Microbial agent and application thereof in grass seed screening

Abstract

Bacterial strains PG42 and NH42 are obtained through primary screening from alpine forest litters and soil, bacterial colonial morphology is combined to obtain bacteria, bacterial strains with multiple physiological activities are screened through physiological and biochemical experiments, DNA is extracted from the bacterial strains, 16S rRNA is amplified, and sequence alignment is carried out; the preparation method comprises the following steps: screening a microbial inoculum, namely activating bacillus subtilis MZS1 (B.sub MZS1) and rhodococcus PG42 (Rhodococcus sp.PG42) according to a ratio of 2: 1; and preparing the microbial inoculum. The microbial agent is applied to grass seed screening, the screening efficiency is high, the acting speed is high, the effect of replacing chemical fertilizer is achieved, and pollution to the environment is reduced.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial application technology, specifically relating to a microbial agent and its application in grass seed screening. Background Technology

[0002] Grassland ecosystems are among the most important terrestrial ecosystems, providing abundant resources and ecological services for humankind. Distributed globally, they cover approximately 24% of the Earth's land area, with the majority located in arid regions with low rainfall. Grasslands account for about 40% of China's land area, primarily distributed in Inner Mongolia, Northeast China, Northwest China, and the Qinghai-Tibet Plateau. my country's grasslands can be classified into four types: meadow steppe, typical steppe, desert steppe, and alpine steppe. Grassland ecosystems possess strong carbon sequestration and soil and water conservation capabilities, while also providing habitats for livestock farming, playing a crucial role in global carbon sequestration and mitigating climate change.

[0003] Alpine grasslands, one of the four major types of grasslands, are mainly distributed in areas with high altitudes, low temperatures, and little precipitation. Alpine grassland ecosystems are closely linked to local cultural development, ecological protection, and economic growth. However, due to the high altitudes and the impacts of oxygen scarcity, large diurnal temperature variations, water shortages, and unreasonable human activities, alpine grassland ecosystems are becoming increasingly fragile and degraded. This is mainly manifested in varying degrees of soil vitality and vegetation degradation, leading to a decline in ecosystem multifunctionality, specifically in desertification, salinization, and rodent infestations. The degradation of aboveground vegetation species affects soil water and soil conservation capacity and physicochemical properties, and also severely impacts the quantity and activity of soil microorganisms. Soil microorganisms are closely related to soil material cycling and energy flow, and are an important component of soil nutrients. The deficiencies caused by vegetation degradation further deteriorate the soil itself, creating a vicious cycle. This results in a series of consequences, including decreased ecosystem stability, reduced resistance to disturbance, increased vulnerability, and decreased species diversity. Therefore, how to restore and protect grassland ecosystems, improve their stability and system functions, and maintain ecological balance are urgent issues that need to be explored and resolved.

[0004] Taking the Qinghai-Tibet Plateau as an example, the restoration of alpine grassland ecosystems mainly includes two methods: natural restoration and artificial restoration. Natural restoration, due to the unique geographical location and climate conditions of alpine grasslands, characterized by high altitude, cold, and drought, results in a highly fragile ecosystem, making natural restoration extremely slow. Therefore, artificial restoration is a method worthy of close attention. Among these methods, grass seed selection, a crucial aspect of agricultural production, plays a vital role in improving grassland productivity and quality, enhancing grass species' resilience and adaptability, and protecting the ecological environment. In addressing grassland degradation issues such as reduced grassland area, decreased grass species, and desertification, phytoremediation offers advantages such as low investment, rapid results, short cycles, and diverse utilization methods. Herbaceous plants can quickly stabilize surface and subsurface soils, while also improving soil structure, fertility, and purification. By evaluating grass species based on factors including climatic suitability, drought tolerance, soil physicochemical properties, resilience, and aesthetic appeal, the optimal grass species for the local environment and soil can be selected to improve grassland restoration efficiency.

[0005] Studies have shown that microorganisms play a crucial role in promoting soil biochemical cycling and organic matter decomposition. Furthermore, microorganisms can enhance soil structure stability and aeration, as well as improve soil moisture retention and nutrient content. Therefore, microbial screening methods are highly feasible. Summary of the Invention

[0006] The purpose of this invention is to provide a microbial inoculant and its application in grass seed screening:

[0007] A microbial inoculant,

[0008] 1) Source of microbial strains

[0009] Bacterial strains PG42 and NH42 were initially screened from alpine forest litter and soil. They were merged based on colony morphology to obtain bacteria. Through physiological and biochemical experiments, bacterial strains with multiple physiological activities were screened. DNA was extracted from these bacterial strains, and 16S rRNA was amplified for sequence alignment.

