Deteriorated grassland restoration microbial agent, granular restoration microbial agent and amendment and application thereof

Abstract

The present application relates to the prairie repair technical field, especially in kind of degraded prairie repair fungicide, granular repair fungicide and amendment and application thereof.The present application provides the degraded prairie repair fungicide by the preservation number of fluorescent pseudomonas T6 and the preservation number of the alfalfa sinorhizobium NFB5 of CGMCC NO.33618, and the degraded prairie repair fungicide provided by the present application is composed of the preservation number of fluorescent pseudomonas T6 and the preservation number of the alfalfa sinorhizobium NFB5 of CGMCC No.36876.The fluorescent pseudomonas T6 in the degraded prairie repair fungicide provided by the present application and the alfalfa sinorhizobium NFB5 two compound have the synergistic effect, and the repair effect to the degraded prairie is good, and the strain colonization rate is high.The present application relies on the native resources (sheep manure, peat soil, local microorganism and native grass species) of Inner Mongolia grassland, and gives consideration to ecological repair and livestock production, realizes the integration repair of "soil-microorganism-vegetation" three-dimensional synergy, and has important technical innovation value and practical application significance.

Description

Technical Field

[0001] This invention relates to the field of grassland restoration technology, and in particular to a degraded grassland restoration microbial agent, a particulate restoration microbial agent and a modifier, and their applications. Background Technology

[0002] Grassland ecosystems are an important component of my country's terrestrial ecosystems. my country's total grassland area is approximately 400 million hectares, accounting for 41.7% of the country's total land area, far exceeding the proportion of cultivated land and forests, making it the most widely distributed terrestrial ecosystem. However, in the past 30 years, due to the combined effects of long-term overgrazing, climate change, and limitations of traditional restoration techniques, my country's grasslands have exhibited a trend of "comprehensive and complex degradation," with the degradation area expanding year by year: In terms of community structure, the coverage of dominant grassland species has decreased from 70%–80% in 1990 to the current 30%–40%, while the proportion of miscellaneous grasses (such as *Euphorbia fischeriana* and *Oxytropis henryi*) has increased from 6% to 25%; in terms of soil quality, the available nitrogen content in the topsoil (0–20 cm) of grasslands is generally below 28 mg / kg, only 45%–55% of that in undegraded grasslands, and the soil organic matter content is also low. Carbon content dropped to 16-24 g / kg, and the nitrogen-phosphorus ratio generally deviated from the healthy value (10:1), with some areas even reaching 4:1, resulting in a 30% reduction in vegetation photosynthetic efficiency and a seedling survival rate of less than 25%. In terms of microbial function, the activity of core functional microbial communities in different types of grasslands declined by more than 55% compared to healthy grasslands. The weakened carbon metabolism capacity accelerated the decomposition rate of soil organic matter by 20%-30%, forming a vicious cycle of "decreased soil fertility - declining microbial function - degraded community structure," which seriously threatens the stability of grassland ecosystems and the sustainable development of regional animal husbandry.

[0003] Current grassland restoration technologies have significant limitations and are difficult to adapt to the overall ecological characteristics and management needs: First, single vegetation restoration technologies focus only on a single species or sowing process, without linking soil-microbe synergistic restoration, resulting in poor community stability after restoration and the inability to form a sustainable dominant community; Second, general microbial restoration schemes lack regional adaptability. Existing technologies mostly use exogenous strains, with a colonization rate of less than 22% in local grassland soils, poor symbiotic synergy with local vegetation, and a growth-promoting effect of only 25% to 30% of that of natural microbial communities in healthy grasslands, making it difficult to overcome the bottleneck of "microbial functional failure". Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a degraded grassland restoration microbial agent, a granular restoration microbial agent, and a conditioner, along with their applications. The degraded grassland restoration microbial agent provided by this invention solves the problems of limited restoration dimensions, poor post-restoration community stability, and mismatched microbial resources in existing technologies. It exhibits good restoration effects on degraded grasslands and a high bacterial colonization rate.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a degraded grassland restoration microbial agent, composed of *Pseudomonas fluorescens* (…). Pseudomonas fluorescens T6 and *Alfalfa rhizobium* ( Sinorhizobium meliloti The composition is NFB5; the preservation number of the fluorescent pseudomonad T6 is CGMCC No. 36876, and the preservation number of the alfalfa rhizobium NFB5 is CGMCC NO. 33618.

