Growth-promoting microbial active seeds for repairing degenerated typical grassland and preparation method of growth-promoting microbial active seeds

Abstract

The invention belongs to the technical field of seed treatment, and relates to a growth-promoting microbial active seed for repairing degenerated typical grassland and a preparation method thereof, and the preparation method comprises the following steps: preparing a carrier growth-promoting microbial agent from a specific strain; after grass seeds are disinfected and soaked, a carrier growth-promoting microbial agent is firmly loaded on the surfaces of the seeds by adopting a process of coating the seeds with a functional material layer by layer, and the growth-promoting microbial active seeds are prepared. The method effectively solves the problems of difficult seed field planting, low survival rate, weak seedling stress resistance, soil micro-ecology imbalance and the like in degenerated typical grassland reseeding. After the prepared growth-promoting microbial active seeds are sown, the emergence rate and the survival rate of forage grass can be remarkably increased, plant growth is promoted, rhizosphere soil improvement and ecological function restoration are synchronously achieved, and an efficient and reliable technical means is provided for rapid and sustainable restoration of degenerated typical grassland.

Description

Technical Field

[0001] This invention belongs to the field of seed treatment technology and relates to a growth-promoting microbial active seed for the restoration of degraded typical grasslands and its preparation method. Background Technology

[0002] With the increasing severity of grassland degradation, artificial reseeding has become a key measure for vegetation restoration. However, current reseeding techniques for degraded grasslands face many bottlenecks in practical applications:

[0003] (1) The topsoil of degraded grasslands often suffers from problems such as compaction, infertility, and uneven moisture. Seeds that are traditionally broadcast or simply pelleted and coated are difficult to make stable contact with the soil and germinate effectively. Seedling roots have weak penetration ability, resulting in low transplanting success rate and unstable seedling rate.

[0004] (2) Existing seed coating technologies mostly focus on physical protection (such as increasing weight and regularizing shape) or adding conventional fertilizers and pesticides. Their core is to achieve adaptability to mechanical sowing and control of pests and diseases. This type of coating lacks active ingredients with soil ecological restoration functions and cannot specifically improve the rhizosphere microenvironment or activate the soil microbial community. Therefore, it has limited effect on improving the physiological resistance of seeds under adversity and promoting the recovery of soil health.

[0005] (3) Although existing technologies use specific microbial agents for grassland restoration, the mainstream application method is to apply the agents directly into or spray them onto the soil surface. This method has problems such as spatial separation between the agents and seeds, easy dilution and loss in the soil, low colonization efficiency, and slow establishment of synergy with seedling roots, and fails to achieve direct and precise benefits to seed germination and early seedling growth.

[0006] (4) Existing restoration methods often separate the steps of “seed treatment”, “fertilizer application” and “microbial inoculation”, which is not only cumbersome and costly, but also lacks temporal and spatial coordination between the measures, resulting in low restoration efficiency and difficulty in achieving rapid and sustainable ecological restoration.

[0007] Therefore, there is an urgent need to develop a reseeding method that can efficiently and stably combine plant seeds with specific functional microorganisms and achieve rhizosphere growth promotion and microecological improvement after sowing. Summary of the Invention

[0008] To address the above problems, this invention provides a growth-promoting microbial active seed for the restoration of degraded typical grasslands and its preparation method. The preparation method specifically includes the following steps:

[0009] Step 1: Inoculate *Serratia marcescens* 5, *Enterobacter spp.* 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 26-30°C with shaking at 100-140 rpm until OD... 600 =0.8~1, collect the bacterial solution separately.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] The *Serratia rubidaea* 11 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC NO.65391, deposited on October 31, 2024, at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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 the carrier 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 the carrier-based growth-promoting microbial agent.

[0020] Preferably, the carrier is one of organic fertilizer, biochar, and diatomaceous earth.

[0021] Step 3: Soak the forage seeds in anhydrous ethanol for 10 seconds, soak them in 10% hydrogen peroxide solution for 10 minutes, rinse them with water 4-5 times, and germinate them at 20-25℃ for 24 hours before placing them in a coating machine at a speed of 80 r / min.

[0022] Preferably, the forage seeds are sheepgrass seeds or hyacinth bean seeds.

