A pluriens, a microbial inoculant thereof and application

Abstract

The application relates to the field of microbial technology, and particularly discloses a Priestia megaterium, a microbial agent thereof and application. The Priestia megaterium NPKM-001 has a preservation number of CGMCC No. 32751. The Priestia megaterium and the preparation thereof provided by the application have great application value in crop planting, and are particularly suitable for application of urea to crops. The strain has significant phosphorus solubilization capacity, high urease inhibitor and indole acetic acid yield, and no denitrification function, can realize multiple goals of promoting crop growth, improving yield, increasing planting benefits and reducing environmental pollution, has wide application in agricultural planting, provides a new scheme for green agriculture, helps microbial fertilizer strain updating, and promotes sustainable development of green agriculture.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to a plistrella megaspora, a microbial inoculant thereof and application. BACKGROUND

[0002] In modern agricultural production, although the large use of nitrogen fertilizer plays an important role in improving crop yield, it also brings a series of serious environmental problems and resource waste problems. 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 fertilizer, but also causes air pollution and increases greenhouse gas emissions. At the same time, the 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 utilization rate of urea, the application of urease inhibitors has attracted 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 fertilizer. However, most of the current urease inhibitors are chemically synthesized substances, and long-term use may have adverse effects on soil microbial community structure and function, disrupt soil ecological balance, and may also pose potential risks to agricultural products.

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

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

[0005] At present, although some microorganisms have been found to have the ability to produce urease inhibitors or indole acetic acid, or some microorganisms do not have denitrification function, but microorganisms with the ability to produce urease inhibitors and indole acetic acid and without 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 rate, promoting plant growth and reducing environmental pollution. SUMMARY

[0006] In view of the problem that there is no microorganism capable of simultaneously producing urease inhibitors and indole acetic acid and having no denitrification in the prior art, and the multiple effects of promoting crop growth, improving yield and reducing environmental pollution cannot be achieved simultaneously, the present application provides a Priestia megaterium, a microbial inoculum thereof and application. The Priestia megaterium provided by the present application is used as a fertilizer microorganism, and after being applied to soil, it can produce urease inhibitors and indole acetic acid in the soil. The activity of soil urease and the decomposition rate of urea can be reduced by urease inhibitors, so that the speed of urea hydrolysis to produce ammonia is as close as possible to the speed of crop utilization of ammonia, the utilization rate of urea and other fertilizers is improved, and NH3 volatilization caused by the application of urea and other nitrogen fertilizers is reduced. At the same time, the strain has no denitrification effect, which can effectively reduce N2O emission and reduce air pollution. In addition, the indole acetic acid can promote crop root growth and improve crop yield.

[0007] To solve the above technical problems, the technical scheme provided by the present application is:

[0008] In a first aspect, the present application provides a Priestia megaterium NPKM-001, which has a preservation number of CGMCC No. 32751.

[0009] The Priestia megaterium NPKM-001 is screened from the rhizosphere soil of corn in a corn field with good growth near Ba Jiazi Village (longitude 123.965, latitude 42.770) in Changtu County, Tieling City, Liaoning Province, and is classified and named as Priestia megaterium. It has been preserved in the General Microbiological Center of China Microbial Culture Collection Management Committee (CGMCC) on November 22, 2024, and the strain preservation number is CGMCC No. 32751. The preservation address is: No. 1, Beichen West Road, Yard 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing.

[0010] The biological characteristics of the Priestia megaterium NPKM-001 provided by the present application are as follows: the colony is milky white, the surface is dry, the edge is relatively neat, it is opaque, the texture is relatively hard, the bacterial body is rod-shaped and relatively large, the size of the bacterial body is about (1.2-1.5) μm x (2.0-4.0) μm, and it is usually single or arranged in short chains; the spore is oval, mesopores, the spore diameter is smaller than the bacterial body, and the size of the spore is about (1.0-1.2) μm x (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, urea hydrolysis test are all negative; the oxidase test, catalase test, glucose fermentation test, citrate utilization test, starch hydrolysis test, gelatin liquefaction test, casein test are all positive.

[0012] The present application provides the giant plister bacteria NPKM-001, which belongs to the "bacteria strain exempted from toxicity test" in the "NY / T 1109-2017 Microbial Fertilizer Biological Safety General Technical Guidelines", has stable production performance, belongs to the common strain type of the current microbial fertilizer production enterprise, and can be used for producing microbial fertilizer. It has the characteristics of being significantly different from the existing microbial fertilizer production strains, has high production of urease inhibitor and indole acetic acid, and has no denitrification effect, can realize the multiple goals of promoting crop growth, improving yield, increasing planting benefit and reducing environmental pollution, has wide application in agricultural planting, and has high practical value.

[0013] In the second aspect, the present application also provides the application of the above-mentioned giant plister bacteria NPKM-001 in reducing NH3 and N2O emission in soil and improving nitrogen fertilizer utilization rate.

[0014] Further, the present application also provides the application of the above-mentioned giant plister bacteria NPKM-001 in promoting crop growth and improving crop yield.

[0015] The giant plister bacteria NPKM-001 provided by the present application has unique metabolic advantages and ecological regulation capabilities on the basis of traditional phosphorus and potassium solubilization functions. In the growth and metabolism process, the strain can efficiently synthesize urease inhibitor and indole acetic acid (IAA), and has no denitrification activity. This multi-effect integrated feature provides an innovative solution for agricultural production. In terms of improving fertilizer efficiency, the secreted urease inhibitor can inhibit soil urease activity, significantly slow down the rate of urea hydrolysis into ammonia (ammonium), and also limit the supply of nitrification and NO3 - , thereby reducing the subsequent denitrification rate and reducing the emission of harmful gases such as NH3 and N2O in farmland soil. In terms of plant growth promotion, the indole acetic acid produced by the strain can promote root growth and improve crop yield. At the same time, due to the lack of denitrification function, the strain can optimize the soil microbial community structure and increase the relative abundance of non-denitrification functional flora during colonization and reproduction in the rhizosphere of crops. This optimization of microbial community structure further inhibits the soil denitrification process, reduces the conversion of nitrate, nitrite and ammonium nitrogen to N2O, reduces greenhouse gas emissions, and achieves the dual goals of saving fertilizer and increasing efficiency and ecological protection.

