Microbial ecological restoration method for degenerated typical grassland

Abstract

The invention belongs to the technical field of grassland restoration, and relates to a microbial ecological restoration method for a degenerated typical grassland, which comprises the following steps: selecting the degenerated typical grassland to be treated, ditching, applying a carrier growth-promoting microbial agent, and combining the carrier growth-promoting microbial agent with an arbuscular mycorrhizal fungus agent and / or an azotobacter agent according to the degeneration type. The carrier growth-promoting microbial agent comprises specific strains such as serratia rubra, enterobacter paraphora, cosinia cosinii, bacillus and halomonas. The carrier growth-promoting microbial agent is prepared from the specific strains such as serratia rubra, enterobacter paraphora, cosinia cosinii, bacillus and halomonas. The microbial inoculum is cultured and prepared by being loaded on an organic fertilizer carrier, and the application amount is adjusted according to different degeneration degrees. According to the method, vegetation recovery is remarkably promoted through the synergistic effect of three main microbial functional groups of soil, the biomass and coverage of plant communities are improved, soil nutrients and microbial activity are improved, and the method is suitable for various degeneration types such as grazing degeneration, cradling degeneration and desertification typical grassland; the method has the advantages of being good in remediation effect, environmentally friendly, high in sustainability and the like.

Description

Technical Field

[0001] This invention belongs to the field of grassland restoration technology and relates to a microbial ecological restoration method for degraded typical grasslands. Background Technology

[0002] Grassland degradation is primarily a degenerative succession of ecosystems caused by overexploitation, encompassing both vegetation and soil degradation. However, current methods for restoring degraded grasslands mainly focus on plant community restoration, emphasizing increased plant productivity and native vegetation reconstruction. In the evolutionary history of grasslands, plants, soil, and microorganisms interact synergistically to adapt to climate fluctuations and animal grazing. Therefore, grassland degradation represents a holistic decline in the functions of plants, soil, and microorganisms. Severely degraded grasslands are difficult to restore because of multiple limitations, including those related to plant propagules, nutrients, and microorganisms. In the ecological restoration of degraded grasslands, methods such as no-till reseeding, root pruning, and fertilization are commonly used to address propagule and nutrient limitations, accelerating the vegetation restoration process. However, the restored grassland ecosystems often exhibit poor stability, making it difficult to maintain the sustainability of ecosystem services. Currently, there is relatively little research and solutions for soil microbial limitations in typical degraded grasslands. Existing restoration technologies (such as fencing, reseeding, and fertilization) have limitations such as high cost, slow results, and susceptibility to secondary degradation. While microbial remediation has some applications, it largely relies on single microbial functional groups of agents and lacks efficient composite microbial functional group agent systems and microbial ecological restoration technologies that target multiple typical types of degraded grasslands. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a microbial ecological restoration method for degraded typical grasslands. A typical grassland is defined as a grassland composed of typical xerophytic perennial herbaceous plants. Typical grassland vegetation is widely distributed in the Hulunbuir Plateau, Xilin Gol Plateau, southern foothills of the Yinshan Mountains, and eastern Ordos Plateau of the Inner Mongolia Plateau, forming a vast typical grassland zone, which constitutes the main body of the Inner Mongolia grassland region. Specifically, the method includes the following steps: Step 1, inoculating *Serratia marcescens* 5, *Enterobacter spp.* 24, *Serratia marcescens* 23, *Cossackia cochinchinensis* 11, *Bacillus* 9-3-1, *Bacillus sabovella* 11-5-4, *Bacillus terrapinus* K4, and *Haloxylon ammodendron* L31 into LB liquid medium and incubating at 26-30℃ and 100-140 rpm with shaking until OD... 600 =0.8~1, collect the bacterial solution separately.

[0004] The aforementioned *Serratia marcescens* 5, *Enterobacter septemlobus* 24, *Serratia marcescens* 23, *Cossaconia covanni* 11, *Bacillus* 9-3-1, *Bacillus saffron* 11-5-4, *Bacillus terracotta* K4, and *Haloxymonas* L31 have been disclosed in Chinese patent application CN120966720A.

[0005] The Serratia rubidaea 5 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC NO.65386, deposited on October 31, 2024, at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0006] The Enterobacter hormaechei 24 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC NO.65388, deposited on October 31, 2024, at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0007] The Serratia rubidaea 23 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC NO.65389, deposited on October 31, 2024, at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0008] The Kosakonia cowanii 11 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC NO.65391, deposited on October 31, 2024, at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0009] The Bacillus sp. 9-3-1 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65392, deposited on October 31, 2024, at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0010] The Bacillus safensis 11-5-4 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65394, deposited on October 31, 2024, at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0011] The Bacillus bingmayongensis K4 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65395, deposited on December 23, 2024, at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0012] The Halomonas sp. L31 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65397, on December 23, 2024, at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0013] Step 2: Mix equal volumes of the collected bacterial solutions and inoculate them into LB liquid medium. Incubate at 26-30℃ and 100-140 rpm for 10-14 h to obtain a compound growth-promoting microbial solution. Sterilize organic fertilizer with steam at 121℃ and 101 Pa for 30 min, and mix it with the compound growth-promoting microbial solution at a mass ratio of 100:(10-15). Incubate in a cool, ventilated place for 5 days to obtain a carrier growth-promoting microbial agent.