[0007] Preferably, the ratio of viable counts of the fluorescent pseudomonad T6 to the alfalfa rhizobium NFB5 is 1:1 to 2:1.

[0008] This invention provides a granular remediation microbial agent, comprising a carrier and the degraded grassland remediation microbial agent described in the above-mentioned technical solution; the carrier comprises straw powder, and the effective viable count of the granular remediation microbial agent is ≥1×10⁻⁶. 8 CFU / g.

[0009] Preferably, the particle size of the granular repair microbial agent is 4-6 mm and the moisture content is <10%; the particle size of the straw powder is 0.5-1.5 mm.

[0010] This invention provides a modifier, comprising: a granular modifier and the granular repair microbial agent described in the above technical solution; the granular modifier comprises the following components in parts by weight: 8-16 parts of well-rotted sheep manure, 1-2 parts of peat moss, and 0.5-1 parts of potassium dihydrogen phosphate.

[0011] Preferably, the mass ratio of the granular repair agent to the granular improver is 1:10 to 1:15; the particle size of the granular improver is 4 to 6 mm, and the water content is 10% to 12%.

[0012] This invention provides the application of the degraded grassland restoration microbial agent, the granular restoration microbial agent, or the modifier described in the above-mentioned technical solutions in grassland degradation restoration.

[0013] Preferably, the grassland is the grassland of Inner Mongolia.

[0014] This invention provides a grassland restoration method, comprising the following steps:

[0015] The improver described in the above technical solution is applied to the grassland to be improved, and a microbial activating solution is sprayed on the grassland to be improved 1-2 times every August; the microbial activating solution comprises the following components in parts by volume: 1-2 parts of humic acid solution and 1-2 parts of sucrose solution; the concentration of the humic acid solution is 3g / L, and the concentration of the sucrose solution is 1g / L.

[0016] Sow grass seeds on the grassland to be improved; the grass seeds include the following parts by weight: 4-5 parts of sheepgrass seeds, 2-3 parts of needlegrass seeds and 2-3 parts of alfalfa seeds;

[0017] During the restoration period from July to August of that year, light grazing was implemented during the rapid growth phase of the community, with a carrying capacity of 0.1–0.3 sheep units / hm². 2 Grazing is prohibited from September to May of the following year.

[0018] Preferably, the application rate of the particulate amendment in the amendment is 1200–1500 kg / hm². 2 The application time is within 48 hours after rain during the greening period of Leymus chinensis in May each year; the sowing method is no-till row sowing; the sowing rate is 30-36 kg / hm². 2 .

[0019] Beneficial effects:

[0020] This invention provides a degraded grassland restoration microbial agent composed of *Pseudomonas fluorescens* T6 and *Rhizobium sinense* NFB5 from alfalfa. The preservation number of *Pseudomonas fluorescens* T6 is CGMCC No. 36876, and the preservation number of *Rhizobium sinense* NFB5 is CGMCC No. 33618. The *Pseudomonas fluorescens* T6 in this grassland restoration microbial agent is a dominant native plant symbiotic bacterium isolated from the roots of *Leymus chinensis*, a non-degraded grassland in Xilingol League. *Rhizobium sinense* NFB5 is also isolated from the rhizosphere soil of *Alfalfa* in Inner Mongolia. The combination of these two microorganisms has a synergistic effect, resulting in good grassland restoration and a high colonization rate.

[0021] Furthermore, the restoration method provided by this invention relies on local resources of the Inner Mongolian grasslands (sheep manure, peat soil, native microorganisms and native grass species), takes into account both ecological restoration and animal husbandry production, and achieves integrated restoration of "soil-microorganisms-vegetation" in a three-dimensional synergy. It has become the key to solving the current grassland degradation problem and supporting regional economic development, and has important technological innovation value and practical application significance.

