Prosteria megatherium and microbial agent and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses prelisteria megatherium, a microbial agent thereof and application of the prelisteria megatherium. The preservation number of the presteria megaterium NPKM-001 is CGMCC (China General Microbiological Culture Collection Center) No.32751, and the preservation number of the presteria megaterium NPKM-001 is CGMCC No.32751. The prelisteria megatherium and the preparation thereof, provided by the invention, have extremely high application value in crop planting, and are particularly suitable for applying urea to crops, and the strain has remarkable phosphorus dissolving and solubilizing capacity, high yield of urease inhibitors and indoleacetic acid and no denitrification function, so that the yield of the urease inhibitors and the indoleacetic acid is increased. The microbial fertilizer can achieve multiple purposes of promoting crop growth, increasing yield, increasing planting benefits and reducing environmental pollution, is widely applied to agricultural planting, provides a new scheme for green agriculture, is beneficial to updating microbial fertilizer strains, and promotes sustainable development of the green agriculture.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Priesteria gigantea, a microbial agent thereof and application thereof. Background Art

[0002] In modern agricultural production, the large-scale use of nitrogen fertilizers has played an important role in increasing crop yields, but it has also brought about a series of serious environmental problems and resource waste. Urea, as a widely used nitrogen fertilizer in agricultural production, will be rapidly hydrolyzed under the action of urease after being applied to the soil, resulting in ammonia volatilization loss, which not only reduces the utilization rate of nitrogen fertilizers, but also causes air pollution and increases greenhouse gas emissions. At the same time, excessive accumulation of nitrogen in the soil will cause soil degradation problems such as soil acidification and salinization, further affecting the growth of crops and the sustainable development of the ecological environment. In order to solve the problem of low urea utilization, the application of urease inhibitors has received widespread attention. Urease inhibitors can inhibit the activity of urease, slow down the hydrolysis rate of urea, thereby reducing ammonia volatilization and improving the utilization rate of nitrogen fertilizers. However, most urease inhibitors are currently chemically synthesized substances, and long-term use may have adverse effects on the structure and function of soil microbial communities, destroy the ecological balance of the soil, and may also pose potential risks to agricultural product safety.

[0003] In promoting plant growth, the plant hormone indoleacetic acid (IAA) plays a key role. It can promote plant root growth, cell elongation and division, and improve crop stress resistance and yield. Traditional ways to obtain indoleacetic acid include chemical synthesis and extraction from plants, but chemical synthesis has problems such as high cost, complex process and possible environmental pollution; extraction from plants has limited yield and is difficult to meet the needs of large-scale agricultural production.

[0004] In addition, denitrifying bacteria in the soil will reduce nitrate nitrogen to gaseous nitrogen, resulting in soil nitrogen loss and reducing nitrogen fertilizer utilization. At the same time, the nitrous oxide produced is a potent greenhouse gas that exacerbates global warming. Therefore, screening microbial strains with specific functions and environmental friendliness has become an important research direction for solving the above agricultural production problems.

[0005] At present, although some microorganisms have been found to have the ability to produce urease inhibitors or indoleacetic acid, or some microorganisms do not have denitrification function, microbial strains that have the ability to produce urease inhibitors and indoleacetic acid at the same time and do not have denitrification function are relatively rare. The development of microbial strains with multiple beneficial functions has important practical significance and application value for improving nitrogen fertilizer utilization, promoting plant growth, and reducing environmental pollution. Summary of the invention

[0006] In view of the problem in the prior art that there is a lack of microorganisms that can simultaneously produce urease inhibitors and indole acetic acid and have no denitrification effect, and it is impossible to simultaneously achieve multiple effects of promoting crop growth, increasing yield and reducing environmental pollution, the present invention provides a Priestia megaterium, its microbial agent and application. Using the Priestia megaterium provided by the present invention as a fertilizer microorganism, after it is applied to the soil, it can produce urease inhibitors and indole acetic acid in the soil. The urease inhibitor can reduce the activity of soil urease and the decomposition rate of urea, making the rate of ammonia production by urea hydrolysis as close as possible to the rate of ammonia utilization by crops, improving the utilization rate of fertilizers such as urea, and reducing NH3 volatilization caused by the application of nitrogen fertilizers such as urea; at the same time, this strain has no denitrification effect, can effectively reduce N2O emissions, and reduce air pollution; it can also promote the growth of crop roots through indole acetic acid and increase crop yield.

[0007] To solve the above technical problems, the technical solution provided by the present invention is:

[0008] In the first aspect, the present invention provides a Priestia megaterium NPKM-001, whose preservation number is CGMCC No. 32751.

[0009] The Priestia megaterium NPKM-001 was screened from the rhizosphere soil of corn in a corn field with good growth near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770), and was classified and named as Priestia megaterium. It was deposited in the China General Microbiological Culture Collection Center (CGMCC) on November 22, 2024. The preservation number of the strain is CGMCC No. 32751, and the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

[0010] The biological characteristics of the Priestia megaterium NPKM-001 provided by the present invention are as follows: the colony is milky white, the surface is dry, the edge is relatively neat, opaque, the texture is relatively hard, the cells are rod-shaped, relatively large, and the cell size is about (1.2-1.5) μm × (2.0-4.0) μm, often single or arranged in short chains; the spores are oval, central, the spore diameter is smaller than the cell, and the spore size is about (1.0-1.2) μm × (1.5-2.0) μm.

[0011] The Priestia megaterium NPKM-001 is a Gram-positive bacterium, and the methyl red test, VP test, indole test, and urea hydrolysis test are all negative; the oxidase test, catalase test, glucose fermentation test, citrate utilization test, starch hydrolysis test, gelatin liquefaction test, and casein test are all positive.

[0012] The provided Priestia megaterium NPKM-001 of the present invention belongs to the "strains exempt from toxicology tests" permitted by the "General Technical Guidelines for Biosafety of Microbial Fertilizers (NY / T 1109-2017)". It has stable production performance and belongs to the common types of strains used by current microbial fertilizer production enterprises, and can be used to produce microbial fertilizers. It has characteristics significantly different from the existing strains for producing microbial fertilizers. It has high yields of urease inhibitors and indole acetic acid, and has no denitrification effect, and can achieve multiple goals of promoting crop growth, increasing yield, improving planting benefits, and reducing environmental pollution, and has broad applications in agricultural planting and high practical value.

[0013] In a second aspect, the present invention also provides the application of the above-mentioned Priestia megaterium NPKM-001 in reducing the emissions of NH3 and N2O in soil and improving nitrogen fertilizer utilization efficiency.

[0014] Furthermore, the present invention also provides the application of the above-mentioned Priestia megaterium NPKM-001 in promoting crop growth and increasing crop yield.

[0015] The Priestia megaterium NPKM-001 provided by the present invention demonstrates unique metabolic advantages and ecological regulation capabilities on the basis of the traditional functions of phosphorus and potassium solubilization. During its growth and metabolism process, it can efficiently synthesize urease inhibitors and indole acetic acid (IAA), and does not have denitrification activity. This multi-functional characteristic provides an innovative solution for agricultural production. In terms of enhancing fertilizer efficiency, the secreted urease inhibitors can inhibit the activity of soil urease, significantly slow down the rate of urea hydrolysis to ammonia (ammonium), and at the same time, also limit the supply of nitrification and NO3 - , thus reducing the subsequent denitrification rate and reducing the emissions of harmful gases such as NH3 and N2O in farmland soil. In terms of promoting plant growth, the indole acetic acid produced by the strain can promote root growth and increase crop yield. At the same time, due to the lack of denitrification function of this strain, during the process of colonizing and multiplying in the rhizosphere of crops, it can optimize the soil microbial community structure and increase the relative abundance of the denitrification-free functional bacteria. This optimization of the microbial community structure further inhibits the soil denitrification process, reduces the conversion of nitrate, nitrite and ammonium nitrogen to N2O, and reduces greenhouse gas emissions, achieving the dual goals of fertilizer saving and efficiency increase and ecological protection.

