An organic carrier endo- rhizosphere growth promoting microbial agent for degraded grassland ecological restoration and a preparation method and application thereof

Abstract

The application provides an organic carrier endo- rhizosphere growth promoting microbial agent for degraded grassland ecological restoration and a preparation method and application thereof, and belongs to the technical field of grassland ecological restoration and microbial agents. The organic carrier growth promoting microbial agent comprises four endo- rhizosphere growth promoting bacteria (PGPE), four rhizosphere growth promoting bacteria (PGPR) and an organic carrier; the PGPE comprises B. terrigena K4, B. subtilis 9-3-1, B. safensis 11-5-4 and Halomonas sp. L31, the PGPR comprises Serratia marcescens 5, Serratia marcescens 23, Kosakonia koreensis 11 and Enterobacter cloacae 24, and the organic carrier is carbon-based organic fertilizer, cattle and sheep manure organic fertilizer or biochar. The application of the organic carrier growth promoting microbial agent in degraded grassland can significantly improve the biomass and coverage of the plant community. The organic carrier growth promoting microbial agent can be used for the ecological restoration of degraded grassland.

Description

Technical Field

[0001] This invention relates to the fields of grassland ecological restoration and microbial agents, and in particular to an organic carrier root-rhizosphere growth-promoting microbial agent for the ecological restoration of degraded grasslands, its preparation method, and its application. Background Technology

[0002] Grassland degradation refers to the process of reverse succession in the structure and function of grassland ecosystems, leading to a continuous decline in their productivity, biodiversity, ecological functions, and self-recovery capacity. This includes both vegetation and soil degradation. The main manifestations are the destruction of the original structure and function of grasslands, reduced ecological diversity and ecosystem stability, decreased vegetation cover, a significant reduction in high-quality forage species, deterioration in the quality of various forages, decreased forage productivity and quality, changes in the physical and chemical properties of grassland soils (resulting in degradation, desertification, and salinization), reduced nutrient content, and exacerbated soil erosion. In the past few decades, the restoration of degraded grasslands worldwide has focused on plant community restoration, primarily emphasizing improving plant productivity and rebuilding native vegetation. In the evolutionary history of grasslands, plants, soil, and microorganisms interact collaboratively to adapt to climate fluctuations and animal grazing. Therefore, grassland degradation is a holistic decline in the functions of plants, soil, and microorganisms. The difficulty in restoring severely degraded grasslands stems from multiple constraints, including those related to plant propagation, nutrients, and microorganisms.

[0003] Grassland ecological restoration is the process of restoring degraded and damaged grassland ecosystems to a new, stable, and healthy state. Currently, common grassland ecological restoration methods include no-till reseeding, rational grazing, artificial grassland establishment, fencing, tillage and fertilization, and microbial remediation. However, compared to other restoration measures, research on microbial remediation is relatively limited. Soil microbial diversity is a crucial driver of aboveground plant community development and plays a key role in determining the ecological response of terrestrial ecosystems to environmental changes. Grassland ecological vegetation restoration largely depends on the microbial community in grassland soil. Microbial inoculation can simultaneously promote forage growth and improve the soil environment. Microorganisms also help forage grasses acquire nutrients such as nitrogen and phosphorus from the soil. Degraded grassland soils exhibit significantly reduced microbial diversity and abundance, altering the structure and function of the microbial community, thus limiting the grassland ecological restoration process. Supplementing with beneficial microorganisms is beneficial for the ecological restoration of degraded grasslands. Existing literature indicates that three types of microbial functional groups play important roles in grassland ecological restoration: plant growth-promoting bacteria, arbuscular mycorrhizal fungi, and nitrogen-fixing bacteria. Under stress conditions, they play a vital role in improving soil health and maintaining soil productivity. Among them, plant rhizosphere growth-promoting bacteria can withstand drought, salinity and other stressful environments, helping pasture grasses obtain soil nutrients; plant rhizosphere growth-promoting fungi can decompose and utilize cellulose, pectin and starch to provide nutrients for pasture grasses; symbiotic nitrogen-fixing bacteria and non-symbiotic nitrogen-fixing bacteria also promote the absorption of nutrients and growth of pasture grasses; arbuscular mycorrhizal fungi are beneficial to the formation and stability of soil aggregates, and can also promote the absorption of water and phosphorus nutrients by pasture grasses.

[0004] Plants typically require more than one symbiotic microorganism to resist environmental stress, and inoculating with a single strain under natural conditions is almost always ineffective for plant stress resistance. The rapid development of rhizosphere microbiome research has led to a renewed understanding that plants benefit from the interactions with their microbial communities, rather than from individual members of the community. Therefore, the untapped potential of beneficial microbial communities in enhancing plant adaptability is increasingly recognized. Endophytic bacteria are present in almost all plant tissues, determining the health, growth, development, and secondary metabolism of the host plant and conferring broader environmental adaptability. Endophytic bacteria have a closer relationship with plants, becoming natural components of the plant micro-ecosystem through long-term co-evolution, and are considered to play a more important role in synergistic plant stress resistance. Studies have confirmed that the presence of multiple endophytic bacteria is a highly effective strategy for improving plant stress tolerance, with mechanisms similar to those of rhizosphere bacteria. The host plant can provide nutrients and ecological niches for the growth of endophytic growth-promoting bacteria, reducing competitive pressure and the impact of stressful environments, and maintaining stable colonization within the plant. Therefore, endophytic growth-promoting bacteria play a more lasting and important role in plant resistance to environmental stress, and are more economical and environmentally friendly. Recent research has confirmed that the combined use of various rhizosphere and root-derived growth-promoting bacteria under environmental stress can produce a synergistic effect on plant growth, further enhancing their beneficial effects. Therefore, developing and utilizing combined rhizosphere and root-derived growth-promoting bacteria to improve plant tolerance to adverse environments could open up a new avenue for promoting the ecological restoration of degraded grasslands.

[0005] Under stress conditions, the most crucial factor for inoculating plant growth-promoting bacteria to have a beneficial effect on plant growth is ensuring their colonization and survival within the plant roots and rhizosphere. However, the survival of plant growth-promoting bacteria in stress environments depends not only on the availability of ecological niches but also on their ability to compete with well-adapted native microorganisms. The application of microbial technology in degraded ecosystems in arid and semi-arid regions has not been successful, especially in areas with scarce soil organic matter, primarily due to the inability to provide a suitable substrate environment and nutrient sources for their growth. In fact, a sustained supply of carbon and energy helps to successfully establish beneficial bacteria in the plant rhizosphere, enhancing the competitiveness of plant growth-promoting bacteria. Organic matter has significant advantages as a carrier material. On the one hand, organic matter can directly provide nutrients for plant growth; on the other hand, organic matter, as a carrier, can provide sufficient energy and nutrients, as well as a favorable habitat, for the survival and growth of microbial agents, and protect them from pathogens, thus facilitating the beneficial functions of plant growth-promoting bacteria. Therefore, utilizing Inner Mongolia's unique organic solid waste resources to construct organic carrier microbial agents is fundamental to ensuring the beneficial effects of growth-promoting bacteria in degraded grassland restoration practices.