[0023] Step 4: Add binder to the coating machine, add talc powder after 60 seconds, dry at 30℃ for 3 minutes, add binder again, add 1 / 5 of the carrier growth-promoting microbial agent after 60 seconds, dry at 30℃ for 3 minutes, add binder again, add 1 / 5 of the carrier growth-promoting microbial agent after 60 seconds, dry at 30℃ for 3 minutes, repeat the operation until all the carrier growth-promoting microbial agent is added, and then pass through a 200-mesh sieve and let stand in a dry and ventilated environment for 48 hours to obtain growth-promoting microbial active seeds.

[0024] Preferably, the mass ratio of the seeds, talc powder, carrier-promoting microbial agent, and binder is 25:5:(5~25):65. Most preferably, the binder is a 4% sodium alginate solution.

[0025] Preferably, in the coating machine, binder is added, and after 60 seconds, 1 / 4 mass of attapulgite soil and 1 / 5 mass of carrier-promoting microbial agent are added. The mixture is dried at 30°C for 3 minutes, then binder is added again, and after 60 seconds, 1 / 4 mass of attapulgite soil and 1 / 5 mass of carrier-promoting microbial agent are added. The mixture is dried at 30°C for 3 minutes, and this process is repeated until all the attapulgite soil and carrier-promoting microbial agent are added. Then, binder is added, and after 60 seconds, talc powder is added. The mixture is dried at 30°C for 3 minutes to obtain the final product. Most preferably, the mass ratio of seeds to attapulgite soil is 5:4.

[0026] This invention also provides the application of the aforementioned growth-promoting microbial active seeds in the restoration of degraded typical grasslands and the recovery of vegetation in degraded typical grasslands: the prepared growth-promoting microbial active seeds are applied at a concentration of 10-20 g / m³. 2 The density was sown in degraded typical grasslands.

[0027] The definition of a typical grassland is 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 Yin Mountains, and eastern Ordos Plateau of the Inner Mongolia Plateau, forming a vast typical grassland zone, which is the main body of the Inner Mongolia grassland region.

[0028] The present invention has the following advantages:

[0029] (1) This invention does not simply attach the microbial agent to the seed surface, but constructs a composite functional layer with multiple functions such as physical protection, growth-promoting microbial inoculation, and water and nutrient regulation through specific components and precise mass ratio, so that the growth-promoting microbial active seed becomes a complete micro-repair unit that can work in synergy with the growth-promoting microorganisms after sowing.

[0030] (2) The eight specific functional strains selected in this invention are scientifically matched to form a synergistic symbiotic system. After seed germination, they continue to act on the rhizosphere, directly providing seedlings with easily absorbed nutrients, accelerating early biomass accumulation, stimulating root development, enhancing the root system's ability to absorb water and nutrients, producing antagonistic substances to inhibit soil pathogens, and reducing diseases. Salt-tolerant strains such as Haloxylon ammodendron can alleviate soil salinity stress. This multi-dimensional, endogenous biological growth promotion and stress resistance support enables pasture grasses to maintain strong growth momentum in the adversity of degraded typical grasslands, which is significantly better than traditional coating that relies solely on physical protection or simple nutrient supplementation.

[0031] (3) The growth-promoting microbial active seeds of the present invention integrate physical protection (talc powder, composite growth-promoting microbial functional layer), stress buffering (water and fertilizer retention characteristics of attapulgite soil), and biological growth promotion (multifunctional bacterial agent) triple protection. Experimental examples have shown that they can significantly improve the germination rate, seedling survival rate, and biomass accumulation (especially promoting root development) of seeds in degraded typical grassland soils, effectively overcoming the problems of difficult sowing and low survival rate in traditional sowing and planting.

[0032] (4) After the product of this invention is sown, the functional microorganisms quickly establish a dominant community in the rhizosphere, which can not only directly promote plant growth, but also secrete metabolites, improve soil aggregate structure, and activate nutrients, thereby driving the benign succession of the rhizosphere microecology in sync, realizing the synergistic progress of vegetation restoration and soil improvement, and providing key technical support for the sustainable restoration of degraded grasslands. Detailed Implementation

[0033] 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.

