Preparation method and application of paenibacillus polymyxa microbial inoculum for promoting growth of festuca arundinacea and improving soil

Abstract

The invention discloses a preparation method and application of a paenibacillus polymyxa microbial inoculum for promoting the growth of festuca arundinacea and improving soil, and belongs to the field of microbial agricultural application and grassland ecological restoration, the paenibacillus polymyxa YF is preserved in China General Microbiological Culture Collection Center on December 21, 2020, the preservation number of the paenibacillus polymyxa is CGMCC No.21517. By utilizing the colonization capability and metabolic activity of the paenibacillus polymyxa in soil, on one hand, the plant height, the leaf width, the fresh weight and the dry weight of festuca arundinacea are remarkably increased; on the other hand, the soil pH value can be adjusted, the organic matter content is increased, the soil physical and chemical environment is optimized, meanwhile, the stress resistance of festuca arundinacea is enhanced, and complex climate and soil conditions in Qinghai regions are adapted. The paenibacillus polymyxa microbial inoculum is applied to festuca arundinacea planting, has both economic benefits and ecological benefits, and has wide practical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of microbial agricultural application and grassland ecological restoration, and particularly relates to a preparation method of a Paenibacillus polymyxa microbial agent and application of the Paenibacillus polymyxa microbial agent in Festuca ovina planting BACKGROUND

[0002] Festuca ovina is a perennial herbaceous plant of the genus Festuca in the family Poaceae, has the outstanding characteristics of cold tolerance, drought tolerance, and poor soil tolerance, and has developed root system and strong soil fixation capacity, is one of the core high-quality forage grasses in the grassland regions in northern China, and is also a key grass species for degraded grassland improvement, slope protection, and ecological restoration.

[0003] In Qinghai, Festuca ovina not only provides high-quality forage rich in crude protein and crude fiber for the livestock industry, but also plays an irreplaceable role in maintaining the ecological balance of grasslands, preventing soil erosion, and improving the regional climate. According to statistics, the planting area of Festuca ovina in Qinghai accounts for more than 25% of the total natural pasture area, and the yield and quality directly affect the income of local herdsmen and the stability of the grassland ecosystem.

[0004] However, the current Festuca ovina planting faces two core problems: one is growth limitation and yield deficiency: the soil in Qinghai generally has low organic matter content and poor nutrient availability (such as phosphorus and potassium fixation), and the traditional planting relies on the use of single fertilizers (such as nitrogen and potassium fertilizers), which not only fails to meet the growth needs of Festuca ovina, but also leads to soil compaction and deterioration of physical and chemical properties, further inhibiting the biomass accumulation of Festuca ovina; the other is ecological adaptability and soil degradation: long-term overgrazing or unreasonable planting in some areas leads to grassland degradation and soil fertility decline, and the stress tolerance (such as cold and drought resistance) of Festuca ovina is weakened, and the risk of pathogen accumulation in the soil is increased, indirectly affecting the survival rate and growth quality of Festuca ovina.

[0005] In the prior art, microbial agents have been gradually applied to forage planting, but most single microbial agents have the defect of poor adaptability: either only focusing on a single growth indicator (such as only promoting growth without improving soil), or failing to adapt to the Qinghai alpine environment, and lacking a synergistic optimization scheme with base fertilizer, making it difficult to achieve the dual goals of high yield and soil improvement of Festuca ovina. Therefore, it is an urgent need in the current grassland livestock and ecological restoration field to develop a Paenibacillus polymyxa microbial agent for Festuca ovina that is suitable for Qinghai and has the functions of promoting growth and soil improvement. SUMMARY

[0006] The primary object of the present application is to provide the application of Paenibacillus polymyxa YF or its fermentation liquor in promoting the growth of fescue, wherein the Paenibacillus polymyxa YF is preserved in the China General Microbiological Culture Collection Center on December 21, 2020, and the preservation number is CGMCC No. 21517.

