Bacillus amyloliquefaciens as well as microbial agent and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses bacillus amyloliquefaciens and a microbial agent and application thereof. The preservation number of the bacillus amyloliquefaciens Y-102 is CGMCC (China General Microbiological Culture Collection Center) No.33966, and the preservation number of the bacillus amyloliquefaciens Y-102 is CGMCC No.33966. The strain not only has outstanding phosphorus and potassium solubilizing capacity, but also can generate a urease inhibitor and indoleacetic acid, and has no denitrification function. From the perspective of functions, the produced indoleacetic acid can effectively promote the growth of crops and obviously increase the yield of the crops; the urease inhibitor can reduce the activity of soil urease and greatly reduce the emission of ammonia and nitrous oxide, and in addition, as the bacterial strain has no denitrification effect, the bacterial strain grows and propagates massively at the rhizosphere of crops after being applied to the soil, the proportion of bacteria without denitrification function in the soil can be increased, the denitrification effect of the soil is further reduced, and the emission of nitrous oxide is reduced. And the method has great significance on environmental protection.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a Bacillus amyloliquefaciens, a microbial agent thereof and applications. Background Art

[0002] In the context of sustainable agricultural development, soil health, efficient fertilizer utilization, and plant growth promotion have become key research areas. Microorganisms, as crucial components of the soil ecosystem, possess diverse functions that significantly impact agricultural production. However, current microorganisms used in agricultural production often suffer from limited functionality or struggle to balance multiple beneficial functions.

[0003] The use of urease inhibitors is crucial for improving fertilizer utilization. While some microorganisms can produce urease inhibitors, their production is generally low, making them difficult to effectively inhibit soil urease activity in actual agricultural production. This inability to sufficiently slow the hydrolysis of urea to ammonium nitrogen makes it difficult to significantly increase urea utilization, and soil ammonia emissions can cause environmental pollution. Furthermore, microorganisms that produce urease inhibitors often lack other functional capabilities, such as the ability to promote plant growth, and therefore cannot provide additional growth support for plants.

[0004] Denitrification is a key link in the soil nitrogen cycle, leading to nitrogen loss. Denitrifying microorganisms convert nitrate nitrogen into gaseous nitrogen, which escapes from the soil, reducing soil nitrogen content and weakening the effectiveness of nitrogen fertilizers. The production of greenhouse gases such as nitrous oxide (N2O) also exacerbates environmental problems. While some Bacillus amyloliquefaciens strains have been discovered to possess multiple functions, some strains possess denitrification, which severely limits their widespread application in agricultural production.

[0005] In summary, developing a Bacillus amyloliquefaciens that can produce high levels of urease inhibitors and indoleacetic acid without denitrification function, and making it into a microbial agent for use in agricultural production, has important practical significance and broad application prospects for improving fertilizer utilization, promoting plant growth, reducing nitrogen loss and protecting the environment. Summary of the Invention

[0006] In view of the problem that the existing technology lacks microorganisms that can simultaneously produce high levels of urease inhibitors and indoleacetic acid and have no denitrification effect, and cannot simultaneously achieve the multiple effects of promoting crop growth, increasing yields and reducing environmental pollution, the present invention provides a Bacillus amyloliquefaciens, its microbial agent and application.

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

[0008] In a first aspect, the present invention provides a Bacillus amyloliquefaciens Y-102, whose deposit number is CGMCC No.33966.

[0009] Bacillus amyloliquefaciens Y-102 was screened from the rhizosphere soil of a well-growing corn field near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770). It was classified and named Bacillus amyloliquefaciens and was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, abbreviated as CGMCC, on March 24, 2025. The strain deposit number is CGMCC No. 33966. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The biological characteristics of the Bacillus amyloliquefaciens Y-102 provided by the present invention are as follows: the colonies are round with neat edges, and slightly irregular edges appear when the culture time is long; the colonies are off-white and opaque; the surface is dry and relatively rough, with a raised middle and slightly crater-like shape; the bacteria are straight rod-shaped, with a size of about 0.6 μm×(2.0-5.0) μm, and are often solitary or arranged in short chains; the spores are oval with blunt ends and are mesogenous; the spore capsules are slightly swollen, and the spore size is about 0.8 μm×(1.2-1.5) μm.

[0011] The Bacillus amyloliquefaciens Y-102 is a Gram-positive bacterium, and its oxidase test, methyl red test, indole test, and urea hydrolysis test are all negative; its catalase test, glucose fermentation test, citrate utilization test, VP test, starch hydrolysis test, gelatin liquefaction test, and casein test are all positive.

[0012] The Bacillus amyloliquefaciens Y-102 provided by the present invention is a "strain exempt from toxicology testing" as approved by the "NY / T 1109-2017 General Technical Guidelines for Biosafety of Microbial Fertilizers." In actual production scenarios, this strain demonstrates excellent stability, meeting the process requirements of microbial fertilizer manufacturers. Furthermore, compared to currently known microbial fertilizer production strains, Bacillus amyloliquefaciens Y-102 possesses unique functional properties: it can efficiently synthesize urease inhibitors and indoleacetic acid, while lacking denitrification activity. This property gives this strain multiple applications in agricultural production: on the one hand, by inhibiting soil urease activity, it reduces ammonia and nitrous oxide emissions, thereby reducing nitrogen losses. Combined with the plant growth-promoting effects of indoleacetic acid, it can significantly improve crop growth and yield. On the other hand, its lack of denitrification helps reduce greenhouse gas emissions and mitigate environmental pollution risks. With these advantages, Bacillus amyloliquefaciens Y-102 can not only effectively improve the economic benefits of agricultural planting but also promote the sustainable development of green agriculture, demonstrating broad application prospects and high practical value in the agricultural planting field.

[0013] In a second aspect, the present invention also provides the use of the above-mentioned Bacillus amyloliquefaciens Y-102 in reducing NH3 and N2O emissions in soil and improving nitrogen fertilizer utilization efficiency.

[0014] Furthermore, the present invention also provides the use of the above-mentioned Bacillus amyloliquefaciens Y-102 in promoting crop growth and increasing crop yield.

[0015] The Bacillus amyloliquefaciens Y-102 provided by the present invention has a high production of urease inhibitors and indoleacetic acid, and has no denitrification function, taking into account multiple beneficial functions. The urease inhibitor secreted by it can effectively inhibit the activity of multiple ureases in the soil, slowing down the conversion rate of nitrogen fertilizers such as urea to ammonia (ammonium), thereby significantly reducing NH3 emissions; after the ammonia (ammonium) content is reduced, the soil nitrification is weakened, and NO3 - Limited supply further reduces denitrification and effectively reduces N2O emissions, not only improving urea utilization and fertilizer efficiency but also protecting the ecological environment. Furthermore, this strain has no denitrifying function. When applied to the soil and allowed to grow and multiply within the crop root system, it can increase the proportion of bacteria without denitrifying function in the soil, further reducing soil denitrification and continuously lowering N2O emissions from farmland. Furthermore, the high yield of indoleacetic acid produced by Bacillus amyloliquefaciens Y-102 can effectively stimulate the development of crop roots and above-ground growth. These functions work synergistically to not only improve soil quality, enhance crop adaptability to the environment, and increase crop yields, but also reduce harmful gas emissions. This provides strong technical support for the sustainable development of green agriculture, with significant economic, social, and environmental benefits.