[0014] Step 3: Select typical degraded grasslands to be treated, and directly spray with growth-promoting microbial agents, or dig a 10 cm trench and apply one of the following five combinations into the trench:

[0015] (1) Organic fertilizer 0.6~0.8 kg / m 2 ;

[0016] (2) Carrier-promoting microbial inoculant 0.6~0.8 kg / m 2 ;

[0017] (3) Carrier-promoting microbial inoculant 0.6~0.8 kg / m 2 And arbuscular mycorrhizal fungi inoculant 0.15 g / m 2 ;

[0018] (4) Carrier-promoting microbial inoculant 0.6~0.8 kg / m 2 And nitrogen-fixing bacteria inoculant 1.8~2.4 g / m³ 2 ;

[0019] (5) Carrier-promoting microbial inoculant 0.6~0.8 kg / m 2 Arbuscular mycorrhizal fungi inoculant 0.15 g / m 2 Nitrogen-fixing bacteria inoculant: 1.8~2.4 g / m³ 2 .

[0020] Preferably, for typical grazing and mowing moderately degraded grasslands, the organic fertilizer application rate is 0.6 kg / m². 2 The application rate of the carrier-promoting microbial agent was 0.6 kg / m³. 2 The application rate of nitrogen-fixing bacteria inoculant was 1.8 g / m³. 2 For severely degraded typical grasslands and typical desertified grasslands, the application rate of organic fertilizer is 0.8 kg / m². 2 The application rate of the carrier-promoting microbial agent was 0.8 kg / m³. 2 The application rate of nitrogen-fixing bacteria inoculant was 2.4 g / m³. 2 .

[0021] Preferably, the organic fertilizer is carbon-based organic fertilizer (LOF) or cow / sheep manure organic fertilizer (MOF).

[0022] Preferably, the arbuscular mycorrhizal fungal agent (AMF) is purchased from Qianhe Dingsheng Technology Co., Ltd., including *Aureobasidium simulans*, *Aureobasidium simulans*, *Aureobasidium mosieboldii*, *Aureobasidium sabina*, and *Aureobasidium heteromorpha*.

[0023] Preferably, the nitrogen-fixing bacteria agent (NFB) is purchased from Yangzhou Haicheng Biotechnology Co., Ltd., and is *Azotobacter chrysotrichum*.

[0024] The microbial ecological restoration method provided by this invention significantly promotes vegetation restoration in degraded typical grasslands through the individual, paired (MPA, LPA, MPN, LPN), or combined (MPAN, LPAN) application of organic fertilizer (MOF or LOF), carrier-based growth-promoting microbial agents (LP or MP), arbuscular mycorrhizal fungi (AMF), and nitrogen-fixing bacteria (NFB). Specific advantages include:

[0025] (1) Significantly improve plant community biomass and cover: Both indoor potted and in-situ field experiments showed that the combined treatment of the three (LPAN) had the most significant effect on improving aboveground / underground plant biomass.

[0026] (2) Improve soil physical and chemical properties: Combined treatment can effectively increase soil organic matter content, nitrogen, phosphorus and potassium nutrients, and enhance soil microbial activity.

[0027] (3) Adaptable to multiple degradation types: This method is applicable to multiple degradation types such as severely degraded grasslands due to grazing, moderately degraded grasslands due to grazing, moderately degraded grasslands due to mowing, and typical desertified grasslands. The restoration effect is stable and has strong universality.

[0028] (4) Environmentally friendly and sustainable: The use of chemical fertilizers is reduced through the synergistic effect among microbial functional groups, thus reducing ecological risks and meeting the green development needs of grassland ecological protection. Detailed Implementation

[0029] The technical solutions in the embodiments of the invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] The raw materials for the following embodiments are sourced as follows:

[0031] strain name Collection Number Preservation date Serratia marcescens 5 GDMCC NO.65386 October 31, 2024 Enterobacter 24 GDMCC NO.65388 October 31, 2024 Serratia marcescens 23 GDMCC NO.65389 October 31, 2024 Cossaconia covani 11 GDMCC NO.65391 October 31, 2024 Bacillus 9-3-1 GDMCC NO.65392 October 31, 2024 Bacillus sarfusae 11-5-4 GDMCC NO.65394 October 31, 2024 Bacillus subtilis K4 GDMCC NO.65395 December 23, 2024 Halomonas L31 GDMCC NO.65397 December 23, 2024

[0032] Carbon-based organic fertilizer (LOF) was purchased from Yuantaifeng (Baotou) Biotechnology Co., Ltd., and cow and sheep manure organic fertilizer (MOF) was purchased from Inner Mongolia Gugong Biotechnology Co., Ltd.