[0007] Preferably, the Paenibacillus polymyxa YF is used in combination with a fertilizer.

[0008] Preferably, the fertilizer comprises one or more of chemical fertilizer, potassium fertilizer, and organic fertilizer.

[0009] The second object of the present application is to provide the application of Paenibacillus polymyxa YF or its fermentation liquor in increasing the yield of fescue, wherein the Paenibacillus polymyxa YF is preserved in the China General Microbiological Culture Collection Center on December 21, 2020, and the preservation number is CGMCC No. 21517.

[0010] The third object of the present application is to provide the application of Paenibacillus polymyxa YF or its fermentation liquor in increasing the length and leaf width of fescue, wherein the Paenibacillus polymyxa YF is preserved in the China General Microbiological Culture Collection Center on December 21, 2020, and the preservation number is CGMCC No. 21517.

[0011] The fourth object of the present application is to provide the application of Paenibacillus polymyxa YF or its fermentation liquor in increasing the fresh weight content of the aboveground and underground parts of fescue, wherein the Paenibacillus polymyxa YF is preserved in the China General Microbiological Culture Collection Center on December 21, 2020, and the preservation number is CGMCC No. 21517.

[0012] The fifth object of the present application is to provide the application of Paenibacillus polymyxa YF or its fermentation liquor in increasing the dry weight content of the aboveground and underground parts of fescue, wherein the Paenibacillus polymyxa YF is preserved in the China General Microbiological Culture Collection Center on December 21, 2020, and the preservation number is CGMCC No. 21517.

[0013] The sixth objective of this invention is to provide the application of Paenibacillus polymyxa YF or its fermentation broth in the preparation of fescue growth promoters. The Paenibacillus polymyxa YF was deposited at the China General Microbiological Culture Collection Center on December 21, 2020, with accession number CGMCC No. 21517.

[0014] The seventh objective of this invention is to provide a method for promoting the restoration of degraded grasslands, characterized in that Paenibacillus polymyxa YF and other fertilizers are applied to the degraded grasslands. The Paenibacillus polymyxa YF was deposited at the China General Microbiological Culture Collection Center on December 21, 2020, with accession number CGMCC No. 21517. The fertilizers include organic fertilizers and / or inorganic fertilizers.

[0015] The polymyxin Bacillus inoculant of the present invention, when applied to fescue cultivation, can achieve the following beneficial effects:

[0016] ① Promotes fescue growth and increases biomass

[0017] Organic fertilizer + microbial agent combination: Fescue plant height increased by 14.09%, leaf width increased by 19.37%, fresh weight (per unit area) increased by 17.95%, and dry weight (per unit area) increased by 17.22%; Chemical fertilizer + microbial agent combination: Fescue plant height increased by 9.23%, leaf width increased by 12.15%, fresh weight increased by 10.87%, and dry weight increased by 9.65%. Compared with the single base fertilizer group without microbial agent, the growth indicators of the microbial agent synergistic group generally increased by 15% to 20%.

[0018] ② Improve soil physical and chemical properties and enhance soil fertility

[0019] Organic fertilizer + microbial agent combination: Soil pH increased by 6.61% (approaching the neutral range suitable for fescue), organic matter content increased by 29.08%, available potassium increased by 21.93%, available phosphorus increased by 24.92%, available nitrogen increased by 6.86%, and total nitrogen increased by 12.59% (total potassium decreased by 16.19%, and total phosphorus decreased by 9.26%, which is a normal fluctuation in the nutrient transformation process from "insoluble form to available form"); Chemical fertilizer + microbial agent combination: Soil pH increased by 8.86%, organic matter content increased by 22.37%, available potassium increased by 17.11%, available phosphorus increased by 25.67%, available nitrogen increased by 5.22%, and total nitrogen increased by 9.89% (total potassium decreased by 8.14%, and total phosphorus decreased by 11.55%).

