Organic carrier in-root-rhizosphere growth-promoting microbial agent for ecological restoration of degraded grassland as well as preparation method and application of organic carrier in-root-rhizosphere growth-promoting microbial agent

Abstract

The invention provides an organic carrier in-root-rhizosphere growth-promoting microbial agent for ecological restoration of degraded grassland as well as a preparation method and application of the microbial agent, and belongs to the technical field of grassland ecological restoration and microbial agents. The organic carrier growth-promoting microbial agent disclosed by the invention is prepared from four kinds of growth-promoting bacteria (PGPE) in roots, four kinds of growth-promoting bacteria (PGPR) in roots and an organic carrier, the PGPE comprises terracotta warrior bacillus K4, bacillus 9-3-1, bacillus deserticola 11-5-4 and halomonas L31, the PGPR comprises serratia rubra 5, serratia rubra 23, cosinia cosinii 11 and enterobacter parabarium 24, and the organic carrier is a carbon-based organic fertilizer, a cattle and sheep manure organic fertilizer or biochar. When the organic carrier growth-promoting microbial agent is applied to the degraded grassland, the biomass and coverage of plant communities can be remarkably improved. The organic carrier growth-promoting microbial agent can be used for ecological restoration of degraded grassland.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of grassland ecological restoration and microbial agent, and particularly relates to an organic carrier endo- rhizosphere growth promoting microbial agent for degraded grassland ecological restoration and a preparation method and application thereof. BACKGROUND

[0002] Grassland degradation refers to a process of reverse succession of grassland ecosystem structure and function, leading to continuous decline of productivity, biodiversity, ecological function and self-recovery ability of the grassland, which includes degradation of vegetation and soil. The main performances are that the original structure and function of the grassland are destroyed, the ecological diversity and stability of the ecosystem are reduced, the vegetation coverage is decreased, the excellent grass species are significantly reduced, the quality of various grasses is deteriorated, the productivity and quality of the grasses are decreased, the physicochemical properties of the grassland soil are changed, the soil is degraded, desertified and salinized, the nutrient content is reduced, and the soil erosion is aggravated. In the past few decades, the restoration of the degraded grassland all over the world aims at the restoration of plant community, mainly focusing on improving the plant productivity and reconstruction of local vegetation. In the evolution of the grassland, the plant-soil-microorganism synergistically interact to adapt to the fluctuation of climate and the grazing of animals. Therefore, the grassland degradation is the overall decline of the functions of plants, soil and microorganisms, and the reason why the severely degraded grassland is difficult to restore is that multiple restrictions including plant propagules, nutrients and microorganisms occur.

[0003] Grassland ecological restoration is a process of restoring degraded and damaged grassland to a new long-term stable and healthy state. At present, the common methods of grassland ecological restoration include: no-till and reseeding, rational grazing, artificial grassland planting, fencing, ploughing and fertilization, microbial remediation, etc. However, compared with other restoration measures, the related research of microbial remediation is less. Soil microbial diversity is an important driving force for the development of plant communities on the ground, and it plays a key role in determining the ecological response of terrestrial ecosystems to environmental changes. Grassland ecological vegetation restoration depends largely on the microbial community in the grassland soil. Microbial inoculation can promote the growth of forage grasses and improve the soil environment. Microbes can also help forage grasses obtain nitrogen, phosphorus and other nutrients in the soil. The diversity and abundance of microorganisms in degraded grassland soil will decrease significantly, changing the structure and function of microbial communities, thus limiting the process of grassland ecological restoration. Supplementing beneficial microorganisms can help restore the ecology of degraded grassland. Existing literature shows that three types of microbial functional groups play an important role in the process of grassland ecological restoration, namely plant growth-promoting bacteria, arbuscular mycorrhizal fungi and nitrogen-fixing bacteria, which are essential for improving soil health and maintaining soil productivity in stressful environments. Among them, plant growth-promoting bacteria can help forage grasses obtain soil nutrients in drought, saline-alkali and other stress environments. Plant growth-promoting fungi can decompose and utilize cellulose, pectin and starch to provide nutrients for forage grasses. Symbiotic and non-symbiotic nitrogen-fixing bacteria can promote the absorption and growth of forage 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 forage grasses.

[0004] Plants usually require more than one symbiotic microorganism to resist environmental stress, and inoculation 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 reevaluation of the benefits that plants can derive from interactions with microbial communities rather than from individual members of the community. Therefore, there is an increasing recognition of the untapped potential of beneficial microbial communities in improving plant adaptability. Endophytes exist in almost all plant tissues and determine the health, growth and development, and secondary metabolism of host plants, and confer a wider range of environmental adaptability. Endophytic bacteria are more closely associated with plants and have become a natural component of plant microecosystems through long-term coevolution, and are believed to play a more important role in synergistically resisting stress. Studies have shown that the presence of multiple endophytic bacteria is a very effective strategy for improving plant stress tolerance, and its mechanism is similar to that of rhizosphere bacteria. Host plants can provide nutrients and niches for the growth of endophytic bacteria, reducing competition pressure and the impact of stress environments, and maintaining stable colonization in plants. Therefore, endophytic bacteria play a more persistent and important role in plant resistance to environmental stress, and are more economical and environmentally friendly. Recent studies have shown that the combined use of multiple rhizosphere and endophytic bacteria under environmental stress can have a synergistic effect on plant growth, which is more conducive to the exertion of their beneficial effects. Therefore, the development and use of endophytic and rhizosphere bacteria combined microbial communities to improve plant tolerance to adverse environments can open up a new way to promote the ecological restoration process of degraded grasslands.

[0005] Under stress conditions, the most important thing to ensure that the inoculation of plant growth-promoting bacteria has a beneficial effect on plant growth is to ensure the colonization and survival of plant growth-promoting bacteria in the rhizosphere and rhizosphere of plants. However, the survival of plant growth-promoting bacteria in stress environments depends not only on the availability of niches, but also on the ability of plant growth-promoting bacteria to compete with local microorganisms with good adaptability. The application of microbial technology has not been successful in degraded ecosystems located in arid and semiarid regions, especially in areas with low soil organic matter, and the main reason is that it cannot provide a good substrate environment and nutrient source for its growth. In fact, the continuous supply of carbon and energy helps to successfully establish beneficial bacteria in the root zone of plants and enhance the competitiveness of plant growth-promoting bacteria. Organic matter as a carrier material has obvious advantages. On the one hand, organic matter can provide direct nutrient elements for plant growth; on the other hand, organic matter as a carrier can provide sufficient energy and nutrients for the survival and growth of microbial inoculants and provide a favorable habitat to protect them from pathogens, which helps to exert the beneficial functions of plant growth-promoting bacteria. Therefore, the use of Inner Mongolia characteristic organic solid waste resources to construct organic carrier microbial inoculants is the basis for ensuring the beneficial effects of plant growth-promoting bacteria in the restoration of degraded grasslands.