[0033] Arbuscular mycorrhizal fungi (AMF) were purchased from Qianhe Dingsheng Technology Co., Ltd., including *Rhizomyces simulans*, *Rhizomyces simulans*, *Rhizomyces mossicae*, *Rhizomyces sabina*, and *Rhizomyces heteromorpha*.

[0034] The nitrogen-fixing bacteria inoculant (NFB) was purchased from Yangzhou Haicheng Biotechnology Co., Ltd., and included Azotobacter chrysotrichum.

[0035] Experimental Example 1

[0036] Step 1: Inoculate *Serratia marcescens* 5, *Enterobacter johnsonii* 24, *Serratia marcescens* 23, *Cossacchariformis* 11, *Bacillus* 9-3-1, *Bacillus saffron* 11-5-4, *Bacillus terracotta* K4, and *Haloxylon ammodendron* L31 into LB liquid medium and culture at 28°C with shaking at 120 rpm until OD. 600 =1.0, collect bacterial solutions separately.

[0037] Step 2: Mix the collected bacterial solutions in equal volumes and inoculate them into LB liquid medium. Incubate at 28°C and 120 rpm for 12 h to obtain a compound growth-promoting microbial solution. Sterilize carbon-based organic fertilizer and cow / sheep manure organic fertilizer by steaming at 121°C and 101 Pa for 30 min. Mix them with the compound growth-promoting microbial solution at a mass ratio of 100:10 and incubate in a cool, ventilated place for 5 days to obtain carbon-based organic fertilizer carrier growth-promoting microbial agent (LP) and cow / sheep manure organic fertilizer carrier growth-promoting microbial agent (MP).

[0038] Experimental Example 1

[0039] Soil preparation: Soil samples were taken from typical grasslands in Xilinhot, Inner Mongolia, which were severely degraded due to grazing, moderately degraded due to mowing, and desertified (2 kg / pot).

[0040] Seed treatment: Disinfect alfalfa or sheepgrass seeds with 10% hydrogen peroxide for 10 min, rinse with distilled water, and germinate at 37℃ for 24 hours.

[0041] Sowing: 40 seedlings of sheepgrass per pot, 15 seedlings of flat alfalfa per pot;

[0042] Microbial fertilizer dosage: MOF / MP / LOF / LP 20 g / pot, AMF 0.05 g / pot, NFB 0.6 g / pot, mixed evenly with soil. Experimental group design is shown in Table 1.

[0043] Cultivation conditions: After sowing, cover the surface with 0.15 kg of soil, in a greenhouse at 25℃ with 14 h of light, and water regularly to maintain 80% field water holding capacity. After two months, compare the aboveground and underground dry and fresh weights of plants in each treatment. The results are shown in Tables 2-9.

[0044] Table 1. Pot Experiment Group Design

[0045] Group code Processing instructions CK No action taken PGPB Apply PGPB microbial agent alone AMF Apply AMF microbial agent alone NFB Apply NFB fungicide alone AN AMF+NFB MOF Applying only cow and sheep manure organic fertilizer MP Apply MP bacterial agent alone MPA MP+AMF MPN MP+NFB MPAN MP+AMF+NFB LOF Apply carbon-based organic fertilizer alone LP Apply LP bacterial agent alone LPA LP+AMF LPN LP+NFB LPAN LP+AMF+NFB

[0046] Table 2. Effects of each treatment group on the growth of Leymus chinensis in typical desertified grassland soil (g / pot)

[0047] deal with Fresh weight on the ground Dry weight above ground underground fresh weight Underground dry weight CK 1.53±0.47 0.45±0.02 4.80±0.10 0.73±0.02 PGPB 2.04±0.36 0.66±0.04 6.55±0.62 1.28±0.22 AMF 1.96±0.28 0.60±0.01 5.44±0.11 0.88±0.03 NFB 1.96±0.63 0.62±0.03 5.36±0.65 0.84±0.03 AN 2.05±0.67 0.67±0.07 5.94±0.49 1.04±0.13 MOF 5.97±0.73 1.85±0.04 21.43±0.68 4.56±0.26 MP 6.91±0.26 2.11±0.12 22.67±0.27 5.08±0.07 MPA 7.11±0.26 2.24±0.13 21.82±0.80 4.55±0.29 MPN 7.20±0.27 2.31±0.14 23.65±2.51 4.85±0.61 MPAN 9.79±0.51 3.14±0.15 29.87±0.80 6.36±0.16 LOF 9.24±0.79 3.02±0.03 26.05±0.49 5.45±0.03 LP 11.55±0.90 3.81±0.06 28.79±0.15 6.16±0.03 LPA 12.22±0.21 4.42±0.26 31.28±1.41 6.68±0.23 LPN 11.29±0.25 3.73±0.15 32.15±1.32 6.80±0.21 LPAN 13.62±0.24 5.99±0.19 37.68±0.61 8.33±0.25