[0016] The above-mentioned multiple functions form a superposition effect in improving soil physical and chemical properties, promoting healthy growth of crops, reducing agricultural non-point source pollution, etc., and provide strong technical support for the sustainable development of green agriculture, with significant economic, social and environmental benefits.

[0017] In the third aspect, the present application also provides a microbial inoculant comprising the above-mentioned giant plister bacteria NPKM-001.

[0018] Further, the microbial agent is a liquid microbial agent or a powder microbial agent.

[0019] The production of the Microbacterium sp. NPKM-001 into a microbial agent achieves good storage stability and convenient transportation characteristics. The microbial agent is highly adaptable to the needs of modern agricultural production and exhibits strong flexibility and universality in application. Depending on the differences in crop types, planting systems, and soil characteristics, the microbial agent can be applied in various ways, such as foliar spraying, root irrigation, or as base fertilizer. This diversified application scheme ensures efficient application and widespread promotion of the technology without increasing the cost and complexity of operation for farmers.

[0020] Further, the viable cell count of the Microbacterium sp. NPKM-001 in the liquid microbial agent is ≥1.0×10 9 CFU / mL; and the viable cell count of the Microbacterium sp. NPKM-001 in the powder microbial agent is ≥1.0×10 10 CFU / g.

[0021] As a specific embodiment of the present application, the preparation method of the liquid microbial agent comprises the following steps:

[0022] The fermentation medium is loaded into a ventilated stirring fermenter, the loading coefficient is 0.65-0.75, sterilized at 121℃ for 30 min, and cooled to 40℃ for inoculation. The seed liquid of the Microbacterium sp. NPKM-001 is inoculated into the ventilated stirring fermenter, the inoculation amount is 5%-10%, and the fermentation is carried out at 37℃, a rotation speed of 150-200 r / min, and a ventilation amount of 1.0-2.0 VVM for 48-72 h to obtain a fermentation liquid with a viable cell count of ≥1.0×10 9 CFU / mL, which can be directly used as a liquid microbial agent with a shelf life of 6 months.

[0023] As a specific embodiment of the present application, the preparation method of the powder microbial agent comprises the following steps:

[0024] The fermentation medium is loaded into a ventilated stirring fermenter, the loading coefficient is 0.65-0.75, sterilized at 121℃ for 30 min, and cooled to 40℃ for inoculation. The seed liquid of the Microbacterium sp. NPKM-001 is inoculated into the ventilated stirring fermenter, the inoculation amount is 5%-10%, and the fermentation is carried out at 37℃, a rotation speed of 150-200 r / min, and a ventilation amount of 1.0-2.0 VVM for 48-72 h to obtain a fermentation liquid with a viable cell count of ≥1.0×10 9 CFU / mL;

[0025] The fermentation liquid is continuously centrifuged by a disc centrifuge at 6000-7000 r / min, and concentrated 5-10 times to obtain a bacterial slurry; the bacterial slurry is added with auxiliary materials, and then spray dried to obtain a powdery bacterial agent.

[0026] As a specific embodiment of the present application, the preparation method of the seed liquid of the above-mentioned P. megakarya NPKM-001 includes the following steps:

[0027] ① Flask slant strain preparation

[0028] Several 500 mL flasks are taken, 50 mL of NA medium is added to each flask, sterilized at 121 ℃ for 30 min, and placed into a slant. After solidification, it is ready for use. 5 mL of sterile water is poured into the activated test tube strain slant, the mycelium is scraped off with a sterile bamboo stick and stirred as much as possible, and the bacterial suspension is poured into the flask slant. Slowly shake to evenly inoculate the flask slant, and cultivate at 37 ℃ for 5-7 days to produce a large number of spores for standby use.

[0029] ② Seed liquid preparation

[0030] The seed tank is filled with culture medium, the loading factor is 0.65-0.75, and the medium is sterilized at 121 ℃ for 30 min. After cooling to 40 ℃, inoculation is performed. Take 4 of the above flasks, pour 50 mL of sterile water into each flask, scrape off the mycelium with a sterile bamboo stick, pour it into a 1000 mL inoculation bottle, and inoculate the seed tank by differential pressure method. Cultivate at 37 ℃, 1500-2000 r / min, and 1.0-2.0 VVM for 12-14 h to obtain the seed liquid.

[0031] Specifically, the seed culture medium and the fermentation culture medium include the following components: corn powder 2.0%, bean 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%, and the pH is natural, sterilized at 121 ℃ for 30 min.

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

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

[0034] Further, the microbial agent also includes Paenibacillus mucilaginosus N-002, with the accession number CGMCC No. 32752. Further, the microbial agent also includes Paenibacillus mucilaginosus N-002, with the accession number CGMCC No. 32752.

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

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

[0037] Specifically, the application method of the microbial agent comprises spraying, pouring or using as a base fertilizer.

[0038] In a fourth aspect, the application further provides a microbial fertilizer comprising the above-mentioned giant priestleya NPKM-001.