[0006] Although some microbial inoculants for degraded grassland ecological restoration have been developed, the existing microbial inoculants have the following defects: 1. Due to the possible competitive inhibition of arbuscular mycorrhizal fungi, rhizosphere bacteria, rhizosphere fungi and nitrogen-fixing bacteria, single strain or simple combination of a small number (2-3 strains) is mostly used, and the function is single; 2. When developing microbial inoculants using plant growth-promoting bacteria, most only consider soil or rhizosphere growth-promoting bacteria, ignoring the important role of endo-root growth-promoting bacteria and the synergistic effect of the combination of endo-root growth-promoting bacteria and rhizosphere growth-promoting bacteria; 3. The adaptability of the carrier to the functional microbial flora is insufficient, which affects the colonization activity and long-term effect of the microorganisms, and thus affects the growth-promoting effect of the microbial inoculant; 4. The strains in the microbial inoculant are mostly foreign strains, which have poor adaptability in adverse conditions such as degraded grassland, saline-alkali grassland or sandy grassland, and unstable growth-promoting effect. Therefore, it is necessary to develop a carrier microbial inoculant suitable for degraded grassland ecological restoration in Inner Mongolia. SUMMARY

[0007] Therefore, the present application provides an organic carrier endo-root-rhizosphere growth-promoting microbial inoculant for degraded grassland restoration and a preparation method and application thereof to solve the above problems.

[0008] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions: The present application provides an organic carrier endo-root-rhizosphere growth-promoting microbial inoculant for degraded grassland ecological restoration, which comprises an endo-root-rhizosphere growth-promoting microbial solution and an organic carrier, wherein the endo-root-rhizosphere growth-promoting microbial solution comprises endo-root growth-promoting bacteria and rhizosphere growth-promoting bacteria. The endo-root growth-promoting bacteria comprise B. terrigena K4, B. subtilis 9-3-1, B. safensis 11-5-4 and Halomonas L31. The rhizosphere growth-promoting bacteria comprise S. rubida 5, S. rubida 23, K. kosakonia 11 and E. asburiae 24. The B. terrigena K4, B. subtilis 9-3-1, B. safensis 11-5-4 and Halomonas L31 are preserved in the 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 S. rubida 5, S. rubida 23, K. kosakonia 11 and E. asburiae 24 are preserved in the Guangdong Microbial Culture Collection Center, and the preservation numbers are GDMCC NO.65386, GDMCC NO.65389, GDMCC NO.65391 and GDMCC NO.65388, respectively.

[0009] Preferably, the organic carrier comprises carbon-based organic fertilizer, cattle and sheep manure organic fertilizer or biochar.

[0010] Preferably, the endo- rhizosphere growth promoting microbial liquid is 10-20% of the mass of the carrier.

[0011] Preferably, the effective viable bacterial count in the endo- rhizosphere growth promoting microbial agent is ≥1.4×10 8 cfu / g, and the moisture content is ≤25%.

[0012] Preferably, the carbon-based organic fertilizer is prepared by fermenting lignite.

[0013] Preferably, the cattle and sheep manure organic fertilizer comprises matured cattle manure and / or sheep manure.

[0014] Preferably, the biochar is prepared by calcining a mixture of corn, wheat and peanut straw.

[0015] The application also provides a preparation method of the organic carrier endo- rhizosphere growth promoting microbial agent, characterized by comprising the following steps: S1. Culturing Bacillus figurans K4, Bacillus sp. 9-3-1, Bacillus safensis 11-5-4, Halomonas sp. L31, Serratia rubidaea 5, Serratia rubidaea 23, Kosakonia kwangjuensis 11 and Enterobacter bugandensis 24 to an OD 600 =0.8-1.0 of the bacterial liquid, and collecting the bacterial liquid; S2. Mixing the bacterial liquids in step S1 in equal volumes to obtain a mixed bacterial liquid; S3. Inoculating the mixed bacterial liquid into a fermentation medium and culturing at 26-30°C and 100-140 rpm for 10-14 h to obtain the endo- rhizosphere growth promoting microbial liquid; S4. Mixing the endo- rhizosphere growth promoting microbial liquid with an organic carrier, such as carbon-based organic fertilizer, cattle and sheep manure organic fertilizer or biochar, and culturing at 26-30°C for 4-6 days, and then drying to obtain the endo- rhizosphere growth promoting microbial agent.

[0016] Preferably, the inoculation amount in step S3 is 10-20%.

[0017] Preferably, the fermentation medium is LB liquid medium.

[0018] The application also provides application of the organic carrier endo- rhizosphere growth promoting microbial agent in at least one of the following: (1) Application in repairing degraded grasslands; (2) Application in promoting plant growth; (3) Application in promoting grassland vegetation restoration.

[0019] Preferably, the plants include Suaeda salsa, Puccinellia distans, Sophora alopecuroides, Elymus nutans, Medicago ruthenica or Leymus chinensis.

[0020] Preferably, the degraded grassland includes degraded grassland, desertified grassland and salinized grassland.

[0021] By adopting the technical scheme, the application has the following beneficial effects: the organic carrier endo- rhizosphere growth promoting microbial agent includes four endo- rhizosphere growth promoting bacteria (PGPE), four rhizosphere growth promoting bacteria (PGPR) and an organic carrier; the endo- rhizosphere growth promoting bacteria (PGPE) include Bacillus terrae K4, Bacillus 9-3-1, Bacillus safensis 11-5-4 and Halomonas sp. L31; the rhizosphere growth promoting bacteria (PGPR) include Serratia rubidaea 5, Serratia rubidaea 23, Kosakonia cowanii 11 and Enterobacter hormaechei 24; and the organic carrier is carbon-based organic fertilizer, cow and sheep manure organic fertilizer or biochar. Application of the organic carrier endo- rhizosphere growth promoting microbial agent in the degraded grassland can significantly improve the biomass and coverage of the vegetation community. The organic carrier endo- rhizosphere growth promoting microbial agent can be used for ecological restoration of the degraded grassland.