[0048] Table 3. Effects of each treatment group on the growth of Leymus chinensis in moderately degraded typical grassland soil (g / pot)

[0049] deal with Fresh weight on the ground Dry weight above ground underground fresh weight Underground dry weight CK 2.37±0.15 0.80±0.03 4.87±0.64 0.95±0.10 PGPB 2.49±0.34 0.78±0.19 7.11±0.71 1.31±0.11 AMF 2.66±0.28 0.89±0.11 7.71±0.48 1.35±0.03 NFB 2.57±0.42 0.84±0.16 7.24±0.95 1.35±0.17 AN 2.49±0.08 0.88±0.04 6.77±0.12 1.19±0.07 MOF 5.87±0.98 2.13±0.35 18.23±0.82 4.05±0.59 MP 6.16±0.34 2.19±0.21 19.57±0.23 4.63±0.36 MPA 6.44±0.29 2.08±0.19 17.21±1.39 3.59±0.17 MPN 5.99±0.18 1.88±0.06 18.01±1.40 3.84±0.22 MPAN 6.09±0.20 2.13±0.08 20.98±0.57 4.30±0.30 LOF 6.40±0.67 2.60±0.19 24.61±0.59 5.42±0.06 LP 6.90±0.05 2.77±0.23 26.18±0.42 5.18±0.38 LPA 7.02±0.89 2.60±0.49 26.08±1.51 5.49±0.72 LPN 6.90±0.04 2.66±0.22 25.73±0.33 5.08±0.90 LPAN 7.71±0.32 3.42±0.42 26.78±0.66 6.09±0.23

[0050] Table 4. Effects of each treatment group on the growth of Leymus chinensis in soils of severely degraded typical grazing grasslands (g / pot)

[0051] deal with Fresh weight on the ground Dry weight above ground underground fresh weight Underground dry weight CK 0.90±0.14 0.34±0.06 3.13±0.16 0.45±0.03 PGPB 1.18±0.10 0.4±0.06 4.72±0.32 0.6±0.04 AMF 1.24±0.23 0.36±0.07 3.62±0.27 0.54±0.03 NFB 1.36±0.27 0.26±0.02 3.48±0.14 0.5±0.06 AN 1.90±0.13 0.33±0.06 4.05±0.4 0.77±0.11 MOF 4.35±0.55 1.28±0.14 15.35±1.22 2.65±0.34 MP 5.90±0.32 1.83±0.03 19.06±1.02 3.60±0.03 MPA 5.72±0.70 1.65±0.19 16.46±0.90 3.47±0.11 MPN 5.36±1.08 1.63±0.37 16.39±2.01 3.69±0.24 MPAN 7.55±0.92 2.33±0.29 23.43±3.25 4.24±0.67 LOF 5±0.58 1.52±0.13 19.52±4.15 3.3±0.34 LP 6.7±1.01 2.06±0.25 24.9±3.06 4.27±0.4 LPA 7.08±0.29 2.63±0.32 24.38±1.91 4.5±0.14 LPN 6.81±0.62 2.18±0.15 24.69±1.45 4.22±0.27 LPAN 8.27±0.62 2.73±0.22 30.92±3.67 4.96±0.09

[0052] Table 5. Increase in growth indicators of Leymus chinensis by combined application of microbial agents compared to single application.

[0053]

[0054] Table 6. Effects of each treatment group on the growth of alfalfa in typical desertified grassland soil (g / pot)

[0055] deal with Fresh weight on the ground Dry weight above ground underground fresh weight Underground dry weight CK 3.72±0.18 0.56±0.03 5.18±0.35 0.78±0.05 PGPB 4.93±0.09 0.77±0.01 6.59±0.13 0.95±0.01 AMF 4.64±0.27 0.73±0.05 6.67±0.29 0.96±0.02 NFB 4.46±0.29 0.70±0.05 8.69±0.32 1.15±0.05 AN 4.93±0.21 0.86±0.09 9.35±0.23 1.25±0.02 MOF 15.30±0.51 3.04±0.09 14.92±0.14 2.45±0.04 MP 16.75±0.23 3.42±0.16 19.17±0.35 3.57±0.08 MPA 17.11±0.34 3.59±0.14 18.83±0.41 3.45±0.12 MPN 17.98±0.68 3.80±0.26 21.68±0.19 3.95±0.06 MPAN 18.69±0.40 4.19±0.17 21.95±0.46 4.10±0.13 LOF 20.10±0.12 5.02±0.15 18.43±0.40 3.28±0.13 LP 23.03±0.75 5.72±0.23 21.10±0.39 3.87±0.03 LPA 22.86±0.56 5.68±0.11 20.27±0.26 3.79±0.03 LPN 24.16±0.68 6.04±0.23 27.46±0.77 5.47±0.14 LPAN 25.85±0.34 6.92±0.22 30.56±0.55 6.09±0.13