[0016] In a third aspect, the present invention further provides a microbial agent comprising the above-mentioned Bacillus amyloliquefaciens Y-102.

[0017] Furthermore, the microbial agent is a liquid agent or a powdered agent.

[0018] The preparation of Bacillus amyloliquefaciens Y-102 into a microbial inoculant improves storage stability and transport convenience, facilitating its widespread application in various agricultural scenarios. The inoculant offers flexible application methods, allowing for foliar spraying, root drench application, or basal fertilizer application, tailored to different crop varieties, planting patterns, and soil conditions. This reduces operational complexity for farmers while avoiding additional costs, thus meeting the diverse needs of modern agricultural production.

[0019] Furthermore, the number of viable bacteria of Bacillus amyloliquefaciens Y-102 in the liquid bacterial agent is ≥1.0×10 9 CFU / mL; the number of viable bacteria of Bacillus amyloliquefaciens Y-102 in the powdered bacterial agent is ≥1.0×10 10 CFU / g.

[0020] As a specific embodiment of the present invention, the preparation method of the liquid bacterial agent includes the following steps:

[0021] The fermentation medium was filled into a ventilated stirred fermenter with a filling factor of 0.65-0.75, sterilized at 121°C for 30 minutes, and cooled to 40°C for inoculation. The seed liquid of Bacillus amyloliquefaciens Y-102 was inoculated into the ventilated stirred fermenter with an inoculation amount of 5% to 10%, and fermented for 36 to 48 hours at 37°C, a rotation speed of 150 to 200 r / min, and a ventilation volume of 1.0 to 2.0 VVM to obtain a viable cell count of ≥1.0×10 9 The fermentation broth with CFU / mL can be directly used as a liquid bacterial agent with a shelf life of 6 months.

[0022] As a specific embodiment of the present invention, the preparation method of the powdered bacterial agent includes the following steps:

[0023] The fermentation medium was filled into a ventilated stirred fermenter with a filling factor of 0.65-0.75, sterilized at 121°C for 30 minutes, and cooled to 40°C for inoculation. The seed liquid of Bacillus amyloliquefaciens Y-102 was inoculated into the ventilated stirred fermenter with an inoculation amount of 5% to 10%, and fermented for 36 to 48 hours at 37°C, a rotation speed of 150 to 200 r / min, and a ventilation volume of 1.0 to 2.0 VVM to obtain a viable cell count of ≥1.0×10 9 CFU / mL of fermentation broth;

[0024] The fermentation liquid is continuously centrifuged at 6000-7000 r / min in a disc centrifuge to be concentrated 5-10 times to obtain bacterial sludge; the bacterial sludge is added with auxiliary materials and spray-dried to obtain a powdered bacterial agent.

[0025] As a specific embodiment of the present invention, the method for preparing the seed liquid of the above-mentioned Bacillus amyloliquefaciens Y-102 comprises the following steps:

[0026] ① Preparation of eggplant bottle slant culture

[0027] Take several 500mL eggplant flasks and add 50mL of NA medium to each. Sterilize at 121°C for 30 minutes, arrange them into a slant, and allow to solidify before use. Pour 5mL of sterile water onto the activated test tube slant. Use a sterile bamboo stick to scrape off the bacterial moss and stir as much as possible. Pour the bacterial suspension onto the slant of the eggplant flask and shake gently to ensure even inoculation. Incubate the flask upside down at 37°C for 5-7 days to produce a large number of spores before use.

[0028] ②Seed liquid preparation

[0029] Fill the seed tank with culture medium at a filling factor of 0.65-0.75, sterilize at 121°C for 30 minutes, and inoculate after cooling to 40°C. Take the inclined surfaces of four eggplant bottles, pour 50 mL of sterile water into each, scrape the bacterial moss with a sterilized bamboo stick, and pour it into a 1000 mL inoculation bottle. Inoculate the seed tank using the pressure differential method. Incubate at 37°C, a rotation speed of 1500-2000 r / min, and a ventilation volume of 1.0-2.0 VVM for 10-12 hours to obtain the seed solution.

[0030] Specifically, the seed culture medium and fermentation culture medium include the following components: 2.0% corn flour, 1.5% soybean meal, 0.1% sucrose, 0.1% ammonium sulfate, 0.05% NaH2PO4, 0.02% KH2PO4, 0.05% MgSO4, 0.05% NaCl, 0.2% calcium carbonate, 0.05% MnSO4, natural pH, and sterilized at 121°C for 30 minutes.

[0031] Specifically, the above-mentioned auxiliary materials can be the auxiliary materials commonly used in conventional microbial solid inoculants in the art, such as light calcium carbonate, starch, etc., which can be conventionally selected in the art and are not particularly limited in the present invention.

[0032] More specifically, the number of viable bacteria in the powdered bacterial agent is required to be ≥ 1.0 × 10 10 CFU / g, moisture content ≤10%, shelf life 18 months.

[0033] Furthermore, the microbial agent also includes Paenibacillus mucilaginosus N-002, whose preservation number is CGMCC No.32752.

[0034] Furthermore, the microbial agent also includes Priestia megaterium NPKM-001, whose preservation number is CGMCC No.32751.

[0035] The Bacillus amyloliquefaciens Y-102 provided by the present invention can be used in combination with functional strains such as Bacillus mucilaginosus N-002 and P. giganteus NPKM-001. 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 of 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. It is not only conducive to creating a better rhizosphere microenvironment for the growth of crops, promoting the healthy development of plants, and significantly improving crop yield and quality, but also effectively reducing the emission of harmful gases from farmland, alleviating the ecological pressure on the atmosphere, soil and water bodies, and providing strong support for the sustainable development of ecological agriculture.

[0036] Specifically, the application method of the microbial agent includes spraying, pouring or using as base fertilizer.

[0037] In a fourth aspect, the present invention further provides a microbial fertilizer comprising the above-mentioned Bacillus amyloliquefaciens Y-102.