[0022] Biological preservation instructions Serratia rubidaea 5 is taxonomically named as Serratia rubidaea, which was preserved in the Guangdong Microbial Digital Culture Collection Center on October 31, 2024, with a preservation number of GDMCC NO.65386 and a preservation address of No. 59 Building, 5th Floor, 100 Middle Liangma Street, Guangzhou.

[0023] Enterobacter hormaechei 24 is taxonomically named as Enterobacter hormaechei, which was preserved in the Guangdong Microbial Digital Culture Collection Center on October 31, 2024, with a preservation number of GDMCC NO.65388 and a preservation address of No. 59 Building, 5th Floor, 100 Middle Liangma Street, Guangzhou.

[0024] Serratia rubidaea 23 is taxonomically named as Serratia rubidaea, which was preserved in the Guangdong Microbial Digital Culture Collection Center on October 31, 2024, with a preservation number of GDMCC NO.65389 and a preservation address of No. 59 Building, 5th Floor, 100 Middle Liangma Street, Guangzhou.

[0025] Kosakonia cowanii 11 is taxonomically named as Kosakonia cowanii, which was preserved in the Guangdong Microbial Digital Culture Collection Center on October 31, 2024, with a preservation number of GDMCC NO.65391 and a preservation address of No. 59 Building, 5th Floor, 100 Middle Liangma Street, Guangzhou.

[0026] The taxonomic name of Bacillus sp. 9-3-1 is Bacillus sp., which was preserved in Guangdong Microbial Culture Collection Center on October 31, 2024, with a preservation number of GDMCC NO. 65392 and a preservation address of 5th Floor, Building 59, 100 Middle Lieth Road, Guangzhou.

[0027] The taxonomic name of Bacillus safensis 11-5-4 is Bacillus safensis, which was preserved in Guangdong Microbial Culture Collection Center on October 31, 2024, with a preservation number of GDMCC NO. 65394 and a preservation address of 5th Floor, Building 59, 100 Middle Lieth Road, Guangzhou.

[0028] The taxonomic name of Bacillus bingmayongensis K4 is Bacillus bingmayongensis, which was preserved in Guangdong Microbial Culture Collection Center on December 23, 2024, with a preservation number of GDMCC NO. 65395 and a preservation address of 5th Floor, Building 59, 100 Middle Lieth Road, Guangzhou.

[0029] The taxonomic name of Halomonas sp. L31 is Halomonas sp., which was preserved in Guangdong Microbial Culture Collection Center on December 23, 2024, with a preservation number of GDMCC NO. 65397 and a preservation address of 5th Floor, Building 59, 100 Middle Lieth Road, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is a repair test area of typical grassland with moderate degradation by grazing.

[0031] Figure 2 It is a repair test area of typical grassland with different causes and degrees of degradation.

[0032] Figure 3 It is the repair effect of organic carrier microbial agent on typical grassland degraded by cutting.

[0033] Figure 4 It is the repair effect of organic carrier microbial agent on typical grassland with moderate degradation by grazing.

[0034] Figure 5 It is the repair effect of organic carrier microbial agent on typical grassland with severe degradation by grazing. DETAILED DESCRIPTION

[0035] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0036] Preparation of LB liquid medium: take 10 g of proteose peptone, 5 g of yeast extract, 10 g of sodium chloride, add distilled water to 1000 mL, adjust pH to 7.0, sterilize at 121℃ for 20 min.

[0037] Preparation of LB solid medium: take 10 g of proteose peptone, 5 g of yeast extract, 10 g of sodium chloride, 10 g of agar, add distilled water to 1000 mL, adjust pH to 7.0, sterilize at 121℃ for 20 min.

[0038] The carbon-based organic fertilizer in the embodiment of the application is provided by Yuantaihong (Baotou) Biotechnology Co., Ltd., the matured cow and sheep manure is provided by Inner Mongolia Gujung Biotechnology Co., Ltd., and the biochar is purchased from Pingdingshan Carboner Nu New Material Co., Ltd.

[0039] Example 1. Strain isolation and culture The dominant plant roots and rhizosphere soil in Bayannur City, Wulateqian Banner, Bayannur City, Alashan Banner, Alashan Left Banner, Sunit Right Banner of Xilingol League, Bayannur City, Wulateqian Banner, Bayannur City, Hangjinqi Banner, and Taipusi Banner of Xilingol League were taken, the surface of the dominant plant roots was sterilized, ground into powder, then 1 g of plant root powder and rhizosphere soil was taken into 1 mL of sterile PBS buffer, and mixed uniformly with a pipette gun, to obtain a microbial liquid. Each microbial liquid was diluted by 10 times gradient, and diluted into 10 -2 ~10 -7 Each 0.1 mL of diluted microbial liquid of each concentration was coated on the LB solid medium.

[0040] The culture dish was placed in a 28℃ incubator, and when single colonies appeared on the surface, single bacterial colonies were picked according to the differences in morphology and color of the colonies, and further purified and cultured. The bacterial single colonies were streaked on the LB solid medium and subcultured for several times until the color, size and morphology of the colonies on the medium were completely consistent, and pure culture was obtained. All the obtained pure colonies were stored in 50% glycerol and stored at -80℃.

[0041] Example 2. Strain identification The single colonies were picked into 1.5 mL centrifuge tubes containing 1 mL of LB medium, and cultured at 30℃, 200 rpm for 24 h. The bacterial liquid was used as a template, and universal primers 27F / 1492R were used for 16s rRNA sequence amplification.

[0042] The PCR amplification reaction system was 50 μL, including 25 μL 2xTaq enzyme, 1 μL 27F primer, 1 μL 1492R primer, 1 μL bacterial liquid and 22 μL ddH2O. The amplification conditions were as follows: 95°C pre-denaturation for 10 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 32 cycles, 72°C extension for 5 min, and the amplification product was stored at 4°C. The amplification product was separated and identified by 1% agarose gel electrophoresis, and the PCR product was sent to Beijing Qikexin Biotechnology Co., Ltd. for sequencing. The determination results showed that four strains of intraradical growth promoting bacteria (PGPE) and four strains of rhizosphere growth promoting bacteria (PGPR) were isolated. The four strains of intraradical growth promoting bacteria (PGPE) included one strain of B. terra, one strain of B. sp., one strain of B. safensis and one strain of Halomonas sp.; the four strains of rhizosphere growth promoting bacteria (PGPR) included two strains of Serratia marcescens, one strain of Kosakonia koreensis and one strain of Enterobacter hormaechei.