[0006] Although some microbial agents for the ecological restoration of degraded grasslands have been developed, existing microbial agents have the following shortcomings: 1. Due to the potential competitive inhibition between arbuscular mycorrhizal fungi, rhizosphere bacteria, and nitrogen-fixing bacteria, most agents use a single species or a simple combination of a few (2-3 strains), resulting in limited functionality; 2. Most microbial agents developed using plant growth-promoting bacteria only consider soil or rhizosphere growth-promoting bacteria, neglecting the important role that rhizosphere growth-promoting bacteria may play and the synergistic effects that may occur when combined with rhizosphere growth-promoting bacteria; 3. Insufficient compatibility between the carrier selection and the functional microbial flora affects the colonization activity and long-term effectiveness of the microorganisms, thus affecting the growth-promoting effect of the agent; 4. The strains in the agents are mostly exogenous species, which have poor adaptability to adverse conditions such as degraded grasslands, saline-alkali grasslands, or desertified grasslands, resulting in unstable growth-promoting effects. Therefore, it is necessary to develop a carrier-based microbial agent suitable for the ecological restoration of degraded grasslands in Inner Mongolia. Summary of the Invention

[0007] In view of this, the present invention provides an organic carrier root-rhizosphere growth-promoting microbial agent for the restoration of degraded grasslands, its preparation method and application, in order to solve the above problems.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides an organic carrier root-rhizosphere growth-promoting microbial inoculant for ecological restoration of degraded grasslands, comprising root-rhizosphere growth-promoting microbial inoculant solution and an organic carrier, wherein the root-rhizosphere growth-promoting microbial inoculant solution comprises root-promoting bacteria and rhizosphere-promoting bacteria;

[0010] The root growth-promoting bacteria include Bacillus terracotta warriors K4, Bacillus 9-3-1, Bacillus safortiformis 11-5-4 and Halomonas L31.

[0011] The rhizosphere growth-promoting bacteria include Serratia marcescens 5, Serratia marcescens 23, Cossackia covani 11, and Enterobacter hygroscopicus 24.

[0012] The Bacillus terracotta warriors K4, Bacillus 9-3-1, Bacillus saforticus 11-5-4, and Halomonas L31 are deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession numbers GDMCC NO.65395, GDMCC NO.65392, GDMCC NO.65394, and GDMCC NO.65397, respectively.

[0013] The *Serratia marcescens* 5, *Serratia marcescens* 23, *Cossackia covani* 11, and *Enterobacter hygroscopicus* 24 are deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession numbers GDMCC NO.65386, GDMCC NO.65389, GDMCC NO.65391, and GDMCC NO.65388, respectively.

[0014] Preferably, the organic carrier includes carbon-based organic fertilizer, cow or sheep manure organic fertilizer, or biochar.

[0015] Preferably, the root-rhizosphere growth-promoting microbial solution accounts for 10-20% of the carrier mass.

[0016] Preferably, the effective viable bacteria count in the root-rhizosphere growth-promoting microbial agent is ≥1.4×10⁻⁶. 8 cfu / g, moisture content ≤25%.

[0017] Preferably, the carbon-based organic fertilizer is prepared from lignite through fermentation.

[0018] Preferably, the cow and sheep manure organic fertilizer includes well-rotted cow manure and / or sheep manure.

[0019] Preferably, the biochar is prepared by calcining a mixture of corn, wheat and peanut straw.

[0020] This invention also provides a method for preparing the aforementioned organic carrier root-rhizosphere growth-promoting microbial inoculant, characterized by comprising the following steps:

[0021] S1. Culture *Bacillus terracotta* K4, *Bacillus 9-3-1*, *Bacillus safortiformis* 11-5-4, *Haloxysporum* L31, *Serratia marcescens* 5, *Serratia marcescens* 23, *Cossackia covani* 11, and *Enterobacter hygroscopicus* 24 separately until the OD of the bacterial culture is obtained. 600 =0.8~1.0, collect the bacterial solution;

[0022] S2. Mix equal volumes of the bacterial solutions from step S1 to obtain a mixed bacterial solution;

[0023] S3. Inoculate the mixed bacterial solution into the fermentation medium and culture it at 26-30℃ and 100-140 rpm for 10-14 h to obtain the root-rhizosphere growth-promoting microbial solution.

[0024] S4. Using carbon-based organic fertilizer, cow or sheep manure organic fertilizer, or biochar as the organic carrier, thoroughly mix the root-rhizosphere growth-promoting microbial inoculum with the organic carrier, incubate at 26-30℃ for 4-6 days, and then dry to obtain the product.

[0025] Preferably, the inoculation amount in step S3 is 10-20%.

[0026] Preferably, the fermentation medium is LB liquid medium.

[0027] The present invention also provides the application of the aforementioned organic carrier root-rhizosphere growth-promoting microbial agent in at least one of the following:

[0028] (1) Application in restoring degraded grasslands;

[0029] (2) Application in promoting plant growth;

[0030] (3) Application in promoting grassland vegetation restoration.

[0031] Preferably, the plants include Suaeda salsa, Imperata cylindrica, Sophora flavescens, Leymus chinensis, Alfalfa, or Leymus chinensis.

[0032] Preferably, the degraded grassland includes degraded grassland, desertified grassland and saline-alkali grassland.

[0033] By adopting the above technical solution, the present invention has the following beneficial effects: The organic carrier root-rhizosphere growth-promoting microbial agent of the present invention comprises four root-promoting bacteria (PGPE), four rhizosphere growth-promoting bacteria (PGPR), and an organic carrier; the root-promoting bacteria (PGPE) include Bacillus terracotta K4, Bacillus 9-3-1, Bacillus saforticus 11-5-4, and Halomonas L31; the rhizosphere growth-promoting bacteria (PGPR) include Serratia marcescens 5, Serratia marcescens 23, Cossackia covani 11, and Enterobacter hygroscopicus 24; and the organic carrier is carbon-based organic fertilizer, cow or sheep manure organic fertilizer, or biochar. Applying the organic carrier root-rhizosphere growth-promoting microbial agent of the present invention to degraded grasslands can significantly increase vegetation community biomass and cover. The organic carrier root-rhizosphere growth-promoting microbial agent of the present invention can be used for the ecological restoration of degraded grasslands.