[0034] The raw material sources for the following embodiments are:

[0035] 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

[0036] Sheepgrass seeds and alfalfa seeds were purchased from Inner Mongolia Xiangcao Ecological Technology Co., Ltd. Carbon-based organic fertilizer was purchased from Yuantaifeng (Baotou) Biotechnology Co., Ltd. Biochar was purchased from Henan Dongcui Carbon Materials Technology Co., Ltd. Diatomaceous earth was purchased from Tianjin Zhonglian Chemical Reagent Co., Ltd. Talc powder was purchased from Tianjin Zhonglian Chemical Reagent Co., Ltd. Sodium alginate was purchased from Shandong Zhongchen Biotechnology Co., Ltd. Attapulgite was purchased from Henan Zhuangwei New Materials Co., Ltd.

[0037] Example 1

[0038] 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.

[0039] 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 with steam at 121°C and 101 Pa for 30 min. Mix the carbon-based organic fertilizer with the compound growth-promoting microbial solution at a mass ratio of 100:10. Incubate in a cool and ventilated place for 5 days to obtain a carbon-based organic fertilizer carrier growth-promoting microbial agent.

[0040] Step 3: The seeds are soaked in anhydrous ethanol for 10 seconds, in 10% hydrogen peroxide solution for 10 minutes, rinsed with water 5 times, and soaked at 25°C for 24 hours before being placed in a coating machine at a speed of 80 r / min.

[0041] Step four, the mass ratio of the seeds, talc powder, carrier growth-promoting microbial agent and sodium alginate solution is 25:5:25:65.

[0042] Add 4% sodium alginate solution to the coating machine, add talc powder after 60 seconds, dry at 30℃ for 3 minutes, add sodium alginate solution again, add 1 / 5 of the mass of carbon-based organic fertilizer carrier growth-promoting microbial agent after 60 seconds, dry at 30℃ for 3 minutes, add sodium alginate solution again, add 1 / 5 of the mass of carbon-based organic fertilizer carrier growth-promoting microbial agent after 60 seconds, dry at 30℃ for 3 minutes, repeat the operation until all the carbon-based organic fertilizer carrier growth-promoting microbial agent is added, and the product is obtained. Pass through a 200-mesh sieve and let stand in a dry and ventilated environment for 48 hours to obtain carbon-based organic fertilizer carrier growth-promoting microbial active seeds (LP).

[0043] Example 2

[0044] The difference between this embodiment and Embodiment 1 is that the carrier in this embodiment is biochar. In step four, sodium alginate solution is added to the coating machine. After 60 seconds, 1 / 4 mass of attapulgite clay and 1 / 5 mass of biochar carrier growth-promoting microbial agent are added. The mixture is dried at 30°C for 3 minutes. Then, sodium alginate solution is added again. After 60 seconds, 1 / 4 mass of attapulgite clay and 1 / 5 mass of biochar carrier growth-promoting microbial agent are added. The mixture is dried at 30°C for 3 minutes. This process is repeated until all the attapulgite clay and biochar carrier growth-promoting microbial agent are added. Then, sodium alginate solution is added again. After 60 seconds, talc powder is added. The mixture is dried at 30°C for 3 minutes to complete the process, thus obtaining biochar carrier-promoting microbial active seeds (BP). The mass ratio of seeds, talc powder, carrier growth-promoting microbial agent, attapulgite clay, and sodium alginate solution is 25:5:5:20:65.

[0045] Example 3

[0046] The difference between this embodiment and Embodiment 1 is that the carrier in this embodiment is diatomaceous earth. In step four, sodium alginate solution is added to the coating machine. After 60 seconds, 1 / 4 mass of attapulgite and 1 / 5 mass of diatomaceous earth carrier-promoting microbial agent are added. The mixture is dried at 30°C for 3 minutes. Then, sodium alginate solution is added again. After 60 seconds, 1 / 4 mass of attapulgite and 1 / 5 mass of diatomaceous earth carrier-promoting microbial agent are added. The mixture is dried at 30°C for 3 minutes. This process is repeated until all the attapulgite and diatomaceous earth carrier-promoting microbial agent are added. Then, sodium alginate solution is added again. After 60 seconds, talc powder is added. The mixture is dried at 30°C for 3 minutes to complete the process, thus obtaining diatomaceous earth carrier-promoting microbial active seeds (DP). The mass ratio of seeds, talc powder, carrier-promoting microbial agent, attapulgite, and sodium alginate solution is 25:5:5:20:65.