[0038] The Bacillus amyloliquefaciens Y-102 and its preparation provided by the present invention have multiple functional characteristics and show significant advantages in the crop planting industry. This strain not only has outstanding phosphorus and potassium solubilization capabilities, but also can produce urease inhibitors and indoleacetic acid, and has no denitrification function. These characteristics work together to achieve a win-win situation in ecological and economic benefits. From the functional point of view, the indoleacetic acid it produces can effectively promote the growth of crops and significantly increase crop yields; urease inhibitors can reduce soil urease activity, especially after applying urea, can significantly reduce the emission of ammonia and nitrous oxide, which not only helps to maintain soil nitrogen, improve the utilization efficiency of nitrogen fertilizers such as urea, but also can reduce the pollution of ammonia emissions to the atmosphere, soil and water bodies. In addition, since the strain itself has no denitrification effect, after being applied to the soil, it will grow and reproduce in large quantities in the rhizosphere of crops, increase the proportion of bacteria without denitrification function in the soil, further reduce soil denitrification, reduce nitrous oxide emissions, and have great significance for environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The colony morphology of Bacillus amyloliquefaciens Y-102 of the present invention on the NA medium plate;

[0040] Figure 2 The bacterial body and spore morphology of Bacillus amyloliquefaciens Y-102 of the present invention;

[0041] Figure 3 This is the phylogenetic tree of Bacillus amyloliquefaciens Y-102 of the present invention. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0043] Unless otherwise specified, analytically pure reagents that meet national standards were used in the examples, and the analytical water was grade tertiary water as specified in GB / T6682.

[0044] Culture medium used in the examples:

[0045] Phosphate-solubilizing liquid culture 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 30 min.

[0046] Phosphate-dissolving plate culture 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 30 min.

[0047] 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, and sterilize at 121°C for 30 min.

[0048] LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1000 mL of distilled water, pH 7.4, sterilized at 121°C for 30 min.

[0049] Phosphate-solubilizing fermentation 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 30 min.

[0050] Potassium-solubilizing fermentation medium: sucrose 5.0 g, MgSO4·7H2O 0.5 g, Na2HPO4 2 g, FeCl3 0.005 g, K2HPO4 0.2 g, CaCO3 0.1 g, potassium feldspar 5.0 g, distilled water 1000 mL, pH 7.0-7.5, high temperature sterilization at 121°C for 30 min.

[0051] Urea phenol red bacterial culture medium: urea 2.0%, phenol red 0.1%, peptone 1%, beef extract 0.3%, sodium chloride 0.5%, agar 2%, pH 6.4, sterilization at 121℃ for 30 min.

[0052] 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 the pH to 7.0, and sterilize at 121°C for 30 min.

[0053] Giltay liquid medium: Solution A: 1.0 g KNO3, 1.0 g asparagine, 5 mL of 1% (w / v) bromothymol blue (BTB) alcohol solution, 500 mL of distilled water; Solution B: 8.5 g trisodium citrate, 1 g MgSO4·7H2O, 1 g KH2PO4, 0.05 g FeCl3·6H2O, 0.15 g CaCl2, and distilled water to 500 mL; mix solutions A and B, adjust the pH to 7.0-7.2, and sterilize at 121°C for 30 minutes before use.

[0054] Organophosphorus culture medium: glucose 10 g, (NH4)2SO4 0.5 g, MgSO4·7H2O 0.3 g, NaCl 0.3 g, KCl 0.3 g, FeSO4·7H2O 0.03 g, MnSO4·7H2O 0.03 g, lecithin 2 g, CaCO3 5 g, distilled water 1 L, pH 7.0, high temperature sterilization at 115°C for 30 min.

[0055] Example 1

[0056] Strain screening

[0057] 1. Enrichment of Bacillus phosphate-solubilizing

[0058] 10.0 g of soil was collected from the rhizosphere of a thriving corn field near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770). The soil was added to a flask containing 90 mL of sterile water and glass beads. The suspension was shaken at 200 rpm for 10 minutes, followed by an 80°C water bath for 15 minutes to isolate spore-forming bacteria. After a short stagnant period, 5 mL of this soil suspension was inoculated into a phosphate-solubilizing liquid medium and incubated at 37°C with shaking for 3 days. After stagnant period, 5 mL of the suspension was again inoculated into the phosphate-solubilizing liquid medium. The above steps were repeated to complete the secondary enrichment, which was then used for future use.

[0059] 2. Isolation of Bacillus phosphate-solubilizing

[0060] Use a sterile pipette to take 1.0 mL of the secondary enrichment solution and dilute it to 10 -6 Pipette 0.1 mL of each gradient onto a phosphate-solubilizing plate. Spread evenly with a sterile spreader. Incubate inverted in a 37°C incubator for 2–4 days. Observe for the formation of clear zones around the colonies. If clear zones appear, the strain is a Bacillus phosphate-solubilizing bacterium with a certain degree of phosphate-solubilizing ability.

[0061] The strain with transparent circle was transferred to NA slant medium and agar truffle cake with diameter of 8mm was prepared (area s = r 2 π=4 2 ×3.14=50.24mm 2 ) and inoculated onto phosphate-solubilizing plates; five replicates were grown for each strain. After 2–4 days of culture, the clear zone area (S) was measured and the phosphate-solubilizing ability of the strain was determined as S / s.

[0062] Results: A total of 525 Bacillus strains with clear zones were screened, of which 113 strains had S / s values greater than 5.0, with strain Y-102 having the highest S / s value of 6.9.

[0063] 3. Screening of Bacillus with strong phosphate and potassium solubilization capabilities

[0064] Each of the 113 strains screened above was inoculated into 50 mL of LB medium and incubated at 37°C and 180 rpm for 12 hours. At this point, the culture reached its logarithmic growth phase and served as the seed culture. One mL of the seed culture was added to 100 mL of phosphate-solubilizing fermentation medium. A blank control, containing no inoculated culture medium, served as the blank control. Each treatment was replicated three times, and the culture was continued at 37°C and 150 rpm for 7 days. After incubation, the fermentation broth was centrifuged at 6000 rpm for 10 minutes to obtain the supernatant. 10 mL of the supernatant was added to a 50 mL volumetric flask, diluted to the mark with distilled water, and digested. The available phosphorus content was determined using a molybdenum-phosphorus colorimetric method. The available phosphorus concentration (measured as P₂O₅) was compared with that of the blank control, and the degree of increase in available phosphorus (measured as P₂O₅) for each strain was calculated.

[0065] In the potassium-solubilizing test, the same procedures were followed, except that potassium-solubilizing fermentation medium was used instead of phosphate-solubilizing fermentation medium, and the available potassium content was measured using a flame photometer after digestion. The available potassium concentration (measured as K₂O) was compared with that of the blank control group, and the degree of increase in the available potassium concentration (measured as K₂O) corresponding to each strain was calculated.