[0034] Biological Preservation Instructions

[0035] The taxonomic name of Serratia rubidaea is Serratia rubidaea. It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 31, 2024, with accession number GDMCC NO.65386. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0036] The taxonomic name of Enterobacter hormaechei 24 was entered into the Guangdong Provincial Microbial Culture Collection Center on October 31, 2024, with accession number GDMCC NO.65388. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0037] The taxonomic name of Serratia rubidaea 23 was Serratia rubidaea, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 31, 2024, with accession number GDMCC NO.65389, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0038] The taxonomic name of Kosakonia cowanii 11 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 31, 2024, with accession number GDMCC NO.65391, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0039] The taxonomic name of Bacillus 9-3-1 is Bacillus sp., which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 31, 2024, with accession number GDMCC NO.65392, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0040] The taxonomic name of Bacillus safensis 11-5-4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 31, 2024, with accession number GDMCC NO.65394, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0041] The taxonomic name of Bacillus bingmayongensis K4 is Bacillus bingmayongensis. It was deposited on December 23, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65395, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0042] The taxonomic name of Halomonas L31 is Halomonas sp., which was deposited on December 23, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65397, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0043] Figure 1 This is a pilot area for the restoration of moderately degraded typical grasslands used for grazing.

[0044] Figure 2 This serves as a pilot area for the restoration of typical grasslands with different causes and degrees of degradation.

[0045] Figure 3 This study investigates the restorative effects of organic-carrier microbial agents on mowed and degraded typical grasslands.

[0046] Figure 4 The effect of organic carrier microbial inoculants on the restoration of moderately degraded typical grasslands under grazing.

[0047] Figure 5 The effect of organic carrier microbial inoculants on the restoration of severely degraded typical grasslands due to grazing. Detailed Implementation

[0048] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Preparation of LB liquid culture medium: Take 10 g of peptone, 5 g of yeast extract and 10 g of sodium chloride, add distilled water to make up to 1000 mL, adjust pH to 7.0, and sterilize at 121 ℃ for 20 min.

[0050] Preparation of LB solid medium: Take 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, and 10 g of agar, add distilled water to a final volume of 1000 mL, adjust the pH to 7.0, and sterilize at 121 ℃ for 20 min.

[0051] The carbon-based organic fertilizer in this embodiment of the invention was provided by Yuantaifeng (Baotou) Biotechnology Co., Ltd., the decomposed cow and sheep manure was provided by Inner Mongolia Gugong Biotechnology Co., Ltd., and the biochar was purchased from Pingdingshan Tanernuo New Materials Co., Ltd.

[0052] Example 1. Isolation and culture of bacterial strains

[0053] Dominant plant roots and rhizosphere soil were collected from Urad Front Banner of Bayannur City, Linhe District of Bayannur City, Alxa Left Banner of Alxa League, Wurigentala Town of Sunite Right Banner of Xilingol League, Urad Front Banner of Bayannur City, Dashetai Town of Urad Front Banner of Bayannur City, Hangjin Rear Banner of Bayannur City, and Taipusi Banner of Xilingol League. The surfaces of the dominant plant roots were sterilized and ground into powder. Then, 1g of the plant root powder and rhizosphere soil were placed in 1mL of sterile PBS buffer and mixed thoroughly with a pipette to obtain bacterial suspensions containing microorganisms. Each bacterial suspension was then serially diluted 10-fold to obtain 10... -2 ~10 -7 The bacterial suspensions were prepared. 0.1 mL of each diluted bacterial suspension was taken and spread onto LB solid medium.

[0054] Incubate the petri dishes at 28°C. When single colonies appear on the surface, select individual colonies based on their morphology and color differences for further purification. Streak the single bacterial colonies onto LB agar plates and subculture them multiple times until the colonies on the medium are completely uniform in color, size, and morphology, thus obtaining a pure culture. Store all obtained pure colonies in 50% glycerol at -80°C.

[0055] Example 2. Strain Identification

[0056] A single colony was picked and placed in a 1.5 mL centrifuge tube containing 1 mL of LB medium. The colony was cultured at 30 °C and 200 rpm for 24 h with shaking. The bacterial culture was then used as a template to amplify the 16S rRNA sequence using universal primers 27F / 1492R.

[0057] The PCR amplification reaction system consisted of 50 μL of 2xTaq enzyme, 1 μL of 27F primer, 1 μL of 1492R primer, 1 μL of bacterial culture, and 22 μL of ddH2O. Amplification conditions were as follows: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 32 cycles, followed by a final extension at 72℃ for 5 min. The amplified products were stored at 4℃. The amplified products were separated and identified by 1% agarose gel electrophoresis. The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The results showed that four root growth-promoting bacteria (PGPE) and four rhizosphere growth-promoting bacteria (PGPR) were isolated. The four root growth promoters (PGPE) include one Bacillus terracotta, one Bacillus, one Bacillus saforticus, and one Halomonas; the four rhizosphere growth promoters (PGPR) include two Serratia rubra, one Cossackia covani, and one Enterobacter hygroscopicus.

[0058] Example 3. Determination of salt tolerance, secretion of IAA and ACC deaminase activities, phosphate solubilization, siderophore production, and extracellular polymeric substance (EPS) capacity of each strain (Table 1)

[0059] 1. Salt tolerance assessment of strains: All initially screened strains (glycerol-based bacterial culture solution) were inoculated at a 1:1 (v / v) ratio into sterile LB broth for batch activation (35℃, 130 r·min). -1 LB solid medium with NaCl contents of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and 11% was prepared for incubation at a constant temperature for 12 h. The pH was the basic screening pH condition (i.e., pH 8.0). The activated strains were streaked onto plates using sterile inoculation sticks and incubated at a constant temperature of 35℃ for 24–48 h. The growth of the strains was observed and the results were recorded to determine the salt tolerance of the strains.

[0060] 2. Quantitative determination of the growth-promoting function of the strain

[0061] Before determining the growth-promoting function of the strains, each test strain must be activated and the concentration of the test strains must be consistent.