[0016] Through the synergistic effect of the above multiple functions, a superimposed effect is formed in aspects such as improving soil physical and chemical properties, promoting healthy crop growth, and reducing agricultural non-point source pollution, providing strong technical support for the sustainable development of green agriculture, and having significant economic, social and environmental benefits.

[0017] In a third aspect, the present invention also provides a microbial inoculant containing the above-mentioned Priestia megaterium NPKM-001.

[0018] Furthermore, the microbial inoculant is a liquid inoculant or a powder inoculant.

[0019] Making the Priestia megaterium NPKM-001 into a microbial inoculant has achieved good storage stability and convenient transportation characteristics. This inoculant highly adapts to the needs of modern agricultural production and shows strong flexibility and universality in application. It can flexibly select diversified application methods such as foliar spraying, root watering, or basal application according to the differences in crop types, planting systems, and soil characteristics. This diversified application plan ensures the efficient implementation and wide promotion of the technology application without increasing the usage cost and operation complexity for farmers.

[0020] Furthermore, the viable count of Priestia megaterium NPKM-001 in the liquid inoculant ≥ 1.0×10 9 CFU / mL; the viable count of Priestia megaterium NPKM-001 in the powder inoculant ≥ 1.0×10 10 CFU / g.

[0021] As a specific embodiment of the present invention, the preparation method of the liquid inoculant includes the following steps:

[0022] Load the fermentation medium into a ventilated stirring fermenter, with a filling coefficient of 0.65 - 0.75, sterilize at 121°C for 30 min, and cool to 40°C for inoculation. Inoculate the seed liquid of Priestia megaterium NPKM-001 into the ventilated stirring fermenter, with an inoculation amount of 5% - 10%, and ferment at 37°C, a rotation speed of 150 - 200 r / min, and an aeration rate of 1.0 - 2.0 VVM for 48 - 72 h to obtain a fermentation broth with a viable count ≥ 1.0×10 9 CFU / mL, which can be directly used as a liquid inoculant with a shelf life of 6 months.

[0023] As a specific embodiment of the present invention, the preparation method of the powder inoculant includes the following steps:

[0024] Load the fermentation medium into a ventilated stirring fermenter, with a filling coefficient of 0.65 - 0.75, sterilize at 121°C for 30 min, and cool to 40°C for inoculation. Inoculate the seed liquid of Priestia megaterium NPKM-001 into the ventilated stirring fermenter, with an inoculation amount of 5% - 10%, and ferment at 37°C, a rotation speed of 150 - 200 r / min, and an aeration rate of 1.0 - 2.0 VVM for 48 - 72 h to obtain a fermentation broth with a viable count ≥ 1.0×10 9 CFU / mL;

[0025] The fermentation broth is continuously centrifuged by a disc centrifuge at 6000 - 7000 r / min and concentrated 5 - 10 times to obtain bacterial sludge; after adding auxiliary materials to the bacterial sludge, it is spray-dried to obtain a powdery bacterial agent.

[0026] As a specific embodiment of the present invention, the method for preparing the seed liquid of the above-mentioned Paenibacillus megaterium NPKM-001 includes the following steps:

[0027] ① Preparation of slant culture in eggplant-shaped flasks

[0028] Take several 500 mL eggplant-shaped flasks, add 50 mL of NA medium to each flask, sterilize at 121 °C for 30 min, arrange them into slants, and set aside after solidification. Pour 5 mL of sterile water into the slant of the activated test tube strain, scrape the bacterial mass with a sterile bamboo stick and stir well as much as possible, pour the bacterial suspension into the slant of the eggplant-shaped flask, shake slowly to make the inoculation on the slant of the eggplant-shaped flask uniform, and culture it in an inverted position at 37 °C for 5 - 7 d to produce a large number of spores for standby.

[0029] ② Preparation of seed liquid

[0030] Fill the seed tank with the medium, with a filling coefficient of 0.65 - 0.75, sterilize at 121 °C for 30 min, and inoculate after cooling to 40 °C. Take 4 of the above-mentioned slants in eggplant-shaped flasks, pour 50 mL of sterile water into each, scrape the bacterial mass with a sterilized bamboo stick, pour it into a 1000 mL inoculation bottle, inoculate the seed tank by the differential pressure method, and culture it at 37 °C, a rotation speed of 1500 - 2000 r / min, and an air flow rate of 1.0 - 2.0 VVM for 12 - 14 h to obtain the seed liquid.

[0031] Specifically, the seed medium and the fermentation medium include the following components: corn flour 2.0%, soybean cake powder 2.0%, ammonium sulfate 0.2%, NaH2PO4 0.4%, KH2PO4 0.03%, Na2CO3 0.1%, MgSO4 0.05%, calcium carbonate 0.2%, MnSO4 0.05%, natural pH, sterilize at 121 °C for 30 min.

[0032] Specifically, the above-mentioned auxiliary materials can adopt the commonly used auxiliary materials for conventional microbial solid bacterial agents in the art, such as light calcium carbonate, starch, etc., which can be conventionally selected in the art, and the present invention does not make special limitations.

[0033] More specifically, it is required that the viable bacteria count in the above-mentioned powdery bacterial agent ≥ 1.0×10 10 CFU / g, water content ≤ 10%, and shelf life of 18 months.

[0034] Furthermore, the microbial bacterial agent also includes Paenibacillus mucilaginosus N-002, and its preservation number is CGMCC No. 32752.

[0035] Furthermore, the microbial inoculant further comprises Bacillus amyloliquefaciens Y-102, and the preservation number of Bacillus amyloliquefaciens Y-102 is CGMCC No. 33966.

[0036] The provided Priestia megaterium NPKM-001 of the present invention can be used in combination with functional strains such as Paenibacillus mucilaginosus N-002 and Bacillus amyloliquefaciens Y-102. Through the functional synergy among the strains, the significant improvement of soil improvement effect and crop growth promotion ability can be achieved. Through the synergy among the strains, the urease activity in the rhizosphere soil can be more efficiently inhibited, the volatilization of NH3 and the emission of N2O can be significantly reduced, the nitrogen loss can be effectively reduced, and the fertilizer utilization efficiency can be improved. This is not only beneficial to creating a better rhizosphere microenvironment for the growth of crops, promoting the healthy development of plants, significantly increasing the crop yield and quality, but also can effectively reduce the emission of harmful gases in farmland, reduce the ecological pressure on the atmosphere, soil and water bodies, and provide strong support for the sustainable development of ecological agriculture.

[0037] Specifically, the application methods of the microbial inoculant include spraying, watering or using as base fertilizer.

[0038] In the fourth aspect, the present invention also provides a microbial fertilizer comprising the above-mentioned Priestia megaterium NPKM-001.