[0066] Results: From the 113 strains with S / s values greater than 5.0, a total of 51 strains were screened out that could increase the concentration of available phosphorus (calculated as P2O5) by more than 20 times and the concentration of available potassium (calculated as K2O) by more than 10%. Among them, the Y-102 strain could increase the concentration of available phosphorus (calculated as P2O5) in the fermentation broth by 34.08 times and at the same time increase the concentration of available potassium (calculated as K2O) by 17.46%.

[0067] 4. Screening of Bacillus that do not produce urease

[0068] Use a sterilized bamboo stick to pick up the bacterial mosses of the 51 strains of Bacillus with strong phosphate-dissolving and potassium-dissolving abilities obtained in the above screening, and make cross-inoculations on urea phenol red bacterial culture medium plates. Incubate them upside down at 37°C for 24 hours, observe the color changes around the cross bacterial mosses, select the colonies with yellow surrounding areas (not turning red), inoculate them into NA slant culture medium, and culture them at 37°C for 24 to 48 hours before storage.

[0069] Test Principle: Phenol red is an acid-base indicator that turns yellow in acidic conditions, orange in neutral conditions, and red in alkaline conditions. Its color change range is from pH 6.8 (yellow) to 8.4 (red). If the area surrounding the bacterial lawn remains yellow, it indicates that the strain likely does not produce urease, is not breaking down urea in the culture medium surrounding the lawn, and has not significantly changed the pH. Conversely, if the area surrounding the bacterial lawn turns red, the strain likely produces urease, breaking down urea in the culture medium to produce ammonia, which raises the pH and turns red.

[0070] Results: Twenty-three non-urease-producing Bacillus strains were screened from 51 Bacillus strains with strong phosphate and potassium solubilization abilities, including strain Y-102.

[0071] 5. Screening of Bacillus spp. producing urease inhibitors

[0072] The 23 non-urease-producing strains obtained in the previous step were inoculated into a 250 mL Erlenmeyer flask containing 50 mL of NB medium and cultured in a shaking incubator at 37°C and 150 rpm for 48 h. The fermentation broth was then centrifuged at 6000 rpm for 10 min to obtain the supernatant, which was the test solution for the urease inhibitor.

[0073] Soil was excavated from a well-growing cornfield near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770). Urease activity in the cornfield soil was determined using the sodium phenol-sodium hypochlorite colorimetric method. The principle is that urea is hydrolyzed by soil urease to produce ammonia, which reacts with phenol-sodium hypochlorite at room temperature to form blue indigophenol. The color depth of indigophenol is proportional to the amount of ammonia generated. Therefore, the amount of ammonia can be measured colorimetrically, indicating urease activity. The inhibition rate of urease activity in the cornfield soil can then be calculated using the test solution. The specific test method is as follows.

[0074] (1) Reagents and solutions

[0075] Ammonia standard solution: Accurately weigh 0.4717 g (accurate to 0.0001 g) of ammonium sulfate, dried in a drying oven at 105°C for 3 hours, and dissolve in water. The volume is adjusted to 1000 mL to obtain a stock solution containing 0.1 mg of ammonia per mL. Before use, dilute this solution 10-fold with water to a working solution of 0.01 mg / mL.

[0076] Sodium phenolate solution (1.35 mol / L): Solution A: Weigh 62.50 g of phenol (accurate to 0.01 g) and dissolve it in a small amount of ethanol. Add 2 mL of methanol and 18.5 mL of acetone, and dilute to 100 mL with ethanol. Solution B: Weigh 27.00 g of sodium hydroxide (accurate to 0.01 g), dissolve it in water, and dilute to 100 mL. Store solutions A and B in a refrigerator at 4°C. Before use, mix 20 mL of each solution A and solution B and dilute to 100 mL with water.

[0077] Sodium hypochlorite solution: Dilute the reagent according to the concentration of commercially available sodium hypochlorite solution to an active chlorine concentration of 0.9%.

[0078] Urea solution (100 g / L): Weigh 10.00 g of urea (accurate to 0.01 g), dissolve it in water, and dilute to 100 mL.

[0079] 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 separately in water. Combine the two solutions, adjust the pH to 6.7 with 1 mol / L sodium hydroxide, and dilute to 1000 mL with water.

[0080] (2) Preparation of standard curve

[0081] Before measuring sample absorbance, pipette 0.00mL, 1.00mL, 3.00mL, 5.00mL, 7.00mL, 9.00mL, 11.00mL, and 13.00mL of ammonia working solution (0.1mg / mL) into a 50mL volumetric flask. Then, add 20mL of water, followed by 4mL of sodium phenolate solution and 3mL of sodium hypochlorite solution, shaking thoroughly. After 20 minutes, color will develop. Adjust the volume to a set of standard ammonia concentrations of 0.0μg / mL, 0.2μg / mL, 0.6μg / mL, 1.0μg / mL, 1.4μg / mL, 1.8μg / mL, 2.2μg / mL, and 2.6μg / mL. Compare the colors at 578nm on a spectrophotometer within 1 hour (the blue color should remain stable for 1 hour). Plot a standard curve with ammonia concentration as the horizontal axis and absorbance as the vertical axis.

[0082] (3) Determination of urease inhibition rate of test solution

[0083] ① Determination of soil urease activity

[0084] 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 redistilled water in a 100 mL stoppered Erlenmeyer flask, add 1 mL of toluene, and shake evenly. After 15 minutes, 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 box at 37℃±1℃ for 24 hours.

[0085] After the incubation period, filter the filtrate. Pipette 1.00 mL of the filtrate into a 50 mL volumetric flask. Add 4 mL of sodium phenolate solution and 3 mL of sodium hypochlorite solution, shaking thoroughly. After 20 minutes, color will develop and the volume will be adjusted to 50 mL with water. Repeat the above steps using 10 mL of redistilled water instead of 10 mL of 100 g / L urea solution as a control. Measure the color within 1 hour using a spectrophotometer at a wavelength of 578 nm.

[0086] ②After adding the test solution, soil urease activity was measured

[0087] Except replacing "5.0 mL of redistilled water" in ① with "5.0 mL of the test solution", the other steps are the same as above ①.

[0088] (4) Calculation of urease activity inhibition rate

[0089] ① Calculation of soil urease activity

[0090] Urease activity is expressed as the amount of ammonia produced by hydrolyzing the substrate (urea) in 1 g of air-dried soil in 24 hours, and is calculated as follows:

[0091]

[0092] Where X is the urease content in the sample soil (mg / (g·24h));

[0093] c1 is the amount of ammonia (μg / mL) obtained from the standard curve using the absorbance value of the sample plus urea;

[0094] c2 is the amount of ammonia (μg / mL) obtained from the standard curve using the absorbance value of the sample without urea added;

[0095] V is the color development volume (mL), V = 50mL.

[0096] N is the fractionation multiple, N = leachate volume (mL) / filtrate volume (mL) = 41mL / 1mL = 41.