[0062] To ensure the highest growth activity of each strain, each strain was cultured and activated under optimal pH and temperature conditions. The specific activation method is as follows: 400 μL of glycerol culture from each single strain was added to 40 mL of sterile LB broth (prepared to optimal pH conditions) for activation culture (optimal temperature, 130 r·min). -1 (Constant temperature incubation), incubated until OD 600 Stop culturing when the value is 1.0 and set aside for later use.

[0063] (1) Determination of indoleacetic acid (IAA) content:

[0064] The IAA production capacity of the strain was determined according to the Salkowski colorimetric method, as follows: The activated strain (1 mL) was inoculated into a solution containing L-tryptophan (L-tryptophan, 200 mg·L⁻¹). -1 DF liquid culture medium (40 mL) (with the uninoculated treatment as the control group), 30°C, 180 r·min -1 OD was measured after 48 h of constant temperature incubation. 600 After maintaining consistent absorbance values ​​for all strains, the absorbance was measured at 10000 r·min. -1 Centrifuge for 3 min, take 1 mL of supernatant and add 4 mL of Salkowski colorimetric solution, mix well (the procedure is the same as the control group), let stand at room temperature in the dark for 20 min, and measure OD. 530 The value was calculated based on the standard curve to determine the IAA content (mg·L). -1 ).

[0065] The standard curve is constructed based on the OD value of the 3-indoleacetic acid standard sample. A simplified method is as follows: Prepare the 3-indoleacetic acid standard solution, add 4 mL of Salkowski colorimetric reagent, mix well, and let stand at room temperature in the dark for 20 min. Then measure the OD value. 530 Values. A standard curve was constructed based on the concentration of the 3-indoleacetic acid standard solution and its corresponding absorbance value.

[0066] (2) ACC deaminase content detection:

[0067] Cell sample extraction: Take 7.5 mL of the activated bacterial culture medium and incubate at 30°C and 200 r·min. -1 After incubation at a constant temperature for 24 hours, the samples were then subjected to an incubation at 8000 r·min. -1 Centrifuge for 10 min (4°C) and collect the precipitate; then resuspend the bacterial cells in 5 mL of DF nitrogen-free medium (without (NH4)2SO4) and incubate at 30°C and 200 r·min. -1 After incubation at a constant temperature for 24 h, the precipitate was collected by centrifugation under the same conditions. Then, Tris-HCl buffer (5 mL, 0.1 mol·L⁻¹) was added. -1The cells were resuspended and washed in 7.5 mL of nitrogen-free DF medium (pH 7.6) twice; the cells were then resuspended again in 45 μL of sterile ACC solution (0.5 mol·L⁻¹). -1 Placed on a shaker at 30°C and 200 r·min -1 Incubate at a constant temperature for 24 h (this process induces the strain to produce ACC deaminase); then, increase the temperature of the culture medium at 5000 r·min. -1 Centrifuge for 20 min and collect the precipitate; add Tris-HCl solution (5 mL, 0.1 mol·L⁻¹). -1 The cells were resuspended and washed at pH 7.6, and the process was repeated twice. The precipitate was collected. 100 μL of the cell lysate was taken and dissolved in toluene by shaking. The lysate was stored at 4 °C for later use in the determination of protein content.

[0068] Enzyme activity assay: Take 200 μL of the remaining cell lysate into a 1.5 mL centrifuge tube, add 20 μL of ACC solution (0.5 mol·L⁻¹). -1 Mix the two solutions thoroughly, one without ACC solution, and react at 30 °C for 15 min. Then add 1 mL of HCl solution (0.56 mol·L⁻¹) to each solution. -1 Mix thoroughly at 11000 r·min -1 Centrifuge for 10 min (4°C), and collect the supernatant (800 μL each) into 5 mL centrifuge tubes for later use; add HCl solution (800 μL, 0.56 mol·L⁻¹) sequentially. -1 The reaction solution of 2,4-dinitrophenylhydrazine (300 μL) was reacted at 30 °C for 30 min, and then 22 mL of NaOH solution (2 mol·L⁻¹) was added. -1 Mix well, develop color, and then measure OD. 540 The absorbance values ​​of the samples were used as a blank control (using TrisHCl solution (0.1 M, pH 8.5)). The absorbance values ​​of the samples were substituted into the regression equation of the standard curve to obtain the α-ketobutyric acid content, and the amount of α-ketobutyric acid (μmol) was calculated. The total protein content (mg) in the cell lysate was then determined using the Bradford method. Finally, the amount of α-ketobutyric acid produced per unit protein content of bacterial cells per unit time was defined as the ACC deaminase activity, and the ACC deaminase activity (U·mg) was calculated according to the formula. -1 The calculation formula is as follows. Three replicates are set up for each strain.

[0069]

[0070] The standard curve is constructed based on the OD value of the α-ketobutyric acid standard sample. A simplified method is as follows: use 0.1 mol·L⁻¹... -1Prepare α-ketobutyric acid standard solutions (0, 0.2, 0.4, 0.6, 0.8, 1 mmol·L⁻¹) using Tris-HCl buffer (pH 8.5). -1 ), and 300 μL of 2,4-dinitrophenylhydrazine (2 mol·L⁻¹) was added to each. -1 Dissolved in HCl, with a mass concentration of 2 g·L⁻¹ -1 After thoroughly mixing, place in a 30℃ water bath and react for 30 min; then add 2 mL of NaOH (2 mol·L⁻¹). -1 After the color changes, the OD is measured. 540 The absorbance was measured using a pH 8.5 Tris-HCl buffer solution as a blank control; a standard curve was constructed based on the concentration of α-ketobutyric acid solution and its corresponding absorbance value.

[0071] (3) Determination of phosphorus solubility:

[0072] The phosphorus solubilizing ability of the strain was determined using the molybdenum-antimony colorimetric method. The method was as follows: 1 mL of the activated strain was inoculated into 50 mL of PKO medium. The control group was inoculated with an equal volume of sterile water. The mixture was incubated at 30°C and 150 rpm. -1 Incubate at a constant temperature for 7 days; then incubate at 11000 r·min -1 Centrifuge for 5 min and collect the supernatant. Add 1-2 drops of dinitrophenol indicator to the supernatant (30 mL), and adjust the solution to a slightly yellow color by micro-addition of NaOH solution (1 M, 10 M) and HCl solution (1 M, 5 M). Then accurately add 5 mL of molybdenum-antimony mixed colorimetric solution, shake thoroughly, and dilute to 50 mL with deionized water. Incubate at room temperature (above 15°C) for 30 min (within 8 h), then measure and record the OD. 700 Value. Calculate phosphorus content (mg·L) based on the standard curve. -1 ).