[0039] The Priestia megaterium and its preparations provided by the present invention have great application value in crop planting. Especially when the crop applies urea, this strain has significant phosphorus-solubilizing ability to ensure the phosphorus nutrition of the crop. At the same time, the indole acetic acid produced by the strain can promote the root development and cell growth of the crop and increase the yield. In terms of improving the fertilizer efficiency of urea, the urease inhibitor produced by this strain plays a key role. By reducing the urease activity in the soil and slowing down the hydrolysis rate of urea, the ammonia emission and nitrous oxide emission in the soil after applying urea are significantly reduced. This is not only beneficial to maintaining the soil nitrogen, but also greatly improves the utilization rate of nitrogen fertilizers such as urea. In addition, this strain has no denitrification function. It can grow and reproduce rapidly in the crop rhizosphere, increase the proportion of bacteria without denitrification function in the soil, effectively reduce the denitrification effect of the soil, and reduce the nitrous oxide emission, which is of great significance to environmental protection and improving fertilizer efficiency. The Priestia megaterium and its preparations, through the functional synergy of phosphorus-solubilizing, growth promotion, fertilizer efficiency improvement, emission reduction, etc., provide a new solution for green agriculture, contribute to the renewal of microbial fertilizer strains, and promote the sustainable development of agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the colony morphology of the Priestia megaterium NPKM-001 of the present invention on the NA medium plate;

[0041] Figure 2Morphology of the cells and spores of the huge Priestia megaterium NPKM-001 of the present invention;

[0042] Figure 3 Phylogenetic tree of the huge Priestia megaterium NPKM-001 of the present invention;

[0043] Figure 4 Growth status of inoculating strain N-002 by the cross method on the medium containing strain NPKM-001;

[0044] Figure 5 Growth status of inoculating strain NPKM-001 by the cross method on the medium containing strain N-002. Detailed implementation mode

[0045] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0046] Unless otherwise specified, analytical pure reagents meeting national standards are used in the embodiments, and the analytical water used is the third-grade water specified in GB / T6682.

[0047] Media used in the embodiments:

[0048] Phosphate-solubilizing liquid medium: Glucose 10 g, Ca3(PO4)2 5 g, MgCl2 5 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, distilled water 1000 mL, pH 7.0 - 7.5, sterilized at 115 °C for 20 min.

[0049] Phosphate-solubilizing plate medium: Glucose 10 g, Ca3(PO4)2 5 g, MgCl2 5 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, distilled water 1000 mL, agar 15.0 g, pH 7.0 - 7.5, sterilized at 115 °C for 20 min.

[0050] NA slant medium: Peptone 10.0 g, beef extract powder 3.0 g, sodium chloride 5.0 g, agar 15.0 g, distilled water 1000 mL, adjust pH to 7.0. Sterilized at 121 °C for 30 min.

[0051] LB medium: Tryptone 10 g, yeast extract 5 g, NaCl 10 g, distilled water 1000 mL, pH 7.4. Sterilized at 121 °C for 30 min.

[0052] Phosphate-solubilizing fermentation medium: 10 g of glucose, 5 g of Ca3(PO4)2, 5 g of MgCl2, 0.25 g of MgSO4·7H2O, 0.2 g of KCl, 0.1 g of (NH4)2SO4, 1000 mL of distilled water, pH 7.0 - 7.5. Sterilize at 115 °C for 30 min.

[0053] Potassium-solubilizing fermentation medium: 5.0 g of sucrose, 0.5 g of MgSO4·7H2O, 2 g of Na2HPO4, 0.005 g of FeCl3, 0.2 g of K2HPO4, 0.1 g of CaCO3, 5.0 g of potassium feldspar, 1000 mL of distilled water, pH 7.0 - 7.5. Sterilize at 121 °C for 30 min.

[0054] Urea phenol red bacteria medium: 2.0% urea, 0.1% phenol red, 1% peptone, 0.3% beef extract, 0.5% sodium chloride, 2% agar, pH 6.4. Sterilize at 121 °C for 30 min.

[0055] NB medium: 10.0 g of peptone, 3.0 g of beef extract powder, 5.0 g of sodium chloride, 1000 mL of distilled water, adjust pH to 7.0. Sterilize at 121 °C for 30 min.

[0056] Giltay liquid medium: Solution A: 1.0 g of KNO3, 1.0 g of asparagine, 5 mL of 1% (w / v) bromothymol blue (BTB) alcohol solution, 500 mL of distilled water; Solution B: 8.5 g of trisodium citrate, 1 g of MgSO4·7H2O, 1 g of KH2PO4, 0.05 g of FeCl3·6H2O, 0.15 g of CaCl2, make up to 500 mL with distilled water; Mix solutions A and B, adjust pH to 7.0 - 7.2, use after sterilizing at 121 °C for 30 min.

[0057] Organic phosphorus medium: 10 g of glucose, 0.5 g of (NH4)2SO4, 0.3 g of MgSO4·7H2O, 0.3 g of NaCl, 0.3 g of KCl, 0.03 g of FeSO4·7H2O, 0.03 g of MnSO4·7H2O, 2 g of lecithin, 5 g of CaCO3, 1 L of distilled water, pH 7.0. Sterilize at 115 °C for 30 min.

[0058] Example 1

[0059] Strain screening

[0060] 1. Enrichment of phosphate-solubilizing Bacillus

[0061] Take 10.0 g of soil from the rhizosphere of vigorously growing corn in a farmland near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770), add it to an Erlenmeyer flask containing 90 mL of sterile water and glass beads, shake it on a shaker at a rotation speed of 200 r / min for 10 min, then heat it in a water bath at 80 °C for 15 min to obtain bacteria in the spore state. After standing for a while, take 5 mL of this soil suspension and inoculate it into a phosphorus-solubilizing liquid medium, and culture it with shaking at 37 °C for 3 d. After standing, take 5 mL and inoculate it into the phosphorus-solubilizing liquid medium again, repeat the above operation to complete secondary enrichment, and reserve the secondary enrichment solution.

[0062] 2. Isolation of phosphorus-solubilizing Bacillus

[0063] Use a sterile pipette to take 1.0 mL of the above secondary enrichment solution and dilute it serially to 10 -6 . Pipette 0.1 mL from each dilution and inoculate it onto a phosphorus-solubilizing plate medium, spread it evenly with a sterile spreading rod, and incubate it upside down in a constant temperature incubator at 37 °C for 2 - 4 d, and observe whether a transparent zone forms around the colonies. If a transparent zone appears, it indicates that the strain is a phosphorus-solubilizing Bacillus with certain phosphorus-solubilizing ability.

[0064] Transfer the strain with the above transparent zone to an NA slant medium and make an agar block bacterial cake with a diameter of 8 mm (area s = r 2 π = 4 2 ×3.14 = 50.24 mm 2 ), and inoculate it into the phosphorus-solubilizing plate medium; there are 5 replicates for each strain. After culturing for 2 - 4 d, measure the area S of the transparent zone, and judge the phosphorus-solubilizing ability of the strain by S / s.

[0065] Results: 506 Bacillus strains with transparent zones were screened, among which 128 strains had an S / s value greater than 5.0, and the NPKM-001 strain had the largest S / s value, which was 6.7.