[0039] The giant priestleya and the preparation thereof provided by the application have great application value in crop planting, and are particularly suitable for use when urea is applied to crops. The strain has significant phosphorus solubilization ability, which ensures the phosphorus nutrition of crops. At the same time, the indole acetic acid produced by the strain can promote the root development and cell growth of crops and improve the yield. In terms of improving the efficiency of urea, the urease inhibitor produced by the strain plays a key role. By reducing the urease activity in the soil, the urea hydrolysis rate is slowed down, the ammonia emission and the nitrous oxide emission in the soil after the application of urea are significantly reduced, which is not only beneficial to maintaining the nitrogen in the soil, but also greatly improves the utilization rate of nitrogen fertilizers such as urea. In addition, the strain has no denitrification function, and it can grow and reproduce rapidly in the rhizosphere of crops, increase the proportion of non-denitrification functional bacteria in the soil, effectively reduce the denitrification of the soil, and reduce the emission of nitrous oxide, which is of great significance to environmental protection and improving fertilizer efficiency. The giant priestleya and the preparation thereof can provide a new solution for green agriculture through the functional synergy of phosphorus solubilization, growth promotion, fertilizer efficiency improvement and emission reduction, and can help to update the microbial fertilizer strains and promote the sustainable development of agriculture. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 The colony morphology of the giant priestleya NPKM-001 on the NA culture medium plate;

[0041] Figure 2Fig. 1 is a photograph showing the cell and spore morphology of the B. megaterium NPKM-001 of the present application;

[0042] Figure 3 Fig. 2 is a phylogenetic tree of the B. megaterium NPKM-001 of the present application;

[0043] Figure 4 Fig. 3 is a photograph showing the growth of the N-002 strain inoculated on the medium containing the NPKM-001 strain in a cross pattern;

[0044] Figure 5 Fig. 4 is a photograph showing the growth of the NPKM-001 strain inoculated on the medium containing the N-002 strain in a cross pattern. DETAILED DESCRIPTION

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

[0046] Unless otherwise specified, the reagents used in the examples are analytical pure reagents conforming to the national standards, and the water used for analysis is the third grade water specified in GB / T6682.

[0047] Culture medium used in the examples:

[0048] Phosphorus-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, high-temperature sterilization at 115°C for 20 min.

[0049] Phosphorus-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, high-temperature sterilization 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, pH 7.0. Sterilization 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. Sterilization at 121°C for 30 min.

[0052] Phosphate solubilizing fermentation medium: glucose 10 g, Ca3(P04)2 5 g, MgCl2 5 g, MgS04-7H20 0.25 g, KCl 0.2 g, (NH4)2S04 0.1 g, distilled water 1000 mL, pH 7.0-7.5. Sterilized at 115°C for 30 min.

[0053] Potassium solubilizing fermentation medium: sucrose 5.0 g, MgS04-7H20 0.5 g, Na2HP04 2 g, FeCl3 0.005 g, K2HP04 0.2 g, CaCO3 0.1 g, potassium feldspar 5.0 g, distilled water 1000 mL, pH 7.0-7.5. Sterilized at 121°C for 30 min.

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

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

[0056] Giltay liquid medium: A solution: 1.0 g KNO3, 1.0 g asparagine, 1% (w / v) bromo-muskatol blue (BTB) alcohol solution 5 mL, distilled water 500 mL; B solution: 8.5 g trisodium citrate, 1 g MgS04-7H20, 1 g KH2P04, 0.05 g FeCl3-6H20, 0.15 g CaCl2, distilled water added to 500 mL; mix A and B solutions, adjust pH to 7.0-7.2, sterilized at 121°C for 30 min and then used.

[0057] Organophosphorus medium: glucose 10 g, (NH4)2S04 0.5 g, MgS04-7H20 0.3 g, NaCl 0.3 g, KCl 0.3 g, FeS04-7H20 0.03 g, MnS04-7H20 0.03 g, lecithin 2 g, CaCO3 5 g, distilled water 1 L, pH 7.0. Sterilized at 115°C for 30 min.

[0058] Example 1

[0059] Strain screening

[0060] 1. Enrichment of Bacillus sp. P-1

[0061] Take 10.0g of soil from the rhizosphere of a vigorous corn field near the village of Bajiazi, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770), add it to a triangular flask containing 90mL of sterile water and glass beads, shake on a shaker at a speed of 200r / min for 10min, then 80℃ water bath for 15min, obtain the bacteria in spore state. After standing for a moment, take 5mL of this soil suspension and inoculate it into a phosphorus solubilizing liquid medium, shake and cultivate at 37℃ for 3d. After standing, take 5mL and inoculate it again into a phosphorus solubilizing liquid medium, repeat the above operation, complete the secondary enrichment, and the secondary enrichment liquid is ready for use.

[0062] 2. Isolation of phosphorus-solubilizing Bacillus

[0063] Take 1.0mL of the above secondary enrichment liquid with a sterile pipette, dilute it to 10 -6 . Take 0.1mL of each gradient and inoculate it into a phosphorus solubilizing plate medium, evenly spread it with a sterile spreader, and incubate it in a 37℃ constant temperature incubator for 2-4d, and observe whether a transparent circle forms around the colony. If a transparent circle appears, it indicates that the strain is a phosphorus-solubilizing Bacillus with certain phosphorus-solubilizing ability.

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

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

[0066] 3. Screening of Bacillus with strong phosphorus-solubilizing and potassium-solubilizing ability

[0067] The 128 strains obtained by the above screening were inoculated into 50 mL of LB medium, and cultured at 37°C and 180 r / min for 12 h. The culture was in the logarithmic growth phase, and was used as seed liquid. 1 mL of the seed liquid was added into 100 mL of phosphorus solubilization fermentation medium, and the phosphorus solubilization fermentation medium without inoculation of any bacteria was used as a blank control group. Each treatment was repeated three times, and was cultured at 37°C and 150 r / min for 7 d. After the culture, the fermentation broth was centrifuged at 6000 r / min for 10 min to obtain supernatant. 10 mL of the supernatant was added into a 50 mL volumetric flask, and was diluted with distilled water to the calibration mark. After digestion, the available phosphorus content was determined by the molybdenum-phosphorus colorimetric method. Compared with the available phosphorus (calculated as P2O5) concentration of the blank control group, the increase degree of the available phosphorus (calculated as P2O5) concentration of each strain was calculated.