[0073] Standard curve plotting: Accurately pipette 5 mg·L⁻¹ -1 Dissolve 0, 2, 4, 6, 8, and 10 mL of K₂HPO₄ standard solution in 50 mL volumetric flasks. Simultaneously add an equal volume of blank solution to the sample solution used for colorimetric determination. Add 1-2 drops of dinitrophenol indicator. Adjust the solution to a pale yellow color by micro-addition of NaOH solution (1 M, 10 M) and HCl solution (1 M, 5 M). Then, accurately add 5 mL of molybdenum-antimony mixed colorimetric solution, shake thoroughly, and dilute to 50 mL with deionized water. Incubate at room temperature (above 15°C) for 30 min. Measure the OD corresponding to each standard solution. 700 The standard curve is constructed based on the concentration of the standard solution and its corresponding absorbance value.

[0074] (4) Siderophore synthesis capacity determination: The activated strain (0.5 mL) was inoculated into MKB liquid medium (5 mL), and an equal amount of uninoculated MKB liquid medium (1 mL) was added for the determination of the reference value (Ar). The culture was incubated at 30°C and 150 r·min. -1 After 48 h of incubation, 1 mL of culture medium was taken and mixed with the CAS detection solution at a 1:1 (v / v) ratio. After reacting at room temperature for 1 h, the OD was measured. 630 The values ​​(A for the experimental group and Ar for the reference group) are zeroed using distilled water during measurement. The relative content of siderophores in the sample is ultimately expressed as the A / Ar ratio; the smaller the value, the stronger the strain's ability to produce siderophores. (Note: In the experiment, if the strain produces siderophores, the reaction system is orange-yellow; if it does not produce siderophores, the reaction system remains blue.)

[0075] (5) Determination of extracellular polymeric substances (EPS) content

[0076] EPS content was determined according to the Congo red agar method. The method is as follows: 1 mL of the activated strain was inoculated into 40 mL of LB liquid medium containing 5% salinity and incubated at 37°C and 150 r·min. -1 Incubate overnight at a constant temperature for 72 h; then incubate at 8000 r·min -1 Centrifuge for 10 min to remove bacterial cells. Mix the supernatant with anhydrous ethanol at a ratio of 1:2 (v / v) and incubate overnight at 4°C. Then centrifuge to separate the precipitate, which is EPS. Dry the obtained EPS in hot air at 40°C for 2-3 days to obtain the dry weight, which is the EPS content.

[0077] Table 1. Determination of salt tolerance and growth-promoting potential indicators for each strain

[0078]

[0079] Table 1 shows that all strains exhibited good salt tolerance, with *Cossaconia covanni* 11 showing the best salt tolerance, surviving in a 10% salt medium. All strains possessed the ability to secrete IAA and siderophores, with IAA levels ranging from 2.84 to 15.93 mg / L and siderophore levels ranging from 1.55 to 64.60%. Except for *Enterobacter hygroscopicus* 24, all other strains produced ACC deaminase and extracellular polysaccharides, with ACC deaminase activity ranging from 0.02 to 3.71 U / mg and extracellular polysaccharide content ranging from 0.45 to 24.90 g / L. All strains also possessed phosphate-solubilizing capabilities, with phosphate solubilization amounts ranging from 0.68 to 730.82 mg / L.

[0080] Example 4. Interactions between strains

[0081] (1) Activation of strains: The two strains were inoculated onto LB liquid medium and cultured at 28℃ and 120 rpm / min with shaking for 10-12 h until the OD of each bacterial culture was reached. 600 ≈1.0.

[0082] (2) Cross-inoculation: Prepare LB solid medium, autoclave at 121°C for 15 min, cool to approximately 50°C and pour into 90 mm petri dishes to prepare solid medium. Use an inoculation loop to pick up the activated antagonistic strain and draw two parallel straight lines on the LB solid medium plate. Then use another inoculation loop to pick up the indicator strain and draw two straight lines perpendicular to the first two lines to form a cross-inoculation pattern.

[0083] (3) Culture and observation: Place the inoculated plates in a constant temperature incubator at a suitable temperature for 1 to 3 days. Observe the growth of colonies on the plates, paying particular attention to whether inhibition zones or growth inhibition bands appear in the cross-regional areas.

[0084] (4) Result judgment: If an obvious inhibition zone or growth inhibition zone appears in the cross area, it indicates that there is an antagonistic effect between the two strains; if there is no obvious inhibition, it indicates that there is no antagonistic effect between the two strains.

[0085] (5) Eight strains were paired up for antagonistic experiments, totaling 56 groups, and none of them showed any antagonistic effect.

[0086] Example 5. Indoor Potted Plant Experiment

[0087] (1) Take out 8 plant growth-promoting bacteria from the -80℃ freezer. On the clean bench, use a sterile inoculation loop to pick up a small amount of the bacteria. After drawing the first line on one side of the LB solid medium, ignite the inoculation loop and cool. Rotate the plate and touch the inoculation loop to the line in one area. Then draw a few sparse lines in the new area.

[0088] After reheating and cooling, streak the plates in three- and four-zone configurations (more zones make it easier to obtain single colonies). Invert the plates and incubate them in a constant temperature incubator for 3-5 days. Select morphologically uniform single colonies from the final streaked areas for 16SRNA sequencing and phylogenetic analysis to confirm them as pure cultures.

[0089] (2) Pick a portion of the pure culture and add it to LB liquid medium. Place it on a shaker and incubate at 28℃ and 120 rpm / min for 10-12 h until the OD of each bacterial culture reaches the specified value. 600 ≈1.0.

[0090] (3) Mix the bacterial solutions of 8 strains in equal volumes to obtain a mixed bacterial solution. Add the mixed bacterial solution to LB liquid medium at an inoculation rate of 5% and incubate at 28℃ and 120rpm for 12h to obtain the root-rhizosphere growth-promoting microbial solution.

[0091] (4) Sterilize the carbon-based organic fertilizer at 121℃ and 101Pa for 30 minutes to obtain an organic carrier; mix the organic carrier with 15% of the root-rhizosphere growth-promoting microbial inoculant solution and culture it thoroughly in a cool and ventilated place for 5 days to obtain the root-rhizosphere growth-promoting microbial agent of the organic carrier.

[0092] (5) Sterilize the cow and sheep manure organic fertilizer at 121℃ and 101Pa for 30 minutes to obtain an organic carrier; mix the organic carrier with 15% of the root-rhizosphere growth-promoting microbial inoculant solution and culture it thoroughly in a cool and ventilated place for 5 days to obtain the root-rhizosphere growth-promoting microbial inoculant.

