and then irrigating: Cover with 1-2cm of soil and then spray irrigation until the soil moisture is 40-60%; (d) Reseeding with grass seeds: 7 days later, mix in drought-resistant grass seeds.

8. The repair method according to claim 7, characterized in that, In mildly degraded areas with vegetation coverage of 30-50%, the application rate of microbial agent is 200-250 kg / hectare. In areas with moderate to severe degradation and vegetation coverage of <30%, the application rate of microbial agent is 300-400 kg / hectare, and an additional 500 kg / hectare of well-rotted sheep manure organic fertilizer is applied.

9. The repair method according to claim 7, characterized in that, The drought-resistant grass species mentioned in step (d) are Leymus chinensis and Agropyron cristatum, with a sowing density of 15-20 kg / ha and a mixed planting ratio of 1:

1.

10. The application of the microbial inoculant as described in any one of claims 1-3 in the restoration of degraded grasslands, characterized in that, After 90 days of restoration, vegetation coverage increased by more than 40%; Soil organic matter content increased by 25-40%, and the Shannon diversity index of microorganisms was ≥3.5.

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