[0068] In the potassium solubilization test, the potassium solubilization fermentation medium was used to replace the phosphorus solubilization fermentation medium, and the available potassium content was determined by the flame photometry after digestion. Compared with the available potassium (calculated as K2O) concentration of the blank control group, the increase degree of the available potassium (calculated as K2O) concentration of each strain was calculated.

[0069] Results: From the 128 strains with S / s value greater than 5.0, 42 strains capable of increasing the available phosphorus (calculated as P2O5) concentration by more than 20 times and the available potassium (calculated as K2O) concentration by more than 10% were screened. The NPKM-001 strain could increase the available phosphorus (calculated as P2O5) concentration by 35.71 times and the available potassium (calculated as K2O) concentration by 15.29%.

[0070] 4. Screening of Bacillus strains without urease production

[0071] The bacterial lawns of the 42 Bacillus strains with strong phosphorus solubilization and potassium solubilization capacity obtained by the above screening were picked up with sterilized bamboo sticks, and were cross-inoculated on the urea-phenol red bacterial culture medium plates. The plates were cultured at 37°C for 24 h, and the color change around the cross bacterial lawns was observed. The colonies with yellow color (without changing into red color) around the cross bacterial lawns were picked up, inoculated into NA slant medium, and cultured at 37°C for 24-48 h for preservation.

[0072] Test principle: Phenol red is an acid-base indicator, which is yellow in acidic condition, orange in neutral condition, and red in alkaline condition. The color change range of phenol red is pH 6.8 (yellow) to 8.4 (red). If the color around the bacterial lawn is still yellow, it indicates that the strain may not produce urease, and does not decompose urea in the culture medium around the bacterial lawn, and does not cause significant change of pH. On the contrary, if the color around the bacterial lawn changes into red, it indicates that the strain may produce urease, decomposes urea in the culture medium to produce ammonia, and causes the pH to increase to change into red.

[0073] Results: 19 strains of non-urease-producing Bacillus were screened from 42 strains of Bacillus with strong phosphorus-dissolving and potassium-releasing capacity, including NPKM-001 strain.

[0074] 5. Screening of Bacillus producing urease inhibitor

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

[0076] Soil with a depth of 5-20 cm from a corn field with good growth in Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770) was excavated. The urease activity of the corn field soil was detected by the sodium phenolate-sodium hypochlorite colorimetric method. The principle is that urea is hydrolyzed by soil urease to generate ammonia, which reacts with sodium phenolate-sodium hypochlorite at room temperature to generate blue indophenol, the color depth of which is proportional to the amount of ammonia generated. Therefore, the amount of ammonia can be determined by colorimetry to represent the urease activity, and 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] Ammonia standard solution: 0.4717 g (accurate to 0.0001 g) of ammonium sulfate dried in a drying oven at 105°C for 3 h was accurately weighed, dissolved in water, and made up to 1000 mL to obtain a 1 mL stock solution containing 0.1 mg of ammonia. Before use, the above solution was diluted 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): A solution: 62.50 g of phenol (accurate to 0.01 g) was dissolved in a small amount of ethanol, 2 mL of methanol and 18.5 mL of acetone were added, and ethanol was used to make up to 100 mL. B solution: 27.00 g of sodium hydroxide (accurate to 0.01 g) was dissolved in water and made up to 100 mL. The A and B solutions were stored in a 4°C refrigerator. Before use, 20 mL of each of the A and B solutions were mixed and made up to 100 mL with water.

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

[0081] Urea solution (100 g / L): 10.00 g of urea (accurate to 0.01 g) was dissolved in water and made up 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) into water, respectively, combine the two solutions, adjust the pH to 6.7 with 1 mol / L sodium hydroxide, and dilute to 1000 mL with water.

[0083] (2) Standard curve preparation

[0084] Before measuring the absorbance of the sample, 0.00 mL, 1.00 mL, 3.00 mL, 5.00 mL, 7.00 mL, 9.00 mL, 11.00 mL, and 13.00 mL of the ammonia working solution (0.1 mg / mL) were taken into a 50 mL volumetric flask, respectively, then 20 mL of water was added, followed by the addition of 4 mL of sodium phenolate solution and 3 mL of sodium hypochlorite solution in sequence, while shaking. After 20 min of color development, the solution was diluted to 50 mL. A set of standard 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, and 2.6 μg / mL of ammonia were prepared. Colorimetry was performed at a wavelength of 578 nm within 1 h (blue color remained stable within 1 h). The standard curve was plotted with the ammonia concentration as the abscissa and the absorbance as the ordinate.

[0085] (3) Determination of urease inhibition rate of the test solution

[0086] ① Determination of soil urease activity

[0087] 5 g of soil sample (accurate to 0.0001 g; if the content is high, the amount of sample can be appropriately reduced) and 5.0 mL of heavy water were taken into a 100 mL stoppered flask, 1 mL of toluene was added, and the mixture was shaken uniformly. After 15 min, 10 mL of 100 g / L urea solution and 20 mL of pH 6.7 citrate buffer solution were added, and the mixture was shaken uniformly and then incubated in a 37℃±1℃ incubator for 24 h.

[0088] After incubation, the mixture was filtered to obtain the filtrate. 1.00 mL of the filtrate was taken into a 50 mL volumetric flask, followed by the addition of 4 mL of sodium phenolate solution and 3 mL of sodium hypochlorite solution in sequence, while shaking. After 20 min of color development, the solution was diluted to 50 mL with water. The above operation was repeated with 10 mL of heavy water instead of "10 mL of 100 g / L urea solution" as the control, and colorimetry was performed at a wavelength of 578 nm within 1 h.

[0089] ② Determination of soil urease activity after adding the test solution

[0090] Except that "5.0 mL of heavy water" in ① was replaced by "5.0 mL of test solution", the other steps were the same as in ①.