[0097] V0 is the amount of soil sampled (g), V0 = 5 g.

[0098] t is the sample incubation time, t = 1 unit (unit is 24 h).

[0099] f is the dry matter content of soil sample, %.

[0100] ②Calculation of urease activity inhibition rate

[0101] Urease activity inhibition rate (%) = (soil urease activity - soil urease activity after adding the test solution) / soil urease activity × 100%

[0102] Results: From 23 non-urease-producing Bacillus strains, 5 strains were obtained with an inhibition rate of more than 60% on urease activity in cornfield soil. Among them, the inhibition rate of the fermentation supernatant of strain Y-102 on urease activity in cornfield soil was 89.22% (the urease activity in cornfield soil was 0.529±0.064 mg / (g·24h), and the urease activity after adding the fermentation supernatant was 0.057±0.011 mg / (g·24h).

[0103] The same method was used for testing, and the fermentation supernatant of the Y-102 strain could inhibit the urease activity of soil samples of tidal 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 suburbs of Shenyang City) by 90.16%, 87.84% and 83.99%, respectively.

[0104] 6. Screening of Bacillus sp. that produces high indoleacetic acid (IAA)

[0105] The five strains with urease inhibition rates greater than 60% obtained in the previous step were inoculated into 50 mL of NB medium in a 250 mL Erlenmeyer flask. The culture was shaken at 37°C and 150 rpm for 48 hours, and then centrifuged at 6000 rpm for 10 minutes to obtain the supernatant. The supernatant was used as the IAA test solution. IAA content was determined using the Salksowski colorimetric method as follows.

[0106] (1) Drawing of standard curve

[0107] Prepare IAA standard solutions of 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, and 35 mg / L in distilled water. Mix 2.0 mL of each IAA standard solution with 2.0 mL of Salksowski colorimetric reagent and incubate in a dark water bath at 40°C for 30 minutes. Measure the absorbance at 530 nm using a spectrophotometer. Plot a standard curve with IAA concentration as the horizontal axis and absorbance as the vertical axis.

[0108] (2) Determination of IAA content in fermentation broth

[0109] Pipette 2.0 mL of the test solution and mix with 2.0 mL of Salksowski colorimetric reagent. Incubate in a dark water bath at 40°C for 30 min. Measure the absorbance at 530 nm using a spectrophotometer. Calculate the IAA content in the fermentation broth using the standard curve.

[0110] Salksowski color developing reagent: 1 mL of 0.5 mol / L FeCl3 solution was mixed with 50 mL of 35% HClO4 solution.

[0111] Results: Among the five strains mentioned above, three strains produced IAA, among which the fermentation broth of strain Y-102 had the highest IAA content, reaching 135.8 mg / L.

[0112] 7. Screening of Bacillus without denitrification function

[0113] Use a sterile bamboo stick to transfer the above three Bacillus strains with strong IAA production ability to NA slant culture medium and culture at 37°C for 24 hours for activation.

[0114] In a clean bench, add 10 mL of Giltay liquid medium to a large test tube (20 mm x 200 mm). Place a small Dulbecco's tube (5 mm x 20 mm) upside down into the large test tube to allow the gas in the small test tube to escape. Three activated Bacillus strains with strong IAA production abilities were then inoculated into the tubes. Culture at 37°C for 5-7 days and observe whether bubbles are generated in the tubes. If gas is generated, the strain has denitrification; if not, it does not.

[0115] The test showed that two of the three strains mentioned above had no denitrification function, including strain Y-102.

[0116] Identification of strain species

[0117] 1. Morphological Observation

[0118] Use a sterile bamboo stick to pick a Y-102 strain slant and inoculate it into NB medium. After incubation at 37°C, 150 rpm, and shaking for 24 hours, take 1.0 mL of the culture medium and dilute it appropriately. Then spread it onto a NA medium plate and incubate it inverted at 37°C for 24-48 hours to observe the colony morphology. Take an appropriate amount of the 24-48 hour culture medium for smearing and stain with crystal violet to observe the morphology of the bacteria and spores.

[0119] Results: On NA medium plates, the colonies are round with neat edges. When cultured for a long time, the edges become slightly irregular. They are off-white, opaque, dry and rough on the surface, with a bulge in the middle and a slightly crater-like shape. The bacteria are straight rod-shaped, about 0.6μm×(2.0~5.0)μm in size, often solitary or arranged in short chains, and Gram-positive. The spores are oval, with blunt ends, and are mesogenous. The spore capsule is slightly swollen, and the spore size is about 0.8μm×(1.2~1.5)μm. Figures 1 and 2 .

[0120] 2. Physiological and biochemical identification

[0121] Following the experimental methods in Bergey's Manual of Systematic Bacteriology, strain Y-102 was subjected to physiological and biochemical tests, including oxidase and peroxidase assays, glucose fermentation, citrate utilization, methyl red test, VP test, starch hydrolysis, indole test, gelatin liquefaction, casein test, and urea hydrolysis. The results are shown in Table 1.

[0122] Table 1 Physiological and biochemical characteristics of strain Y-102

[0123]

[0124] Note: “+” indicates a positive reaction; “-” indicates a negative reaction.

[0125] 3. Molecular Biological Identification

[0126] Total DNA from strain Y-102 was extracted using a bacterial genomic DNA extraction kit (Biomiga, catalog number BW-GD2411-01) according to the instructions. PCR amplification was performed using universal primers for the prokaryotic 16S rDNA gene sequence (upstream 5′-ACTGGAGGAAGGTGGGGA-3′, downstream 5′-AGGAGGTGATCCAACCGCA-3′) to obtain the amplified product. Sequencing of the amplified product was commissioned to BGI. The 16S rDNA sequencing results were as follows:

[0127]

[0128] The sequencing results were aligned at NCBI, and a phylogenetic tree was constructed using MEGA 11.0 based on the Neighbor-Joining method. Figure 3 Based on 16S rDNA sequence similarity analysis, and referring to the results of physiological and biochemical tests and colony and bacterial morphology, the Y-102 strain was identified as Bacillus amyloliquefaciens.

[0129] The Bacillus amyloliquefaciens was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, abbreviated as CGMCC, on March 24, 2025. The culture deposit number is CGMCC No. 33966, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0130] Phosphate solubilization effect of Y-102 strain on organic phosphorus

[0131] A sterile bamboo skewer was used to pick a Y-102 strain slant and inoculated into 50 mL of LB medium. The culture was shaken at 37°C and 180 rpm for 12 hours to obtain a seed solution. In the experimental group, the seed solution was inoculated into an organophosphate medium at a 3% inoculum concentration. In the control group, the seed solution was first inactivated by heating at 121°C for 20 minutes. All other procedures were the same as for the experimental group, with three replicates per group. After inoculation, the culture was shaken at 37°C and 180 rpm for 5 days. The culture was centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected and the available phosphorus content was determined using the molybdenum antimony colorimetric method.