[0047] Experimental Example 1

[0048] Seeds of Leymus chinensis and Leymus chinensis were treated using the methods described in Examples 1-3, with seeds without the composite growth-promoting microbial functional layer serving as a blank control. The results of physical indicators such as the pass rate, thousand-seed weight, thickness, disintegration time, breakage rate, seed content, and single-seed rate of seeds with active growth-promoting microbial functional layer and seeds without the composite growth-promoting microbial functional layer are shown in Tables 1-2.

[0049] Experimental steps: Take 3 samples, 200 seeds in each sample. Seeds with a coverage area of ​​more than 80% of the compound growth-promoting microbial functional layer are considered to have qualified coverage. Calculate the seed coating qualification rate, H=h / 200×100%, where H is the seed coating coverage qualification rate and h is the number of qualified seeds.

[0050] Three seed samples without the composite growth-promoting microbial functional layer and three seed samples with active growth-promoting microorganisms were randomly selected, with 1000 seeds in each sample. The weight of each sample of 1000 seeds was weighed using an electronic balance (accurate to 0.01 g), and the average value and standard error of the three measurements were calculated.

[0051] Thirty seeds without the composite growth-promoting microbial functional layer and 30 seeds with growth-promoting microbial activity were randomly selected. The thickness of each seed was measured with vernier calipers (measured at the thickest part of the seed, accurate to 0.01 cm). The average value and standard error of the seed thickness were calculated.

[0052] Place two layers of moistened filter paper (soaked in distilled water and drained of excess water) at the bottom of a petri dish and position it horizontally on the experimental table. Randomly select 30 active seeds of growth-promoting microorganisms. Gently place each seed into the petri dish lined with moistened filter paper using tweezers. Start a stopwatch and observe the time taken for the functional layer of the compound growth-promoting microorganisms in each seed to disintegrate (the disintegration endpoint is defined as the functional layer of the compound growth-promoting microorganisms absorbing water, swelling, and beginning to detach). Record the disintegration time for each seed. Repeat each treatment three times and calculate the mean and standard error.

[0053] Three groups of growth-promoting microbial active seeds were randomly selected for each treatment, with 100 seeds in each group. Each group of seeds was placed on a 200-mesh sieve and rinsed with water until the functional layer of the compound growth-promoting microorganisms disintegrated. The individual seed count was checked after disintegration, and the number of pellets containing only one seed in each group was counted. The single-seed rate was calculated using the formula: Single-seed rate = (Number of single-seed pellets / Number of test seeds) × 100%.

[0054] Using the same method as the single-seed rate determination, three groups of samples (100 seeds per group) were used for each treatment. The samples were rinsed with water through a 200-mesh sieve until the functional layer of the compound growth-promoting microorganisms disintegrated. Each seed was examined after disintegration to determine if it contained a real seed, and the number of seed-containing pellets in each group was counted. The seed-containing rate was calculated using the formula: Seed-containing rate = (Number of seed-containing pellets / Number of tested seeds) × 100%.

[0055] Accurately weigh the seeds before treatment (M1) and after treatment and before shaking (M2) (both accurate to 0.01 g). Place the seeds with the growth-promoting microbial activity in a shaker and shake at 220 r / min for 2 min, then weigh the seeds after shaking (M3) (accurate to 0.01 g). Breakage rate calculation formula: Breakage rate = (M2 - M3) / (M2 - M1) × 100%

[0056] Table 1 Physical properties of Leymus chinensis seeds with growth-promoting microbial activity

[0057] deal with Pass rate (%) 1000-grain weight / g Thickness / cm Disintegration time / s Breakage rate (%) Seed yield (%) Single seed rate (%) CK - 1.67±0.03c 0.96±0.02d - - - - LP 95.50±0.29b 3.16±0.07b 1.35±0.03b 33.33±1.67a 0.25±0.16c 100.00±0.00a 100.00±0.00a BP 93.67±0.60b 3.03±0.08b 1.19±0.03c 22.00±1.53b 2.08±0.58b 98.00±0.82a 99.67±0.27a DP 99.17±0.17a 3.46±0.06a 1.46±0.01a 23.33±1.67b 6.19±0.29a 98.33±0.47a 99.33±0.47a

[0058] Table 2 Physical properties of alfalfa seeds with growth-promoting microbial activity