[0022] Biological Preservation Instructions

[0023] Fluorescent Pseudomonas T6, classified and named Pseudomonas fluorescens It was deposited on December 2, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 36876;

[0024] *Alfalfa rhizobium* NFB5, classified and named *Alfalfa rhizobium* Sinorhizobium melilotiIt was deposited on February 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.33618. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0026] Figure 1 The colony culture results of Pseudomonas fluorescens T6;

[0027] Figure 2 The results show the antagonistic activity between *Pseudomonas fluorescens* T6 and *Rhizobium sinense* NFB5 from alfalfa. Detailed Implementation

[0028] This invention provides a degraded grassland restoration microbial agent, composed of *Pseudomonas fluorescens* (…). Pseudomonas fluorescens T6 and *Alfalfa rhizobium* ( Sinorhizobium meliloti The composition is NFB5; the preservation number of the fluorescent pseudomonad T6 is CGMCC No. 36876, and the preservation number of the alfalfa rhizobium NFB5 is CGMCC NO. 33618.

[0029] In one embodiment, the viable count ratio of *Pseudomonas fluorescens* T6 to *Rhizobium sinense* NFB5 from alfalfa is 1:1 to 2:1. In another embodiment, the degraded grassland restoration agent comprises *Pseudomonas fluorescens* T6 and *Rhizobium sinense* NFB5 solutions, with a volume ratio of 1:1, and the effective viable count of the *Pseudomonas fluorescens* T6 solution is ≥1×10⁻⁶. 8 CFU / mL; the effective viable count of the *Rhizobium sinense* NFB5 bacterial suspension in alfalfa is ≥1×10⁻⁶. 8 CFU / mL.

[0030] This invention provides a granular remediation microbial agent, comprising a carrier and the degraded grassland remediation microbial agent described in the above-mentioned technical solution; the carrier comprises straw powder, and the effective viable count of the granular remediation microbial agent is ≥1×10⁻⁶. 8 CFU / g.

[0031] In one embodiment, the granular remediation agent has a particle size of 4-6 mm and a moisture content of <10%; the straw powder has a particle size of 0.5-1.5 mm. In another embodiment, the remediation agent has a particle size of 5 mm, suitable for mechanized application.

[0032] This invention provides a method for preparing the particulate remediation bacterial agent described in the above technical solution, comprising:

[0033] The degraded grassland restoration microbial agent is activated to obtain an activated microbial solution; the activated microbial solution is mixed with straw powder, granulated, and dried to obtain the granular restoration microbial agent.

[0034] In one embodiment, the mass ratio of the activated bacterial solution to straw powder is 10:1 to 15:1, and the viable count of the activated bacterial solution is >1×10⁻⁶. 8 CFU / mL. As one implementation method, the drying method includes natural air drying.

[0035] This invention provides a modifier, comprising: a granular modifier and the granular remediation microbial agent described in the above-mentioned technical solution; the granular modifier comprises the following components in parts by weight: 8-16 parts of well-rotted sheep manure, 1-2 parts of peat moss, and 0.5-1 parts of potassium dihydrogen phosphate. As one embodiment, the granular modifier comprises the following components in parts by weight: 8 parts of well-rotted sheep manure, 1.5 parts of peat moss, and 0.5 parts of potassium dihydrogen phosphate.

[0036] In one embodiment, the mass ratio of the granular remediation microbial agent to the granular amendment is 1:10 to 1:15; the particle size of the granular amendment is 4 to 6 mm, and the moisture content is 10% to 12%. In another embodiment, the mass ratio of the granular remediation microbial agent to the granular amendment is 1:10. The granular amendment provided by this invention can increase soil available nitrogen to 45 to 50 mg / kg and organic matter to 15% to 20%, and can avoid soil compaction caused by traditional chemical fertilizers; in addition, the granular amendment provides a carbon source for microorganisms, which helps the colonization of native microorganisms.

[0037] This invention provides the application of the degraded grassland restoration microbial agent, the granular restoration microbial agent, or the modifier described in the above-mentioned technical solutions in grassland degradation restoration.

[0038] In one implementation method, the grassland is the Inner Mongolian grassland.