[0093] (6) Sterilize biochar at 121℃ and 101Pa for 30 min to obtain organic carrier; mix 15% of the root-rhizosphere growth-promoting microbial inoculum of organic carrier with organic carrier, and mix and culture thoroughly in a cool and ventilated place for 5 days to obtain organic carrier root-rhizosphere growth-promoting microbial inoculum.

[0094] Potted Plant Experiment 1

[0095] Select plump, uniformly sized seeds of *Suaeda salsa* and *Suaeda salsa*, soak them in 10% hydrogen peroxide for 10 minutes to disinfect, and then rinse them four times with distilled water. Soak the seeds in ultrapure water and place them in a 37°C constant temperature incubator for germination.

[0096] After germination, the seeds were sown in pots (23 cm × 18 cm × 21.5 cm) containing 1.85 kg of soil. The soil was taken from the saline-alkali grassland of Xilinhot, Inner Mongolia, with a pH of 8.38 and a total salt content of 2.92 g / kg. Each pot contained 50 plants of *Suaeda salsa* and 40 plants of *Suaeda salsa*. 20 g of root-rhizosphere growth-promoting microbial inoculant (coated with cow or sheep manure organic fertilizer) was added to each pot (evenly mixed into the soil). The following treatments were also implemented:

[0097] Treatment with organic carrier root-rhizosphere growth-promoting microbial inoculant (MP): Load 2 mL of the root-rhizosphere growth-promoting microbial inoculant prepared in step (3) onto 20 g of sterilized organic fertilizer, and then add it to each flower pot;

[0098] Treatment with only bacterial inoculation solution (PGPB): Add 2 mL of the root-rhizosphere growth-promoting microbial inoculation solution prepared in step (3);

[0099] Treatment with only organic carrier (MOF): Add 20 g of sterilized cow and sheep manure organic fertilizer;

[0100] Control group (CK): No additives.

[0101] After sowing, spread 0.15 kg of soil evenly and water to control the soil's maximum water holding capacity to 80%.

[0102] Two months later, the aboveground and belowground biomass of plants in each treatment were compared, and the growth indicators and soil nutrient indicators of potted plants in each group were calculated compared with the control group. The results are shown in Tables 2, 3, 4, and 5.

[0103] Table 2 Biomass data of potted plants in each group

[0104]

[0105] Table 3. Increase in biomass of potted plants in each group compared to the control group.

[0106]

[0107] Table 4. Soil nutrient index data for each group of potted plants

[0108]

[0109] Table 5. Increase in soil nutrient indices of each group of potted plants compared to the control group.

[0110]

[0111] The results in Tables 2 and 3 show that in saline-alkali grassland soils, the root-rhizosphere growth-promoting microbial inoculant (MP) with cow and sheep manure organic fertilizer carrier significantly promoted the growth of *Suaeda salsa* and *Suaeda salsa*. Compared with the blank control (CK), the single sterilized cow and sheep manure organic fertilizer carrier (MOF), and the single root-rhizosphere growth-promoting microbial inoculant solution (PGPB), this inoculant treatment significantly increased the aboveground / belowground fresh weight and dry weight of both plants, with increases ranging from 53.17% to 2115.38%.

[0112] The results in Tables 4 and 5 show that in saline-alkali grassland soils, the root-rhizosphere growth-promoting microbial inoculant (MP) with cow and sheep manure organic fertilizer carrier significantly affected the soil organic matter and available nutrient content of *Suaeda salsa* and *Suaeda salsa*. Compared with the blank control (CK), single sterilized cow and sheep manure organic fertilizer carrier (MOF), and single root-rhizosphere growth-promoting microbial inoculant solution (PGPB), this inoculant treatment increased the soil organic matter content by 2.18–73.29% and the available nutrient content by 1.68–241.33%.

[0113] Potted Plant Experiment 2

[0114] Select plump, uniformly sized alfalfa and sheepgrass seeds, soak them in 10% hydrogen peroxide for 10 minutes to disinfect, and then rinse them 3-5 times with distilled water. Soak the forage seeds in ultrapure water and place them in a 37℃ constant temperature incubator for germination.

[0115] After germination, the seeds were sown in flowerpots (23 cm × 18 cm × 21.5 cm) containing 1.85 kg of soil. The soil in the flowerpots was taken from a typical grassland in Xilinhot, Inner Mongolia, characterized by severe degradation due to grazing and mowing. Each pot contained 40 plants of Leymus chinensis and 15 plants of Alfalfa. 20 g of a carbon-based organic fertilizer carrier for root-rhizosphere growth promotion was added to each flowerpot (evenly mixed into the soil). The following treatments were also implemented:

[0116] Add carbon-based organic fertilizer carrier root-rhizosphere growth-promoting microbial inoculant (LP): Take 2 mL of the composite microbial inoculant prepared in step (3) of Example 5, load it onto 20 g of sterilized biochar, and then add it to each flower pot;

[0117] Treatment with only bacterial inoculation solution (PGPB): Add 2 mL of the root-rhizosphere growth-promoting microbial inoculation solution prepared in step (3);

[0118] Treatment with added organic carrier only (LOF): Add 20 g of sterilized carbon-based organic fertilizer;

[0119] Control group (CK): No additives.

[0120] After sowing, spread 0.15 kg of soil evenly and water to control the soil's maximum water holding capacity to 80%.

[0121] Two months later, the aboveground and belowground biomass of plants in each treatment were compared, and the growth index and soil nutrient index of potted plants in each group were calculated compared with the control group. The results are shown in Tables 6, 7, 8 and 9.

[0122] Table 6 Biomass data of potted plants in each group

[0123]

[0124] Table 7. Increase in biomass of potted plants in each group compared to the control group.

[0125]

[0126] Table 8. Soil nutrient index data for each group of potted plants

[0127]

[0128] Table 9. Increase in soil nutrient indices of each group of potted plants compared to the control group.

[0129]

[0130] Tables 6-7 show that in typical grassland soils severely degraded by grazing and mowing, the root-rhizosphere growth-promoting microbial inoculant (LP) with carbon-based organic fertilizer carrier significantly promoted the growth of alfalfa and sheepgrass. Compared with the blank control (CK), single sterilized carbon-based organic fertilizer carrier (LOF), and single root-rhizosphere growth-promoting microbial inoculant solution (PGPB), this inoculant treatment significantly increased the aboveground / belowground fresh weight and dry weight of both plants, with increases ranging from 31.70% to 582.68%.