[0066] 3. Screening of Bacillus strains with strong phosphorus and potassium solubilizing abilities

[0067] The 128 strains obtained through the above screening were respectively inoculated into 50 mL of LB medium and cultured at 37°C and 180 r / min for 12 h. At this time, the culture was in the logarithmic growth phase, and it was used as the seed liquid. Take 1 mL of the seed liquid and add it to 100 mL of phosphate-solubilizing fermentation medium. The phosphate-solubilizing fermentation medium without inoculating any bacterial liquid was used as the blank control group; each treatment had 3 replicates and was cultured at 37°C and 150 r / min for 7 d. After the culture ended, the fermentation broth was centrifuged at 6000 r / min for 10 min to obtain the supernatant. Take 10 mL of the supernatant and add it to a 50 mL volumetric flask, and make up the volume to the scale with distilled water. After digestion, the available phosphorus content was determined by the molybdenum phosphorus colorimetric method. It was respectively compared with the available phosphorus (calculated as P2O5) concentration of the blank control group, and the improvement degree of the available phosphorus (calculated as P2O5) concentration corresponding to each strain was calculated.

[0068] In the potassium-solubilizing test, except that the phosphate-solubilizing fermentation medium was replaced with the potassium-solubilizing fermentation medium and the available potassium content was determined by a flame photometer after digestion, the rest of the operations were the same as above. It was respectively compared with the available potassium (calculated as K2O) concentration of the blank control group, and the improvement degree of the available potassium (calculated as K2O) concentration corresponding to each strain was calculated.

[0069] Results: From the above 128 strains with S / s value greater than 5.0, a total of 42 strains were screened that could increase the available phosphorus (calculated as P2O5) concentration by more than 20 times and increase the available potassium (calculated as K2O) concentration by more than 10%. Among them, the NPKM-001 strain could increase the available phosphorus (calculated as P2O5) concentration in the fermentation broth by 35.71 times and increase the available potassium (calculated as K2O) concentration by 15.29% at the same time.

[0070] 4. Screening for urease-negative Bacillus

[0071] Use a sterilized bamboo stick to pick the bacterial colonies of the 42 strains of Bacillus with strong phosphate-solubilizing and potassium-solubilizing abilities obtained through the above screening, and make cross-streak inoculations on the urea phenol red bacterial medium plates respectively. Incubate them upside down at 37°C for 24 h, observe the color change around the cross-shaped bacterial colonies, screen and pick the colonies that are still yellow (do not turn red) around, inoculate them into the NA slant medium, and preserve them for standby after culturing at 37°C for 24 - 48 h.

[0072] Test principle: Phenol red is an acid-base indicator, yellow in acidic, orange in neutral, and red in alkaline, and its color change range is pH 6.8 (yellow) - 8.4 (red). If the area around the bacterial colony is still yellow, it indicates that the strain may not produce urease, does not decompose the urea in the medium around the bacterial colony, and does not cause a significant change in pH; on the contrary, if the area around the bacterial colony turns red, it indicates that the strain may produce urease, decomposes urea in the medium to produce ammonia, and causes the pH to rise and turn red.

[0073] Result: 19 urease-negative Bacillus strains were screened out from 42 Bacillus strains with strong phosphate-solubilizing and potassium-solubilizing abilities, including strain NPKM-001.

[0074] 5. Screening of Bacillus strains producing urease inhibitors

[0075] The 19 urease-negative strains obtained in the above step were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of NB medium and cultured on a shaker at 37 °C and 150 r / min for 48 h. Then, the fermentation broth was centrifuged at 6000 r / min for 10 min to obtain the supernatant, which was the test solution for urease inhibitors.

[0076] Soil with a depth of 5 - 20 cm from the surface was dug from a well-growing corn field near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770). The urease activity of the corn field soil was detected by the phenol-sodium hypochlorite colorimetric method. The principle is as follows: Urea is hydrolyzed by soil urease to produce ammonia, and ammonia reacts with phenol-sodium hypochlorite at room temperature to produce blue indophenol. The color depth is proportional to the amount of ammonia produced. Therefore, the amount of ammonia can be determined by colorimetry to represent the urease activity, and then the inhibition rate of the test solution on the urease activity of the corn field soil can be calculated. The specific detection method is as follows.

[0077] (1) Reagents and solutions

[0078] Standard ammonia solution: Accurately weigh 0.4717 g (accurate to 0.0001 g) of ammonium sulfate dried in an oven at 105 °C for 3 h, dissolve it in water and make up the volume to 1000 mL to obtain a stock solution containing 0.1 mg of ammonia per 1 mL. Before use, dilute the above solution 10 times with water to prepare a working solution with a concentration of 0.01 mg / mL.

[0079] Sodium phenolate solution (1.35 mol / L): Solution A: Weigh 62.50 g of phenol (accurate to 0.01 g), dissolve it in a small amount of ethanol, add 2 mL of methanol and 18.5 mL of acetone, and make up the volume to 100 mL with ethanol. Solution B: Weigh 27.00 g of sodium hydroxide (accurate to 0.01 g), dissolve it in water and make up the volume to 100 mL. Store solutions A and B in a 4 °C refrigerator. Before use, mix 20 mL of solution A and 20 mL of solution B and make up the volume to 100 mL with water.

[0080] Sodium hypochlorite solution: Dilute the reagent according to the concentration of commercially available sodium hypochlorite solution to a concentration of active chlorine of 0.9%.

[0081] Urea solution (100 g / L): Weigh 10.00 g of urea (accurate to 0.01 g), dissolve it in water, and make up the volume to 100 mL.

[0082] Citrate buffer (pH 6.7): Weigh 184.00 g of citric acid (accurate to 0.01 g) and 147.50 g of potassium hydroxide (accurate to 0.01 g) and dissolve them in water respectively. Combine the two solutions, adjust the pH to 6.7 with 1 mol / L sodium hydroxide, and make up the volume to 1000 mL with water.

[0083] (2) Preparation of standard curve

[0084] Before measuring the absorbance of the sample, pipette 0.00 mL, 1.00 mL, 3.00 mL, 5.00 mL, 7.00 mL, 9.00 mL, 11.00 mL, 13.00 mL of ammonia working solution (0.1 mg / mL) into 50 mL volumetric flasks respectively. Then add 20 mL of water, and successively add 4 mL of sodium phenolate solution and 3 mL of sodium hypochlorite solution, shaking well while adding. After 20 min, develop the color, make up the volume, and prepare a set of standard concentrations with ammonia concentrations of 0.0 μg / mL, 0.2 μg / mL, 0.6 μg / mL, 1.0 μg / mL, 1.4 μg / mL, 1.8 μg / mL, 2.2 μg / mL, 2.6 μg / mL. Colorimetric determination is carried out at a wavelength of 578 nm on a spectrophotometer within 1 h (the blue color remains stable within 1 h). Plot the standard curve with ammonia concentration as the abscissa and absorbance as the ordinate.

[0085] (3) Determination of urease inhibition rate of the solution to be measured

[0086] ① Determination of soil urease activity

[0087] Take 5 g of soil sample (accurate to 0.0001 g; if the content is high, the sample weight can be appropriately reduced) and 5.0 mL of double-distilled water into a 100 mL stoppered Erlenmeyer flask, add 1 mL of toluene, shake well, and after 15 min, add 10 mL of 100 g / L urea solution and 20 mL of pH 6.7 citrate buffer solution, shake well and incubate in a constant temperature incubator at 37 °C ± 1 °C for 24 h.

[0088] After the incubation, filter to obtain the filtrate. Pipette 1.00 mL of the filtrate into a 50 mL volumetric flask, and then successively add 4 mL of sodium phenolate solution and 3 mL of sodium hypochlorite solution, shaking well while adding. After 20 min, develop the color, and make up the volume to 50 mL with water. Repeat the above operation with 10 mL of double-distilled water replacing "10 mL of 100 g / L urea solution" as the control, and colorimetric determination is carried out at a wavelength of 578 nm on a spectrophotometer within 1 h.