[0039] This invention provides a method for restoring grasslands in Inner Mongolia, comprising the following steps:

[0040] The improver described in the above technical solution is applied to the grassland to be improved, and a microbial activating solution is sprayed on the grassland to be improved 1-2 times every August; the microbial activating solution comprises the following components in parts by volume: 1-2 parts of humic acid solution and 1-2 parts of sucrose solution; the concentration of the humic acid solution is 3g / L, and the concentration of the sucrose solution is 1g / L.

[0041] Sow grass seeds on the grassland to be improved; the grass seeds include the following parts by weight: 4-5 parts of sheepgrass seeds, 2-3 parts of needlegrass seeds and 2-3 parts of alfalfa seeds;

[0042] During the restoration period from July to August of that year, light grazing was implemented during the rapid growth phase of the community, with a carrying capacity of 0.1–0.3 sheep units / hm². 2 Grazing is prohibited from September to May of the following year.

[0043] In one embodiment, the application rate of the particulate amendment in the amendment is 1200–1500 kg / hm². 2 The best time to apply fertilizer is within 48 hours after rain during the greening period of sheepgrass in May each year. It can be applied using a tractor-pulled fertilizer spreader.

[0044] In one implementation method, the grass seeds are sown using no-till row sowing; the sowing rate is 30–36 kg / hm². 2 .

[0045] The restoration method provided by this invention addresses problems such as degradation of dominant grassland plant communities, soil nitrogen and phosphorus imbalance, and decline in microbial function. It overcomes the limitations of existing technologies, which suffer from single restoration dimensions, poor post-restoration community stability, misallocation of microbial resources, disconnection from grazing management, and lack of lightweight technical solutions that are compatible with both restoration and grazing. The invention constructs a closed-loop technical system of "soil fertility regulation → core microbial enhancement → native community optimization," which has a good effect on the restoration of degraded grasslands.

[0046] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a degraded grassland restoration microbial agent, a particulate restoration microbial agent, and an amendment provided by the present invention, and their applications, should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] The dominant native plant symbiotic bacterium *Pseudomonas fluorescens* was isolated from the root system of *Leymus chinensis*, a non-degraded grassland in Xilingol League. Pseudomonasfluorescens T6, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36876, was isolated and identified as follows:

[0049] (1) Isolation and purification of Pseudomonas fluorescens

[0050] Take the roots of *Leymus chinensis*, place them in 20–30 mL of sterile water, and vortex for 5 minutes. Remove the roots, take 0.5 mL of the supernatant, add it to 4.5 mL of sterile water, vortex to mix, and then dilute to 10⁻⁶. -1 10 -2 10 -3 10 -4The diluted suspensions were spread onto TSA medium and incubated upside down in a 28°C incubator for 48 hours. Single colonies that were clearly identifiable and grew uniformly were selected from the medium and transferred to TSA medium for three subcultures. Single colonies were then picked and purified on LB purification medium to obtain pure cultured bacteria, which were designated as T6.

[0051] (2) Physiological and biochemical characteristics of Pseudomonas fluorescens

[0052] Physiological and biochemical experiments were conducted on the purified T6 strain. Under a microscope, the colony morphology of the strain after initial division or subculturing was short rod-shaped, slightly yellowish, with a smooth, moist surface and regular edges. Figure 1 The bacteria showed negative Gram staining under an optical microscope.

[0053] (3) Molecular identification of Pseudomonas fluorescens strains

[0054] Total DNA was extracted from the above-mentioned strains, and PCR amplification was performed using universal 16S rDNA primers. The samples were then sent to Sangon Biotech Co., Ltd. for sequencing, yielding a gene sequence fragment of 1437 bp (SEQ ID NO. 1). Sequence analysis and homology comparison were performed in GenBank. The similarity between strain T6 and *Pseudomonas fluorescens* was 99%. A phylogenetic tree was constructed and analyzed. Morphological identification confirmed that the strain was *Pseudomonas fluorescens*. Strain T6 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 33618.

