Composition with nitrogen fixation effect, paenibacillus graminis used by composition and application of composition and paenibacillus graminis

Abstract

The invention discloses a composition with a nitrogen fixation effect, paenibacillus graminis used by the composition and application of the paenibacillus graminis. The technical problem to be solved by the invention is how to reduce the application of chemical nitrogen fertilizer, reasonably fix nitrogen and promote plant growth. The strain number of the paenibacillus graminis is LCAH615, and the preservation number of the paenibacillus graminis in the China General Microbiological Culture Collection Center is CGMCC No.29549. The activity determination of the strain finds that the paenibacillus graminis has strong nitrogen fixation capability, and can be used as an active component to prepare a microbial nitrogen fertilizer. Corn plant and soil indexes are analyzed through a corn pot experiment, and it is found that the prepared liquid nitrogen fertilizer can not only improve the soil environment, but also promote crop growth. Therefore, the paenibacillus graminis can be used as a green nitrogen fertilizer, plays a role in reducing the dosage of a chemical nitrogen fertilizer, and has important significance in improving the ecological environment.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, specifically relating to compositions with nitrogen-fixing properties, the Bacillus species used in them, and their applications. Background Technology

[0002] As an essential element for life, nitrogen plays an indispensable role in maintaining and sustaining life on Earth. Nitrogen fertilizer plays a crucial role in increasing crop yield and efficiency, and is an important chemical fertilizer for crop cultivation. Currently, nitrogen fertilizer is applied in the form of urea, and its rational application is one of the important measures to improve corn yield and quality. However, in agricultural cultivation, excessive application of nitrogen fertilizer has led to problems such as soil compaction, acidification, low crop yield, low nitrogen utilization rate, and nutrient imbalance.

[0003] Microbial nitrogen fixation is mainly classified into three forms: autotrophic nitrogen fixation, associative nitrogen fixation, and symbiotic nitrogen fixation. The application of microbial nitrogen fixation involves preparing nitrogen-fixing microorganisms into inoculum, dry powder, or liquid form, which is then directly applied to crops. Microbial nitrogen-fixing inoculants are a finished product that can effectively serve the development of green agriculture. Multiple studies have shown that applying microbial fertilizers can not only reduce chemical fertilizer use but also promote plant growth and increase crop yields. Therefore, developing new types of microbial nitrogen fertilizers to reduce chemical fertilizer use provides a new impetus for achieving green and sustainable agricultural development. Summary of the Invention

[0004] The problem this invention aims to solve is how to reduce the application of chemical nitrogen fertilizers and rationally fix nitrogen to promote plant growth.

[0005] Therefore, the present invention provides a bacterium containing Bacillus cereus (Bacillus subtilis). Paenibacillus graminis The composition of the Bacillus cereus strain is numbered LCAH615 and has the accession number CGMCC No.29549 at the China General Microbiological Culture Collection Center.

[0006] The composition may be any one of the following: A1) Compositions for nitrogen fixation, A2) Compositions that promote plant growth. A3) Soil-improving compositions.

[0007] This invention also protects a strain of Bacillus, wherein the Bacillus is a herbaceous Bacillus (Bacillus spp.) Paenibacillus graminis The strain is numbered LCAH615 and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 29549.

[0008] This invention also protects the use of the above-mentioned Bacillus in the preparation of any of the following bacterial agents: B1) Microbial agents used for nitrogen fixation, B2) Microbial agents that promote plant growth. B3) Soil-improving microbial agents.

[0009] The aforementioned soil improvement methods can increase soil carbon and nitrogen content, as well as increase soil organic matter content.

[0010] The present invention also provides a method for preparing the above composition, the method comprising the step of using the above-mentioned Bacillus as a component of the composition.

[0011] The above composition can be used to prepare fertilizer.

[0012] The above-mentioned microbial agents can also be used to prepare fertilizers. The active ingredient in the above composition may be the Bacillus or / and the metabolites of the Bacillus or / and the culture of the Bacillus.

[0013] The culture can be a substance obtained by culturing the Bacillus in a microbial culture medium (i.e., a fermentation product, such as a fermentation broth containing the Bacillus and a substance secreted into a liquid culture medium, or a solid fermentation product containing the Bacillus and a substance secreted into a solid culture medium).