[0089] ② Determination of soil urease activity after adding the solution to be measured

[0090] Except for replacing "5.0 mL of double-distilled water" in ① with "5.0 mL of the solution to be measured", the other steps are the same as ① above.

[0091] (4) Calculation of urease activity inhibition rate

[0092] ① Calculation of soil urease activity

[0093] Urease activity is expressed as the number of mg of ammonia produced by hydrolyzing the substrate (urea) in 1 g of air-dried soil in 24 h, and is calculated according to the following formula:

[0094]

[0095] In the formula, X is the content of urease in the sample soil (mg / (g·24 h));

[0096] c1 is the amount of ammonia (μg / mL) obtained from the absorbance value of the sample plus urea from the standard curve;

[0097] c2 is the amount of ammonia (μg / mL) obtained from the absorbance value of the sample without adding urea from the standard curve;

[0098] V is the volume of color development and constant volume (mL), V = 50 mL.

[0099] N is the aliquot multiple, N = volume of leachate (mL) / volume of filtrate aspirated (mL) = 41 mL / 1 mL = 41.

[0100] V0 is the amount of soil sampled (g), V0 = 5 g.

[0101] t is the sample incubation time, t = 1 unit (the unit is 24 h).

[0102] f is the dry matter content of the soil sample, %.

[0103] Calculation of urease activity inhibition rate

[0104] Urease activity inhibition rate (%) = (soil urease activity - soil urease activity after adding the solution to be tested) / soil urease activity × 100%

[0105] Results: A total of 4 strains with a urease activity inhibition rate greater than 60% were obtained from 19 urease-negative Bacillus strains in cornfield soil. Among them, the supernatant of the fermentation broth of strain NPKM-001 had a urease activity inhibition rate of 84.92% on cornfield soil. Using the same method for detection, the urease activity inhibition rates of the supernatant of the fermentation broth of strain NPKM-001 on fluvo-aquic soil (collected from the western part of Mancheng District, Baoding City), cinnamon soil (collected from the western part of Mancheng District, Baoding City), and black soil (collected from the northern suburbs of Shenyang City) were 87.93%, 85.97%, and 91.72% respectively.

[0106] 6. Screening of Bacillus strains with high indole-3-acetic acid (IAA) production

[0107] The 4 strains with urease activity inhibition rate greater than 60% obtained in the above steps were respectively inoculated into 250 mL Erlenmeyer flasks containing 50 mL of NB medium, cultured on a shaker at 37 °C and 150 r / min for 48 h, and centrifuged at 6000 r / min for 10 min to obtain the supernatant; the supernatant was the test solution for IAA. The IAA content was determined by the Salksowski colorimetric method, and the specific operation was as follows.

[0108] (1) Standard curve drawing

[0109] Prepare IAA standard solutions with concentrations of 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, and 35 mg / L using distilled water. Pipette 2.0 mL of IAA standard solutions with different concentrations and mix them with 2.0 mL of Salksowski colorimetric reagent, and place them in a 40 °C water bath in the dark for 30 min. Use a spectrophotometer to measure the absorbance at a wavelength of 530 nm. Draw a standard curve with the IAA concentration as the abscissa and the absorbance value as the ordinate.

[0110] (2) Determination of IAA content in the fermentation broth

[0111] Pipette 2.0 mL of the above test solution and mix it with 2.0 mL of Salksowski colorimetric reagent, and place them in a 40 °C water bath in the dark for 30 min. Use a spectrophotometer to measure the absorbance at a wavelength of 530 nm. Calculate the IAA content in the fermentation broth using the standard curve.

[0112] Salksowski colorimetric reagent: Mix 1 mL of 0.5 mol / L FeCl3 solution with 50 mL of 35% HClO4 solution.

[0113] Result: Among the above 4 strains, 3 strains produced IAA, and the fermentation broth of strain NPKM-001 had the highest IAA content, reaching 105.6 mg / L.

[0114] 7. Screening of Bacillus strains without denitrification function

[0115] Use a sterile bamboo stick to transfer the above 3 Bacillus strains with strong IAA-producing ability to an NA slant medium and culture them at 37 °C for 24 h for activation.

[0116] In a laminar flow hood, 10 mL of Giltay liquid medium was added to a large test tube (20 mm × 200 mm). A small Durham tube (5 mm × 20 mm) was inverted and placed into the large test tube to expel all the gas inside the small tube. Then, three Bacillus strains with strong IAA-producing ability after activation were inoculated respectively. They were cultured at 37 °C for 5 - 7 d, and whether there were bubbles in the small tube was observed. If gas was produced, it indicated that the strain had denitrification ability; if no gas was produced, it meant that the strain had no denitrification ability.

[0117] The tests showed that two of the above three strains had no denitrification function, including strain NPKM - 001.

[0118] Strain species identification

[0119] 1. Morphological observation

[0120] Using a sterile bamboo stick to pick the slant culture of strain NPKM - 001, inoculating it into NB medium, culturing it on a shaker at 37 °C and 150 r / min for 24 h. Then, 1.0 mL of the culture solution was taken, appropriately diluted, and spread on the NA medium plate. It was cultured upside down at 37 °C for 24 - 48 h to observe the colony morphology. An appropriate amount of the culture solution at 24 - 48 h was smeared, and then crystal violet staining was carried out to observe the morphology of the bacteria and spores.

[0121] Results: On the NA medium plate, the colonies were milky white, with a dry surface, relatively regular edges, opaque, hard texture, and easy to pick. The bacteria were rod-shaped, relatively large, with a size of about (1.2 - 1.5) μm × (2.0 - 4.0) μm, often single or arranged in short chains, and Gram-positive. The spores were oval, central, with a diameter smaller than that of the bacteria, and the spore size was about (1.0 - 1.2) μm × (1.5 - 2.0) μm. See Figures 1 to 2 。

[0122] 2. Physiological and biochemical identification

[0123] According to the experimental methods in Bergey's Manual of Systematic Bacteriology, physiological and biochemical tests such as oxidase, peroxidase, glucose fermentation, citrate utilization, methyl red test, V.P test, starch hydrolysis, indole test, gelatin liquefaction, casein test, and urea hydrolysis were carried out on strain NPKM - 001. The results are shown in Table 1.

[0124] Table 1 Physiological and biochemical characteristics of strain NPKM - 001

[0125]

[0126]

[0127] Note: "+" indicates a positive reaction; "-" indicates a negative reaction.

[0128] 3. Molecular Biology Identification

[0129] The total DNA of strain NPKM-001 was extracted using a bacterial genomic DNA extraction kit (Biomiga, catalog number BW-GD2411-01) and following the steps in its instruction manual. The prokaryotic 16S rDNA gene sequence universal primers (forward 5′-ACTGGAGGAAGGTGGGGA-3′, reverse 5′-AGGAGGTGATCCAACCGCA-3′) were used for PCR amplification to obtain the amplification product. The sequencing of the amplification product was entrusted to BGI. The sequencing result of 16S rDNA was as follows:

[0130]

[0131] The sequencing results were aligned in NCBI. Based on the Neighbor-Joining method, a phylogenetic tree was constructed using MEGA 11.0, as shown in Figure 3 . According to the 16S rDNA sequence similarity analysis, and referring to the results of physiological and biochemical tests and the colony and cell morphology, the strain NPKM-001 was identified as Priestia megaterium, namely Bacillus megaterium.