[0010] 2) Screening of microbial agents

[0011] The bacteria were activated using a mixture of Bacillus subtilis MZS1 and Rhodococcus sp. PG42 in a 2:1 ratio.

[0012] 3) Preparation of microbial inoculants

[0013] The activated bacterial agent obtained in step 2 was inoculated into the bacterial fermentation medium and cultured on a shaker at 28°C and 180 rpm for 24 hours. Then, it was inoculated into the fermentation medium at a rate of 20% and cultured at room temperature for 3 days. The mixture was used at a ratio of 2:1.

[0014] Application method of microbial inoculant: After the seeds germinate, dilute the prepared inoculant and spray it.

[0015] The preservation number of Bacillus subtilis MZS1 is CCTCC NO: M20251008;

[0016] This bacterium is deposited at the China Center for Type Culture Collection on May 9, 2025, and its classification name is Bacillus subtilis MZS1. The deposit address is Wuhan University. The preservation number of Bacillus subtilis PG42 is CCTCC NO: M20251009. This bacterium is deposited at the China Center for Type Culture Collection on May 9, 2025, with the taxonomic name Rhodococcus sp. PG42, and the deposit address is Wuhan University.

[0017] The fermentation medium for the strain in step 3) contains 0.5‰ potassium dihydrogen phosphate, 0.6‰ dipotassium hydrogen phosphate, 1‰ ammonium sulfate, 0.2‰ magnesium sulfate, 0.1‰ yeast extract, 0.02‰ sodium chloride, 0.05‰ calcium chloride, 1 ml boron and molybdenum elements, and 1 L water.

[0018] Application of microbial inoculants in grass seed screening: Microbial inoculants have significant effects in grass seed screening, effectively weakening or even offsetting the negative impact of drought stress on grass seeds, and improving indicators such as grass seed survival rate, soil urease activity, bacterial diversity, soil moisture content, biomass and soil organic matter.

[0019] This invention offers the following advantages: First, it avoids the drawbacks of traditional grass seed screening methods, such as cumbersome procedures, long processing times, high investment costs, and limited, often wasteful, and environmentally polluting results. Microbial screening is highly efficient and fast-acting, allowing for comprehensive analysis of grass seed characteristics, replacing the effects of chemical fertilizers and reducing environmental pollution. Second, microorganisms have nitrogen-fixing and organic matter-decomposing capabilities, providing nutrients to plants and inhibiting the spread of plant pathogens. Specifically: 1. Regarding grass seed survival rate, microbial agents not only counteract the effects of adverse living environments on plant adaptability but can also significantly improve the adaptability and resistance of grass seeds. The effects are most significant on sedges and Kentucky bluegrass. Survival rate is an important indicator for assessing the adaptability and survival ability of grass seeds.

[0020] 2. Regarding soil urease activity in grass species, microbial inoculants can significantly reduce the inhibition of drought stress and greatly increase soil urease activity. Among them, the effects of old awned wheat and sedge are the most significant, increasing the nitrogen content and soil organic matter content in the soil.

[0021] 3. Regarding soil bacterial diversity, microbial inoculants have a slight mitigating effect on the reduction of bacterial diversity under drought stress conditions, but their promoting effect on bacterial diversity relative to normal levels is not significant.

[0022] 4. Regarding soil moisture content, microbial inoculants can significantly increase the moisture content of herbaceous soils and mitigate the adverse effects of drought, with the most significant effects on old wheat and early-maturing bluegrass.

[0023] 5. Regarding aboveground biomass, it is mainly used to assess the biological characteristics of grassland or pasture ecosystems, such as productivity, material cycling, and energy flow. In terms of the effect of microbial inoculants, drought-stressed plants showed a significant increase in aboveground biomass after treatment with microbial inoculants, essentially returning to the level of the control group. The effect was most significant in shawlgrass, reflecting its growth rate and benefits.

[0024] 6. Regarding soil organic matter, the effects and survival rates are similar; microbial inoculants can alleviate drought inhibition and achieve higher organic matter content than normal levels. Among them, sedge has the most significant effect.