[0014] In the above text, the metabolite may be a product obtained by removing the Bacillus from the culture, such as culturing the Bacillus in a liquid fermentation medium, collecting the fermentation broth (containing the Bacillus and substances secreted into the liquid culture medium), removing the Bacillus from the fermentation broth, collecting the remaining components of the fermentation broth, and obtaining the metabolite of the Bacillus.

[0015] The active ingredients of the above composition may also contain other biological or non-biological components, and those skilled in the art can determine the other active ingredients of the above composition based on the effects of the composition.

[0016] The above composition can also be a microbial agent or a microbial fertilizer.

[0017] The above-mentioned microbial fertilizers can be used for soil application.

[0018] The aforementioned microbial agents refer to live microbial preparations made by using a carrier as an adsorbent to adsorb the fermentation broth or solid fermentation products of the target microorganisms after propagation.

[0019] The above-mentioned microbial agents can be in various dosage forms, including but not limited to liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.

[0020] Depending on the needs, the microbial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0021] The above-mentioned microbial fertilizer is a microbial nitrogen fertilizer with a strong nitrogen-fixing ability.

[0022] The present invention also protects fertilizers containing the above-described composition and / or the above-described Bacillus cereus.

[0023] The plant may be any of the following: C1) Monocotyledons, C2) Plants of the order Poales, C3) Gramineae plants, C4) Plants of the genus *Zea*. C5) Corn.

[0024] Microbial fertilizers have broad application prospects. While reducing the application of chemical nitrogen fertilizers, the combined application of microbial nitrogen fertilizers can promote crop growth and improve soil. The preparation of microbial nitrogen fertilizers using strain LCAH615 can further reduce the application of chemical fertilizers and provide theoretical reference and data support for the green and sustainable development of agriculture.

[0025] The beneficial effects of this invention are: 1. Promotes crop growth and improves soil environmental conditions.

[0026] 2. Increasing soil microbial biomass can promote the accumulation of dry matter in crops.

[0027] 3. The content of total soil carbon, total organic carbon, and available nitrogen has increased.

[0028] This invention utilizes Bacillus subtilis to prepare a liquid microbial nitrogen fertilizer with strong nitrogen-fixing capabilities. Besides increasing maize plant height and total biomass and promoting maize growth, it also improves soil environmental conditions, increases soil microbial biomass, and promotes the accumulation of crop dry matter. After fertilizer application, the soil's total carbon, total organic carbon, and available nitrogen content increased. These results indicate that Bacillus subtilis and its compositions can serve as green nitrogen fertilizers, reducing the use of chemical nitrogen fertilizers and playing a significant role in improving the ecological environment.

[0029] Preservation Instructions Bacterial strain name: Bacillus subtilis Latin name: Paenibacillus graminis Strain number: LCAH615 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: January 9, 2024 CGMCC Registration Number: CGMCC No. 29549 Attached Figure Description Figure 1 Bacillus subtilis Paenibacillus graminis Genetic phylogenetic tree.

[0030] Figure 2 The figures show the growth curves and nitrogenase activities of Bacillus cereus, where A is the growth curve of Bacillus cereus and B is the nitrogenase activity of Bacillus cereus.

[0031] Figure 3 The parameters for measuring maize plant indicators are as follows: A is maize plant height, B is maize plant biomass, C is maize plant pigment content, and D is total chlorophyll content of maize plant.

[0032] Figure 4 For soil index determination, A is soil carbon content, B is soil organic matter content, C is soil ammonium nitrogen content, and D is soil nitrate nitrogen content.

[0033] Figure 5 For comparison of corn potted plants in different treatments, from left to right, the treatments are: 20% nitrogen fertilizer reduction without inoculation, full fertilizer without inoculation, and inoculation treatment.

[0034] Figure 6 This is a 16S rRNA PCR amplification fragment of Bacillus subtilis.

[0035] Figure 7 This is an amplified fragment of nifH from Bacillus cereus.

[0036] Figure 8 The morphological characteristics of Bacillus cereus colonies as seen with the naked eye.

[0037] Figure 9 Morphological characteristics of Bacillus cereus under a scanning electron microscope. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] The following examples used one-way ANOVA with IBM SPSS Statistics 24, at a significance level of P < 0.05. The analysis results were plotted using Origin 2021.

[0041] The method for isolating Bacillus cereus in this study involves preparing a soil suspension, serially diluting it, and then uniformly spreading it onto the corresponding culture medium.

[0042] The nitrogenase activity of the strain was determined by acetylene reduction method.

[0043] Liquid microbial nitrogen fertilizer was prepared by inoculating 1% of Bacillus cereus into LB liquid medium and fermenting for 1 day.