[0055] The T6 sequence of *Pseudomonas fluorescens* is shown in SEQ ID NO.1:

[0056]

[0057] Example 2

[0058] (1) Detection of antagonism between Pseudomonas fluorescens T6 and Rhizobium sinense NFB5 in alfalfa

[0059] The activated Pseudomonas fluorescens T6 strain and Rhizobium sinense NFB5 strain obtained from Example 1 were streaked on LB solid medium plates and cultured at 30°C for 1-2 days. The growth of the two strains at the cross-streaked areas was observed.

[0060] If the two strains grow relatively weakly or not at the crossover point, it indicates that there is an antagonistic effect between the two strains; if both bacteria grow well at the crossover point, it indicates that there is no antagonistic effect between the two strains and they can be used for mixed culture.

[0061] Antagonism detection results showed that no sterile zone was formed between *Pseudomonas fluorescens* strain T6 and *Rhizobium sinense* strain NFB5, indicating that they could grow in contact and therefore did not inhibit each other. Figure 2 ).

[0062] (2) Preparation of microbial agents for the restoration of degraded grassland

[0063] The fluorescent Pseudomonas T6 isolated in Example 1 was inoculated into LB liquid medium and cultured at 28°C and 150 rpm for 48 h with shaking to obtain T6 bacterial suspension (effective viable count of 6.4 × 10⁻⁶). 8 (CFU / mL).

[0064] The *NFB5* rhizobium spp. from alfalfa was inoculated into LB liquid medium and cultured at 28°C and 150 rpm for 48 h with shaking to obtain an NFB5 bacterial suspension (effective viable count of 6.4 × 10⁻⁶). 8 (CFU / mL).

[0065] T6 bacterial solution and NFB5 bacterial solution were mixed at a volume ratio of 2:1 and then freeze-dried at low temperature (-40℃) (to maintain more than 90% activity) to obtain a degraded grassland restoration bacterial agent.

[0066] Comparative Example 1

[0067] A degraded grassland restoration microbial agent similar to that in Example 2, the difference being that the T6 bacterial solution is subjected to low-temperature freeze-drying (-40℃) (maintaining more than 90% activity) to obtain the degraded grassland restoration microbial agent.

[0068] Comparative Example 2

[0069] A degraded grassland restoration microbial agent similar to that in Example 2, the difference being that the NFB5 bacterial solution is subjected to low-temperature freeze-drying (-40℃) (maintaining more than 90% activity) to obtain the degraded grassland restoration microbial agent.

[0070] Comparative Example 3

[0071] A degraded grassland restoration microbial agent similar to that in Example 2, except that the volume ratio of T6 microbial solution to NFB5 microbial solution is 1:1.

[0072] Comparative Example 4

[0073] A degraded grassland restoration microbial agent similar to that in Example 2, except that the volume ratio of T6 microbial solution to NFB5 microbial solution is 1:2.

[0074] Test Example 1

[0075] Determination of seedling growth indicators

[0076] Select uniformly sized and plump seeds of Leymus chinensis and Alfalfa, soak them in sterile water for 5 minutes, then disinfect them by soaking in 1% sodium hypochlorite solution for 30 seconds, rinse them three times with sterile water, and soak them in 5 mL of cultured bacterial suspension for 6 hours. Place the treated seeds in petri dishes lined with two layers of filter paper (wetted with sterile water) for germination. One week later, select seedlings with uniform growth and transplant them into flowerpots (11 cm high × 12 cm long × 8 cm wide) containing 500 g of sterilized soil. Add bacteria with a final concentration of OD value (600 nm) = 0.02. Use sterile water treatment as a control. Each group has four replicates. Grow the seeds for 3 months under 25℃ and 16 h light conditions. Measure the plant height, root length, stem weight, root weight, and total weight of Leymus chinensis and Alfalfa.

[0077] Table 1 Effects of different inoculants on Leymus chinensis seedlings

[0078]

[0079] Note: Data in the same column of the table marked with the same lowercase letter indicates that the data is in the same column. P The difference was not significant at the 0.05 level. Those labeled with different lowercase letters indicate that... P The differences were significant at the <0.05 level, and the same applies to the table below.