[0025] 7. From the perspective of biological indicators, microbial inoculants can eliminate or offset the losses to plants caused by drought stress to varying degrees, and even promote them. The promoting effect on grass seed survival rate and soil urease activity is particularly obvious, far exceeding the normal level without treatment. Attached Figure Description

[0026] Figure 1 The effects of different microbial agents on soil organic carbon;

[0027] Figure 2 A comparative graph showing the impact of different treatments on grass seed survival rate;

[0028] Figure 3 Comparison of the effects of different treatments on soil urease activity;

[0029] Figure 4 Comparison of the effects of different treatments on bacterial diversity;

[0030] Figure 5 Comparison of the effects of different treatments on the moisture content of herbaceous soils;

[0031] Figure 6Comparison of the effects of different treatments on aboveground biomass;

[0032] Figure 7 Comparison of the effects of different treatments on soil organic matter in grass species. Detailed Implementation

[0033] The following is in conjunction with the appendix Figure 1-7 The present invention will be further described in detail below with reference to the embodiments:

[0034] Example 1

[0035] 1. Source of microbial strains

[0036] Initial screening of bacterial strains MZS1, CDN1, PG42, and NH42 from alpine forest litter and soil yielded approximately 50 bacterial strains. Physiological and biochemical experiments were conducted to screen four bacterial strains with diverse physiological activities. DNA was extracted from these four strains, and 16S rRNA was amplified for sequence alignment (Table 1).

[0037] Table 1. Four multifunctional strains obtained through screening.

[0038]

[0039] Note: + indicates that it has the ability to decompose. - indicates that it does not have the ability to decompose.

[0040] 2. Screening of microbial inoculants

[0041] Seven different formulations of the four strains were prepared according to different proportions, and a field application optimization experiment of the multifunctional microbial inoculant was carried out in Tangke Town, Ruoergai County.

[0042] In-situ plot experiments were conducted (plot area 8m × 8m), and different strain combinations were selected based on comprehensive considerations: WC1 (MZS1, CDN1; 2:1); WC2 (MZS1, NH42; 2:1); WC3 (MZS1, CDN1, NH42; 2:1:1); WC4 (CDN1, NH42; 2:1); WC5 (CDN1, PG42; 2:1); WC6 (PG42, NH42; 2:1); WC7 (MZS1, PG42; 2:1). The inoculum agent usage for each plot was calculated at 1L / acre, and the agent was diluted and sprayed after seed germination.

[0043] Soil sampling and analysis at the pasture maturity stage showed that the WC7 strain configuration had a significantly greater effect on promoting soil organic matter production compared to other groups (P<0.05). Figure 1Therefore, the initial selection was to use Bacillus subtilis MZS1 and Rhodococcus sp. PG42 in a 2:1 ratio for grass seed screening experiments.

[0044] 3. Cultivation Experiment

[0045] Based on literature review and field investigation, an area with similar site conditions, disturbance intensity, and stand conditions was selected as the study area. Herbaceous experimental plots were set up, with sowing strips 0.30m wide. Land preparation was carried out before sowing to a depth of 15-20cm, followed by thin soil covering and regular weeding. Information such as plot elevation, slope position, and aspect was recorded. After sowing, three treatment groups were established: conventional treatment (CK), drought treatment with 50% deficit irrigation (GH), and drought + microbial inoculant treatment (GG). Three replicates were set up for each plant and each treatment. The GH treatment began two weeks after sowing, with weighing and replenishment of 50% evaporative losses before each irrigation. The GG treatment involved spraying 50 ml of diluted inoculant onto each pot after irrigation. Samples were collected after the forage matured.

[0046] 3.1 Indoor Testing

[0047] Before harvesting the herbs, the herb cover of each strip was measured, and the survival rate was recorded. The aboveground parts of the herbs were cut at ground level within each strip and packaged into labeled envelopes. After being brought back to the laboratory, they were placed in an oven at 108℃ for blanching, and then dried at 65℃ to constant weight. The weight obtained was the dry weight of the aboveground biomass of the plants. The plant roots were dug up with a soil hoe, bagged, brought back to the laboratory, cleaned, weighed, and recorded. The result was the fresh weight of the roots.

[0048] 3.2 Results and Analysis

[0049] 3.2.1 Effects of different treatments on grass seed survival rate

[0050] according to Figure 2 The results show that in the control group (CK), *Poa annua* and *Sedge* had the highest survival rates, both above 69.0%, followed by *Leymus chinensis* and *Leymus chinensis*, with survival rates slightly lower at 68% and 67.7% respectively. *Juncus effusus* had the lowest survival rate at only 41.34%. Under drought stress (GH) treatment, *Sedge* had the highest survival rate at 50.0%, followed by *Leymus chinensis* at 48.67%, then *Poa annua* and *Leymus chinensis* at 42.34% and 41.67% respectively, with *Juncus effusus* having the lowest at 27.0%. Under drought plus fungicide (GG) treatment, *Sedge* had the highest survival rate at 93.34%, followed by *Poa annua*, *Leymus chinensis*, and *Leymus chinensis*, all with survival rates between 87.34% and 88.34%, while *Juncus effusus* had the lowest survival rate at 65.34%.