[0132] This Priestia megaterium was deposited in the China General Microbiological Culture Collection Center (CGMCC) on November 22, 2024. The accession number is CGMCC No. 32751, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0133] Phosphorus solubilization effect of strain NPKM-001 on organic phosphorus

[0134] A sterilized bamboo stick was used to pick the slant bacterial plaque of strain NPKM-001 and inoculated into 50 mL of LB medium. It was cultured with shaking at 37 °C and 180 r / min for 12 h to obtain the seed liquid. In the experimental group, the seed liquid was inoculated into the organic phosphorus medium at an inoculation amount of 3%. In the control group, the seed liquid was first inactivated by heating at 121 °C for 20 min, and the remaining operations were the same as those in the experimental group, with 3 replicates in each group. After inoculation, it was cultured with shaking at 37 °C and 180 r / min for 5 d, centrifuged at 5000 r / min for 10 min, and the supernatant was decanted. The available phosphorus content in the supernatant was determined by the molybdenum antimony anti-colorimetric method.

[0135] Results: The content of available phosphorus in the control group was (0.066 ± 0.010) mg / L; the content of available phosphorus in the experimental group was (1.720 ± 0.082) mg / L, which was 25.91 times that of the control group. This indicates that strain NPKM-001 can release the phosphorus in part of the lecithin (organic phosphorus) in the medium.

[0136] Example 2

[0137] This example provides the preparation of a microbial fertilizer:

[0138] (1) Fermentation medium: corn flour 2.0%, soybean cake powder 2.0%, ammonium sulfate 0.2%, NaH2PO4 0.4%, KH2PO4 0.03%, Na2CO3 0.1%, MgSO4 0.05%, calcium carbonate 0.2%, MnSO4 0.05%, natural pH, sterilized at 121 °C for 30 min.

[0139] (2) Preparation of seed liquid:

[0140] Take several 500 mL eggplant bottles, add 50 mL of NA medium to each bottle, sterilize at 121 °C for 30 min, arrange them into slopes, and set aside after solidification. Pour 5 mL of sterile water into the slant of the activated NPKM-001 strain test tube, scrape the bacterial lawn with a sterile bamboo stick and stir well as much as possible. Pour the bacterial suspension into the slant of the eggplant bottle, shake slowly to evenly inoculate the slant of the eggplant bottle, and incubate upside down at 37 °C for 5 - 7 d to produce a large number of spores for standby.

[0141] Fill a 100 L ventilated and stirred seed tank with fermentation medium, with a filling coefficient of 0.65 - 0.75, sterilize at 121 °C for 30 min, and inoculate after cooling to 40 °C. Take 4 of the above-mentioned slants of eggplant bottles, pour 50 mL of sterile water into each, scrape the bacterial lawn with a sterilized bamboo stick, pour it into a 1000 mL inoculation bottle, and inoculate the seed tank by the differential pressure method. Incubate at 37 °C, a rotation speed of 1500 r / min, and an aeration rate of 1.0 - 2.0 VVM for 12 - 14 h to obtain the seed liquid.

[0142] (3) Add the above fermentation medium to a 1 M 3 ventilated and stirred fermentation tank, with a filling coefficient of 0.65 - 0.75, sterilize at 121 °C for 30 min, inoculate the above seed liquid after cooling to 40 °C, with an inoculation amount of 5% - 10%, and ferment at 37 °C, 150 r / min, and an aeration rate of 1.0 - 2.0 VVM for 48 - 72 h to obtain the fermentation broth. Detect the viable bacteria content in the fermentation broth by the gradient dilution plate coating method, and observe and calculate the spore rate under the microscope by the smear staining method.

[0143] Result: The spore content in the fermentation broth is between (1.0 - 3.0)×10 9 CFU / mL. Therefore, a liquid microbial fertilizer (liquid inoculant) with a viable bacteria (spore) content higher than 1.0×10 9 CFU / mL can be prepared, and the shelf life is 6 months.

[0144] (4) Continuously centrifuge the above fermentation broth at 6000 r / min by a disc centrifuge, concentrate it 5 - 10 times to obtain bacterial sludge. After adding auxiliary materials to the bacterial sludge, spray dry it to obtain bacterial powder.

[0145] Result: The spore content in the bacterial sludge can reach (5.0 - 15.0)×10 9 CFU / mL. The spore content in the bacterial powder can reach (1.0 - 3.0)×10 11 CFU / g, which can be used to prepare a spore (viable bacteria) content of ≥1.0×10 10 CFU / g or ≥1.0×10 11Powdery microbial fertilizer (powdered inoculant) with CFU / g, water content ≤ 10%, and shelf life of 18 months.

[0146] The technical parameters for preparing the microbial fertilizer of strain NPKM-0001 are shown in Table 2.

[0147] Table 2 Using 1M 3 Technical parameters for preparing NPKM-0001 inoculant using a 1M fermenter

[0148]

[0149]

[0150] Example 3

[0151] 1. Pot experiment on ammonia emission reduction effect of liquid inoculant of Megasphaera elsdenii NPKM-001

[0152] Take 28 plastic flowerpots with an inner diameter × height of 15 cm × 18 cm, fill them with soil (take soil from a depth of 5 - 15 cm in farmland), and make the soil depth in the pots about 15 cm. Select 28 pepper seedlings that have been raised for one week and are basically the same in size, thickness, and number of leaves, and plant them into the flowerpots, one plant per pot. Divide them into 4 groups: CK group, experimental group I, experimental group II, and experimental group III, with 7 pots in each group. Apply fertilizer at the beginning of the pot experiment, and the experimental period is 15 days in total. The experimental scheme is shown in Table 3.

[0153] Table 3 Experimental scheme for pepper pot experiment with NPKM-001 inoculant

[0154]

[0155] Place the flowerpots of the 4 groups into transparent glass tanks with a length × width × height of 0.5 m × 0.5 m × 0.5 m respectively, and conduct daily irrigation by supplying water to the bottom of the tank and through the water-permeable holes at the bottom of the flowerpots. Keep other management the same for each group, such as maintaining the soil water holding rate at 40% - 60%, keeping the average air temperature in the tank at 25°C, and having 8 - 10 hours of light per day. Seal the glass tanks with a sealing cover to form a closed static chamber, and install 3 PVC plastic pipes with valves on the sealing cover. One PVC plastic pipe is used for supplying water to the bottom of the tank, 1 PVC plastic pipe is used for air intake, controlling the air intake volume at about 60 L / h (using a small blower to continuously supply air into the tank), and the third PVC plastic pipe is a three-way pipe. One end can lead the discharged gas into 200 mL of 0.01 mol / L sulfuric acid absorption solution, and the other end can insert a 20-cm-long needle to suck the gas in the glass tank for sampling and measuring the N2O emission.

[0156] The ammonia absorbed by the absorption solution was determined by the method described in "HJ533-2009 Ambient Air and Exhaust Gas - Determination of Ammonia - Nessler's Reagent Spectrophotometry". The principle is that ammonia in the air is absorbed by a dilute sulfuric acid solution, and the generated ammonium ions react with Nessler's reagent to form a yellowish-brown complex. The absorbance of this complex is proportional to the ammonia content. The absorbance is measured at a wavelength of 420 nm, and the ammonia content in the air is calculated based on the absorbance. The test results are shown in Table 4.