[0131] Tables 8-9 show that in severely degraded typical grassland soils under grazing conditions, the effects of carbon-based organic fertilizer carrier-rhizosphere growth-promoting microbial inoculant (LP) on soil nutrients were significant. Compared with the blank control (CK), single sterilized carbon-based organic fertilizer carrier (LOF), and single rhizosphere growth-promoting microbial inoculant solution (PGPB), this inoculant treatment significantly increased soil organic matter and available nutrients for the two plant species, with increases ranging from 22.04% to 43.49% and 21.97% to 96.11%, respectively.

[0132] Potted Plant Experiment 3

[0133] Select plump, uniformly sized alfalfa and sheepgrass seeds, soak them in 10% hydrogen peroxide for 10 minutes to disinfect, and then rinse them 3-5 times with distilled water. Soak the forage seeds in ultrapure water and place them in a 37℃ constant temperature incubator for germination.

[0134] After germination, the seeds were sown in flowerpots (23 cm × 18 cm × 21.5 cm) containing 1.85 kg of soil. The soil in the flowerpots was taken from severely degraded typical grasslands and desertified grasslands in Xilinhot, Inner Mongolia. Each pot contained 40 seedlings of Leymus chinensis and 15 seedlings of Alfalfa. 20 g of biochar carrier root-rhizosphere growth-promoting microbial inoculant was added to each flowerpot (evenly mixed into the soil). The following treatments were also implemented:

[0135] Add biochar carrier root-rhizosphere growth-promoting microbial inoculant (BP): Take 2 mL of the composite microbial inoculant prepared in step (3) of Example 5, load it with 20 g of sterilized biochar, and then add it to each flower pot;

[0136] Treatment with only bacterial inoculation solution (PGPB): Add 2 mL of the root-rhizosphere growth-promoting microbial inoculation solution prepared in step (3);

[0137] Treatment with only organic carrier added (BC): Add 20 g of sterilized biochar;

[0138] Control group (CK): No additives.

[0139] After sowing, spread 0.15 kg of soil evenly and water to control the soil's maximum water holding capacity to 80%.

[0140] Two months later, the aboveground and belowground biomass of plants in each treatment were compared, and the growth index of each potted plant group was calculated compared with the control group. The results are shown in Tables 10 and 11.

[0141] Table 10 Biomass data of potted plants in each group

[0142]

[0143] Table 11. Increase in biomass of potted plants in each group compared to the control group.

[0144]

[0145] Tables 10-11 show that in severely degraded typical grasslands and desertified grassland soils, the biochar-carrier root-rhizosphere growth-promoting microbial inoculant (BP) significantly promoted the growth of alfalfa and sheepgrass. Compared with the blank control (CK), sterilized biochar carrier alone (BC), and root-rhizosphere growth-promoting microbial inoculant solution alone (PGPB), this inoculant treatment significantly increased the aboveground / belowground fresh weight and dry weight of both plants, with increases ranging from 11.35% to 265.59%.

[0146] Example 6. The growth-promoting effect of organic carrier-based root-rhizosphere growth-promoting microbial agents on degraded grassland vegetation.

[0147] The experiment was conducted in Xilinhot, Inner Mongolia in June 2024, in typical grasslands with moderate degradation due to grazing, severe degradation due to grazing, and degradation due to mowing. The preparation method of the organic carrier root-rhizosphere growth-promoting microbial inoculant was the same as in Example 5. In each type of grassland, the experiment was divided into five groups: carbon-based organic fertilizer carrier root-rhizosphere growth-promoting microbial inoculant (LP), cow / sheep manure organic fertilizer carrier root-rhizosphere growth-promoting microbial inoculant (MP), AMF fungal inoculant (AMF), Aijia No. 3 organic-inorganic composite carrier microbial inoculant (Aijia No. 3), and a control (CK) group. The LP group and MP group were treated with root-rhizosphere growth-promoting microbial inoculants using carbon-based organic fertilizer and cow / sheep manure organic fertilizer as carriers, respectively. The application rate of the organic carrier root-rhizosphere growth-promoting microbial inoculant was 0.4 kg / m². 2 The application method was strip application; the AMF inoculant was purchased from Qianhe Dingsheng Technology Co., Ltd., and consisted of *Claroideoglomus claroideum*, *Claroideogloms etunicatum*, *Funneliformis mosseae*, *Septoglomus deserticola*, and *Rhizophagus irregularis*, with an application rate of 0.15 g / m³.2 Aijia No. 3 organic-inorganic composite carrier microbial agent was purchased from Harbin Baidu Technology Development Co., Ltd., and the application rate was 30g / m³. 2 The control group (CK) received no treatment. The application method was as follows: a heavy-duty tractor pulled a hydraulic disc root cutter at the front to cut into the soil (10 cm deep), severing old roots, breaking up compaction, and creating loose soil cracks. A fertilizer applicator (strip applicator type) followed closely behind, precisely applying each group's microbial inoculants into the soil cracks created by the root cutter. The tractor should be driven at a constant speed (approximately 6 km / h) to ensure stable root cutting depth, even fertilization, and close coordination between the two operations, allowing the microbial inoculants to effectively penetrate the root zone and reduce volatilization loss. Three months after applying the microbial inoculants, the aboveground dry weight, underground dry weight, and cover of all plants within a 1-square-meter sample plot for each group were measured. The growth indicators of the grassland vegetation treated with the organic carrier root-rhizosphere growth-promoting microbial inoculants were calculated compared to the control group and other microbial inoculants, as shown in Tables 12-15. The increases in organic matter, available nitrogen, available phosphorus, and available potassium in soil treated with organic-carrier root-rhizosphere growth-promoting microbial agents compared to the control group were statistically analyzed, and the results are shown in Tables 16 and 17.

[0148] Table 12 Growth index data of grassland vegetation using organic carrier microbial inoculants

[0149]

[0150] Table 13. Increase in growth indicators of grassland vegetation compared to the control group using organic carrier microbial inoculants.

[0151]

[0152] Table 14. Increase in growth indicators of grassland vegetation compared to AMF inoculant using organic carrier microbial agents.

[0153]

[0154] Table 15. Increase in growth indicators of grassland vegetation compared to Aijia No. 3 microbial inoculant using organic carrier microbial agents.

[0155]

[0156] Table 16 Soil nutrient index data of grasslands with organic carrier microbial inoculants

[0157]

[0158] Table 17. Increase in soil nutrient indices in grasslands treated with organic-carrier microbial inoculants compared to the control group.