[0080] Table 2 Effects of different inoculants on alfalfa seedlings

[0081]

[0082] The experimental results show that the remediation bacterial agent prepared using Example 2 can significantly ( P<0.05) increased seedling height, root length, and weight, indicating that the remedial bacterial agent has a significant growth-promoting effect (Table 1-2). Among them, the T6 bacterial solution and NFB5 bacterial solution mixed at a volume ratio of 2:1 showed the best effect.

[0083] In summary, the repair agent prepared in Example 2 can promote the growth of seedlings and roots of Leymus chinensis and Alfalfa.

[0084] Example 3

[0085] A particulate remedial bacterial agent is prepared by the following method:

[0086] Mix the bacterial agent, brown sugar water, and sterile water in a ratio of 1:1:100 (by volume), and activate the mixture at 25–30°C in the dark for 24–48 hours, stirring once every 8 hours until the bacterial solution produces foam and has a slightly sweet and sour taste (indicating a large proliferation of bacteria). The activated bacterial solution is obtained. The bacterial agent is the degraded grassland restoration bacterial agent prepared in Example 2. The concentration of the brown sugar water is 1% (w / v).

[0087] Mix sheepgrass straw powder (crushed to 1mm) with activated bacterial solution at a ratio of 10:1 to 15:1 (by mass) to saturate the straw powder with the activated bacterial solution. Extrude the sheepgrass straw powder with the activated bacterial solution through a sieve with a pore size of 3-5mm, or feed the mixed material into the disc granulator in batches, spraying a small amount of water while rotating (to maintain the material's stickiness). Collect the granules promptly when they reach approximately 5mm in size to prevent them from becoming too large. Spread the wet granules evenly on a drying ground and air dry naturally for 2-3 days until the moisture content of the granules drops below 10%, obtaining the granular repair agent. The effective viable bacteria count of the granular repair agent is ≥1×10⁻⁶. 8 CFU / g.

[0088] Example 4

[0089] An improver is composed of the following components in parts by weight: 10 parts of granular improver and 1 part of the granular repair bacteria agent described in Example 3.

[0090] The granular amendment is composed of the following components in parts by weight: 8 parts of well-rotted sheep manure, 1.5 parts of peat soil (taken from Duolun County to supplement organic matter) and 0.5 parts of potassium dihydrogen phosphate.

[0091] The method for preparing the decomposed sheep manure is as follows: After collecting the pasture residue from the sheepfold, remove the manure from the pen. Select a high-lying, well-drained site, level it, and lay a 10 cm thick layer of pasture residue as a base. Layer the manure and pasture residue, stacking each layer to a height of 20-30 cm before adding a layer of pasture residue, compacting each layer. The top of the pile should be arched to prevent rainwater accumulation. After 2-3 months at natural temperature, when the pile turns dark brown or black, has a loose texture, no odor, and a slight earthy fragrance, it is considered fully decomposed. In winter, this process can be extended to 4-5 months. After decomposition, it can be used directly as compost.

[0092] Preparation of granule conditioner: Select well-rotted sheep manure and peat moss with a moisture content of 25%–30%. Crush them using a pulverizer and pass them through a 20-mesh sieve to remove impurities such as stones and grass roots, ensuring uniform material fineness. Weigh the materials according to the following mass ratio: 80 kg of well-rotted sheep manure, 15 kg of peat moss, and 5 kg of potassium dihydrogen phosphate (containing 52% P2O2 and 34% K2O). First, manually stir the well-rotted sheep manure and peat moss for 5–8 minutes until the color is uniform; then slowly spray the potassium dihydrogen phosphate solution and wet mix for 10–15 minutes, stabilizing the material moisture content at 25%–28% (the material should clump together when squeezed by hand but crumble easily when lightly pinched). Alternatively, use a disc granulator to feed the mixed material into the disc in batches, spraying a small amount of water while rotating (to maintain the material's viscosity); collect the granules promptly when they reach approximately 5 mm in size to avoid excessively large granules. Spread the wet granules evenly on the drying ground and let them air dry naturally for 2 to 3 days until the moisture content of the granules drops to 10% to 12% (at which point the granules reach the required hardness and are not easily broken).