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

[0092] ① Soil urease activity calculation

[0093] Urease activity is expressed as mg of ammonia generated from 24h, 1g of air-dried soil hydrolysis substrate (urea), calculated as follows:

[0094]

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

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

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

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

[0099] N is the fractionating multiple, N = the volume of leaching liquid (mL) / the volume of suctioned filtrate (mL) = 41 mL / 1 mL = 41.

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

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

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

[0103] Urease activity inhibition rate calculation

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

[0105] Results: Among the 19 non-urease-producing Bacillus strains, 4 strains had an inhibition rate of corn field soil urease activity greater than 60%, and the fermentation supernatant of NPKM-001 strain had an inhibition rate of corn field soil urease activity of 84.92%. Using the same method for detection, the fermentation supernatant of NPKM-001 strain had an inhibition rate of urease activity of 87.93%, 85.97% and 91.72% for the soil samples of moist soil (taken from the western part of Mancheng District, Baoding City), brown soil (taken from the western part of Mancheng District, Baoding City) and black soil (taken from the northern suburb of Shenyang City), respectively.

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

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

[0108] (1) Standard curve drawing

[0109] 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 were prepared with distilled water. 2.0 mL of IAA standard solutions with different concentrations were mixed with 2.0 mL of Salksowski color reagent, and incubated at 40°C in a dark water bath for 30 min. The absorbance was measured at a wavelength of 530 nm using a spectrophotometer. The standard curve was drawn with the IAA concentration as the horizontal coordinate and the absorbance as the vertical coordinate.

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

[0111] 2.0 mL of the above test solution was mixed with 2.0 mL of Salksowski color reagent, and incubated at 40°C in a dark water bath for 30 min. The absorbance was measured at a wavelength of 530 nm using a spectrophotometer. The IAA content in the fermentation broth was calculated using the standard curve.

[0112] Salksowski color reagent: 1 mL of 0.5 mol / L FeCl3 solution was mixed with 50 mL of 35% HCIO4 solution.

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

[0114] 7. Screening of Bacillus without denitrification function

[0115] The above 3 Bacillus strains with strong IAA-producing ability were transferred to NA slant medium with sterile bamboo sticks, and incubated at 37°C for 24 h for activation.

[0116] In the super-clean bench, 10 mL of Giltay liquid medium was added into a large test tube (20 mm x 200 mm), a small test tube (5 mm x 20 mm) was placed upside down in the large test tube to exhaust the gas in the small test tube, and three strains of Bacillus with strong IAA-producing ability were inoculated into the small test tube after activation, and then the small test tube was incubated at 37°C for 5-7 days. Whether bubbles were generated in the small test tube was observed. If gas was generated, the strain had denitrification function; if no gas was generated, the strain had no denitrification function.

[0117] The test showed that two of the above three strains had no denitrification function, and the NPKM-001 strain was included.

[0118] Strain species identification

[0119] 1. Morphological observation

[0120] The NPKM-001 strain slant was picked with a sterile bamboo stick and inoculated into NB medium, which was then cultured at 37°C and 150 r / min on a shaking table for 24 h. Then 1.0 mL of the culture solution was appropriately diluted and spread on NA medium plates, which were then incubated at 37°C for 24-48 h to observe the colony morphology. A smear of the 24-48 h culture solution was made and then stained with crystal violet to observe the morphology of the bacterial cells and spores.

[0121] Results: On the NA medium plate, the colony was milky white, dry on the surface, with a relatively neat edge, opaque, and hard in texture, and easy to pick. The bacterial cells were rod-shaped, large, about (1.2-1.5) μm x (2.0-4.0) μm, often single or in short chains, and gram-positive. The spores were oval, mesosporic, with a diameter smaller than that of the bacterial cells, about (1.0-1.2) μm x (1.5-2.0) μm. Figures 1-2 .

[0122] 2. Physiological and biochemical identification

[0123] According to the experimental methods in the Bergey's Systematic Bacteriology Manual, the NPKM-001 strain was subjected to 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. The results are shown in Table 1.

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

[0125]

[0126]

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

[0128] 3. Molecular biological identification

[0129] The total DNA of the NPKM-001 strain was extracted using a bacterial genomic DNA extraction kit (Biomiga, item number BW-GD2411-01) and following the steps in the instructions. The universal primer for prokaryotic 16S rDNA gene sequence (upstream 5'-ACTGGAGGAAGGTGGGGA-3', downstream 5'-AGGAGGTGATCCAACCGCA-3') was used for PCR amplification to obtain the amplification product. The sequencing of the amplification product was completed by Huada Gene. The sequencing result of the 16S rDNA is as follows:

[0130]

[0131] The sequencing results were aligned in NCBI, and the phylogenetic tree was constructed based on the Neighbor-Joining method using MEGA 11.0, see Figure 3 According to the 16S rDNA sequence similarity analysis, and referring to the physiological and biochemical test results and the colony and cell morphology, the NPKM-001 strain was determined as Priestia megaterium, i.e. Bacillus megaterium.

[0132] The Priestia megaterium has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 22, 2024, with the strain preservation number of CGMCC No. 32751, and the preservation address of No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard 3rd.

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

[0134] The slant mycelium of NPKM-001 strain was picked up with a sterilized bamboo stick and inoculated into 50 mL of LB medium, which was cultured at 37°C with 180 r / min shaking for 12 h to obtain a seed liquid. In the test group, the seed liquid was inoculated into the organic phosphorus medium at a 3% inoculation amount. In the control group, the seed liquid was first inactivated by heating at 121°C for 20 min, and the rest of the operations were the same as those in the test group, with 3 parallel groups. After inoculation, the medium was cultured at 37°C with 180 r / min shaking for 5 days, and then centrifuged at 5000 r / min for 10 min. The supernatant was poured out, and the effective phosphorus content in the supernatant was determined by the molybdenum-antimony anti-colorimetric method.