[0051] Data analysis revealed that under drought stress (GH), the survival rates of all experimental grass species were lower than the control group (CK), all below 50%. However, after treatment with microbial agents, the survival rates significantly increased. Furthermore, the survival rates of the five herbaceous species used in this experiment showed a pattern: drought + microbial agent treatment (GG) > conventional control treatment (CK) > drought treatment with 50% deficit irrigation (GH). The most significant differences were observed in Kentucky bluegrass and Eriocaulon buergerianum, where the survival rate increased by as much as 46.0% after microbial agent treatment compared to drought stress. In addition, the lowest survival rate in the drought + microbial agent treatment group (GG) (Juncus effusus, 65.34%) was greater than the highest survival rate in the drought treatment group (GH) (Carex spp., 50.0%). This indicates that drought stress significantly inhibits the survival rate of grass species, while microbial agent treatment can largely eliminate the effects of drought, even achieving a higher survival rate than the untreated control, thus exhibiting a promoting effect.

[0052] 3.2.2 Effects of different treatments on soil urease activity

[0053] according to Figure 3 The data show that in the conventional control group (CK), *Poa annua* had the highest soil urease activity, reaching 1.47 (unit: mg / (g*d), hereinafter the same), followed by *Leymus chinensis*, *Leymus chinensis*, and *Carex spp.*, whose soil urease activities were all second only to *Poa annua*, ranging from 1.27 to 1.30. In contrast, *Juncus effusus* had the lowest activity.

[0054] Soil urease activity was 1.12. Under drought stress (GH), *Triticum aestivum* and *Carex spp.* had the highest soil urease activity, both at 0.85, followed by *Leymus chinensis* at 0.84, *Poa annua* at 0.78, and *Juncus effusus* at the lowest, with a value of 0.63. Under the drought + fungicide (GG) treatment, *Triticum aestivum* had the highest soil urease activity at 2.16, followed by *Poa annua*, *Carex spp.*, and *Leymus chinensis*, all with high soil urease activities of 2.02, 2.03, and 1.90, respectively, while *Juncus effusus* had the lowest activity at 1.58.

[0055] This indicates that soil urease activity varied significantly among the tested herbaceous plants under different treatments. However, the overall trend was: drought + microbial inoculant treatment (GG) > conventional control (CK) > drought treatment with 50% deficit irrigation (GH). After drought treatment, soil urease activity decreased to varying degrees compared to the control for all herbaceous plants, but showed a significant increase after inoculant treatment. The most significant increase was observed in *Poa annua*, where soil urease activity (GG) was 2.57 times that under GH conditions. Even *Leymus chinensis*, which showed the least effect, still exhibited a 2.27-fold increase in urease activity after inoculant treatment compared to drought conditions. This demonstrates that microbial inoculant treatment can mitigate the effects of drought stress and promote the recovery of aboveground biomass to normal levels, even exceeding normal levels, for all tested herbaceous plants. Therefore, microbial inoculants have a significant effect on increasing soil urease activity in herbaceous plants.

[0056] 3.2.3 The impact of different treatments on bacterial diversity

[0057] according to Figure 4 The data show that in the conventional control group (CK), the bacterial diversity index (H) in the soil of the five experimental grass species was relatively uniform, with no particularly significant differences. Except for Juncus effusus, whose mean bacterial diversity index was 8.30, the mean values ​​of the other four grass species were distributed in the range of 9.40 ± 0.40. Similarly, under drought stress (GH), the diversity index distribution of the five experimental grass species was also relatively even. Kentucky bluegrass had the highest mean value of 8.25, followed by sedge, wheatgrass, and Juncus effusus, with mean bacterial diversity indices of 8.24, 8.21, and 8.07, respectively. Juncus effusus had the lowest mean value of only 7.89. In the drought + microbial agent treatment group (GG), sedge had the highest bacterial diversity in the soil, with a mean value of 9.71, followed by Kentucky bluegrass (9.67), wheatgrass (9.46), and wheatgrass (9.32). Juncus effusus had the lowest mean value of 9.08.