[0043] Example 3. Determination of salt tolerance, IAA secretion, ACC deaminase activity, phosphorus solubilization and iron carrier and extracellular polymer EPS production of each strain (Table 1) 1. Identification of salt tolerance of strains: all the primary screening strains (glycerol bacteria preservation liquid) were inoculated in the base sterilized LB liquid medium according to 1:1 (v / v) for batch activation (35°C, 130 r·min -1 12 h), and LB solid culture medium with NaCl content of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% and 11% was configured, all with pH of the basic screening strain pH condition (i.e. pH 8.0), the activated strains were inoculated with sterilized inoculation rod, respectively, and were plated and cultured at 35°C for 24-48 h, the growth of the strains was observed and the results were recorded, and the salt tolerance of the strains was determined.

[0044] 2. Quantitative determination of growth promoting function of strains Before determining the growth promoting function of the strains, each strain to be tested needs to be activated and the concentration of the strain to be tested needs to be ensured.

[0045] In order to ensure the highest growth activity of each strain, each strain was cultured and activated under the optimum pH and temperature conditions. The specific activation method is as follows: take each single strain glycerol bacterial liquid (400 μL) in sterilized LB liquid medium (40 mL, the medium is prepared under the optimum pH condition) for activation culture (the optimum temperature, 130 r·min -1 constant temperature culture), and the culture is stopped when the OD 600 value is 1.0 for standby use.

[0046] (1) Indole acetic acid (IAA) content determination: The ability of the strains to produce IAA was determined by the Salkowski colorimetric method as follows: the activated strains (1 mL) were inoculated into DF liquid medium (40 mL) containing L-tryptophan (200 mg·L -1 ) (with no inoculation as a control group), and incubated at 30°C, 180 r·min -1 for 48 h. The OD 600 value was determined after keeping the absorbance value of each strain consistent. After centrifugation at 10,000 r·min -1 for 3 min, 1 mL of supernatant was taken and mixed with 4 mL of Salkowski color developing solution (the control group was operated consistently). The mixture was placed at room temperature in the dark for 20 min, and the OD 530 value was determined. The IAA content (mg·L -1 ) was calculated according to the standard curve.

[0047] The standard curve was prepared according to the OD value of the 3-indoleacetic acid standard sample. The method is as follows: the 3-indoleacetic acid standard solution was prepared, mixed with 4 mL of Salkowski color developing solution, and placed at room temperature in the dark for 20 min. The OD 530 value was determined. The standard curve was prepared according to the concentration of the 3-indoleacetic acid standard solution and the corresponding absorbance value.

[0048] (2) ACC deaminase content detection: Cell sample extraction: 7.5 mL of the activated strain culture solution was incubated at 30°C, 200 r·min -1 for 24 h. After centrifugation at 8000 r·min -1 for 10 min (4°C), the precipitate was collected. Then, the bacterial cells were resuspended in 5 mL of DF nitrogen-free medium (without (NH4)2SO4 component) and incubated at 30°C, 200 r·min -1 for 24 h. After centrifugation under the above conditions, the precipitate was collected. Then, the bacterial cells were resuspended and washed twice with Tris-HCl buffer solution (5 mL, 0.1 mol·L -1 , pH 7.6). The bacterial cells were resuspended again in 7.5 mL of DF nitrogen-free medium, and 45 μL of sterile ACC solution (0.5 mol·L -1 ) was added. The mixture was incubated at 30°C, 200 r·min -1 for 24 h (this process induced the production of ACC deaminase by the strains). After centrifugation at 5000 r·min -1 for 20 min, the precipitate was collected. Tris-HCl solution (5 mL, 0.1 mol·L -1, pH 7.6) to resuspend the cells, and repeat twice, collect the precipitate; take 100 μL of the cell lysate (toluene oscillation dissolution) and store at 4 °C for later use in protein content determination.

[0049] Enzyme activity determination: take 200 μL of the remaining cell lysate in a 1.5 mL centrifuge tube, add ACC solution (20 μL, 0.5 mol·L -1 ), mix well, and another one without ACC solution, and then react at 30 °C for 15 min, and then add HCl solution (1 mL, 0.56 mol·L -1 ) each, mix well, centrifuge at 11000 r·min -1 for 10 min (4 °C), take the supernatant (800 μL each) in a 5 mL centrifuge tube for later use; sequentially add HCl solution (800 μL, 0.56 mol·L -1 ), 2,4-dinitrophenylhydrazine reaction solution (300 μL), and react at 30 °C for 30 min, and then add NaOH solution (22 mL, 2 mol·L -1 ), mix well, and after color development, measure the OD 540 value (Tris-HCl solution (0.1 M, pH 8.5) as blank control). Substitute the absorbance value of the sample into the regression equation of the standard curve to obtain the content of α-ketobutyric acid, and calculate the amount of substance (μmol) of α-ketobutyric acid, and then determine the total protein content (mg) in the cell lysate according to the Bradford method. Finally, the amount of α-ketobutyric acid produced by the bacterial cells per unit protein content per unit time is defined as the ACC deaminase activity, and the ACC deaminase activity (U·mg -1 ) is calculated according to the formula, and the formula is as follows. Set 3 parallel samples for each strain.

[0050]

[0051] The standard curve is drawn according to the OD value of the α-ketobutyric acid standard sample, and the brief method is as follows: prepare α-ketobutyric acid standard solution (0, 0.2, 0.4, 0.6, 0.8, 1 mmol·L -1 ) with 0.1 mol·L -1 Tris-HCl buffer (pH 8.5), and add 300 μL of 2,4-dinitrophenylhydrazine (2 mol·L -1 HCI dissolution, mass concentration 2 g·L -1 ) to each, mix well, and then react in a 30 °C water bath for 30 min; then add 2 mL of NaOH (2 mol·L -1 ), and after color development, measure the OD 540Values (using Tris-HCl buffer of pH 8.5 as a blank control); a standard curve was prepared according to the concentration of the a-ketobutyric acid solution and its corresponding absorbance value.