[0059] deal with Pass rate (%) 1000-grain weight / g Thickness / cm Disintegration time / s Breakage rate (%) Seed yield (%) Single seed rate (%) CK - 2.79±0.02d 1.67±0.03b - - - - LP 95.50±0.29b 3.36±0.03c 2.09±0.07a 42.67±1.45b 0.61±0.22b 100.00±0.00a 98.33±0.47a BP 92.67±0.44c 4.13±0.11b 2.22±0.07a 51.00±2.08a 6.30±0.41a 99.33±0.82a 84.67±1.45b DP 98.33±0.44a 5.71±0.07a 2.22±0.06a 32.67±1.45c 5.23±1.48a 95.67±1.45b 55.33±2.05c

[0060] Table 1 shows that the pass rate of Leymus chinensis seeds with growth-promoting microorganisms was greater than 90% in all treatments (LP, BP, DP), and the thousand-seed weight was significantly higher than that of the control (CK) (1.67 g), with increases ranging from 81.44% (BP, 3.03 g) to 107.19% (DP, 3.46 g). This indicates that the compound growth-promoting microorganism functional layer can significantly increase the weight of Leymus chinensis seeds and improve seed plumpness. The seed thickness of Leymus chinensis seeds with growth-promoting microorganisms was generally increased compared to those without the compound growth-promoting microorganism functional layer. The DP treatment (1.46 cm) showed the largest increase (52.08%), while the BP treatment (1.19 cm) showed the smallest increase (23.96%), indicating that the compound growth-promoting microorganism functional layer can enhance the thickness of the seed's physical structure and may improve its resistance to mechanical damage. The BP treatment had the shortest disintegration time (22.00 s), indicating significant differences in water solubility among different compound growth-promoting microorganism functional layer formulations. The BP treatment may be more likely to disintegrate and release seeds in the soil. The DP treatment (6.19%) had a significantly higher breakage rate than other treatments, while the LP treatment (0.25%) had the lowest breakage rate. This indicates that the composite growth-promoting microbial functional layer of the LP formulation has better resilience and can effectively reduce the risk of breakage during seed processing or transportation. The seed-bearing rate and single-seed rate of the LP treatment were both 100%, while the BP and DP treatments showed a slight decrease but still remained at a high level (≥97.67%). This indicates that the composite growth-promoting microbial functional layer has little impact on the seed integrity of Leymus chinensis seeds, and most treatments can ensure seed availability.

[0061] Table 2 shows that the pass rate of alfalfa seeds with active growth-promoting microorganisms was greater than 90% in all treatments (LP, BP, DP), and the thousand-seed weight was significantly higher than that of CK (2.79 g), with increases ranging from 20.43% (LP, 3.36 g) to 104.66% (DP, 5.71 g). The DP treatment showed the most significant improvement. The overall increase in alfalfa seed weight due to the compound growth-promoting microorganism functional layer was greater than that of Leymus chinensis. The thickness of all treatments was greater than that of CK (1.67 cm), with the BP and DP treatments (2.22 cm) showing the largest increase (32.93%). The compound growth-promoting microorganism functional layer significantly enhanced the physical thickness of alfalfa seeds and improved structural stability. Disintegration time: The BP treatment (51.00 s) had a significantly longer disintegration time than the other treatments, while the DP treatment (32.67 s) had a shorter disintegration time. This indicates that BP in the compound growth-promoting microorganism functional layer formulation has poor water solubility, while DP is more easily disintegrated in the soil. The BP (6.30%) treatment had a higher breakage rate, while the LP (0.61%) and DP (5.23%) treatments had lower breakage rates. The LP formulation provided the best protection against breakage of alfalfa seeds. The LP treatment achieved 100% seed retention and maintained a single-seed rate of 98.33%.

[0062] In summary, the compound growth-promoting microbial functional layer significantly improved the thousand-seed weight and thickness of both Leymus chinensis and Leymus chinensis, indicating that the compound growth-promoting microbial functional layer can generally enhance the physical quality of forage seeds and improve their germination and stress resistance potential. The formulations exhibit specific differences; different compound growth-promoting microbial functional layer formulations (LP, BP, DP) showed species-specific effects on the disintegration time, breakage rate, and effectiveness (seed yield, single seed yield) of the two types of forage seeds with active growth-promoting microorganisms.