[0132] Results: The available phosphorus content in the control group was (0.071±0.012) mg / L, while that in the experimental group was (1.936±0.105) mg / L, 27.27 times that of the control group. This suggests that strain Y-102 can decompose and release phosphorus from some lecithin (organic phosphorus) in the culture medium.

[0133] Example 2

[0134] This embodiment provides a preparation of a microbial fertilizer:

[0135] (1) Fermentation medium: corn flour 2.0%, soybean meal 1.5%, sucrose 0.1%, ammonium sulfate 0.1%, NaH2PO4 0.05%, KH2PO4 0.02%, MgSO4 0.05%, NaCl 0.05%, calcium carbonate 0.2%, MnSO4 0.05%, pH natural, sterilized at 121℃ for 30 min.

[0136] (2) Preparation of seed solution:

[0137] Take several 500mL eggplant flasks and add 50mL of NA medium to each. Sterilize at 121°C for 30 minutes, arrange them into a slope, and allow to solidify before use. Pour 5mL of sterile water onto the slope of the activated Y-102 culture tube. Use a sterile bamboo stick to scrape off the bacterial moss and stir as much as possible. Pour the bacterial suspension onto the slope of the eggplant flask and shake gently to ensure even inoculation. Incubate the flask upside down at 37°C for 5-7 days to produce a large number of spores before use.

[0138] Fill a 100L ventilated stirring seed tank with fermentation medium at a filling factor of 0.65-0.75, sterilize at 121°C for 30 minutes, and inoculate after cooling to 40°C. Take the inclined surfaces of four eggplant bottles, pour 50mL of sterile water into each, scrape the bacterial moss with a sterilized bamboo stick, and pour it into a 1000mL inoculation bottle. Inoculate the seed tank using the pressure differential method. Incubate at 37°C, a rotation speed of 1500 r / min, and a ventilation volume of 1.0-2.0 VVM for 10-12 hours to obtain the seed solution.

[0139] (3) Add 1M 3 In a ventilated stirred fermenter, the mixture was sterilized at 121°C for 30 minutes at a charge factor of 0.65-0.75. After cooling to 40°C, the above-mentioned seed solution was inoculated at an inoculum level of 5%-10%. Fermentation was continued at 37°C, 150 rpm, and an aeration rate of 1.0-2.0 VVM for 36-48 hours to obtain a fermentation broth. The viable bacterial content in the fermentation broth was determined by the gradient dilution plate spread method, and the spore rate was calculated by microscopic observation using the smear staining method.

[0140] Results: The spore formation rate in the fermentation broth was 85% to 95%, and the spore content was (2.0 to 5.0)×10 9 CFU / mL, so the content of viable bacteria (spores) ≥1.0×10 9 CFU / mL liquid bacterial agent, that is, liquid microbial fertilizer (liquid bacterial agent), has a shelf life of 6 months.

[0141] (4) The fermentation broth was continuously centrifuged at 6000 r / min in a disc centrifuge to concentrate 5 to 10 times to obtain bacterial sludge. After adding auxiliary materials to the bacterial sludge, the mixture was spray-dried to obtain bacterial powder.

[0142] Results: The spore content in the sludge can reach (1.0~5.0)×10 10 CFU / mL. The spore content in bacterial powder can reach (1.0~3.0)×10 11 CFU / g, can be used to prepare spore (live bacteria) content ≥1.0×10 10 CFU / g or ≥1.0×10 11CFU / g powdered microbial fertilizer (powdered bacterial agent), moisture content ≤ 10%, shelf life 18 months.

[0143] The technical parameters for preparing the microbial fertilizer of strain Y-102 are shown in Table 2.

[0144] Table 2 uses 1M 3 Technical parameters of Y-102 bacterial agent prepared in fermentation tank

[0145]

[0146] Example 3

[0147] 1. Experimental study on the effect of Bacillus amyloliquefaciens Y-102 liquid inoculant on ammonia emission reduction in potted plants

[0148] Twenty-eight plastic flower pots (15 cm inner diameter x height x 18 cm) were filled with soil (from farmland at a depth of 5 to 15 cm) to a depth of approximately 15 cm. Twenty-eight pepper seedlings, approximately uniform in size, thickness, and leaf count, were selected and potted one week after seedling cultivation. Each seedling had its own pot. These seedlings were divided into four groups: CK, Experiment I, Experiment II, and Experiment III, each containing seven pots. Fertilization was initiated at the start of potting, and the experimental period lasted 15 days. The experimental plan is shown in Table 3.

[0149] Table 3 Y-102 fungal agent pepper pot test plan

[0150]

[0151]

[0152] The flower pots from each of the four groups were placed in a transparent glass jar measuring 0.5 m (length × width × height) × 0.5 m (height × height). Daily watering was provided through water supply to the bottom of the jar and through drainage holes in the bottom of the pots. All other management procedures remained consistent across the groups, including maintaining soil water retention at 40%–60%, an average temperature of 25°C, and 8–10 hours of sunlight per day. The jars were sealed with a lid to create an airtight static chamber. Three PVC plastic tubes with valves were attached to the lid. One PVC tube was used to supply water to the bottom of the jar, another to provide airflow at a controlled rate of approximately 60 L / h (using a small blower to continuously supply air). The third PVC plastic tube was a tee-shaped pipe. One end of the pipe allowed the exhaust gas to be introduced into 200 mL of 0.01 mol / L sulfuric acid absorption solution. The other end could be inserted with a 20 cm long needle to aspirate the gas inside the jar for sampling and measurement of N2O emissions.

[0153] Ammonia absorbed by the absorption liquid was determined using the method outlined in "HJ533-2009: Determination of Ammonia in Ambient Air and Waste Gases - Nessler's Reagent Spectrophotometric Method." The principle is that ammonia in the air is absorbed by a dilute sulfuric acid solution. The resulting ammonium ions react with Nessler's reagent to form a yellow-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.

[0154] Table 4 Effect of applying Y-102 liquid microbial agent on ammonia emission reduction in potted pepper soil

[0155]

[0156]

[0157] The experimental results show that: as shown in Table 4, urea application can significantly increase the amount of ammonia volatilization in the soil during the experimental period; compared with Group I (urea application), Group III increased the amount of ammonia volatilization in the soil after the application of the same amount of urea (15g / m 2 ) under the condition of no urea application, the application of Y-102 liquid microbial agent can reduce the ammonia emission of soil by 75.43%; compared with the CK group (no urea application), the application of Y-102 liquid microbial agent in group II can reduce the ammonia emission of soil by 56.48% under the condition of no urea application. Therefore, the liquid microbial agent can significantly reduce the ammonia emission of soil, especially the soil irrigated with urea. The reason for the reduction of ammonia emission is that the Y-102 strain can produce urease inhibitors, which can inhibit the conversion of urea to ammonium (ammonia), so that urea exists mainly in the form of amide nitrogen in the soil, reducing the NH4 + / NH3 concentration, thereby reducing ammonia emissions.