[0052] (3) Phosphorus release capacity determination: The phosphorus release capacity of the strain was determined by reference to the molybdenum-antimony color development method. The method is as follows: the activated strain (1 mL) was inoculated into PKO medium (50 mL), and the control group was inoculated with an equal amount of sterile water, and placed in a 30°C, 150 r·min -1 constant temperature culture for 7 days; then centrifuged at 11000 r·min -1 for 5 min to take the supernatant. Add 1-2 drops of dinitrophenol indicator to the supernatant (30 mL), and adjust to just slightly yellow with a small amount of NaOH solution (1 M, 10 M), HCl solution (1 M, 5 M), then accurately add 5 mL of molybdenum-antimony mixed color development solution, shake well, and dilute to 50 mL with deionized water. React for 30 min at room temperature above 15°C (within 8 h), and measure and record the OD 700 value. According to the standard curve, the phosphorus content (mg·L -1 ) was calculated.

[0053] Standard curve preparation: accurately take 5 mg·L -1 K2HPO4 standard solution 0, 2, 4, 6, 8, 10 mL in a 50 mL volumetric flask, and add the same volume of blank solution as the sample solution used in color development determination. Add 1-2 drops of dinitrophenol indicator, and adjust to just slightly yellow with a small amount of NaOH solution (1 M, 10 M), HCl solution (1 M, 5 M), then accurately add 5 mL of molybdenum-antimony mixed color development solution, shake well, and dilute to 50 mL with deionized water. React for 30 min at room temperature above 15°C, and measure the OD 700 value of each standard solution, and prepare a standard curve according to the concentration of the standard solution and its corresponding absorbance value.

[0054] (4) Iron carrier synthesis capacity determination: inoculate the activated strain (0.5 mL) into MKB liquid medium (5 mL), and at the same time, add an equal amount of MKB liquid medium (1 mL) without inoculating the strain for reference value (Ar) determination, and culture at 30°C, 150 r·min -1 for 48 h. Take 1 mL of the culture, and mix with the detection solution of CAS at 1:1 (v / v). After reacting at room temperature for 1 h, measure the OD 630The value (the experimental group is recorded as A, and the reference is recorded as Ar), and the distilled water is used for zero setting when measuring. Finally, the relative content of siderophore in the sample is expressed by the ratio of A / Ar, and the smaller the value, the stronger the ability of the strain to produce siderophore. (Note: In the experiment, if the strain produces siderophore, the reaction system is orange; if it does not produce siderophore, the reaction system is still blue).

[0055] (5) Determination of extracellular polymeric substance (EPS) content The EPS content is determined by referring to the Congo red agar method. The method is as follows: the activated strain (1 mL) is inoculated in LB liquid medium (40 mL) containing 5% salinity, and cultured at 37°C, 150 r·min -1 for 72 h overnight. Then, the bacterial cells are removed by centrifugation at 8000 r·min -1 for 10 min. The supernatant is mixed with anhydrous ethanol at a ratio of 1:2 (v / v) and incubated at 4°C overnight. Then, the EPS is obtained by centrifugal separation of the precipitate. The obtained EPS is dried in hot air at 40°C for 2-3 d to obtain the dry weight, which is the EPS content.

[0056] Table 1: Determination of salt tolerance and growth-promoting potential of each strain

[0057] As can be seen from Table 1, each strain has good salt tolerance, and Kosakonia koreensis 11 has the best salt tolerance and can survive in a medium containing 10% salt. Each strain has the function of secreting IAA and siderophore, and the content of IAA is 2.84-15.93 mg / L, and the content of siderophore is 1.55-64.60%. Except for Enterobacter hogei 24, the other strains can produce ACC deaminase and extracellular polysaccharide, and the activity of ACC deaminase is 0.02-3.71 U / mg, and the content of extracellular polysaccharide is 0.45-24.90 g / L. All strains have the function of phosphorus solubilization, and the amount of phosphorus solubilization is 0.68-730.82 mg / L.

[0058] Example 4: Interaction between strains (1) Strain activation: two strains are inoculated in LB liquid medium, and cultured at 28°C, 120 rpm / min, and oscillated for 10-12 h until the OD 600 ≈1.0 of each bacterial liquid is reached.

[0059] (2) Cross-shaped inoculation: LB solid medium was prepared, sterilized by high-pressure steam at 121°C for 15 min, and cooled to about 50°C, then poured into a 90 mm culture dish to prepare the solid medium. An activated antagonistic strain was taken with an inoculation loop and two parallel straight lines were drawn on the LB solid medium plate. Then another inoculation loop was used to take the indicator strain and draw two straight lines perpendicular to the previous two, forming a cross-shaped pattern.

[0060] (3) Culture observation: The inoculated plate was placed in a constant-temperature incubator at an appropriate temperature for 1-3 days. The growth of the colonies on the plate was observed, and the focus was on whether there were inhibition zones or growth inhibition bands in the cross region.

[0061] (4) Result judgment: If there are obvious inhibition zones or growth inhibition bands in the cross region, it indicates that there is antagonism between the two strains; if there is no obvious inhibition, it means that there is no antagonism between the two strains.

[0062] (5) Eight strains were combined in pairs for antagonistic experiments, a total of 56 groups, all without antagonistic effect.

[0063] Example 5. Indoor pot experiment (1) Take out the 8 plant growth promoting bacteria from the -80°C refrigerator, and use a sterile inoculation loop to take a small amount of bacterial inoculum on a clean bench. After drawing the first line on one side of the LB solid medium, burn the inoculation loop and cool it. Rotate the plate and touch the inoculation loop on the first line, then draw a few lines in the new area.

[0064] Burn and cool again, and draw lines in three and four areas (the more areas, the easier to obtain single colonies). Invert the plate and place it in a constant-temperature incubator for 3-5 days. Select single colonies with uniform morphology in the final drawing area for 16sRNA sequencing and phylogenetic analysis to identify pure cultures.

[0065] (2) Take a portion of the pure culture and add it to LB liquid medium in a shaker at 28°C, 120 rpm / min, and shake for 10-12 h until the OD 600 ≈1.0.

[0066] (3) Mix equal volumes of the 8 strains to obtain a mixed bacterial solution. Add the mixed bacterial solution to the LB liquid medium at a 5% inoculation amount, and incubate at 28°C and 120 rpm for 12 h to obtain the rhizosphere and rhizosphere growth promoting microbial solution.