[0063] Experimental Example 2

[0064] Select plump and uniformly sized seeds of Leymus chinensis and Leymus chinensis. Treat the Leymus chinensis and Leymus chinensis seeds with the methods of Examples 1-3 respectively. Use seeds without the composite growth-promoting microbial functional layer as a blank control. Sow the growth-promoting microbial active seeds in flower pots (23 cm × 18 cm × 21.5 cm) containing 2 kg of soil. The soil was taken from the severely degraded typical grassland of Xilinhot, Inner Mongolia. The seeds were randomly spread on the soil. 200 Leymus chinensis seeds were sown in each pot and 100 Leymus chinensis seeds were sown in each pot. Each treatment was replicated 3 times.

[0065] Detection indicators: After 14 days of cultivation, the germination rate, germination potential, germination index and vigor index of Leymus chinensis and Alfalfa were measured. At the end of the experiment, 6 seedlings were randomly selected from the three replicates to measure the seedling length and root length of Leymus chinensis and Alfalfa. The results are shown in Tables 3-6.

[0066] Germination potential GE (%) = (Number of normally germinated seeds on day 4 of the germination test / Number of tested seeds) × 100%;

[0067] Germination rate GP (%) = (Number of normally germinated seeds on day 10 of the germination test / Number of tested seeds) × 100%;

[0068] Germination index GI = ΣGt / Dt × 100%, where Dt represents the corresponding number of germination days and Gt is the number of seeds germinating per day corresponding to Dt;

[0069] Vitality index VI = GI × S, where GI is the germination index and S is the average radicle length of the seedling.

[0070] Table 3. Effects of different carriers on the activity of growth-promoting microorganisms on seed germination rate and seedling growth of Leymus chinensis.

[0071] deal with Seedlings grow Root length Germination rate GP (%) Germination potential GE (%) Germination Index (GI) Vitality Index VT CK 7.23±0.50b 6.39±0.49b 9.67±1.88b 2.83±0.67b 2.40±0.43b 19.43±4.29b LP 11.79±0.66a 7.67±0.75a 19.33±1.83a 9.33±2.09a 5.03±0.54a 59.69±9.03a BP 8.02±0.30b 7.42±0.35ab 11.83±3.77ab 3.50±2.08b 2.78±0.99ab 22.31±7.82b DP 8.22±0.42b 6.41±0.40b 18.00±1.32a 4.00±1.16b 4.08±0.29a 29.20±0.36b

[0072] Table 4. Germination rate and seedling growth increase of Leymus chinensis seeds with different carriers promoting microbial activity

[0073] index LP growth BP increase DP growth Seedlings grow 63.07% 10.93% 13.69% Root length 20.03% 16.12% 0.31% Germination rate GP 99.90% 22.3% 86.14% Germination potential GE 229.68% 23.67% 41.34% Germination Index (GI) 109.58% 15.83% 70.00% Vitality Index VT 207.20% 14.82% 50.28%

[0074] Table 5. Effects of different carriers on the activity of growth-promoting microorganisms on foetida seed germination rate and seedling growth.

[0075] deal with Seedlings grow Root length Germination rate GP (%) Germination potential GE (%) Germination Index (GI) Vitality Index VT CK 2.36±0.08b 7.61±0.37c 35.33±0.67b 33.00±2.52ab 9.62±0.68ab 22.72±2.25b LP 3.41±0.12a 12.95±0.73a 65.67±1.20a 50.33±0.88a 12.60±0.55a 43.16±4.30a BP 2.60±0.11b 10.90±0.45b 31.00±11.72b 25.00±9.45b 6.23±2.47b 16.72±6.72b DP 3.38±0.07a 9.90±0.53b 49.33±4.37ab 44.00±2.89a 11.65±0.69a 39.36±2.55a

[0076] Table 6. Germination rate and seedling growth increase of alfalfa bean seeds with different growth-promoting microbial activity on different carriers.

[0077] index LP growth BP increase DP growth Seedlings grow 44.49% 10.17% 43.22% Root length 70.17% 43.23% 30.09% Germination rate GP 85.88% -12.26% 39.63% Germination potential GE 52.52% -24.24% 33.33% Germination Index (GI) 30.98% -35.24% 21.10% Vitality Index VT 89.96% -26.41% 73.24%