[0044] The obtained liquid inoculant was applied to maize cultivation by seed soaking to study its effects on maize growth and soil environment.

[0045] Example 1 Bacillus subtilis ( Paenibacillus graminis Nitrogen fixation capacity determination of LCAH615, CGMCC No. 29549 Bacillus subtilis ( Paenibacillus graminis The nitrogen fixation capacity of strain LCAH615, CGMCC No. 29549 (hereinafter referred to as strain LCAH615) was verified, and the steps are as follows: After activating strain LCAH615, it was inoculated into liquid LB at a ratio of 1:100 and incubated at 200 rpm and 30°C. OD was measured at regular intervals. 600 Continue until the bacterial culture stabilizes. Use no inoculation as a blank control. Perform three parallel experiments. Plot the growth curve with time on the x-axis. OD 600 Using the vertical axis as the ordinate, the final calculation of the strain's growth generations is used to better understand the strain's growth status.

[0046] 2) Acetylene reduction method for nitrogenase activity determination: Activated strain LCAH615 was inoculated into LB medium at a ratio of 1:100 and cultured overnight. The cells were collected by centrifugation at 8000 rpm for 5 min, washed twice with deionized water, and resuspended in nitrogen-limiting medium to OD0.05. 600 The concentration was 0.3-0.5. 10 mL was transferred to an anaerobic tube, aerated, and filled with nitrogen. After stressing at 30℃ and 200 rpm for 1-2 hours, 5 mL was injected into an anaerobic tube containing 10% acetylene gas. 100 μL of gas was injected into the gas chromatograph, and the time was recorded. The zero point was set with the gas from the first acetylene injection. Uninoculated culture medium was used as a control. Samples were taken at corresponding time intervals, and the sampling time and ethylene peak area were recorded. Three replicates were performed each time. Protein content was determined according to the Bradford method protein concentration assay kit instructions.

[0047] The results of the acetylene reduction test are shown below Figure 2 strain B. The nitrogenase activity of this strain is 57.1. + 0.2 C2H4 / mg / min / protein.

[0048] As described above, the nitrogen-fixing ability of Bacillus cereus has been verified, and the culture medium formulation is as follows: Prepare high-concentration stock solutions of the following reagents: 26.3g Na₂HPO₄·2H₂O, 3.4g KH₂PO₄, 10µg biotin, 26mg CaCl₂·2H₂O, 30mg MgSO₄, 0.33mg MnSO₄·H₂O, 36mg ferric citrate, 7.6mg Na₂MoO₄·2H₂O, 10µg p-aminobenzoic acid, 4g glucose, and 0.3g glutamic acid. Filter and sterilize the glutamic acid solution. Prepare a high-concentration stock solution of glucose and sterilize it separately. Sterile the ferric citrate and CaCl₂·2H₂O separately. Mix the remaining culture media thoroughly and sterilize at 115℃ for 30 minutes. Finally, bring the volume to 1L.

[0049] Example 2: Preparation and application of strain LCAH615 inoculant 2.1 Preparation of inoculum from strain LCAH615 1) Inoculate strain LCAH615 at a rate of 1% into LB liquid medium and incubate at 200 rpm and 30°C for 1 day to obtain the fermentation broth.

[0050] 2) Collect the fermentation broth cells and resuspend the OD in sterile water. 600 The fermentation broth was prepared by fermentation in step 1) to a final volume of 0.5. The LB medium formula is as follows: 10g peptone, 5g yeast extract, 10g sodium chloride, and 15-20g agar added to the solids, with a final volume adjusted to 1L. The medium was then autoclaved at 121℃ for 20 minutes using distilled water.

[0051] 2.2 Application of microbial nitrogen-fixing agents containing strain LCAH615 The test soil was selected from Keshan black soil (N 48.01', E 125.82') in Qiqihar City, Heilongjiang Province. For the maize pot experiment, cylindrical brown flowerpots with a diameter of 19 cm were used. The soil was air-dried and sieved through a 2 mm sieve, with 1.6 kg per pot. This experiment included three different treatments: a control of full fertilizer without inoculation (N100); 80% nitrogen fertilizer without inoculation (N80); and 80% nitrogen fertilizer inoculated with the strain Paenibacillus graminis. Each experiment was repeated three times.