[0067] (4) Sterilize the carbon-based organic fertilizer at 121°C, 101 Pa for 30 min to obtain an organic carrier. Mix the organic carrier with 15% of the rhizosphere and rhizosphere growth promoting microbial solution and the organic carrier in a well-ventilated cool place for 5 days to prepare the organic carrier rhizosphere and rhizosphere growth promoting microbial inoculant.

[0068] (5) Sterilize the cow and sheep manure organic fertilizer at 121℃, 101 Pa for 30 min to obtain an organic carrier; mix the organic carrier with 15% of the rhizosphere-promoting microbial liquid by mass to the organic carrier, and fully mix and culture in a cool and ventilated place for 5 days to obtain the rhizosphere-promoting microbial inoculum of the organic carrier.

[0069] (6) Sterilize the biochar at 121℃, 101 Pa for 30 min to obtain an organic carrier; mix the organic carrier with 15% of the rhizosphere-promoting microbial liquid by mass to the organic carrier, and fully mix and culture in a cool and ventilated place for 5 days to obtain the rhizosphere-promoting microbial inoculum of the organic carrier.

[0070] Pot experiment 1 Select seeds of Puccinellia tenuiflora and Suaeda salsa with full grains and uniform size, soak them in 10% hydrogen peroxide for 10 min, and then rinse them with distilled water for 4 times. Soak the seeds of Puccinellia tenuiflora and Suaeda salsa in ultrapure water, and place them in a 37℃ constant temperature incubator for germination.

[0071] After the germination is completed, sow the seeds in flowerpots containing 1.85 kg of soil (specifications: 23 cm x 18 cm x 21.5 cm), and the soil in the flowerpots is taken from a saline-alkali grassland in Xilinhot, Inner Mongolia, with a soil pH of 8.38 and a total salt content of 2.92 g / kg. There are 50 Puccinellia tenuiflora plants and 40 Suaeda salsa plants in one pot. The addition amount of the rhizosphere-promoting microbial inoculum of the cow and sheep manure organic fertilizer carrier in each flowerpot is 20 g (uniformly mixed into the soil); at the same time, the following treatments are set: Treatment of adding the rhizosphere-promoting microbial inoculum of the organic carrier (MP): load 2 mL of the rhizosphere-promoting microbial liquid prepared in step (3) on 20 g of sterilized organic fertilizer, and then add it to each flowerpot; Treatment of only inoculating the microbial liquid (PGPB): add 2 mL of the rhizosphere-promoting microbial liquid prepared in step (3); Treatment of only adding the organic carrier (MOF): add 20 g of sterilized cow and sheep manure organic fertilizer; Control treatment CK: no additive.

[0072] After sowing, lay 0.15 kg of soil again, and control the maximum water holding capacity of the soil at 80%.

[0073] After two months, compare the aboveground biomass and underground biomass of each treatment, and calculate the growth index and soil nutrient index of each group of potted plants compared with the control group, and the results are shown in Tables 2, 3 and 4, and 5.

[0074] Table 2 Biomass data of each group of potted plants

[0075] Table 3 The biomass of each group of potted plants compared to the control group

[0076] Table 4 Soil nutrient index data of each group of potted plants

[0077] Table 5 The soil nutrient index of each group of potted plants compared to the control group

[0078] The results in Tables 2-3 show that in the saline-alkali grassland soil, the endo- rhizosphere growth promoting microbial agent (MP) carried by the organic fertilizer of cattle and sheep manure has a significant growth promoting effect on Puccinellia distans and Suaeda salsa. Compared with the blank control (CK), the single sterilized organic fertilizer of cattle and sheep manure (MOF) and the single endo-rhizosphere growth promoting microbial solution (PGPB), the microbial agent treatment significantly increased the aboveground and underground fresh weight and dry weight of the two plants, with an increase range of 53.17% to 2115.38%.

[0079] The results in Tables 4-5 show that in the saline-alkali grassland soil, the endo- rhizosphere growth promoting microbial agent (MP) carried by the organic fertilizer of cattle and sheep manure has a significant effect on the soil organic matter and available nutrient content of Puccinellia distans and Suaeda salsa. Compared with the blank control (CK), the single sterilized organic fertilizer of cattle and sheep manure (MOF) and the single endo-rhizosphere growth promoting microbial solution (PGPB), the microbial agent treatment increased the soil organic matter content by 2.18-73.29%, and significantly increased the available nutrient content by 1.68-241.33%.

[0080] Pot experiment 2 Select seeds of Medicago ruthenica and Leymus chinensis that are full and uniform in size, soak them in 10% hydrogen peroxide for 10 minutes, and then rinse them 3-5 times with distilled water. Soak the grass seeds in ultrapure water and place them in a 37°C constant temperature incubator for germination.

[0081] After germination, sow the seeds in pots containing 1.85 kg of soil (size 23 cm x 18 cm x 21.5 cm). The soil in the pots is taken from the heavily degraded and mowed degraded typical grassland in Xilinhot, Inner Mongolia. Sow 40 plants of Leymus chinensis and 15 plants of Medicago ruthenica in each pot. Add 20g of carbon-based organic fertilizer carrier endo-rhizosphere growth promoting microbial agent to each pot (mix evenly into the soil); at the same time, set up the following treatments: Add carbon-based organic fertilizer carrier endo-rhizosphere growth promoting microbial agent (LP): take 2 mL of the composite microbial solution prepared in step (3) of Example 5, load it on 20g of sterilized biochar, and then add it to each pot; Treatment of inoculating bacteria liquid only (PGPB): 2 mL of the prepared endo-rhizosphere growth promoting microbial liquid in step (3) was added; Treatment of adding organic carrier only (LOF): 20 g of sterilized carbon-based organic fertilizer was added; Control treatment CK: no additives.

[0082] After sowing, 0.15 kg of soil was spread and watered to control the maximum water holding capacity of the soil at 80%.

[0083] After two months, the aboveground biomass and underground biomass of each treatment were compared, and the growth index and soil nutrient index of each group of pots were calculated, and the increase compared with the control group was calculated, and the results are shown in Tables 6, 7 and 8, 9.