[0078] Tables 3-4 show the general advantages of the compound growth-promoting microbial functional layer. All treatments (LP, BP, DP) improved the germination rate, germination potential, and germination index of Leymus chinensis, and promoted seedling or root growth, indicating that the compound growth-promoting microbial functional layer has a positive effect on Leymus chinensis seed germination and seedling growth. The compound growth-promoting microbial functional layer formulations showed specific differences: the LP formulation showed significant promoting effects on germination indicators (germination rate, germination potential, germination index), vigor index, seedling length, and root length, and was the optimal formulation. Tables 5-6 show the general advantages of the compound growth-promoting microbial functional layer: all treatments (LP, DP, BP) improved the germination rate, germination potential, germination index, and vigor index of Alfalfa oleifera, and promoted seedling and root growth, indicating that the compound growth-promoting microbial functional layer has a positive effect on Alfalfa oleifera seed germination and seedling growth. The compound growth-promoting microbial functional layer formula has specific differences. The LP formula shows a very significant promoting effect on germination indicators (germination rate, germination potential, germination index), vitality index, seedling length, and root length, and is the optimal formula for alfalfa.

[0079] Experimental Example 3

[0080] Soil samples (2 kg / pot) were collected from severely degraded and desertified typical grasslands in Xilinhot, Inner Mongolia. Seeds of *Leymus chinensis* and *Leymus chinensis* were treated using the methods described in Examples 1-3. 40 *Leymus chinensis* plants / pot and 15 *Leymus chinensis* plants / pot were sown. The plants were kept in a greenhouse at 25℃ / 14 h light, with regular watering to maintain 80% field capacity.

[0081] Monitoring indicators: Aboveground / underground biomass was measured after 60 days of cultivation, and the increase in the number of active seeds of growth-promoting microorganisms compared with the control group was calculated. The results are shown in Tables 7-10.

[0082] Table 7. Biomass of Leymus chinensis in potted plants of each group

[0083]

[0084] Table 8. Increase in Leymus chinensis biomass in each potted group relative to the control group

[0085]

[0086] Table 9. Biomass of Alfalfa in potted plants of each group

[0087]

[0088] Table 10. Increase in biomass of alfalfa in potted plants relative to the control group.

[0089]

[0090] Tables 7-8 show that all treatments (LP, BP, DP) significantly increased the aboveground and belowground biomass of Leymus chinensis in severely degraded and desertified typical grassland soils, indicating that the composite growth-promoting microbial functional layer can effectively enhance the growth potential of Leymus chinensis in degraded typical grassland soils. Compared with the control (CK), these treatments significantly increased the aboveground and belowground fresh and dry weights of Leymus chinensis. Among them, LP showed the most significant increase in severely degraded typical grassland soils (aboveground dry weight increased by 166.67%, and belowground dry weight increased by 871.05%), while BP performed best in desertified typical grassland soils (aboveground dry weight increased by 146.00%, and belowground dry weight increased by 716.67%), with the overall increase in dry weight ranging from 74.00% to 871.05%.

[0091] Tables 9-10 show that all treatments (LP, BP, DP) significantly increased the aboveground and belowground biomass of *Alfalfa* in severely degraded and desertified typical grassland soils, indicating that the composite growth-promoting microbial functional layer can effectively enhance the growth potential of *Alfalfa* in degraded typical grassland soils (severe grazing and desertification). In both severely degraded and desertified typical grassland soils, specific treatments (LP, DP) showed significant growth-promoting effects on *Alfalfa*. Compared with the control (CK), these treatments significantly increased the aboveground and belowground fresh and dry weights of alfalfa. Among them, LP showed outstanding performance in both soil types. In the typical grassland soil of severely degraded grazing, the aboveground dry weight increased by 114.04% and the belowground dry weight increased by 104.76%. In the typical desertified grassland soil, the aboveground dry weight increased by 251.72% and the belowground dry weight increased by 229.07%. In the typical desertified grassland soil, DP increased the aboveground dry weight by 182.76% and the belowground dry weight increased by 23.26%. The overall increase in dry weight ranged from 14.29% to 251.72%.

[0092] Test Example 4

[0093] In June 2025, artificial furrow sowing of alfalfa (treated according to Examples 1-3) was conducted at Maodeng Ranch, Grassland Ecology Base of Inner Mongolia University, Xilinhot City, Inner Mongolia Autonomous Region. The seeds were covered with soil but not compacted, and no fertilizer was applied. 15 g (seed weight before treatment) was sown in each plot, which was divided into 6 rows, with 2.5 g per row. There were 4 treatments and 3 replicates. The plot size was 1 m × 1 m. A randomized block design was used. In August 2025, the aboveground and belowground biomass of plants in each plot was investigated and analyzed. The results are shown in Tables 11-12.