[0093] The granular modifier and the granular repair agent are mixed evenly at a mass ratio of 1:10 to obtain the modifier.

[0094] Example 5

[0095] A three-dimensional collaborative restoration method for typical grasslands in Inner Mongolia, comprising the following steps:

[0096] The amendment described in Example 4 should be evenly applied within 48 hours after rain during the greening period of Leymus chinensis in May each year; the application rate of the granular amendment should be 1500 kg / hm² each time. 2 According to the "Inner Mongolia Grassland Degradation Classification Standard" (DB15 / T 1954-2020), mildly degraded areas (dominant species coverage 30%~36%) should be fertilized once every 2 years, moderately degraded areas (dominant species coverage 18%~30%) should be fertilized once a year, and severely degraded areas (dominant species coverage ≤18%) should be fertilized twice a year, with an application interval of 10 days.

[0097] Post-activation: Spray the microbial activator solution once a year in August (during the flowering period of Stipa), maintaining microbial activity until the frost period, at a rate of 6000 L / hm.2 The microbial activation solution is composed of 0.3% (w / v) humic acid solution and 0.1% (w / v) sucrose solution; the volume ratio of the humic acid solution to the sucrose solution is 1:1.

[0098] Lightweight reseeding: Mix sheepgrass seeds, needlegrass seeds, and alfalfa seeds, and clean them with a 1.5 mm mesh sieve to remove impurities, shriveled seeds, and broken seeds, retaining plump seeds; the mass ratio of sheepgrass seeds, needlegrass seeds, and alfalfa seeds is 5:3:2, and the sowing rate is 30 kg / hm. 2 Use a no-till planter to sow in rows with a row spacing of 30 cm and a sowing depth of 2-3 cm.

[0099] Time-series compatibility: Fixed the issue of light grazing during the rapid growth period of the community in July and August of the current year, with the stocking rate controlled at 0.2 sheep units / hm². 2 Grazing is prohibited from September to May of the following year, which ensures the recovery of the community and meets the summer grazing needs of herders.

[0100] Community control: Every autumn, 10% to 15% of invasive species are manually removed to maintain the dominant position of Leymus chinensis-Stipa var. chinensis and prevent weeds from crowding out resources.

[0101] Example 6

[0102] A method similar to Example 5, except that the reseeding method for moderately degraded grassland is as follows: the mass ratio of Leymus chinensis seeds, Stipa krill seeds, and Alfalfa seeds is 2:1:1, and the sowing rate is 32 kg / hm². 2 .

[0103] Example 7

[0104] A method similar to Example 5, except that the reseeding method for severely degraded grassland is as follows: the mass ratio of Leymus chinensis seeds, Stipa krill seeds, and Alfalfa seeds is 4:2:3, and the sowing rate is 36 kg / hm². 2 .

[0105] Comparative Example 5

[0106] A method similar to Example 5, except that only the particulate modifier prepared in Example 4 is used, and the application rate is 1500 kg / hm². 2 .

[0107] Comparative Example 6

[0108] A method similar to Example 5, except that only the particulate remediation bacterial agent prepared in Example 3 is used, and the application rate is 150 kg / hm. 2 .

[0109] Comparative Example 7

[0110] A method similar to Example 5, except that the application rate of the degraded grassland conditioner is 1200 kg / hm². 2 .

[0111] Comparative Example 8

[0112] A method similar to Example 5, except that the modifier prepared in Example 4 is replaced with a comparative modifier; the preparation method of the comparative modifier is similar to that in Example 4, except that the mass ratio of the granular repair bacteria agent to the granular modifier is 1:15.

[0113] Comparative Example 9

[0114] Without taking any action, the degraded grassland is fenced off and managed as a closed-off area.

[0115] Test Example 2

[0116] In 2024, restoration was carried out in typical grasslands of Xilin Gol League using methods from Examples 5-7 and Comparative Examples 5-9. Examples 5-7 restored mildly, moderately, and severely degraded grasslands, respectively, while Comparative Examples 5-9 restored mildly degraded grasslands. In August 2025, the aboveground biomass and soil nutrient content of the degraded grasslands were measured, and the results are shown in the table below:

[0117] Table 3. Impacts of three-dimensional restoration of degraded grassland on aboveground biomass and soil properties.