[0135] Results: The content of effective phosphorus in the control group was (0.066±0.010) mg / L; the content of effective phosphorus in the test group was (1.720±0.082) mg / L, which was 25.91 times that of the control group. It showed that the NPKM-001 strain could decompose and release part of the phosphorus in the lecithin (organic phosphorus) in the medium.

[0136] Example 2

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

[0138] (1) Fermentation medium: corn flour 2.0%, soybean meal 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%, pH natural, 121°C sterilization for 30 min.

[0139] (2) Preparation of seed liquid:

[0140] Take several 500 mL flasks, add 50 mL of NA medium to each flask, sterilize at 121°C for 30 min, and place on an inclined plane. After solidification, reserve for use. Pour 5 mL of sterile water into the activated NPKM-001 strain test tube inclined plane, scrape the mycelium with a sterile bamboo stick and mix as evenly as possible. Pour the bacterial suspension into the flask inclined plane, shake slowly, and inoculate evenly. Incubate at 37°C for 5-7 days, and produce a large amount of spores for reserve.

[0141] Fill the 100 L ventilated and stirred seed tank with fermentation medium, with a loading factor of 0.65-0.75. Sterilize at 121°C for 30 min, and cool to 40°C before inoculation. Take 4 of the above flasks, pour 50 mL of sterile water into each, scrape the mycelium with a sterile bamboo stick, and pour into a 1000 mL inoculum flask. Inoculate the seed tank using the differential pressure method, incubate at 37°C, 1500 r / min, and 1.0-2.0 VVM for 12-14 h to obtain the seed liquid.

[0142] (3) Add the above fermentation medium to 1 M 3 Ventilated and stirred fermentation tank, loading factor 0.65-0.75, 121°C sterilization for 30 min, cool to 40°C before inoculation with the above seed liquid, inoculation amount 5%-10%, fermentation at 37°C, 150 r / min, ventilation 1.0-2.0 VVM for 48-72 h to obtain the fermentation broth. Detect the viable bacterial content in the fermentation broth using gradient dilution plate coating method, and observe and calculate the spore rate under a microscope using smear staining method.

[0143] Results: The spore content in the fermentation broth is between (1.0-3.0) x 10 9 CFU / mL, so a liquid microbial agent (liquid microbial fertilizer) with a viable bacterial (spore) content of more than 1.0 x 10 9 CFU / mL can be prepared, with a shelf life of 6 months.

[0144] (4) The above fermentation broth is continuously centrifuged at 6000 r / min using a disc centrifuge, concentrated 5-10 times to obtain a bacterial slurry. Add adjuvants to the bacterial slurry, and spray dry to obtain a bacterial powder.

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

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

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

[0148]

[0149]

[0150] Example 3

[0151] 1. Pot experiment for ammonia emission reduction effect of P. megaspora NPKM-001 liquid microbial inoculant

[0152] Twenty-eight plastic flowerpots with inner diameter x height = 15 cm x 18 cm were filled with soil (soil taken from 5-15 cm deep in farmland) to a depth of about 15 cm in the pots. Twenty-eight pepper seedlings, which were one week old and had basically the same size, thickness, and number of leaves, were selected and planted in the pots, one seedling per pot. They were divided into a CK group, a test I group, a test II group, and a test III group, a total of 4 groups, 7 pots per group. Fertilization was performed at the beginning of the pot experiment, and the test period was 15 days. The test scheme is shown in Table 3.

[0153] Table 3 NPKM-001 microbial inoculant pepper pot experiment scheme

[0154]

[0155] The flowerpots of the four groups were placed in a transparent glass jar with length x width x height = 0.5 x 0.5 x 0.5 m, and daily watering was performed by adding water to the bottom of the jar and the water-permeable holes at the bottom of the pots. Other management was consistent among the groups, such as maintaining the soil water holding rate at 40%-60%, maintaining an average air temperature of 25°C in the jar, and providing 8-10 hours of light per day. The glass jar was sealed with a sealed lid to form a closed static chamber, and three PVC pipes with valves were installed on the sealed lid. One PVC pipe was used for water addition at the bottom of the jar, one PVC pipe was used for air intake with a control air intake of about 60 L / h (using a small air blower to continuously introduce air into the jar), and the third PVC pipe was a three-way pipe with one end connected to a 200 mL 0.01 mol / L sulfuric acid absorption solution for discharging gas and the other end connected to a 20 cm long needle for sampling and measuring N2O emission.

[0156] The ammonia absorbed by the absorption solution was determined by the method shown in "HJ533-2009 Determination of ammonia in ambient air and waste gas by Nessler's reagent spectrophotometry". The principle is that dilute sulfuric acid solution is used to absorb ammonia in the air, ammonium ions generated are reacted with Nessler's reagent to generate a yellow-brown complex, the absorbance of the complex is proportional to the content of ammonia, the absorbance is measured at 420 nm wavelength, and the content of ammonia in the air is calculated according to the absorbance. The test results are shown in Table 4.

[0157] Table 4 Effect of NPKM-001 liquid inoculant on ammonia emission reduction of pot-grown pepper soil

[0158]

[0159] The test results show that: the application of urea can significantly increase the ammonia volatilization amount of soil during the test period; compared with group I (application of urea), group III can reduce the ammonia emission of soil by 71.32% under the condition of the same urea application amount (15 g / m 2 ); compared with CK group (without application of urea), group II can reduce the ammonia emission of soil by 52.52% under the condition of not applying urea. Therefore, the liquid inoculant can significantly reduce the ammonia emission of soil, especially the soil applied with urea. The reason for ammonia emission reduction is that NPKM-001 strain can produce urease inhibitor, which can inhibit the conversion process of urea to ammonium (ammonia), so that urea mainly exists in the form of amide nitrogen in soil, reducing the concentration of NH4 + / NH3 in soil solution, thereby reducing ammonia emission.