[0020] ③ Green and environmentally friendly, reducing ecological risks

[0021] It reduces the use of chemical fertilizers by more than 30% (achieved through the enhancement of microbial agents), has no pesticide residues, and meets the needs of grassland ecological protection and green animal husbandry development.

[0022] ④ Easy to operate and suitable for large-scale promotion

[0023] The preparation process is mature (it can be achieved with conventional fermentation equipment), and the application method is in line with the local fescue planting habits in Qinghai. It does not require additional operating costs for farmers and is suitable for large-scale application in degraded grassland improvement projects. Attached Figure Description

[0024] Figure 1 The effect of different treatments on the dry weight of fescue in this invention;

[0025] Figure 2 The effect of different treatments on the fresh weight of fescue in this invention;

[0026] Figure 3 The effect of different treatments on fescue leaf width in this invention;

[0027] Figure 4 The effect of different treatments on fescue plant height in this invention;

[0028] Figure 5 The effects of different treatments on the pH of fescue soil in this invention;

[0029] Figure 6 The effects of different treatments on available nitrogen in fescue soil in this invention;

[0030] Figure 7 The effects of different treatments on available potassium in fescue soil in this invention;

[0031] Figure 8 The effects of different treatments on available phosphorus in fescue soil in this invention;

[0032] Figure 9 The effects of different treatments in this invention on total nitrogen in fescue soil;

[0033] Figure 10 The effects of different treatments on total potassium in fescue soil in this invention;

[0034] Figure 11 The effects of different treatments on total phosphorus in fescue soil in this invention;

[0035] Figure 12 This invention illustrates the effects of different treatments on the organic matter content of fescue soil. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] It should be noted that, unless otherwise specified, the methods described in the following embodiments are all conventional methods, and the reagents described are all commercially available.

[0038] It should be noted that in the following embodiments, the *Paenibacillus polymyxa* YF is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 21517 and contact number 010-64807355. In the following embodiments, *Paenibacillus polymyxa* YF will be abbreviated as *Paenibacillus polymyxa* YF.

[0039] In the following examples, the Bacillus megaterium de Bary was isolated by the inventor's research group and preserved in the laboratory.

[0040] In the following embodiments, the *Brevibacillus laterosporus* was isolated by the inventor's research group and preserved in the laboratory.

[0041] Example 1: Preparation of Bacillus polymyxa YF inoculum

[0042] Includes the following steps:

[0043] (1) Activation of strain: The slant culture of Bacillus polymyxa YF was streaked on LB solid plates and cultured at 30℃ for 24h, and the activation was repeated twice.

[0044] (2) Seed culture: The activated strain was inoculated into YF optimal seed culture medium (15 g / L peptone, 15 g / L sucrose, 3 g / L NaCl, 5 g / L magnesium sulfate, pH 7.0~7.2) and cultured at 30℃ and 180 rpm for 24 h to obtain a seed culture with a viable count ≥1.0×109 CFU / mL.

[0045] (3) Fermentation culture: The seed culture was inoculated into the fermentation medium (60g sucrose, 60g peptone, 6.0g yeast extract, 0.26g MgSO4, 0.2g K2HPO4, 0.4g NaCl, 0.05g CaCl2) at a 5% inoculation rate, pH 7.0-7.2, at 30℃ and 200rpm for 48h, with dissolved oxygen content controlled at ≥20%.

[0046] (4) Finished product preparation: The fermentation broth is filtered through a 200-mesh filter to obtain a liquid inoculum with a viable count ≥1.0×10⁻⁶. 8 After dispensing at CFU / mL.

[0047] Example 2: A method for promoting fescue growth and improving planting soil using a Bacillus polymyxa YF-base fertilizer compound.