[0058] Analysis of the data in the figure shows that under drought stress, the bacterial diversity of all five experimental grass species decreased, but the decrease was not significant. After treatment with microbial agents, although the bacterial diversity index of each plant showed signs of increasing, the increase was slight. Among them, the highest increase was seen in sedge, with a bacterial diversity index that increased by 117.71% compared to the drought stress condition. However, the data for the five experimental grass species in this group did not consistently show a pattern of drought + microbial agent treatment (GG) > conventional control treatment (CK). For example, in sedge and wheatgrass, the bacterial diversity in the soil of Kentucky bluegrass, crested wheatgrass, and rush after microbial agent treatment only slightly exceeded that of the conventional treatment group. This indicates that microbial agent treatment can slightly alleviate the adverse effects of drought stress, barely restoring the soil to normal levels, and its effect on drought relief is not very significant.

[0059] 3.2.4 Effects of different treatments on soil moisture content in herbaceous soils

[0060] Based on in-situ cultivation experiments and soil moisture content analysis of various tested herbaceous plants under different treatments, from Figure 5 It can be seen that in the control group (CK), *Leymus chinensis* had the highest soil moisture content at 10.42%, followed by *Agropyron cristatum* and *Poa annua*, with soil moisture contents of 10.27% and 10.03%, respectively, not much different from *Leymus chinensis*. Next were *Juncus effusus* and *Carex spp.*, with the lowest soil moisture contents at 8.37% and 8.60%, respectively. Under drought stress (GH) treatment, the soil moisture contents of the five experimental grass species showed little difference, distributed within the range of 8.50 ± 0.25. *Leymus chinensis* had the highest soil moisture content at 8.64%, and *Poa annua* had the lowest at 8.26%. In the drought + fungicide treatment group (GG), *Agropyron cristatum* had a soil moisture content of 11.53%, followed by *Poa annua*, *Carex spp.*, and *Leymus chinensis*, at 11.26%, 11.17%, and 10.98%, respectively, with *Juncus effusus* having the lowest at 10.10%. After treatment with microbial agents, the average moisture content of herbaceous soil increased to 11.01%.

[0061] Therefore, the soil moisture content of the five grass species remained relatively stable under normal conditions, drought conditions, and drought + microbial inoculant conditions, indicating that drought had a mild inhibitory effect on the soil moisture content of the five experimental grass species. Compared to the normal control group, the soil moisture content of the grass species decreased to varying degrees under drought stress, while the soil moisture content increased significantly under the action of microbial inoculants. Overall, the root fresh weight of each tested herb also showed a trend of drought + microbial inoculant treatment (GG) > control group (CK) > drought treatment group (GH), and the soil moisture content after microbial inoculant treatment was higher than that of the normal control group. This indicates that microbial inoculant treatment can alleviate the disadvantages of soil drought to a certain extent, with a moderate degree of effectiveness.

[0062] 3.2.5 Effects of different treatments on aboveground biomass

[0063] Through in-situ cultivation experiments and statistical analysis of the aboveground biomass of various tested herb species under different treatments, Figure 6It was found that in the control group (CK), the highest aboveground biomass was found in *Leymus chinensis*, reaching 2.67 kg / m², followed by *Leymus chinensis* and *Carex spp.*, with aboveground biomass of 2.62 kg / m² and 2.41 kg / m², respectively. *Poa annua* ranked fourth with an aboveground biomass of 2.18 kg / m². Among the five tested grass species, *Juncus effusus* had the lowest aboveground biomass, at only 1.01 kg / m². In the drought stress treatment group (GH), the highest aboveground biomass was found in *Carex spp.*, reaching 2.61 kg / m², followed by *Leymus chinensis*, *Poa annua*, and *Leymus chinensis*, with biomass values ​​of 1.6 kg / m², 1.26 kg / m², and 1.25 kg / m², respectively. The grass species with the lowest aboveground biomass was *Juncus effusus*, at only 0.69 kg / m². In the drought + fungicide treatment group (GH), the aboveground biomass of *Leymus chinensis* was the highest at 2.62 kg / m2, followed by *Agrostis spp.* and *Carex spp.* at 2.59 kg / m2 and 2.61 kg / m2, respectively. *Poa annua* was the second highest at 2.29 kg / m2, and *Juncus effusus* had the lowest at 1.09 kg / m2.