[0084] Table 6: Biomass data of each group of pots

[0085] Table 7: Increase of biomass of each group of pots compared with the control group

[0086] Table 8: Soil nutrient index data of each group of pots

[0087] Table 9: Increase of soil nutrient index of each group of pots compared with the control group

[0088] Tables 6-7 show that in the typical grassland soil of heavy degradation by grazing and degradation by cutting, the carbon-based organic fertilizer carrier endo-rhizosphere growth promoting microbial agent (LP) has a significant effect on the growth promotion of Medicago ruthenica and Leymus chinensis. Compared with the blank control (CK), single sterilized carbon-based organic fertilizer carrier (LOF) and single endo-rhizosphere growth promoting microbial liquid (PGPB), the agent treatment significantly increases the aboveground and underground fresh weight and dry weight of the two plants, with an increase range of 31.70%-582.68%.

[0089] Tables 8-9 show that in the typical grassland soil of heavy degradation by grazing, the carbon-based organic fertilizer carrier endo-rhizosphere growth promoting microbial agent (LP) has a significant effect on soil nutrients. Compared with the blank control (CK), single sterilized carbon-based organic fertilizer carrier (LOF) and single endo-rhizosphere growth promoting microbial liquid (PGPB), the agent treatment significantly increases the soil organic matter and available nutrients of the two plants, with an increase range of 22.04-43.49% and 21.97-96.11%.

[0090] Pot experiment 3 The seeds of Astragalus complanatus and Leymus chinensis with full grains and uniform size were selected, soaked with 10% hydrogen peroxide for 10 min, and then washed with distilled water for 3-5 times. The grass seeds were soaked in ultrapure water and placed in a 37°C constant temperature incubator for germination.

[0091] After the germination, the seeds were sown in flowerpots containing 1.85 kg of soil (specification: 23 cm x 18 cm x 21.5 cm). The soil in the flowerpots was taken from the severely degraded typical grassland and desertified grassland in Xilinhot, Inner Mongolia. There were 40 plants of Leymus chinensis and 15 plants of Astragalus complanatus in each pot. The amount of biochar carrier endo-rhizosphere growth promoting microbial inoculant added in each pot was 20 g (uniformly mixed into the soil). The following treatments were also set up: Biochar carrier endo-rhizosphere growth promoting microbial inoculant (BP): 2 mL of the composite microbial inoculum prepared in step (3) of Example 5 was loaded on 20 g of sterilized biochar, which was then added to each flowerpot; Inoculation with microbial inoculum only (PGPB): 2 mL of the endo-rhizosphere growth promoting microbial inoculum prepared in step (3) was added; Treatment with organic carrier only (BC): 20 g of sterilized biochar was added; Control treatment CK: no additive.

[0092] After sowing, 0.15 kg of soil was laid flat, and water was added to control the maximum water holding capacity of the soil at 80%.

[0093] After two months, the aboveground and underground biomass of the plants in each treatment was compared, and the growth index of each group of potted plants was calculated relative to the control group. The results are shown in Tables 10 and 11.

[0094] Table 10: Biomass data of each group of potted plants

[0095] Table 11: Increase in biomass of each group of potted plants relative to the control group

[0096] Tables 10-11 show that in the severely degraded typical grassland and desertified grassland soil, the biochar carrier endo-rhizosphere growth promoting microbial inoculant (BP) has a significant effect on the growth of Astragalus complanatus and Leymus chinensis. Compared with the blank control (CK), single sterilized biochar carrier (BC), and single endo-rhizosphere growth promoting microbial inoculum (PGPB), the microbial inoculant treatment significantly increased the aboveground and underground fresh weight and dry weight of the two plants, with an increase range of 11.35-265.59%.

[0097] Example 6: Promoting effect of organic carrier endo-rhizosphere growth promoting microbial inoculant on degraded grassland vegetation The experiment was conducted in June 2024 in Xilinhot, Inner Mongolia, in typical grasslands of moderate degradation under grazing, severe degradation under grazing, and degradation under mowing, and the preparation method of the organic carrier endo-rhizosphere growth promoting microbial inoculant was the same as that in Example 5. In each type of grassland, the experiment was divided into five groups: carbon-based organic fertilizer carrier endo-rhizosphere growth promoting microbial inoculant (LP) group, cow and sheep manure organic fertilizer carrier endo-rhizosphere growth promoting microbial inoculant (MP) group, AMF fungal inoculant (AMF) group, Aijia No. 3 organic-inorganic composite carrier microbial inoculant (Aijia No. 3) group, and control (CK) group. The LP group and the MP group were applied with carbon-based organic fertilizer carrier endo-rhizosphere growth promoting microbial inoculant and cow and sheep manure organic fertilizer carrier endo-rhizosphere growth promoting microbial inoculant, respectively. The use amount of the organic carrier endo-rhizosphere growth promoting microbial inoculant was 0.4 kg / m 2 , and the application method was strip application; the AMF inoculant was purchased from Kenhe Dingcheng Technology Co., Ltd., and was composed of Claroideoglomus claroideum, Claroideogloms etunicatum, Funneliformis mosseae, Septoglomus deserticola, and Rhizophagus irregularis, and the application amount was 0.15 g / m 2 ; the Aijia No. 3 organic-inorganic composite carrier microbial inoculant was purchased from Harbin Baidu Science and Technology Development Co., Ltd., and the application amount was 30 g / m 2 ; the CK group did not add any treatment. The application method was as follows: a heavy-duty tractor was used, a hydraulic disc root cutter was configured at the front end to cut into the soil (depth 10 cm), old roots were cut off, soil compaction was broken, and soil cracks were formed; a fertilizer applicator (strip application type) was followed at the rear end, and each group of microbial inoculants was precisely strip applied into the soil cracks generated by the root cutter. The operation needed to maintain a uniform speed of the tractor (about 6 km / h) to ensure that the root cutting depth was stable, the fertilizer was uniformly applied, and the two operations were closely coordinated, so that the microbial inoculants could be effectively buried in the root zone and the loss due to volatilization was reduced. After three months of applying the microbial inoculants, the aboveground dry weight, underground dry weight, and coverage of all plants in the 1 square meter range of each group were measured, and the growth index of the organic carrier endo-rhizosphere growth promoting microbial inoculant grassland vegetation was calculated compared with the control group and other microbial inoculants, as shown in Tables 12-15. The increase of organic matter, available nitrogen, available phosphorus, and available potassium in the soil treated with the organic carrier endo-rhizosphere growth promoting microbial inoculant compared with the control group was statistically analyzed, and the results are shown in Tables 16 and 17.