[0158] 2. Effect of Bacillus amyloliquefaciens Y-102 liquid inoculant on urease activity in potted pepper soil

[0159] At the completion of the potted plant experiment (day 15), the urease activity of the soil in each pot was measured using the "T / NAIA 011-2020 Soil Urease Activity Determination - Sodium Phenol-Sodium Hypochlorite Colorimetric Method" method. The urease activity of the soil in the pots treated with Bacillus amyloliquefaciens Y-102 inoculum was compared with that in the pots not treated with Bacillus amyloliquefaciens Y-102 inoculum. For the specific detection method, see Example 1. The specific test results are shown in Table 5.

[0160] Table 5 Effects of applying Y-102 liquid microbial agent on urease activity in soil of potted peppers

[0161]

[0162] Note: The mean in the table is the average of 7 corresponding values after removing the highest and lowest values; different capital letters after the mean indicate differences at the P<0.01 level.

[0163] The experimental results showed that compared with group I (irrigation with urea), group III had a higher yield when irrigated with the same amount of urea (15 g / m 2 ), application of Y-102 liquid inoculant reduced soil urease activity by 67.45% (P < 0.01). Compared with the CK group (no urea application), application of Y-102 liquid inoculant in Group II, also without urea application, reduced soil urease activity by 63.87% (P < 0.01). Therefore, regardless of urea application, this liquid inoculant significantly reduced soil urease activity, thereby significantly reducing fertilizer losses caused by ammonia volatilization after urea fertilization and improving urea utilization and efficiency.

[0164] 3. Effect of Bacillus amyloliquefaciens Y-102 liquid inoculant on N2O emission reduction in potted pepper soil

[0165] On the 0th, 3rd, 6th, 9th, 12th, and 15th day after fertilization, air intake and exhaust were stopped for 4 hours between 8:00 AM and 12:00 PM. Each time, at 12:00 PM, 50 mL of air was drawn from the glass cylinder using a syringe and injected into a 12 mL headspace tube evacuated with a vacuum pump. N₂O content was determined using an Agilent 7890A gas chromatograph with an ECD detector, a detection temperature of 300°C, a column temperature of 60°C, and a carrier gas of 95% argon and 5% methane at a flow rate of 40 mL / min. A standard gas with a concentration of 5 mg / L was selected based on the N₂O sample concentration to establish a calibration curve. Manual injection was used: 40 μL of sample was drawn using a 100 μL gas-tight syringe and injected into the analyzer within 1 second.

[0166] To maintain the growth of the pepper seedlings, the glass jars required a constant supply of air. However, during the four hours when air intake and exhaust were stopped, the jars became a confined space, confining N2O emitted from the soil in the flowerpots to the jars. Therefore, by comparing the N2O concentrations within each group of glass jars, we could assess the N2O reduction effectiveness of the Y-102 liquid inoculant (microbial fertilizer). The results are shown in Table 6.

[0167] Table 6 Effect of applying Y-102 liquid microbial agent on N2O emission reduction in potted pepper soil

[0168]

[0169] Results: Group I and Group III had a common pattern: after urea application, the soil nitrogen content was significantly increased, and then the N2O emission was significantly increased through nitrification and denitrification. The emission peak appeared around the third day, and the emission gradually decreased after 3 days, which may be related to the gradual utilization of urea. However, there were also significant differences between Group I and Group III: on the 0th, 3rd, 6th, 9th, 12th, and 15th day after fertilization, compared with Group I (irrigation with urea), Group III, under the condition of irrigating the same amount of urea, could reduce the soil N2O emission by 1.9%, 34.9%, 29.6%, 22.8%, 21.6% and 1.2%, respectively. The results were published in the journal PLOS ONE on Day 0, 2017. The results were published in the Journal of Pepper Research ... Applying Y-102 bacteria agent without urea also has a certain emission reduction effect. The reasons are mainly two-fold. First, the Y-102 bacteria can produce urease inhibitors, which inhibit the rate of urea decomposition into ammonia (ammonium), reduce the generation and emission of NH3, and also lead to nitrification and NO3 - The supply of nitrogen oxides is limited, which in turn reduces the subsequent denitrification rate, thereby reducing the emission of N2O. Secondly, because the Y-102 strain has no denitrification ability, after being applied to the soil, it grows and reproduces in the plant root system, thereby increasing the number of bacteria without denitrification ability in the soil, especially the root soil, reducing the denitrification of the soil, and hindering the conversion of nitrate, nitrite, ammonium salts, etc. to N2O, thereby reducing the emission of N2O.

[0170] 4. Effect of Bacillus amyloliquefaciens Y-102 liquid inoculant on available phosphorus content in pepper potted soil

[0171] The available phosphorus content in the soil of each experimental group was determined before and 15 days after potting using the method described in "HJ 704-2014 Determination of Available Phosphorus in Soil - Sodium Bicarbonate Extraction-Molybdenum Antimony Antiphoretic Spectrophotometry." The results are shown in Table 7.

[0172] Table 7 Effects of applying Y-102 liquid microbial agent on available phosphorus content in potted pepper soil

[0173]

[0174] Note: The mean in the table is the average of 7 corresponding values after removing the highest and lowest values; different capital letters after the mean indicate differences at the P<0.01 level.

[0175] Results: Compared with Group I (urea application), Group III, when treated with the same amount of urea, increased soil available phosphorus content by 62.94% (P < 0.01) after applying Y-102 liquid inoculant. This also increased available phosphorus by 53.76% (P < 0.01) compared to pre-potting levels. Compared with Group CK (no urea application), Group II, when treated with Y-102 liquid inoculant, increased soil available phosphorus content by 59.12% (P < 0.01) after applying Y-102 liquid inoculant. This also increased available phosphorus by 48.37% (P < 0.01) compared to pre-potting levels. This indicates that regardless of whether urea was applied, applying Y-102 liquid inoculant significantly increased soil available phosphorus content, providing sufficient phosphorus for the growth of crops and even the next crop season.

[0176] 5. Effects of Bacillus amyloliquefaciens Y-102 liquid inoculant on the growth performance of potted pepper seedlings

[0177] After the experiment, the pepper seedling pots were removed from the glass jars. Most of the soil surrounding the roots was carefully removed. The roots were then washed in a basin of tap water and placed on absorbent paper to dry. Plant height was measured with a ruler (accurate to mm). The roots and aboveground parts of the pepper seedlings were cut with scissors at the boundary between the aboveground and underground parts. Stem diameter was measured with a vernier caliper. Root and aboveground fresh weights were measured using a balance (accurate to 0.01 g). The results are shown in Table 8.