[0056] Table 7. Effects of each treatment group on the growth of alfalfa in moderately degraded typical grassland soil (g / pot)

[0057] deal with Fresh weight on the ground Dry weight above ground underground fresh weight Underground dry weight CK 3.74±0.23 0.64±0.02 4.87±0.68 1.18±0.20 PGPB 3.40±0.18 0.66±0.13 5.51±0.72 1.21±0.19 AMF 3.84±0.90 0.64±0.16 5.96±0.81 1.33±0.17 NFB 5.28±0.90 1.06±0.17 7.19±0.30 1.52±0.11 AN 3.73±0.58 0.71±0.21 5.34±0.09 1.23±0.04 MOF 8.98±1.01 2.54±0.23 9.84±0.38 2.47±0.24 MP 9.90±0.07 2.82±0.20 11.14±1.36 2.68±0.09 MPA 11.19±0.18 3.41±0.22 13.06±0.97 2.91±0.21 MPN 9.35±0.60 2.96±0.17 12.70±0.55 3.14±0.22 MPAN 9.23±0.75 2.96±0.51 11.74±0.77 2.94±0.35 LOF 14.2±0.81 3.34±0.57 15.22±0.70 3.58±0.15 LP 15.31±0.73 4.15±0.52 17.09±0.66 4.09±0.39 LPA 16.33±0.57 4.52±0.72 17.01±0.55 4.18±0.17 LPN 15.29±0.42 3.95±0.53 17.54±0.61 3.99±0.11 LPAN 15.36±0.73 4.22±0.40 17.49±0.75 4.49±0.55

[0058] Table 8. Effects of each treatment group on the growth of alfalfa in soils of severely degraded typical grazing grassland (g / pot)

[0059] deal with Fresh weight on the ground Dry weight above ground underground fresh weight Underground dry weight CK 0.66±0.06 0.14±0.01 1.83±0.17 0.18±0.03 PGPB 1.55±0.25 0.20±0.01 2.44±0.19 0.27±0.08 AMF 1.35±0.24 0.22±0.01 2.26±0.30 0.26±0.03 NFB 1.43±0.35 0.21±0.01 2.40±0.24 0.29±0.06 AN 2.61±0.29 0.26±0.03 3.01±0.39 0.31±0.07 MOF 10.37±0.93 1.72±0.13 10.562±1.12 1.81±0.57 MP 12.60±0.69 1.85±0.08 12.394±0.89 2.12±0.55 MPA 13.19±0.76 1.97±0.14 11.48±0.85 2.29±0.23 MPN 12.56±1.03 2.06±0.17 11.19±0.85 2.65±0.16 MPAN 14.56±0.76 2.42±0.10 15.11±0.31 3.16±0.70 LOF 11.12±1.28 2.09±0.08 10.90±1.62 2.61±0.06 LP 14.08±1.28 2.63±0.54 14.82±0.89 2.54±0.19 LPA 15.27±1.23 3.08±0.37 15.84±0.84 3.02±0.30 LPN 15.06±1.21 2.96±0.48 14.35±0.55 2.55±0.05 LPAN 17.32±1.05 3.48±0.20 17.92±1.90 3.21±0.41

[0060] Table 9. Increase in growth indicators of alfalfa beans by combined application of microbial agents compared to single application.

[0061]

[0062] As shown in Tables 2-9, in typical grassland soils characterized by desertification, moderate degradation due to mowing, and severe degradation due to grazing, the combined treatment of carbon-based organic fertilizer carrier-promoting microbial inoculant LP with AMF and NFB (LPAN) had the most significant effect on the aboveground / belowground dry and fresh weight of Leymus chinensis and Alfalfa. Compared with the blank control (CK), single inoculant (AMF / NFB), and single carbon-based organic fertilizer carrier-promoting microbial inoculant (LP), the combined treatment (LPAN) increased the aboveground / below groundwater dry / fresh weight of Leymus chinensis by 11.74%–1231.11% and the belowground / below groundwater dry / fresh weight by 2.29%–1041.10%, and increased the aboveground / below groundwater dry / fresh weight of Alfalfa by 0.33%–2524.24% and the belowground / below groundwater dry / fresh weight by 2.34%–1683.33%, and the overall effect was significantly better than the combined treatment of cow and sheep manure organic fertilizer carrier with AMF and NFB (MPAN).