[0160] 2. Effect of NPKM-001 liquid inoculant of P. megaspora on urease activity of pot-grown pepper soil

[0161] When the above pot experiment was completed (15d), the urease activity of soil in each pot was detected by the method of "T / NAIA 011-2020 Determination of soil urease activity by sodium phenolate-sodium hypochlorite colorimetry", and the urease activity of soil in pots with and without NPKM-001 inoculant was compared. The specific detection method is shown in Example 1. The specific test results are shown in Table 5.

[0162] Table 5 Effect of NPKM-001 liquid inoculant on urease activity of pot-grown pepper soil

[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 P<0.01 level difference.

[0166] The test results show that compared with group I (urea irrigation), group III can reduce soil urease activity by 64.09% (P<0.01) under the condition of irrigating the same amount of urea (15 g / m 2 ) and applying NPKM-001 liquid bacterial agent; compared with CK group (no urea irrigation), group II can reduce soil urease activity by 67.71% (P<0.01) under the condition of not irrigating urea and applying NPKM-001 liquid bacterial agent. Therefore, whether urea is irrigated or not, the liquid bacterial agent can significantly reduce the urease activity of the soil, thereby significantly reducing the loss of fertilizer efficiency caused by ammonia volatilization after urea fertilization, and improving the utilization rate and efficiency of urea.

[0167] 3. N2O emission reduction effect of NPKM-001 liquid bacterial agent of Pantoea agglomerans on soil of potted pepper

[0168] At 8:00-12:00 on the 0th, 3rd, 6th, 9th, 12th, and 15th days after fertilization, the air inlet and outlet were stopped for 4 h. At 12:00, 50 mL of air in the glass jar was sucked by a syringe and injected into a 12 mL headspace tube that had been vacuumed by a vacuum pump. The N2O content was determined by Agilent 7890A gas chromatography, the detector was ECD, the detection temperature was 300℃, the column temperature was 60℃, 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 curve was established by selecting a standard gas with a concentration of 5 mg / L. Artificial sampling was used, 40 μL of sample was extracted by a 100 μL gas-tight syringe, and injected into the analyzer within 1 s.

[0169] In order to supply air to the pepper seedlings, the glass jar becomes a closed space within the 4 h of stopping air inlet and outlet, and the N2O emitted by the soil in the flowerpot can only accumulate in the glass jar. Therefore, the concentration of N2O in each group of glass jars is detected and compared to obtain the emission reduction effect of NPKM-001 liquid bacterial agent (microbial fertilizer). The results are shown in Table 6.

[0170] Table 6 Emission reduction effect of NPKM-001 liquid bacterial agent on N2O in soil of potted pepper

[0171]

[0172]

[0173] Results: Group I and group III can significantly increase the soil nitrogen content after pouring urea, and then significantly increase the emission of N2O through nitrification and denitrification. The peak of emission appeared around the 3rd day, and the emission gradually decreased after 3 days, which may be related to the gradual utilization of urea. But there are significant differences between group I and group III: compared with group I (pouring urea), group III can reduce the soil N2O emission by 4.1%, 31.1%, 25.2%, 18.4%, 17.3% and 14.8% respectively on the 0th, 3rd, 6th, 9th, 12th and 15th day after fertilization, and the average reduction degree is 21.4% from the 3rd to the 15th day (except that the urease inhibitor contained in the liquid bacterial agent has the effect of reducing N2O emission, the spores are also the active components of the liquid bacterial agent, and the spores reaching the pepper roots have not germinated on the 0th day, so they do not have the corresponding effect of reducing N2O emission, so the calculation starts from the 3rd day). Compared with CK group (without pouring urea), group II can reduce the soil N2O emission by 1.5%, 12.6%, 11.4%, 9.1%, 9.2% and 8.1% respectively on the 0th, 3rd, 6th, 9th, 12th and 15th day after fertilization, and the average reduction degree is 10.1% from the 3rd to the 15th day. Therefore, while pouring urea, applying NPKM-001 bacterial agent (microbial fertilizer) can significantly reduce the soil N2O emission, and the best reduction effect is more than 30% on the 3rd day of the test. Applying NPKM-001 bacterial agent without pouring urea also has a certain reduction effect. The main reasons are as follows: first, NPKM-001 strains can produce urease inhibitors to inhibit the speed of urea decomposition into ammonia (ammonium), reduce the emission of NH3, and at the same time, limit the nitrification and NO3 - supply, thereby reducing the subsequent denitrification rate and reducing the emission of N2O; second, because NPKM-001 strains have no denitrification ability, they grow and reproduce in the soil after being applied, thereby increasing the number of non-denitrifying bacteria in the soil, especially in the root soil, reducing the denitrification of the soil, and hindering the conversion of nitrate, nitrite and ammonium to N2O, ultimately reducing the emission of N2O.

[0174] 4. Effect of NPKM-001 liquid bacterial agent of P. megaspora on the available phosphorus content of pepper potting soil

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

[0176] Table 7 Effect of NPKM-001 liquid inoculant on the content of available phosphorus in the soil of potted pepper

[0177]

[0178]

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

[0180] Results: Compared with group I (watering with urea), group III can increase the content of available phosphorus in the soil by 50.03% under the condition of watering with equal amount of urea, and increase the content of available phosphorus by 39.38% compared with before potting; compared with CK group (without watering with urea), group II can increase the content of available phosphorus in the soil by 46.29% under the condition of not watering with urea, and increase the content of available phosphorus by 37.79% compared with before potting. It shows that whether watering with urea or not, applying NPKM-001 liquid inoculant can significantly increase the content of available phosphorus in the soil. It provides sufficient phosphorus elements for the growth of crops and even the next season crops.