[0048] The method includes the following steps:

[0049] Step 1: Base fertilizer preparation: Formula 1: Pour the chemical fertilizer (agricultural grade, 10 kg / mu) and potassium fertilizer (agricultural grade, 10 kg / mu) into a mixing container, and stir at 180-200 rpm for 20 minutes at room temperature to ensure that the particles are evenly mixed and free of lumps; If formula 2 is selected, first pour the organic fertilizer (fully decomposed, crushed to a particle size ≤5 mm, 300 kg / mu) into the container, then add the chemical fertilizer and potassium fertilizer, and stir at 200-220 rpm for 30 minutes at room temperature to fully integrate the organic matrix and inorganic nutrients;

[0050] Step 2: Preparation of *Bacillus polymyxa* YF inoculum: Using LB liquid medium (1% peptone, 0.5% yeast extract, 1% sodium chloride, balance sterile water), inoculate *Bacillus polymyxa* YF into the medium. Control the fermentation conditions at 30-32℃, 180-200 rpm, and shake for 36-48 hours until the OD600 value of the inoculum reaches 1.5-1.8 and the viable cell concentration is ≥10⁻⁶. 9 CFU / mL, prepared as a liquid microbial inoculant;

[0051] Step 3: Seed treatment: Take the Bacillus polymyxa YF liquid inoculant prepared in Step 2, pour it into the seed soaking container, and completely immerse the fescue seeds in the inoculant. Control the ambient temperature at 20-25℃ and soak for 24 hours, stirring gently once every 8 hours. After soaking, transfer the seeds to a cool and ventilated place to dry, avoiding direct sunlight to prevent germination or mold.

[0052] Step 4: Base fertilizer application and sowing preparation: The base fertilizer prepared in Step 1 is evenly spread on the surface of the experimental field soil. Mechanical tillage is used to mix the base fertilizer with the 0-20cm soil layer, ensuring even nutrient distribution and laying the foundation for subsequent sowing and microbial colonization. Comparative Example 1: A method for promoting fescue growth and improving planting soil using a Bacillus megaterium-base fertilizer composite combination.

[0053] The method includes the following steps:

[0054] The other methods are the same as in Example 1, except that Bacillus polymyxa YF is replaced with Bacillus megaterium.

[0055] Comparative Example 2: A method for promoting fescue growth and improving planting soil using a Bacillus laterosporus-base fertilizer composite combination.

[0056] The method includes the following steps:

[0057] The other methods are the same as in Example 1, except that Bacillus polymyxa YF is replaced with Bacillus lateralis.

[0058] Example 3

[0059] The following experiments were conducted using the composite combinations prepared in Example 1, Comparative Example 1, and Comparative Example 2, respectively.

[0060] Experimental location: Forage planting base in the No. 7 Factory Area of ​​Haiyan Atomic City Scenic Area (36°57′27″N, 100°51′33″E), with an experimental area of ​​10 mu (approximately 667 square meters). The soil type is chestnut calcareous soil. Initial physicochemical indicators: pH 7.5, organic matter 15.2 g / kg, available potassium 120 mg / kg, and total nitrogen 0.8 g / kg.

[0061] Experimental groups (2 acres per group, 3 replicates, randomized block design):

[0062] CK group: No bacterial agent or base fertilizer applied;

[0063] Fertilizer group: Apply only "10 kg / mu of chemical fertilizer + 10 kg / mu of potassium fertilizer";

[0064] Fertilizer + microbial agent group: Apply "10 kg / mu of chemical fertilizer + 10 kg / mu of potassium fertilizer + Bacillus polymyxa YF microbial agent (seed soaking treatment)";

[0065] Organic fertilizer group: Apply only "300 kg / mu of organic fertilizer";

[0066] Organic fertilizer + microbial agent group: Apply "300 kg / mu of organic fertilizer + Bacillus polymyxa YF microbial agent (seed soaking treatment)";

[0067] Planting procedure: Sow on May 10, 2025. Fescue seeds are local Qinghai varieties (purity 96%, germination rate 88%). After soaking in fungicide for 24 hours (22℃), they are dried. The sowing rate is 1.8 kg / mu. After broadcasting, lightly rake and cover with 1-2 cm of soil.