[0098] Table 12 Growth index data of organic carrier microbial inoculant grassland vegetation

[0099] Table 13 Growth index of grassland vegetation of the organic carrier microbial inoculant compared with the increase of the control group

[0100] Table 14 Growth index of grassland vegetation of the organic carrier microbial inoculant compared with the increase of the AMF inoculant

[0101] Table 15 Growth index of grassland vegetation of the organic carrier microbial inoculant compared with the increase of the Aiga No. 3 microbial inoculant

[0102] Table 16 Soil nutrient index data of the grassland of the organic carrier microbial inoculant

[0103] Table 17 Soil nutrient index of the grassland of the organic carrier microbial inoculant compared with the increase of the control group

[0104] Tables 12-15 show that the carbon-based organic fertilizer carrier endo- rhizosphere growth promoting microbial inoculant (LP) group and the cattle and sheep manure organic fertilizer carrier endo-rhizosphere growth promoting microbial inoculant (MP) group can effectively promote the growth of vegetation in the actual degraded grassland. Compared with the control group (CK), the two kinds of organic carrier endo-rhizosphere growth promoting microbial inoculants significantly increased the above / underground dry weight of vegetation and vegetation coverage, with an increase of 1.50-227.27% and 38.89-79.25%, respectively; compared with the AMF inoculant treatment, the two kinds of organic carrier endo-rhizosphere growth promoting microbial inoculants significantly increased the above / underground dry weight of vegetation and vegetation coverage, with an increase of 11.63-94.59% and 24.37-43.93%, respectively; compared with the Aiga No. 3 organic-inorganic composite carrier microbial inoculant, the two kinds of organic carrier endo-rhizosphere growth promoting microbial inoculants significantly increased the aboveground dry weight of vegetation and vegetation coverage, with an increase of 20.87-84.82% and 12.66-49.01%, respectively, and the growth promoting effect of the organic carrier microbial inoculant of the present application is better than that of the commercial inoculants AMF inoculant and Aiga No. 3 organic-inorganic composite carrier microbial inoculant.

[0105] Tables 16-17 show that the carbon-based organic fertilizer carrier endo-rhizosphere growth promoting microbial agent (LP) group and the sheep and cattle manure organic fertilizer carrier endo-rhizosphere growth promoting microbial agent (MP) group can effectively improve the soil nutrient content in the actual degraded grassland. Compared with the control group (CK), the two kinds of organic carrier endo-rhizosphere growth promoting microbial agents significantly improve the soil organic matter and available nutrient content, and the increase amplitude is 12.95-47.15% and 4.63-266.56%, respectively, and the improvement effect of the organic carrier microbial agent of the present application on the soil is better than that of the commercial microbial agent AMF agent and the Aijia No. 3 organic-inorganic composite carrier microbial agent.

[0106] From the above examples, it can be seen that the present application provides an organic carrier endo-rhizosphere growth promoting microbial agent for degraded grassland ecological restoration and a preparation method and application thereof. The organic carrier endo-rhizosphere growth promoting microbial agent of the present application can significantly improve the aboveground dry weight and vegetation coverage of the vegetation community of the degraded grassland, thereby promoting the ecological restoration of the degraded grassland.

[0107] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An organic carrier root-rhizosphere growth-promoting microbial agent for ecological restoration of degraded grasslands, characterized in that, It includes a root-rhizosphere growth-promoting microbial solution and an organic carrier, wherein the root-rhizosphere growth-promoting microbial solution includes root-promoting bacteria and rhizosphere-promoting bacteria; The root growth-promoting bacteria include Bacillus terracotta warriors K4, Bacillus 9-3-1, Bacillus safortiformis 11-5-4 and Halomonas L31. The rhizosphere growth-promoting bacteria include Serratia marcescens 5, Serratia marcescens 23, Cossackia covani 11, and Enterobacter hygroscopicus 24. The Bacillus terracotta warriors K4, Bacillus 9-3-1, Bacillus saforticus 11-5-4, and Halomonas L31 are deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession numbers GDMCC NO.65395, GDMCC NO.65392, GDMCC NO.65394, and GDMCC NO.65397, respectively. The *Serratia marcescens* 5, *Serratia marcescens* 23, *Cossaconia covanni* 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.

2. The organic carrier root-rhizosphere growth-promoting microbial agent according to claim 1, characterized in that, The organic carrier includes carbon-based organic fertilizer, cow and sheep manure organic fertilizer, or biochar.

3. The organic carrier root-rhizosphere growth-promoting microbial agent according to claim 1, characterized in that, The root-rhizosphere growth-promoting microbial inoculum is 10-20% of the carrier mass.

4. The organic carrier root-rhizosphere growth-promoting microbial agent according to claim 1, characterized in that, The effective viable bacteria count in the organic carrier root-rhizosphere growth-promoting microbial agent is ≥1.4×10⁻⁶. 8 cfu / g, moisture content ≤25%.

5. The organic carrier root-rhizosphere growth-promoting microbial agent according to claim 2, characterized in that, The carbon-based organic fertilizer carrier is prepared from lignite through fermentation; the biochar is prepared from a mixture of corn, wheat and peanut straw through calcination.

6. The organic carrier root-rhizosphere growth-promoting microbial agent according to claim 2, characterized in that, The cow and sheep manure organic fertilizer carrier includes well-rotted cow manure and / or sheep manure.

7. The method for preparing the organic carrier root-rhizosphere growth-promoting microbial inoculant according to any one of claims 1 to 6, characterized in that, Includes the following steps: 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; S2. Mix equal volumes of the bacterial solutions from step S1 to obtain a mixed bacterial solution; 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. 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.

8. The preparation method according to claim 7, characterized in that, The inoculum size in step S3 is 10-20%; the fermentation medium is LB liquid medium.

9. The use of the organic carrier root-rhizosphere growth-promoting microbial agent according to any one of claims 1 to 6 in at least one of the following: (1) Application in the restoration of degraded grasslands, desertified grasslands and salinized grasslands; (2) Application in promoting plant growth; (3) Application in promoting grassland vegetation restoration.

10. The application according to claim 9, characterized in that, The plants mentioned include Suaeda salsa, Imperata cylindrica, Sophora flavescens, Leymus chinensis, Alfalfa, or Leymus chinensis.

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