[0052] N100 (Full Fertilizer, No Inoculation Group): The base fertilizer application amounts are 0.2g of nitrogen fertilizer (urea) per pot, 0.2g of phosphorus fertilizer (superphosphate) per pot, and 0.1g of potassium fertilizer (potassium chloride) per pot. After thoroughly mixing the fertilizer with the soil and watering overnight, sow 3 seeds per pot. After a few days, when seedlings emerge, keep only one seedling of uniform height per pot and remove the other seedlings. Water with 250mL of water every 5 days. Cultivate at room temperature with 14 hours of light and 10 hours of darkness. Take samples after 50 days of cultivation.

[0053] N80 (20% reduced nitrogen fertilizer, no bacterial inoculation): The base fertilizer application amounts are 0.16g nitrogen fertilizer (urea) / pot, 0.2g phosphate fertilizer (superphosphate) / pot, and 0.1g potassium fertilizer (potassium chloride) / pot. After thoroughly mixing the fertilizer with the soil and watering overnight, sow 3 seeds per pot. After a few days, when seedlings emerge, keep only one seedling of uniform height per pot and remove the other seedlings. Water with 250mL of water every 5 days. Cultivate at room temperature with 14 hours of light and 10 hours of darkness. Take samples after 50 days of cultivation.

[0054] Inoculation group: Base fertilizer application consisted of 0.16g nitrogen fertilizer (urea) / pot, 0.2g phosphorus fertilizer (superphosphate) / pot, and 0.1g potassium fertilizer (potassium chloride) / pot. The fertilizer was thoroughly mixed with the soil and watered overnight. Corn seeds of Ful 116 were inoculated at a concentration of 0.115 × 10⁻⁶. 8 Soak the corn in a solution of strain LCAH615 (CFU / ml) for 2 hours before inoculation. Apply topdressing 14 days after corn growth, using a solution of 0.023 × 10⁻⁶ CFU / ml. 8 The concentration of CFU / ml was increased to 10mL per basin, while the control group was given 10mL of sterile water.

[0055] Sow 3 seeds per pot. After a few days, when seedlings emerge, keep only one seedling of uniform height per pot, removing the others. Water with 250mL of water every 5 days. Perform a cultivation treatment at room temperature with 14 hours of light and 10 hours of darkness. Take samples after 50 days of cultivation. See [link to plant growth chart] for details. Figure 5 .

[0056] 2.3 Sample Collection and Processing: 1) Sample collection: a) Plant sampling: Cut the plant from the above-ground part for later use; b) Soil sampling: Remove the corn roots, gently shake off the soil from the root zone, wash them clean with water and dry them with paper, then mix the soil thoroughly for later use.

[0057] 2) Sample processing: a) Plant height measurement: The plant height is measured by measuring the height of the plant cut off from the ground using a soft measuring tape; b) Aboveground and belowground biomass: Place the plants and roots at 105℃ for 30 minutes to kill the green, and dry them at 70℃ until constant weight. Weigh the dry weight of each, which is the biomass. c) Plant pigment content: determined using the 80% acetone method; d) Soil carbon and nitrogen determination: Soil ammonium nitrogen and nitrate nitrogen: determined using a water flow analyzer; Soil carbon content: determined using a TOC analyzer; Sample pretreatment: Air-dried soil was passed through a 2mm mesh sieve, mixed thoroughly, and weighed to 10g. Extraction was performed using 0.5mol / L K₂SO₄ at a liquid-to-soil ratio of 4:1, with shaking at a constant temperature of 25℃ for 40 min. The mixture was then filtered through a 0.45μm filter membrane. The sample was then ready for instrument use. A blank sample was prepared.

[0058] e) Soil organic matter content: Soil organic matter = total soil organic carbon * coefficient 1.724.

[0059] Compared with the experimental group without inoculation (80% nitrogen fertilizer), the maize plant height in the inoculated group was significantly higher than that in the inoculated group (e.g., ...). Figure 3 (A) The corn plant height in the inoculated group was significantly higher than that in the group with 80% nitrogen fertilizer but no inoculation by 15.1%. Although the corn plant height in the inoculated group was not significantly higher than that in the group with full fertilizer but no inoculation, it was still 8.8% higher than that in the group with full fertilizer but no inoculation. This indicates that the strain has a certain growth-promoting effect on crops. On the other hand, the reduction of chemical nitrogen fertilizer also has the effect of soil improvement.