[0068] Field management: Water once a week (30m) after sowing. 3 / mu), weeding was done once in mid-June, and no diseases or pests occurred;

[0069] Indicator monitoring: From July 20 to August 10, 2025, five quadrats (1m×1m) were randomly selected from each group to measure fescue growth indicators; at the same time, soil samples from 0 to 20cm were collected to measure eight major soil physicochemical parameters (pH, organic matter, available potassium, available phosphorus, available nitrogen, total potassium, total phosphorus, and total nitrogen).

[0070] (1) Festuca growth monitoring experiment

[0071] The growth indicators of fescue in each treatment group were measured on July 22, 2025 (2 months after sowing) and August 15, 2025 (3 months after sowing, harvest period): plant height (from the ground to the top of the plant) was measured with a ruler, and leaf width (maximum width of the third pair of fully unfolded leaves at the top) was measured with a vernier caliper. After harvest, the fresh weight of each plant was weighed, and the dry weight was measured after drying at 65℃ to constant weight. Three quadrats (1m×1m) were randomly selected from each group, and 10 plants were measured in each quadrat, and the average value was taken.

[0072] Test results of different strains, such as Figures 1-4 As shown in Tables 1 and 2, compared with other groups, the combined application of organic fertilizer and Bacillus polymyxa inoculant during the forage planting process increased the height, leaf width, fresh weight and dry weight of fescue by 14.09%, 19.37%, 17.95% and 17.22%, respectively. The growth data show that the Bacillus polymyxa YF-base fertilizer compound combination has a significant promoting effect on fescue growth.

[0073] Table 1. Effects of microbial inoculant addition on plants under inorganic fertilizer treatment.

[0074]

[0075] Table 2. Effects of adding microbial agents on plants under organic fertilizer treatment.

[0076]

[0077] (2) Soil physicochemical index monitoring test

[0078] On July 22, 2025, three sampling points were randomly selected from each treatment group to collect rhizosphere soil samples from 0-20 cm (top leaves removed, five-point sampling method was used). The samples were mixed and passed through a 2 mm sieve, and the eight major physicochemical properties of the soil were determined using conventional methods.

[0079] The test results are as follows Figures 5-12 As shown in Tables 3 and 4, compared to the control group, the combined application of inorganic fertilizer and Bacillus polymyxa resulted in an 8.86% increase in pH, a 22.37% increase in organic matter, a 17.11% increase in available potassium, a 25.67% increase in available phosphorus, and a 5.22% increase in available nitrogen in fescue soil, while a 8.14% decrease in total potassium, an 11.55% decrease in total phosphorus, and a 9.89% increase in total nitrogen. Conversely, compared to the control group, the combined application of organic fertilizer and Bacillus polymyxa resulted in a 6.61% increase in pH, a 29.08% increase in organic matter, a 21.93% increase in available potassium, a 24.92% increase in available phosphorus, and a 6.86% increase in available nitrogen in *Leymus chinensis*, while a 16.19% decrease in total potassium, a 9.26% decrease in total phosphorus, and a 12.59% increase in total nitrogen. Organic fertilizer achieved superior results compared to inorganic fertilizer.

[0080] Table 3. Effects of microbial inoculant addition on soil physicochemical properties under inorganic fertilizer treatment.

[0081]

[0082] Table 4. Effects of adding microbial agents on soil physicochemical properties under organic fertilizer treatment.