[0159]

[0160] Tables 12-15 show that both the carbon-based organic fertilizer carrier root-rhizosphere growth-promoting microbial inoculant (LP) group and the cattle and sheep manure organic fertilizer carrier root-rhizosphere growth-promoting microbial inoculant (MP) group can effectively promote vegetation growth on actual degraded grasslands. Compared to the control group (CK), the two organic carrier root-rhizosphere growth-promoting microbial inoculants significantly increased the aboveground / belowground dry weight and vegetation cover, with increases of 1.50%~227.27% and 38.89%~79.25%, respectively. Compared to the AMF inoculant treatment, the two organic carrier root-rhizosphere growth-promoting microbial inoculants significantly increased the aboveground / belowground dry weight and vegetation cover, with increases of 11.63~94.59% and 24.37~43.93%, respectively. Compared to the Aijia No. 3 organic-inorganic composite carrier microbial inoculant, the two organic carrier root-rhizosphere growth-promoting microbial inoculants significantly increased the aboveground dry weight and vegetation cover, with increases of 20.87~84.82% and 12.66~49.01%, respectively. Moreover, the growth-promoting effect of the organic carrier microbial inoculant of this invention is superior to that of the commercially available AMF inoculant and the Aijia No. 3 organic-inorganic composite carrier microbial inoculant.

[0161] Tables 16-17 show that both the carbon-based organic fertilizer carrier root-rhizosphere growth-promoting microbial inoculant (LP) group and the cow / sheep manure organic fertilizer carrier root-rhizosphere growth-promoting microbial inoculant (MP) group effectively increased soil nutrient content in actual degraded grasslands. Compared with the control group (CK), the two organic carrier root-rhizosphere growth-promoting microbial inoculants significantly increased soil organic matter and available nutrient content, with increases of 12.95-47.15% and 4.63-266.56%, respectively. Furthermore, the organic carrier microbial inoculant of this invention showed better soil improvement effects than the commercially available inoculant AMF and the Aijia No. 3 organic-inorganic composite carrier microbial inoculant.

[0162] As can be seen from the above embodiments, the present invention provides an organic carrier root-rhizosphere growth-promoting microbial agent for the ecological restoration of degraded grasslands, its preparation method, and its application. The organic carrier root-rhizosphere growth-promoting microbial agent of the present invention can significantly increase the aboveground dry weight and vegetation cover of degraded grassland vegetation communities, thereby promoting the ecological restoration of degraded grasslands.

[0163] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An organic carrier endo-rhizosphere growth promoting microbial inoculant for degraded prairie ecological restoration, characterized in that, The package comprises endo- and rhizosphere growth promoting microbial liquid and organic carrier, wherein the endo- and rhizosphere growth promoting microbial liquid comprises endo- and rhizosphere growth promoting bacteria; The endo- and rhizosphere growth promoting bacteria comprises Bacillus arrosporesporus K4, Bacillus 9-3-1, Bacillus safensis 11-5-4 and Halomonas L31; The rhizosphere growth promoting bacteria comprises Serratia marcescens 5, Serratia marcescens 23, Kosakonia kwangjuensis 11 and Enterobacter cloacae 24; The Bacillus arrosporesporus K4, Bacillus 9-3-1, Bacillus safensis 11-5-4 and Halomonas L31 are preserved in Guangdong Microbial Culture Collection Center, and the preservation numbers are GDMCC NO. 65395, GDMCC NO. 65392, GDMCC NO. 65394 and GDMCC NO. 65397, respectively; The Serratia marcescens 5, Serratia marcescens 23, Kosakonia kwangjuensis 11 and Enterobacter cloacae 24 are preserved in Guangdong Microbial Culture Collection Center, and the preservation numbers are GDMCC NO. 65386, GDMCC NO. 65389, GDMCC NO. 65391 and GDMCC NO. 65388, respectively. The organic carrier comprises carbon-based organic fertilizer, cattle and sheep manure organic fertilizer or biochar. The endo- and rhizosphere growth promoting microbial liquid is 10-20% of the carrier mass.

2. The organic carrier endo-rhizosphere growth promoting microbial inoculum of claim 1, wherein, The carbon-based organic fertilizer carrier is prepared by fermenting lignite; and the biochar is prepared by calcining a mixture of corn, wheat and peanut straw.

3. The organic carrier endo-rhizosphere growth promoting microbial inoculum of claim 1, wherein, The cattle and sheep manure organic fertilizer carrier comprises matured cattle and sheep manure.

4. The organic carrier endo-rhizosphere growth promoting microbial inoculum of claim 1, wherein, The effective viable cell number in the organic carrier endo-rhizosphere growth promoting microbial inoculant is ≥1.4×10 8 cfu / g, and the moisture content is ≤25%.

5. The organic carrier endo-rhizosphere growth promoting microbial inoculum of claim 2, wherein, 7. A preparation method of the organic carrier endo- and rhizosphere growth promoting microbial agent according to any one of claims 1-6, comprising the following steps:

6. The organic carrier endo-rhizosphere growth promoting microbial inoculum of claim 2, wherein, S2. Mixing the microbial liquids in step S1 in equal volume to obtain mixed microbial liquid; S3. Inoculating the mixed microbial liquid into fermentation medium and culturing at 26-30°C and 100-140 rpm for 10-14 h to obtain the endo- and rhizosphere growth promoting microbial liquid; S4. Mixing the endo- and rhizosphere growth promoting microbial liquid and the organic carrier, which is carbon-based organic fertilizer, cattle and sheep manure organic fertilizer or biochar, at 26-30°C for 4-6 days, and then drying to obtain the product. S1. Cultured the B. vallismortis K4, B. pumilus 9-3-1, B. safensis 11-5-4, Halomonas sp. L31, Serratia rubidaea 5, Serratia rubidaea 23, Kocuria rhodococcomas 11 and Enterobacter neohylicus 24 to the OD of the bacterial liquid 600 = 0.8 ~ 1.0, and collected the bacterial liquid; The inoculation amount in step S3 is 10-20%, and the fermentation medium is LB liquid medium.

9. Application of the organic carrier endo- and rhizosphere growth promoting microbial agent according to any one of claims 1-6 in at least one of the following: (1) Application in repairing degraded grassland, desertified grassland and salinized grassland; 8. The preparation method according to claim 7, characterized in that, (2) Application in promoting plant growth; (3) Application in promoting grassland vegetation restoration. The plants include Suaeda salsa, Puccinellia distans, Sophora alopecuroides, Elymus nutans, Medicago ruthenica or Leymus chinensis. ​ ​ 10. Use according to claim 9, characterized in that, ​

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