[0060] Compared with the full-fertilizer-without-inoculation group and the 80% nitrogen-fertilizer-without-inoculation group, the soil aboveground biomass, belowground biomass, and total biomass of the inoculated group increased (see...). Figure 3 In the group B inoculated with the microbial agent, the biomass was the most significant, with a total biomass increase of 61.7% compared to the group without inoculation and with 20% reduced nitrogen fertilizer. This indicates that the strain can not only improve the nutritional conditions of crops and soil but also improve the soil environment and promote crop growth. Therefore, the application of strain LCAH615 can increase soil microbial biomass and plant biomass, and also promote the accumulation of dry matter in crops, thereby increasing crop yield.

[0061] according to Figure 3 In sections C and D, compared with the full-fertilizer-without-inoculation group and the 80% nitrogen-fertilizer-without-inoculation group, the content of various pigments in maize plants in the inoculated groups was significantly increased. Compared with the full-fertilizer-without-inoculation group, the chlorophyll a, chlorophyll b, and total chlorophyll in the inoculated groups increased significantly by 18.3%, 129.8%, and 34.7%, respectively, while compared with the 80% nitrogen-fertilizer-without-inoculation group, they increased significantly by 32.7%, 149.3%, and 53.4%, respectively.

[0062] The soil total carbon, total organic carbon, and organic matter content all increased in the inoculated group. Figure 4 In both groups (A and B), the soil total carbon, total organic carbon, and organic matter content in the inoculated group were significantly higher than those in the 80% nitrogen fertilizer-free group (without inoculation). However, the total carbon content in the inoculated group did not show a significant increase compared to the full-fertilizer-free group. Compared to the 80% nitrogen fertilizer-free group, the soil organic carbon, total carbon, and organic matter content in the inoculated group were significantly increased, by 14.4%, 15.1%, and 14.4%, respectively.

[0063] Compared with the full-fertilizer-without-inoculation group and the 80% nitrogen-fertilizer-without-inoculation group, the soil ammonium nitrogen and nitrate nitrogen contents in the inoculated group were both increased. Figure 4 (C and D). Compared with the group without inoculation (80% nitrogen fertilizer), the soil ammonium nitrogen and nitrate nitrogen contents in the inoculated group were significantly increased by 18.7% and 48.1%, respectively. Compared with the group without inoculation (full fertilizer), the soil ammonium nitrogen content in the inoculated group did not increase significantly, while the soil nitrate nitrogen content in the inoculated group increased significantly by 55.4%. These results indicate that this strain can effectively increase the soil free nitrogen content, thereby promoting the absorption and utilization of available nitrogen by plants.

[0064] The overall analysis above shows that reducing nitrogen levels and applying microbial nitrogen fertilizer containing strain LCAH615 as an active ingredient can increase maize plant height and total biomass, promoting maize growth. It can also increase soil carbon and nitrogen content, improving soil environmental conditions.

[0065] Example 3: Isolation and Identification of Strain LCAH615 3.1 Strain Screening Methods Soil samples were passed through a 2mm mesh sieve. 1g of soil (collected on October 10, 2021, from Langjia Village, Xingsheng Township, Wuchang City, Heilongjiang Province; soil type: thin-layered sandy meadow albic soil) was weighed and placed in 9mL of physiological saline. The soil suspension was prepared by shaking overnight at 30℃ and 200rpm, and then allowed to stand at room temperature for 15min. 100µL of the suspension was then added to 900µL of sterile water, and serially diluted 10-10 times. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Take 10 respectively -3 -10 -6100 μL of soil suspension was evenly spread on ammonified medium (5 g peptone, 5 g KH₂PO₄, 0.5 g K₂HPO₄, 0.5 g MgSO₄·7H₂O; 20 g agar was added for solids; pH = 7.0; autoclaved at 121℃ for 30 min; after the medium cooled to 40-50℃, 1 mL of 50 mol / L nystatin was added to inhibit mold growth). Each gradient was replicated in triplicate. The plates were inverted and placed in a 30℃ incubator. Colony growth was observed periodically, and single colonies were streaked and purified three times on the corresponding plates. Single clones were then selected, enriched, and stored at 4℃, hereinafter referred to as strain LCAH615.

[0066] 3.2 Strain Identification 1) Strain morphological characteristics The LCAH615 strain, isolated and purified in 1.1 above, which was in the logarithmic growth phase and had stable colony size, was observed as a single colony. The strain was plated on LB medium; it appeared white with clearly defined colony sizes (e.g., ...). Figure 8 The strain morphology was observed using scanning electron microscopy; it was rod-shaped (e.g., ...). Figure 9 ).