[0083]

[0084] In summary, the method described in this invention can promote fescue growth and increase biomass: the combination of organic fertilizer and microbial agent increased fescue plant height by 14.09%, leaf width by 19.37%, fresh weight (per unit area) by 17.95%, and dry weight (per unit area) by 17.22%; the combination of chemical fertilizer and microbial agent increased fescue plant height by 9.23%, leaf width by 12.15%, fresh weight by 10.87%, and dry weight by 9.65%. Compared with the single base fertilizer group without microbial agent, the growth indicators of the microbial agent synergistic group generally increased by 15% to 20%. Improving soil physicochemical properties and enhancing soil fertility: Organic fertilizer + microbial agent combination: Soil pH increased by 6.61% (approaching the suitable neutral range for fescue), organic matter content increased by 29.08%, available potassium increased by 21.93%, available phosphorus increased by 24.92%, available nitrogen increased by 6.86%, and total nitrogen increased by 12.59% (total potassium decreased by 16.19%, and total phosphorus decreased by 9.26%, which is a normal fluctuation during nutrient transformation from "insoluble to available"). Chemical fertilizer + microbial agent combination: Soil pH increased by 8.86%, organic matter content increased by 22.37%, available potassium increased by 17.11%, available phosphorus increased by 25.67%, available nitrogen increased by 5.22%, and total nitrogen increased by 9.89% (total potassium decreased by 8.14%, and total phosphorus decreased by 11.55%). Environmentally friendly and reduces ecological risks: Reduces chemical fertilizer use by more than 30% (achieved through microbial synergy enhancement), with no pesticide residues, meeting the needs of grassland ecological protection and green animal husbandry development. Easy to operate and suitable for large-scale promotion: The preparation process is mature (can be achieved with conventional fermentation equipment), and the application method aligns with local fescue planting habits in Qinghai, requiring no additional operating costs for farmers, making it suitable for large-scale application in degraded grassland improvement projects.

Claims

1. Application of Paenibacillus polymyxa YF or its fermentation broth in promoting the growth of fescue, wherein the Paenibacillus polymyxa YF was deposited at the China General Microbiological Culture Collection Center on December 21, 2020, with accession number CGMCC No. 21517.

2. The application as described in claim 1, characterized in that, The aforementioned polymyxa Bacillus YF is used in combination with fertilizer.

3. The application as described in claim 1, characterized in that, The fertilizers mentioned include one or more of chemical fertilizers, potash fertilizers, and organic fertilizers.

4. Application of Paenibacillus polymyxa YF or its fermentation broth in increasing fescue yield, wherein the Paenibacillus polymyxa YF was deposited at the China General Microbiological Culture Collection Center on December 21, 2020, with accession number CGMCC No. 21517.

5. Application of Paenibacillus polymyxa YF or its fermentation broth in increasing the length and leaf width of fescue, wherein the Paenibacillus polymyxa YF was deposited at the China General Microbiological Culture Collection Center on December 21, 2020, with accession number CGMCC No. 21517.

6. Application of Paenibacillus polymyxa YF or its fermentation broth in increasing the fresh weight content of the aboveground and underground parts of fescue, wherein the Paenibacillus polymyxa YF was deposited at the China General Microbiological Culture Collection Center on December 21, 2020, with accession number CGMCC No. 21517.

7. Application of Paenibacillus polymyxa YF or its fermentation broth in increasing the dry weight content of the aboveground and underground parts of fescue, wherein the Paenibacillus polymyxa YF was deposited at the China General Microbiological Culture Collection Center on December 21, 2020, with accession number CGMCC No. 21517.

8. Application of Paenibacillus polymyxa YF or its fermentation broth in the preparation of fescue growth promoter, wherein the Paenibacillus polymyxa YF was deposited at the China General Microbiological Culture Collection Center on December 21, 2020, with accession number CGMCC No. 21517.

9. A method for promoting the restoration of degraded grasslands, characterized in that, The degraded grassland was treated with Paenibacillus polymyxa YF and other fertilizers. Paenibacillus polymyxa YF was deposited at the China General Microbiological Culture Collection Center on December 21, 2020, with accession number CGMCC No. 21517. The fertilizers included organic fertilizers and / or inorganic fertilizers.

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