[0094] Table 11 Biomass of *Alfalfa* seeds with different growth-promoting microbial activity in field experiments

[0095] deal with <![CDATA[Aboveground dry weight (g / m 2 )]]> <![CDATA[Underground dry weight (g / m 2 )]]> CK 73.18±7.98c 28.55±3.50c LP 667.43±30.20a 150.28±8.13a BP 414.75±28.57b 92.63±6.36b DP 297.12±71.31b 73.88±13.86b

[0096] Table 12. Increase in biomass of alfalfa seeds relative to the control group in field experiments using different carriers to promote growth microbial activity.

[0097] index LP group compared to CK group BP vs. CK group DP compared to CK group Dry weight above ground 812.04% 466.75% 306.01% Underground dry weight 426.48% 224.45% 158.77%

[0098] Tables 11-12 show that, under field experimental conditions, all treatments (LP, BP, DP) significantly increased the aboveground and underground dry weight of alfalfa beans, indicating that the compound growth-promoting microbial functional layer had a good promoting effect on the growth of alfalfa beans under field conditions. The specific treatment (LP) showed a significant growth-promoting effect on alfalfa beans. Compared with the control (CK), these treatments significantly increased both the aboveground and underground dry weight of alfalfa beans, with the LP treatment showing the most significant increase (812.04% increase in aboveground dry weight and 426.48% increase in underground dry weight).

[0099] 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 type of proliferating microbial active seed for the restoration of degraded typical grasslands, characterized in that, It includes forage seeds and a composite growth-promoting microbial functional layer covering the surface of the seeds. The composite growth-promoting microbial functional layer is composed of a carrier growth-promoting microbial agent, a binder, talc powder, and attapulgite clay. The mass ratio of the forage seeds, talc powder, carrier growth-promoting microbial inoculant, binder and attapulgite soil is 25:5:(5~25):65:20; The growth-promoting microbial agents in the carrier 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 *Haloxylon ammodendron* 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 growth-promoting microbial active seed for the restoration of degraded typical grasslands according to claim 1, characterized in that, The forage seeds are sheepgrass seeds or hyacinth bean seeds.

3. The growth-promoting microbial active seed for the restoration of degraded typical grasslands according to claim 1, characterized in that, The adhesive is a 4% sodium alginate solution by mass.

4. The growth-promoting microbial active seed for the restoration of degraded typical grasslands according to claim 1, characterized in that, The method for preparing the carrier-promoting microbial agent is as follows: 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 are inoculated into LB liquid medium and cultured until OD600 = 0.8~1, and the bacterial solutions are collected respectively. The collected bacterial solutions were mixed in equal volumes and inoculated into LB liquid medium. The mixture was then incubated at 26-30℃ and 100-140 rpm for 10-14 h to obtain a compound growth-promoting microbial solution. This solution was then mixed with a carrier at a mass ratio of (10-15):100 and incubated in a cool, ventilated place for 5 days to obtain a carrier-promoting microbial agent.

5. The growth-promoting microbial active seed for the restoration of degraded typical grasslands according to claim 4, characterized in that, The carrier is one of organic fertilizer, biochar, and diatomaceous earth.

6. The method for preparing the growth-promoting microbial active seeds according to any one of claims 1-5, characterized in that, Disinfected and soaked forage seeds are placed in a coating machine, binder is added, and after 60 seconds, 1 / 4 mass of attapulgite soil and 1 / 5 mass of carrier growth-promoting microbial agent are added. The mixture is dried, binder is added again, and the process is repeated until all the carrier growth-promoting microbial agent is added. The mixture is then sieved and dried to obtain growth-promoting microbial active seeds.

7. The preparation method according to claim 6, characterized in that, The disinfection and soaking treatments consist of soaking the forage seeds in anhydrous ethanol for 10 seconds, soaking them in a 10% hydrogen peroxide solution for 10 minutes, rinsing them with clean water 4-5 times, and soaking them at 20-25°C for 24 hours.

8. The application of the growth-promoting microbial active seeds according to any one of claims 1-5 in the restoration of degraded typical grasslands, characterized in that, Seeds containing growth-promoting microorganisms were sown in degraded typical grasslands at a sowing density of 10–20 g / m². 2 .

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