[0181] 5. Effect of NPKM-001 liquid inoculant of P. macrospiderm on the growth performance of potted pepper seedlings

[0182] After the end of the test, the flowerpots planted with pepper seedlings were taken out from the glass jars, and most of the root soil of the pepper seedlings was carefully removed. The roots were washed in a tap water pot and dried on an absorbent paper. The height of the pepper seedlings was measured with a ruler (accurate to mm), and the roots and aboveground parts were cut with scissors at the underground and aboveground boundary. The stem diameter was measured with a vernier caliper, and the fresh weight of the roots and aboveground parts was measured with a balance (accurate to 0.01 g). The results are shown in Table 8.

[0183] Table 8 Effect of NPKM-001 liquid inoculant on the growth promotion test of potted pepper seedlings

[0184]

[0185] Note: The average in the table is the average of 7 corresponding values after removing one highest value and one lowest value; different capital letters indicate P < 0.01 level difference, and different small letters indicate P < 0.05 level difference.

[0186] Results: By measuring the plant height, stem diameter, leaf number, root fresh weight and aboveground fresh weight of the four groups of peppers, it can be known that the liquid inoculant can significantly promote the growth of peppers. Compared with the CK group (without pouring urea), the average values of plant height, stem diameter, leaf number, root fresh weight and aboveground fresh weight of peppers in the II group without pouring urea increased by 26.80%, 16.44%, 2.91%, 75.36% and 40.83%, respectively. Compared with the I group (pouring urea), the average values of plant height, stem diameter, leaf number, root fresh weight and aboveground fresh weight of peppers in the III group with equal amount of urea increased by 19.44%, 22.19%, 2.59%, 46.23% and 44.01%, respectively. In summary, whether urea is poured or not, the NPKM-001 liquid inoculant can significantly increase the plant height, stem diameter, root fresh weight and aboveground fresh weight of peppers, and promote the growth of crops, except that it has no significant effect on the leaf number. The reason is that urea is N fertilizer, which can provide nutrients for peppers and promote growth; in addition to containing natural plant hormones such as IAA, the spores of the liquid inoculant can also produce IAA and other substances after growing and reproducing in the root system of peppers. Therefore, the application of the inoculant can significantly promote the growth of pepper seedlings.

[0187] Example 4

[0188] The present embodiment provides a composite microbial inoculant, which comprises Priestia megaterium NPKM-001 and Bacillus mucilaginosus N-002, and the preservation number of the Bacillus mucilaginosus N-002 is CGMCC No. 32752.

[0189] (1) NPKM-001 and N-002 strains do not inhibit each other

[0190] By using the plate confrontation method test, it can be proved that the two tested strains of NPKM-001 and N-002 do not inhibit each other. The specific test steps are as follows: pour 5 mL of sterile water into a slope of one of the tested strains, scrape the bacterial lawn with a bamboo stick, and transfer the bacterial suspension into a sterile empty test tube. Use a vortex shaker to oscillate for 2 min to become a uniform bacterial suspension, immediately pour into 100 mL of sterilized NA medium which has not yet solidified (50-60°C), mix well, and immediately pour the plate. After solidification, the plate is placed in a 4°C refrigerator overnight. The next day, inoculate the bacterial lawn of the other tested strain on the plate in a cross method, and cultivate at 37°C for 24-48 h. 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 status of NPKM-001 and N-002 strains on the same plate is shown in Figures 4-5 From the figure, it can be seen that the two tested strains of NPKM-001 and N-002 have the characteristics of not inhibiting each other.

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

[0193] The pot experiment was carried out by using NPKM-001 and N-002 liquid inoculants in combination, and the same pot experiment as in Example 3 was carried out. It was found that the two inoculants had mutual synergistic or additive effects in reducing the urease activity in the rhizosphere soil, reducing the soil NH3 and N2O emissions, promoting plant growth, and improving the fertilizer efficiency of urea and other nitrogen fertilizers.

[0194] Under the condition of pot experiment with 0.26 g of urea, 0.13 mL of NPKM-001 liquid inoculant was irrigated, which could 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 0.26 g of urea, 0.13 mL of Bacillus mucilaginosus N-002 liquid inoculant was irrigated, which could 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 pot experiment with 0.26 g of urea per pot, 0.065 mL of NPKM-001 liquid inoculant and 0.065 mL of N-002 liquid inoculant were irrigated at the same time, which could reduce the soil urease activity by 77.13%, the soil NH3 emission by 75.82%, and the average N2O emission by 29.86%, which was 13.04 percentage points, 4.50 percentage points, and 8.46 percentage points higher than the effect of irrigating NPKM-001 alone, respectively; and was 9.98 percentage points, 10.00 percentage points, and 7.86 percentage points higher than the effect of irrigating N-002 alone, respectively.

[0196] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, or improvement within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Megapristus sp. (M. latus) NPKM-001, characterized by, Priestia megaterium )NPKM-001, characterized by, and has a preservation number of CGMCC No. 32751.

2. The use of the Priestia megaterium NPKM-001 of claim 1 in reducing NH3 and N2O emissions in soil, and improving nitrogen fertilizer utilization.

3. The use of the Priestia megaterium NPKM-001 of claim 1 in promoting crop growth, and improving crop yield.

4. A microbial inoculant, characterized in that, A microbial inoculant comprising the Priestia megaterium NPKM-001 of claim 1.

5. The microbial inoculant of claim 4, wherein, The microbial inoculant is a liquid inoculant or a powder inoculant.

6. The microbial inoculant of claim 5, wherein, The viable cell number of P. magnus NPKM-001 in the liquid inoculant is ≥ 1.0 x 10 9 CFU / mL; and the viable cell number of P. magnus NPKM-001 in the powder inoculant is ≥ 1.0 x 10 10 CFU / g.

7. The microbial inoculant of claim 4, wherein The method of applying the microbial inoculant comprises spraying, watering, or using as a base fertilizer.

8. A microbial fertilizer, characterized by, A microbial inoculant comprising the Priestia megaterium NPKM-001 of claim 1.

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