[0157] Table 4 Ammonia Emission Reduction Effect of NPKM-001 Liquid Bacterial Agent on Potted Pepper Soil by Pouring

[0158]

[0159] The test results show that pouring urea can significantly increase the ammonia volatilization amount in the soil during the test period; compared with Group I (pouring urea), in the case of the same urea application amount (15 g / m 2 ), applying NPKM-001 liquid bacterial agent can reduce soil ammonia emissions by 71.32%; compared with the CK group (without pouring urea), in the case of not pouring urea, applying NPKM-001 liquid bacterial agent to Group II can reduce soil ammonia emissions by 52.52%. Therefore, this liquid bacterial agent can significantly reduce the ammonia emissions in the soil, especially in the soil where urea is poured. The reason for ammonia emission reduction is that the NPKM-001 strain can produce urease inhibitors, which can inhibit the conversion process of urea to ammonium (ammonia), making urea mainly exist in the form of amide nitrogen in the soil, reducing the concentration of NH4 + / NH3 in the soil solution, thus reducing ammonia emissions.

[0160] 2. Effect of NPKM-001 Liquid Bacterial Agent of Megasphaera elsdenii on Urease Activity in Potted Pepper Soil

[0161] When the above potted test was completed (on the 15th day), the urease activity of the soil in each flower pot was detected by the method of "T / NAIA 011-2020 Determination of Soil Urease Activity - Phenol Sodium-Sodium Hypochlorite Colorimetric Method", and the urease activities of the soil in the flower pots with and without NPKM-001 bacterial agent were compared. The specific detection method is referred to in Example 1. The specific test results are shown in Table 5.

[0162] Table 5 Effect of NPKM-001 Liquid Bacterial Agent on Urease Activity in Potted Pepper Soil by Pouring

[0163]

[0164]

[0165] Note: The mean value in the table is the average value after removing one highest value and one lowest value from 7 corresponding values; different capital letters after the mean value indicate significant differences at the P<0.01 level.

[0166] The test results showed that: compared with Group Ⅰ (applying urea by irrigation), when applying the same amount of urea (15 g / m 2 ) in Group Ⅲ, applying the NPKM-001 liquid microbial agent could reduce the soil urease activity by 64.09% (P < 0.01); compared with the CK group (without applying urea by irrigation), when not applying urea by irrigation in Group Ⅱ, applying the NPKM-001 liquid microbial agent could reduce the soil urease activity by 67.71% (P < 0.01). Therefore, whether applying urea by irrigation or not, this liquid microbial agent could significantly reduce the soil urease activity, thus significantly reducing the fertilizer efficiency loss caused by ammonia volatilization after urea fertilization and improving the utilization rate and fertilizer efficiency of urea.

[0167] 3. Emission reduction effect of the NPKM-001 liquid microbial agent of Megasphaera elsdenii on N2O in the soil of potted peppers

[0168] At 8:00 - 12:00 on the 0th, 3rd, 6th, 9th, 12th, and 15th days after fertilization, the air intake and exhaust were accurately stopped for 4 h. Each time, at 12:00, 50 mL of air in the glass cylinder was sucked with a syringe and injected into a 12 mL headspace vial that had been evacuated with a vacuum pump. The N2O content was determined by an Agilent 7890A gas chromatograph. The detector was ECD, the detection temperature was 300 °C, the column temperature was 60 °C, the carrier gas was 95% argon and 5% methane, and the flow rate was 40 mL / min. According to the concentration of the N2O sample, a standard gas with a concentration of 5 mg / L was selected to establish a standard curve. Manual injection was used. 40 μL of the sample was drawn with a 100 μL gastight syringe and injected into the analytical instrument within 1 s.

[0169] To ensure the growth of pepper seedlings, air needs to be continuously supplied to the glass cylinder. However, within the 4 h of stopping air intake and exhaust, the glass cylinder becomes a closed space, and the N2O emitted from the soil in the flower pot can only accumulate in the glass cylinder. Therefore, by detecting and comparing the N2O concentrations in the glass cylinders of each group, the emission reduction effect of the NPKM-001 liquid microbial agent (microbial fertilizer) can be obtained. The results are shown in Table 6.

[0170] Table 6 Emission reduction effect of applying the NPKM-001 liquid microbial agent on N2O in the soil of potted peppers

[0171]

[0172]

[0173] Results: After urea was watered in groups I and III, the soil nitrogen content could be significantly increased, and then the N2O emissions were significantly increased through nitrification and denitrification. The emission peak appeared around the 3rd day, and the emissions gradually decreased after 3 days, which might be related to the gradual utilization of urea. However, there were also significant differences between groups I and III: on the 0th, 3rd, 6th, 9th, 12th, and 15th days after fertilization, compared with group I (watered with urea), when the same amount of urea was watered in group III, the application of NPKM-001 liquid microbial agent could reduce the soil N2O emissions by 4.1%, 31.1%, 25.2%, 18.4%, 17.3%, and 14.8% respectively. The average emission reduction from the 3rd day to the 15th day was 21.4% (except that the urease inhibitor contained in the liquid microbial agent had the effect of reducing N2O emissions, and spores were also the active components of the liquid microbial agent. The spores reaching the pepper roots did not germinate on the 0th day and could not play the corresponding role in reducing N2O emissions. Therefore, the calculation started from the 3rd day). Compared with the CK group (without watering urea), in group II without watering urea, the application of NPKM-001 liquid microbial agent could reduce the soil N2O emissions by 1.5%, 12.6%, 11.4%, 9.1%, 9.2%, and 8.1% respectively on the 0th, 3rd, 6th, 9th, 12th, and 15th days after fertilization. The average emission reduction from the 3rd day to the 15th day was 10.1%. Therefore, when watering urea, the application of NPKM-001 microbial agent (microbial fertilizer) could significantly reduce the soil N2O emissions, and the emission reduction effect was the best on the 3rd day of the experiment, reaching more than 30%. When applying NPKM-001 microbial agent without watering urea, there was also a certain emission reduction effect. Analyzing the reasons, there are mainly two aspects. One is that the NPKM-001 strain can produce urease inhibitors, which inhibit the rate of urea decomposition into ammonia (ammonium), reduce the NH3 emissions. At the same time, it also restricts the supply of nitrification and NO3 - , and then reduces the subsequent denitrification rate, thereby reducing the N2O emissions; the other is that because the NPKM-001 strain has no denitrification ability, after being applied to the soil, it grows and reproduces in the plant roots, thus increasing the number of non-denitrifying bacteria in the soil, especially in the root zone soil, reducing the denitrification of the soil, blocking the conversion of nitrate, nitrite, ammonium salt, etc. to N2O, and finally reducing the N2O emissions.

[0174] 4. Effects of the liquid microbial agent of Megasphaera elsdenii NPKM-001 on the available phosphorus content in the soil of pepper potted plants

[0175] The method described in "HJ 704-2014 Determination of available phosphorus in soil - Sodium bicarbonate extraction - Molybdenum antimony resistance spectrophotometry" was used to determine the available phosphorus content in the soil of each test group before potting and 15 days after potting. The results are shown in Table 7.

[0176] Table 7 Effects of Applying NPKM-001 Liquid Bacterial Agent on the Available Phosphorus Content in the Soil of Potted Chili Peppers

[0177]

[0178]

[0179] Note: The mean value in the table is the average value after removing one highest value and one lowest value from the 7 corresponding values; different capital letters after the mean value indicate significant differences at the P<0.01 level.