[0178] Table 8 Effect of applying Y-102 liquid microbial agent on the growth promotion of potted pepper seedlings

[0179]

[0180] Note: The mean in the table is the average of 7 corresponding values after removing the highest and lowest values; different capital letters indicate differences at the P < 0.01 level, and different lowercase letters indicate differences at the P < 0.05 level.

[0181] Results: Measurements of plant height, stem diameter, leaf number, and total plant fresh weight of peppers in the four groups showed that the liquid inoculant significantly promoted pepper growth. Compared with group CK (no urea application), group II (no urea application) increased plant height, stem diameter, leaf number, and total plant fresh weight by 25.04%, 25.17%, 3.12%, and 49.13%, respectively. Compared with group I (urea application), group III (treatment with the same amount of urea) increased plant height, stem diameter, leaf number, and total plant fresh weight by 22.99%, 17.40%, 4.14%, and 44.62%, respectively. In summary, regardless of urea application, the Y-102 inoculant significantly increased plant height, stem diameter, and total plant fresh weight of peppers (but had no significant effect on leaf number), promoting crop growth. The main reason for this is that urea is a nitrogen fertilizer that provides nutrition to peppers and promotes their growth. In addition to containing natural plant growth hormones such as IAA, the liquid inoculant also produces IAA after its spores germinate and reproduce in the pepper root system. Therefore, applying this inoculant can significantly promote the growth of pepper seedlings.

[0182] 5. Effects of Bacillus amyloliquefaciens Y-102 liquid inoculant on the growth performance of potted corn

[0183] In order to verify the growth-promoting effect of Y-102 liquid microbial agent on gramineous crops, a corn pot experiment was carried out in this study.

[0184] The corn variety tested was Zhengdan 958. Twenty-eight plastic flower pots (with several drainage holes at the bottom) measuring 15 cm in diameter × height = 18 cm were filled with soil (from farmland) to a depth of 5–15 cm. 500 mL of tap water was added to each pot. Five corn seeds were sown overnight in each pot. One week after seedling emergence, the seedlings were thinned out, with two plants per pot. The 28 pots were then randomly divided into four groups of seven: a control group (CK), experimental groups I, II, and III. After fertilization according to Table 9, all pots were immediately moved to a leveled bed in the greenhouse. This bed was watered weekly through the drainage holes in the pots' bottoms, and the greenhouse temperature was maintained at 25–30°C. After 20 days of growth in the greenhouse, seedling height, stem diameter, and total plant fresh weight were measured for each group. After removing the corn seedlings from their pots, carefully wash the roots with tap water and place them on absorbent paper to dry the surface moisture. Measure the above-ground length (seedling height) with a ruler, the stem diameter at the base with a vernier caliper, and the fresh weight of the entire plant using a scale. The test results are shown in Table 10.

[0185] Table 9 Test plan for potted corn using Y-102 liquid microbial agent

[0186]

[0187] Table 10 Results of the test on the growth promotion of potted corn seedlings by applying Y-102 liquid microbial agent

[0188] Group Fertilization situation per pot (see Table 9 for details) Plant height (cm) Stem diameter (mm) Whole plant fresh weight (g) CK No fertilizer, watering 200mL of water 38.65±3.04b 8.92±0.61b 116.24±6.14Bb Ⅰ Apply 200mL urea solution 44.82±3.25ab 10.54±0.73ab 135.03±8.05Bab Ⅱ Apply 200mL of bacterial agent dilution 45.01±4.06ab 10.63±0.64ab 138.06±9.11Bab Ⅲ Apply 200mL of urea-bacteria mixture 52.18±4.68a 12.16±0.91a 179.35±12.07Aa

[0189] Note: The mean in the table is the average of 14 corresponding values after removing the 2 highest and 2 lowest values; different capital letters after the mean indicate differences at the P < 0.01 level, and different lowercase letters indicate differences at the P < 0.05 level.

[0190] Results: Plant height, stem diameter, and total plant fresh weight of maize in the four groups were measured, indicating that Y-102 liquid inoculant significantly promoted maize growth. Compared with group CK (no urea application), plant height, stem diameter, and total plant fresh weight increased by 16.46%, 19.17%, and 18.77%, respectively, in group II (no urea application). Compared with group I (urea application), plant height, stem diameter, and total plant fresh weight increased by 16.42%, 15.37%, and 32.82%, respectively, in group III (administered with the same amount of urea and concurrently with Y-102 liquid inoculant). In summary, Y-102 liquid inoculant significantly increased plant height, stem diameter, and total plant fresh weight of maize, promoting crop growth, regardless of urea application. This is primarily because urea, as a nitrogen fertilizer, provides nutrients to maize, promoting growth. Y-102 liquid microbial inoculant contains natural plant growth hormones such as IAA, and its live bacteria grow and reproduce in the corn root system, also producing IAA. It also contains (or produces in the root system) a urease inhibitor, which improves the fertilizer efficiency of urea. Therefore, applying this inoculant can significantly promote the growth of corn seedlings.

[0191] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Bacillus amyloliquefaciens Y-102, characterized in that Its deposit number is CGMCC No.33966.

2. Use of the Bacillus amyloliquefaciens Y-102 according to claim 1 in reducing NH3 and N2O emissions in soil and improving nitrogen fertilizer utilization efficiency.

3. Use of the Bacillus amyloliquefaciens Y-102 according to claim 1 in promoting crop growth and increasing crop yield.

4. A microbial agent, characterized in that: The invention comprises the Bacillus amyloliquefaciens Y-102 according to claim 1.

5. The microbial agent according to claim 4, wherein The microbial agent is a liquid agent or a powdered agent.

6. The microbial agent according to claim 5, wherein The number of viable bacteria of Bacillus amyloliquefaciens Y-102 in the liquid bacterial agent is ≥1.0×10 9 CFU / mL; the number of viable bacteria of Bacillus amyloliquefaciens Y-102 in the powdered bacterial agent is ≥1.0×10 10 CFU / g.

7. The microbial agent according to claim 4, wherein Also included is Paenibacillus mucilaginosus N-002, whose deposit number is CGMCC No.32752.

8. The microbial agent according to claim 4 or 7, wherein Also included is Priestia megaterium NPKM-001, whose deposit number is CGMCC No.32751.

9. The microbial agent according to claim 4, wherein The application method of the microbial agent includes spraying, pouring or using as base fertilizer.

10. A microbial fertilizer, characterized in that: The invention comprises the Bacillus amyloliquefaciens Y-102 according to claim 1.

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