[0064] Therefore, except for Juncus effusus, the aboveground biomass of all tested herbaceous species showed little difference among the control group (CK), drought treatment group (GH), and drought + microbial agent treatment group (GG), indicating high stability. The aboveground biomass of Juncus effusus was significantly lower than that of the other three herbaceous plants in all three groups. Overall, drought stress severely affected the aboveground biomass of the tested herbaceous plants, while microbial agent treatment could mitigate the effects of drought stress and promote the recovery of aboveground biomass to normal levels for all tested herbaceous plants, with some species even exceeding normal levels, such as Kentucky bluegrass, Carex spp., and Juncus effusus. Among them, Leymus chinensis showed the most significant increase in aboveground biomass under microbial agent treatment, exceeding that under drought stress by 107.23% compared to other herbaceous species.

[0065] 3.2.6 Effects of different treatments on soil organic matter in grass species

[0066] according to Figure 7The results show that in the control group (CK), *Elymus sibiricum* had the highest soil organic matter content at 72.26 g / kg, followed by *Poa annua*, *Carex spp.*, and *Leymus chinensis*, with soil organic matter contents of 71.97 g / kg, 71.15 g / kg, and 70.25 g / kg, respectively. In the drought stress treatment group (GH), the highest soil organic matter content was found in *Carex spp.* at 65.73 g / kg, followed by *Elymus sibiricum* at 62.93 g / kg, then *Elymus sibiricum* at 62.83 g / kg, and then *Leymus chinensis* at 60.83 g / kg. The lowest was found in *Juncus effusus* at 58.57 g / kg. In the drought + microbial agent treatment group (GH), the highest soil organic matter content was found in Kentucky bluegrass at 89.36 g / kg, followed by sedge, crested wheatgrass, and old wheatgrass, with values ​​from highest to lowest being 86.23 g / kg, 84.53 g / kg, and 82.28 g / kg, respectively, while the lowest was found in rush pith at 72.61 g / kg.

[0067] Data analysis reveals that under drought stress, the decrease in soil organic matter content among the five experimental grass species was not significant, with the largest decrease in *Leymus chinensis* (eelgrass) being only 12%, indicating a relatively small impact of drought on soil organic matter content. However, after treatment with microbial inoculants, all species showed substantial increases, with *Poa annua* (Kentucky bluegrass) showing the largest increase, rising by 30.0% compared to drought conditions and 19.46% compared to conventional conditions. Furthermore, all five experimental groups exhibited the following pattern: drought + microbial inoculant treatment (GG) > conventional control (CK) > drought treatment with 50% deficit irrigation (GH). This demonstrates that microbial inoculants are highly effective in mitigating soil organic matter deficits caused by drought conditions, achieving levels exceeding normal levels.

[0068] The above experiments show that the optimal ratio proposed in this invention is a 2:1 ratio of Bacillus subtilis MZS1 and Rhodococcus sp. PG42.

[0069] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A microbial inoculant, characterized in that, 1) Source of microbial strains Bacterial strains MZS1 and PG42 were initially screened from alpine forest litter and soil. They were merged based on colony morphology to obtain bacteria. Through physiological and biochemical experiments, bacterial strains with multiple physiological activities were screened. DNA was extracted from these bacterial strains, and 16S rRNA was amplified for sequence alignment. 2) Screening of microbial agents The bacteria were activated using a mixture of Bacillus subtilis MZS1 and Rhodococcus sp. PG42 in a 2:1 ratio. 3) Preparation of microbial inoculants The activated bacterial agent obtained in step 2 was inoculated into the bacterial fermentation medium and cultured on a shaker at 28°C and 180 rpm for 24 hours. Then, 20% of the inoculum was added to the fermentation medium and cultured at room temperature for 3 days. The mixture was then used at a ratio of 2:

1.

2. The microbial inoculant according to claim 1, characterized in that, The preservation number of Bacillus subtilis MZS1 is CCTCC NO: M20251008.

3. The microbial inoculant according to claim 1, characterized in that, The preservation number of Bacillus subtilis PG42 is CCTCC NO: M20251009.

4. The microbial inoculant according to claim 1, characterized in that, The fermentation medium for the strain in step 3) contains 0.5‰ potassium dihydrogen phosphate, 0.6‰ dipotassium hydrogen phosphate, 1‰ ammonium sulfate, 0.2‰ magnesium sulfate, 0.1‰ yeast extract, 0.02‰ sodium chloride, 0.05‰ calcium chloride, 1 ml boron and molybdenum elements, and 1 L water.

5. The application of the microbial agent according to claim 1 in grass seed screening.

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