[0063] Experimental Example 2

[0064] Implementation time: May each year (plant growing season).

[0065] Plot setup: Experimental treatment groups were set up for typical grasslands in Xilinhot City, Inner Mongolia Autonomous Region, with moderate to severe degradation due to grazing, moderate degradation due to mowing, and desertification (see Table 10).

[0066] Experimental steps: A heavy-duty tractor pulls a hydraulic trencher to a depth of 10 cm to break up soil compaction and create loose soil cracks. A strip-applying fertilizer applicator follows closely behind to apply the microbial agent into the loose soil cracks (tractor speed 6 km / h). The operation requires the tractor to maintain a constant speed to ensure stable trenching depth, uniform fertilization, and close coordination between the two operations, so that the microbial agent can be effectively buried in the root zone; the growth-promoting microbial agent PGPB is sprayed directly.

[0067] Dosage of microbial agent: AMF 0.15 g / m³ 2 The dosages of PGPB, NFB, LP, and LOF are as follows:

[0068] Grazing and mowing of moderately degraded typical grasslands: PGPB bacterial solution: 160 mL / m² 2 NFB: 1.8g / m 2 LP and LOF: 0.6 kg / m 2 .

[0069] Grazing of severely degraded typical grasslands and desertified typical grasslands: PGPB bacterial solution: 200 mL / m2 NFB: 2.4 g / m 2 LP and LOF: 0.8 kg / m 2 .

[0070] Three months after the application of microbial inoculants, the aboveground dry and fresh weight and cover of all plants within a 1-square-meter quadrat area of ​​each group were measured, as shown in Tables 11-15. Soil from the top 0-20 cm of quadrats of CK, LP, AM, NFB, and LPAN was collected using the five-point sampling method, and the contents of organic matter, available nitrogen, available phosphorus, and available potassium were measured, as shown in Tables 16-20.

[0071] Table 10 Field Experiment Group Design

[0072] Group code Processing instructions CK No action taken PGPB Apply PGPB microbial agent alone LOF Apply carbon-based organic fertilizer alone LP Apply LP bacterial agent alone AMF Apply AMF microbial agent alone NFB Apply NFB fungicide alone LPA LP+AMF LPN LP+NFB AN AMF+NFB LPAN LP+AMF+NFB

[0073] Table 11 Effects of each treatment group on biomass and cover of plant communities in typical desertified grassland

[0074] deal with <![CDATA[Fresh weight on the ground (g / m 2 )]]> <![CDATA[Aboveground dry weight (g / m 2 )]]> Coverage (%) CK 311.45±19.14 122.239±8.98 27.17±2.32 PGPB 452.93±45.49 145.27±7.71 34.33±4.97 LOF 591.55±45.16 186.66±2.34 42.5±4.81 LP 713.74±54.58 207.03±5.99 49.33±5.57 AMF 441.42±23.61 143.39±3.43 32.83±3.31 NFB 446.96±27.50 146.27±10.92 31±5.62 LPA 754.94±45.6 222.18±6.68 55±1.67 LPN 760.59±62.50 226.43±12.95 60±3.16 AN 460.45±49.49 148.00±4.67 33.17±4.31 LPAN 948.22±37.15 267.32±8.05 70.5±6.29

[0075] Table 12 Effects of each treatment group on biomass and cover of plant communities in moderately degraded mowed typical grassland

[0076] deal with <![CDATA[Fresh weight on the ground (g / m 2 )]]> <![CDATA[Aboveground dry weight (g / m 2 )]]> Coverage (%) CK 217.62±36.37 113.76±20.37 31.83±5.88 PGPB 256.23±49.73 132.32±19.15 37.83±4.88 LOF 308.82±58.15 152.74±28.10 43.67±2.16 LP 324.74±21.63 162.28±17.40 43.67±4.50 AMF 250.94±64.63 133.55±27.61 37.67±5.01 NFB 241.61±50.01 131.40±26.56 37.17±3.13 LPA 386.54±28.22 191.27±11.57 46.50±5.43 LPN 323.12±30.51 164.76±19.84 43.33±6.90 AN 245.87±43.55 132.19±22.30 37.17±5.56 LPAN 440.63±42.17 214.84±16.60 48.17±4.40

[0077] Table 13 Effects of each treatment group on biomass and cover of plant communities in moderately degraded grazing grassland

[0078] deal with <![CDATA[Fresh weight on the ground (g / m 2 )]]> <![CDATA[Aboveground dry weight (g / m 2 )]]> Coverage (%) CK 228.57±39.34 140.56±15.85 40.83±2.71 PGPB 341.41±35.95 179.33±8.99 48.83±5.85 LOF 409.36±14.06 215.1±13.38 49.67±2.07 LP 501.77±60.42 239.29±12.75 53±5.06 AMF 370.09±48.76 186.05±16.17 48.33±6.35 NFB 359.67±39.05 183.65±15.28 46.83±7.22 LPA 526.45±57.41 249.83±26.85 58.83±5.85 LPN 477.21±30.78 240.96±12.85 54.17±3.87 AN 374.64±49.52 192.93±10.17 46.83±5.42 LPAN 581.85±51.49 274.75±21.48 64±5.66