[0067] 2) Physiological and biochemical characteristics The physiological and biochemical characteristics of strain LCAH615 were determined according to *Microbiology Experiments* (Shen Ping, Fan Xiurong, Li Guangwu. *Microbiology Experiments* (3rd Edition). Beijing: Higher Education Press, 1999.) and *Handbook of Systematic Identification of Common Bacteria* (Dong Xiuzhu, Cai Miaoying. *Handbook of Systematic Identification of Common Bacteria*. Beijing: Science Press, 2011.). The results of the physiological and biochemical characteristics determination of strain LCAH615 are as follows: Table 1. Identification of physiological and biochemical characteristics of strain LCAH615

[0068] 3) 16S rRNA identification 16S rRNA genera were identified from single colonies using PCR amplification with primers 27F (5'-AGAGTTGATCCTGGCTCAG-3') and 1492R (5'-CTACGGCTACCTTGTTACGA-3'). The PCR reaction mixture consisted of 8 μL ddH2O, 10 μL 2×M5HiPer plus Taq HiFi, 0.5 μL upstream primer, 0.5 μL downstream primer, and 1 μL bacterial culture. After the reaction, 3 μL was collected for 1% agarose gel electrophoresis to confirm the PCR amplified fragments (see [link to reaction details]). Figure 6The PCR products were sent to the company for DNA sequencing, and the obtained strain sequences were compared and analyzed in the Ezbiocloud database. A phylogenetic tree was constructed using the 16S rDNA sequence of this strain via Neighbor-joining. Analysis showed that the 16S rRNA of *Bacillus cereus* and the 16S rRNA of strain LCAH615 had 99.99% similarity. Therefore, strain LCAH615 was ultimately identified as *Bacillus cereus*. Paenibacillus graminis (See Figure 1 The strain LCAH615 was deposited on January 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 29549.

[0069] The 16S rRNA sequence (SEQ NO.1, 1420bp) of strain LCAH615 is as follows:

[0070] according to Figure 2 The data from sample A showed that this strain grew rapidly, with an OD value of [value missing] within 24 hours. 600 The result can reach 1.0 or higher. By downloading the strain sequence from NCBI and searching for the nifH gene, primers were designed simultaneously upstream and downstream of the nifH gene. PCR confirmed that the strain possesses the nifH gene (see...). Figure 7 ).

[0071] Primers for the nifH gene of strain LCAH615: nifH-UP:5'-TTATTGGCCGGAAGCCTCA-3', nifH-down: 5'-TTTCTACGGTAAAGGCGGTA-3'.

[0072] PCR amplification was performed using a 20 μL reaction system: 8 μL ddH2O, 10 μL 2×M5 HiPer plus Taq HiFi, 0.5 μL upstream primer, 0.5 μL downstream primer, and 1 μL bacterial culture.

[0073] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A composition, characterized in that, The composition contains Bacillus cereus (Bacillus) Paenibacillus graminis The strain is numbered LCAH615 and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 29549.

2. The composition according to claim 1, characterized in that: The composition is a culture, which is a substance obtained by culturing the Bacillus cereus in a microbial culture medium.

3. The composition according to claim 1 or 2, characterized in that: The composition is a microbial agent.

4. The composition according to any one of claims 1-3, characterized in that, The composition is any one of the following: A1) Compositions for nitrogen fixation, A2) Compositions that promote plant growth. A3) Soil-improving compositions.

5. Bacillus, characterized in that, The Bacillus is a type of Bacillus cereus (Bacillus species) Paenibacillus graminis The strain is numbered LCAH615 and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 29549.

6. The use of the Bacillus of claim 5 in the preparation of any one of the following bacterial agents: B1) Microbial agents used for nitrogen fixation, B2) Microbial agents that promote plant growth. B3) Soil-improving microbial agents.

7. A method for preparing the composition, characterized in that, The composition is the composition according to any one of claims 1-4, and the method includes the step of using the Bacillus of claim 5 as a component of the composition.

8. The use of the composition according to claims 1-4 or the Bacillus according to claim 5 in the preparation of fertilizer.

9. A fertilizer containing the composition of claims 1-4 or the Bacillus of claim 5.

10. The composition according to claim 4 or the application according to claim 6, characterized in that, The plant is any one of the following: C1) Monocotyledons, C2) Plants of the order Poales, C3) Gramineae plants, C4) Plants of the genus *Zea*. C5) Corn.

Images