[0118]

[0119] Note: Different lowercase letters in the same column indicate significant differences between treatments. P <0.05).

[0120] As shown in Table 3, Example 5 significantly improved compared to the control group. P <0.05) increased aboveground biomass and soil nutrient content; Examples 6 and 7, compared to Comparative Example 9, significantly ( P The increase in aboveground biomass and soil microbial biomass (carbon, nitrogen, and phosphorus) (<0.05) indicates that the remediation method provided by this invention can enhance the microbial activity of degraded grasslands, thereby improving soil fertility and grassland productivity. These results demonstrate that the remediation method provided by this invention, through three consecutive steps—targeted regulation of soil fertility, strengthening of native microbial communities, and optimization of native communities—can significantly improve the productivity and soil fertility of degraded grasslands, achieving three-dimensional synergistic remediation of vegetation, soil fertility, and soil microorganisms, providing a basis for the rapid remediation of degraded grasslands.

[0121] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A degraded grassland restoration microbial agent, characterized in that, By fluorescent Pseudomonas ( Pseudomonas fluorescens T6 and *Alfalfa rhizobium* ( Sinorhizobium meliloti The composition is NFB5; the preservation number of the fluorescent Pseudomonas T6 is CGMCC No. 36876, and the preservation number of the alfalfa rhizobium NFB5 is CGMCC NO. 33618; the live count ratio of the fluorescent Pseudomonas T6 to the alfalfa rhizobium NFB5 is 2:

1.

2. A granular remediation bacterial agent, characterized in that, The degraded grassland restoration microbial agent of claim 1 and a carrier; the carrier comprises straw powder, and the effective viable cell number of the granular restoration microbial agent is ≥1×10 8 CFU / g.

3. The particulate remediation bacterial agent according to claim 2, characterized in that, The particle size of the granular repair agent is 4-6 mm, and the moisture content is <10%; the particle size of the straw powder is 0.5-1.5 mm.

4. A modifier, characterized in that, include: The granular modifier and the granular remediation microbial agent as described in claim 2 or 3; the granular modifier comprises the following components in parts by weight: 8-16 parts of well-rotted sheep manure, 1-2 parts of peat moss, and 0.5-1 parts of potassium dihydrogen phosphate.

5. The modifier according to claim 4, characterized in that, The mass ratio of the granular repair agent to the granular improver is 1:10 to 1:15; the particle size of the granular improver is 4 to 6 mm, and the water content is 10% to 12%.

6. The application of the grassland degradation restoration microbial agent according to claim 1, or the granular restoration microbial agent according to claim 2 or 3, or the modifier according to claim 4 or 5 in grassland degradation restoration.

7. The application according to claim 6, characterized in that, The grassland in question is the Inner Mongolia grassland.

8. A grassland restoration method, characterized in that, Includes the following steps: The amendment described in claim 4 or 5 is applied to the grassland to be improved, and a microbial activating solution is sprayed on the grassland to be improved 1 to 2 times every August; the microbial activating solution comprises the following components in parts by volume: 1 to 2 parts of humic acid solution and 1 to 2 parts of sucrose solution; The concentration of the humic acid solution is 3 g / L, and the concentration of the sucrose solution is 1 g / L. Sow grass seeds on the grassland to be improved; the grass seeds include the following parts by weight: 4-5 parts of sheepgrass seeds, 2-3 parts of needlegrass seeds and 2-3 parts of alfalfa seeds; The repair in July and August of the same year, in the community fast growing period of light grazing, stocking rate of 0.1~0.3 sheep unit / hm 2 , September to next May grazing.

9. The grassland restoration method according to claim 8, characterized in that, The application rate of the particulate amendment in the amendment is 1200–1500 kg / hm. 2 The application time is within 48 hours after rain during the greening period of Leymus chinensis in May each year; the sowing method is no-till row sowing; the sowing rate is 30-36 kg / hm². 2 .

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