[0079] Table 14 Effects of each treatment group on biomass and cover of plant communities in severely degraded grazing grassland

[0080] deal with <![CDATA[Fresh weight on the ground (g / m 2 )]]> <![CDATA[Aboveground dry weight (g / m 2 )]]> Coverage (%) CK 312.13±64.38 131.85±8.96 25±5.62 PGPB 467.56±80.63 186.01±11.08 36.17±5.27 LOF 622.35±135.49 213.87±12.67 41.5±1.22 LP 672.1±105.58 248.23±10.06 44.5±3.94 AMF 448.02±26.72 172.32±8.99 37.5±5.01 NFB 463.53±61.94 161.4±13.2 35.5±3.83 LPA 680.69±82.3 261.72±19.74 45.83±6.62 LPN 770.1±136.7 264.66±30.61 47.17±7.08 AN 530.37±81.73 196.01±15.63 35.33±3.78 LPAN 1022.52±143.13 303.33±40.4 49.83±2.04

[0081] Table 15. Increase in grassland vegetation growth indicators by combined application of microbial agents compared to single application.

[0082]

[0083] Table 16 Effects of combined application of microbial agents compared to single application on soil nutrients in typical desertified grasslands

[0084] Soil Indicators CK LP AMF NFB LPAN Organic matter (mg / g) 10.01±0.16 12.48±0.25 17.85±0.22 12.42±0.28 20.91±0.17 Available nitrogen (mg / kg) 70.49±0.10 79.83±0.10 78.10±0.20 76.72±0.10 87.56±0.26 Available phosphorus (mg / kg) 1.17±0.18 2.98±0.14 1.89±0.04 1.22±0.11 3.55±0.33 Available potassium (mg / kg) 170.50±4.23 201.27±5.95 220.00±7.90 191.43±5.29 255.90±12.08

[0085] Table 17 Effects of combined application of microbial agents compared to single application on soil nutrients in moderately degraded typical mowed grassland

[0086] Soil Indicators CK LP AMF NFB LPAN Organic matter (mg / g) 15.79±0.78 18.29±0.09 17.32±0.92 17.29±0.60 20.41±1.52 Available nitrogen (mg / kg) 19.15±0.64 26.54±0.99 24.21±1.04 26.30±1.34 35.20±1.49 Available phosphorus (mg / kg) 13.00±0.05 20.78±0.46 12.18±1.15 14.75±0.58 33.93±0.16 Available potassium (mg / kg) 1027.50±57.12 1507.50±49.57 1365.50±19.58 1436.83±43.10 1699.83±9.51

[0087] Table 18 Effects of combined application of microbial agents compared to single application on soil nutrients in a typical moderately degraded pastureland.

[0088] Soil Indicators CK LP AMF NFB LPAN Organic matter (mg / g) 6.04±0.66 10.21±1.91 7.79±0.21 8.16±0.41 15.48±2.24 Available nitrogen (mg / kg) 27.97±2.80 37.49±2.32 33.07±1.50 33.68±2.62 38.44±1.52 Available phosphorus (mg / kg) 139.67±15.07 217.85±26.41 166.8±15.22 182.57±10.03 274.63±28.01 Available potassium (mg / kg) 64.16±6.28 102.25±4.97 87.16±6.29 89.95±6.17 133.93±13.16

[0089] Table 19 Effects of combined application of microbial agents compared to single application on soil nutrients in a typical severely degraded grazing grassland

[0090] Soil Indicators CK LP AMF NFB LPAN Organic matter (mg / g) 3.79±0.41 4.91±0.75 7.18±0.63 10.35±0.96 13.26±2.46 Available nitrogen (mg / kg) 31.60±1.70 41.55±2.01 36.10±2.49 37.05±2.85 44.15±1.95 Available phosphorus (mg / kg) 15.85±2.58 31.14±5.10 23.35±2.87 27.69±3.19 36.81±3.13 Available potassium (mg / kg) 51.37±3.64 73.45±10.19 55.51±5.03 64.77±7.03 83.65±11.27

[0091] Table 20. Increase in soil nutrient indicators in degraded grasslands by combined application of microbial agents compared to single application.