[0180] Results: Compared with Group Ⅰ (applying urea by watering), when applying the same amount of urea by watering, applying NPKM-001 liquid bacterial agent in Group Ⅲ can increase the available phosphorus content in the soil by 50.03%, and at the same time increase the available phosphorus content by 39.38% compared with that before potting; compared with the CK group (not applying urea by watering), when not applying urea by watering in Group Ⅱ, applying NPKM-001 liquid bacterial agent can increase the available phosphorus content in the soil by 46.29%, and at the same time increase the available phosphorus content by 37.79% compared with that before potting. This shows that whether applying urea by watering or not, applying NPKM-001 liquid bacterial agent can significantly increase the available phosphorus content in the soil, providing relatively sufficient phosphorus elements for the growth of crops and even the next season's crops.

[0181] 5. Effects of Megasphaera elsdenii NPKM-001 Liquid Bacterial Agent on the Growth Performance of Potted Chili Pepper Seedlings

[0182] After the experiment ended, take out the flowerpots with chili pepper seedlings from the glass tanks, carefully remove most of the soil from the roots of the chili pepper seedlings, wash the roots in a tap water basin, dry them on absorbent paper, measure the plant height of the chili pepper seedlings with a ruler (accurate to mm), cut the roots and shoots from the ground-underground boundary with scissors, measure the stem diameter with a vernier caliper, and measure the fresh weight of the roots and the fresh weight of the shoots with an electronic balance (accurate to 0.01 g). The results are shown in Table 8.

[0183] Table 8 Effects of Applying NPKM-001 Liquid Bacterial Agent on the Growth Promotion of Potted Chili Pepper Seedlings

[0184]

[0185] Note: The mean value in the table is the average value after removing one highest value and one lowest value from the 7 corresponding values; different capital letters indicate significant differences at the P<0.01 level, and different lowercase letters indicate significant differences at the P<0.05 level.

[0186] Results: By measuring the plant height, stem diameter, number of leaves, fresh root weight, and aboveground fresh weight of 4 groups of peppers, it can be seen that this liquid microbial agent can significantly promote the growth of peppers. Compared with the CK group (without urea application), in the II group without urea application, the average values of plant height, stem diameter, number of leaves, fresh root weight, and aboveground fresh weight of peppers increased by 26.80%, 16.44%, 2.91%, 75.36%, and 40.83% respectively. Compared with the I group (with urea application), in the III group with the same amount of urea application, the average values of plant height, stem diameter, number of leaves, fresh root weight, and aboveground fresh weight of peppers increased by 19.44%, 22.19%, 2.59%, 46.23%, and 44.01% respectively. In summary, regardless of whether urea is applied or not, except for the insignificant effect on the number of leaves, the NPKM-001 liquid microbial agent can significantly increase the plant height, stem diameter, fresh root weight, and aboveground fresh weight of peppers and promote the growth of crops. The reason is mainly that urea is a nitrogen fertilizer, which can provide nutrients for peppers and promote growth; in addition to containing natural plant growth hormones such as IAA in the liquid microbial agent, its spores grow and reproduce after germinating in the pepper roots and can also produce IAA, etc. Therefore, applying this microbial agent can significantly promote the growth of pepper seedlings.

[0187] Example 4

[0188] This example provides a compound microbial agent, including Priestiamegaterium NPKM-001 and Bacillus mucilaginosus N-002, and the preservation number of Bacillus mucilaginosus N-002 is CGMCC No. 32752.

[0189] (1) There is no mutual inhibition between NPKM-001 and N-002 strains

[0190] Using the plate confrontation method test, it can be proved that there is no mutual inhibition between the two test strains NPKM-001 and N-002. The specific test steps are as follows: Pour 5 mL of sterile water into the slant of one test strain, scrape the bacterial lawn with a bamboo stick, transfer the bacterial suspension into a sterile empty test tube, and shake it with a vortex oscillator for 2 min to form a uniform bacterial suspension. Immediately pour it into 100 mL of sterilized and not yet solidified (50 - 60 °C) NA medium, mix well and immediately pour the plate. After solidification, place the plate in a 4 °C refrigerator and refrigerate overnight. The next day, inoculate the bacterial lawn of the other test strain on the plate by the cross method, and culture it at 37 °C for 24 - 48 h, and observe whether there is an inhibition zone around the cross bacterial lawn. If the cross bacterial lawn grows well and there is no inhibition zone around it, it indicates that the two strains do not inhibit each other.

[0191] The growth conditions of NPKM-001 and N-002 on the same plate are shown in Figures 4 to 5 . It can be seen from the figure that the two test strains NPKM-001 and N-002 have the characteristic of not inhibiting each other.

[0192] (2) Strains NPKM-001 and N-002 have a synergistic effect

[0193] The combined application of two liquid bacterial agents, NPKM-001 and N-002, in the same pot experiment as in Example 3 above shows that the two bacterial agents have a mutually synergistic or synergistic effect in reducing the urease activity in rhizosphere soil, reducing soil NH3 and N2O emissions, promoting plant growth, and improving the fertilizer efficiency of nitrogen fertilizers such as urea.

[0194] Under the condition of applying 0.26 g of urea in the pot experiment, irrigating 0.13 mL of liquid bacterial agent NPKM-001 can reduce the soil urease activity by 64.09%, the NH3 emission by 71.32%, and the average N2O emission from the 3rd day to the 15th day by 21.4%. Under the condition of applying 0.26 g of urea, irrigating 0.13 mL of liquid bacterial agent N-002, a Bacillus mucilaginosus strain, can reduce the soil urease activity by 67.15%, the NH3 emission by 65.82%, and the average N2O emission from the 3rd day to the 15th day by 22.0%.

[0195] Under the condition of applying 0.26 g of urea per pot in the pot experiment, when 0.065 mL of liquid bacterial agent NPKM-001 and 0.065 mL of liquid bacterial agent N-002 are irrigated simultaneously, the soil urease activity can be reduced by up to 77.13%, and the soil NH3 emission and the average N2O emission are reduced by 75.82% and 29.86% respectively, which are 13.04 percentage points, 4.50 percentage points, and 8.46 percentage points higher than the effect of irrigating NPKM-001 alone; and 9.98 percentage points, 10.00 percentage points, and 7.86 percentage points higher than the effect of irrigating N-002 alone.

[0196] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Priestia megaterium NPKM-001, characterized in that, Its preservation number is CGMCC No. 32751.

2. Use of the Priestia megaterium NPKM-001 according to claim 1 in reducing NH3 and N2O emissions in soil and improving nitrogen fertilizer utilization rate.

3. Use of the Priestia megaterium NPKM-001 according to claim 1 in promoting crop growth and increasing crop yield.

4. A microbial inoculant, characterized in that, It contains the Priestia megaterium NPKM-001 according to claim 1.

5. The microbial inoculant according to claim 4, wherein The microbial inoculant is a liquid inoculant or a powdery inoculant.

6. The microbial inoculant according to claim 5, characterized in that, The viable count of Priestia megaterium NPKM-001 in the liquid microbial inoculum ≥ 1.0×10 9 CFU / mL; the viable count of Priestia megaterium NPKM-001 in the powdered microbial inoculum ≥ 1.0×10 10 CFU / g.

7. The microbial inoculum according to claim 4, wherein It also includes Paenibacillus mucilaginosus N-002, whose preservation number is CGMCC No. 32752.

8. The microbial inoculum according to claim 4 or 7, characterized in that, It also includes Bacillus amyloliquefaciens Y-102, and the preservation number of the Bacillus amyloliquefaciens Y-102 is CGMCC No. 33966.

9. The microbial inoculant according to claim 4, wherein The application method of the microbial inoculant includes spraying, drenching or using it as a base fertilizer.

10. A microbial fertilizer, characterized in that, It contains the Priestia megaterium NPKM-001 according to claim 1.

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