[0092]

[0093] As shown in Tables 11-15, in typical grasslands with moderate degradation due to desertification, mowing, and grazing, the combined treatment of carbon-based organic fertilizer carrier-based growth-promoting microbial inoculant LP with AMF and NFB (LPAN) significantly affected plant community biomass and cover. Compared with the blank control (CK), single inoculant (AMF / NFB), and single carbon-based organic fertilizer carrier-based growth-promoting microbial inoculant (LP), the LPAN treatment increased the aboveground fresh weight of the plant community by 15.96%–227.59%, the aboveground dry weight by 14.82%–130.06%, and the vegetation cover by 10.30%–159.48%, showing the best vegetation restoration effect in all types of typical degraded grasslands.

[0094] As shown in Tables 16-20, in typical grassland soils of desertification, moderate degradation due to mowing, and moderate / severe degradation due to grazing, the combined treatment of carbon-based organic fertilizer carrier-promoting microbial inoculant LP with AMF and NFB (LPAN) significantly affected soil organic matter and available nitrogen, phosphorus, and potassium content. Compared with the blank control (CK), single inoculant (AMF / NFB), and single carbon-based organic fertilizer carrier-promoting microbial inoculant (LP), the LPAN treatment increased soil organic matter content by 11.59%–249.87%, available nitrogen content by 2.53%–83.81%, available phosphorus content by 18.21%–203.42%, and available potassium content by 12.76%–108.74%, effectively achieving synergistic improvement of soil fertility in typical degraded grasslands.

[0095] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for microbial ecological restoration of degraded typical grasslands, characterized in that, The combination of carrier-based growth-promoting microbial agents, arbuscular mycorrhizal fungal agents, and nitrogen-fixing bacteria agents in degraded typical grasslands to be restored, with the arbuscular mycorrhizal fungal agents and nitrogen-fixing bacteria agents used in combination with the carrier-based growth-promoting microbial agents, produces a synergistic effect in restoring degraded typical grasslands, promoting vegetation restoration and soil improvement. For grazing and mowing of moderately degraded typical grasslands, apply 0.15 g / m² of arbuscular mycorrhizal fungal inoculant. 2 Nitrogen-fixing bacteria inoculant 1.8 g / m³ 2 Carrier-promoting microbial agent 0.6 kg / m 2 ; For severely degraded typical grasslands and desertified typical grasslands, apply arbuscular mycorrhizal fungal inoculant at a rate of 0.15 g / m². 2 Nitrogen-fixing bacteria inoculant 2.4 g / m³ 2 Carrier-promoting microbial agent 0.8 kg / m 2 ; The microorganisms in the carrier-promoting microbial agent include Serratia marcescens 5, Enterobacter spp. 24, Serratia marcescens 23, Cossackia covani 11, Bacillus 9-3-1, Bacillus saffron 11-5-4, Bacillus terracotta K4, and Halomonas L31, with accession numbers GDMCC NO.65386, GDMCC NO.65388, GDMCC NO.65389, GDMCC NO.65391, GDMCC NO.65392, GDMCC NO.65394, GDMCC NO.65395, and GDMCC NO.65397, respectively.

2. The method for microbial ecological restoration of degraded typical grassland according to claim 1, characterized in that, The method for preparing the carrier-promoting microbial agent is as follows: *Serratia marcescens* 5, *Enterobacter johnsonii* 24, *Serratia marcescens* 23, *Cossacchariformis* 11, *Bacillus* 9-3-1, *Bacillus saffron* 11-5-4, *Bacillus terracotta* K4, and *Haloxylon ammodendron* L31 are respectively inoculated into LB liquid medium and cultured until OD... 600 =0.8~1, collect bacterial solutions separately; The collected bacterial solutions were mixed in equal volumes and inoculated into LB liquid medium and cultured at 26-30℃ and 100-140 rpm for 10-14 h to obtain a compound growth-promoting microbial solution. After sterilizing the organic fertilizer, it was mixed with the compound growth-promoting microbial solution at a mass ratio of 100:(10-15) and cultured in a cool and ventilated place for 5 days to obtain a carrier growth-promoting microbial agent.

3. The method for microbial ecological restoration of degraded typical grassland according to claim 2, characterized in that, The organic fertilizer is a carbon-based organic fertilizer or cow / sheep manure organic fertilizer.

4. The microbial ecological restoration method for degraded typical grasslands according to claim 1, characterized in that, The arbuscular mycorrhizal fungi in the inoculum agent include *Aureobasidium spp.* Claroideoglomus claroideum ), young scabies near the scabies ( Claroideoglomus etunicatum ), Moses tube handle cyst ( Funneliformis mosseae ), Sandy wasteland shriveling cysts ( Septoglomis deserticolg ) and Heterogeneous Rhizocystis ( Rhizophagus irregularis ).

5. The microbial ecological restoration method for degraded typical grasslands according to claim 1, characterized in that, The nitrogen-fixing bacteria in the nitrogen-fixing inoculum is *Azotobacter chrysophagus* ( ). Azotobacter chroococcum ).

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