A type of Bacillus amyloliquefaciens, its microbial inoculant and its application

Abstract

This invention relates to the field of microbial technology, specifically disclosing a Bacillus amyloliquefaciens strain, its microbial inoculant, and its applications. The Bacillus amyloliquefaciens strain Y-102 has the preservation number CGMCC No. 33966. This strain not only possesses outstanding phosphorus and potassium solubilizing abilities but also produces urease inhibitors and indoleacetic acid, while lacking denitrification capabilities. Functionally, the indoleacetic acid it produces effectively promotes crop growth and significantly increases crop yield; the urease inhibitor reduces soil urease activity, substantially reducing ammonia and nitrous oxide emissions. Furthermore, since the strain itself lacks denitrification capabilities, after being applied to the soil, it grows and multiplies extensively in the crop rhizosphere, increasing the proportion of non-denitrifying bacteria in the soil, further reducing soil denitrification and nitrous oxide emissions, which is of great significance for environmental protection.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bacillus amyloliquefaciens, its microbial agents, and their applications. Background Technology

[0002] Against the backdrop of sustainable agricultural development, soil health, efficient fertilizer utilization, and plant growth promotion have become key research directions. Microorganisms, as an important component of the soil ecosystem, have a significant impact on agricultural production due to their diverse functions. However, current microorganisms used in agricultural production generally suffer from limited functionality or difficulty in simultaneously performing multiple beneficial functions.

[0003] The application of urease inhibitors is crucial for improving fertilizer utilization. While some microorganisms can produce urease inhibitors, their yields are generally low, making it difficult to effectively suppress soil urease activity in actual agricultural production. This fails to adequately slow the hydrolysis of urea into ammonium nitrogen, hindering a significant improvement in urea utilization and causing environmental pollution due to soil ammonia emissions. Furthermore, urease inhibitor-producing microorganisms often lack other functional deficiencies, such as the ability to promote plant growth, thus failing to provide additional growth support to plants simultaneously.

[0004] In the soil nitrogen cycle, denitrification is a crucial step leading to nitrogen loss. Microorganisms capable of denitrification convert nitrate nitrogen into gaseous nitrogen, which escapes from the soil, reducing soil nitrogen content and weakening the effectiveness of nitrogen fertilizers. Simultaneously, the resulting greenhouse gases, such as nitrous oxide (N₂O), exacerbate environmental problems. While some Bacillus amyloliquefaciens strains possess multiple functions, some exhibit denitrification capabilities, severely limiting their widespread application in agricultural production.

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

[0006] To address the problem that existing technologies lack microorganisms capable of simultaneously producing high levels of urease inhibitors and indoleacetic acid without denitrification, thus failing to achieve multiple effects of promoting crop growth, increasing yield, and reducing environmental pollution, this invention provides Bacillus amyloliquefaciens, its microbial agents, and their applications.

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

[0008] In a first aspect, the present invention provides a Bacillus amyloliquefaciens Y-102, which has the accession number CGMCC No.33966.

[0009] Bacillus amyloliquefaciens Y-102 was isolated from the rhizosphere soil of a well-grown cornfield near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770). It was classified and named Bacillus amyloliquefaciens and deposited on March 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33966. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The biological characteristics of Bacillus amyloliquefaciens Y-102 provided by this invention are as follows: the colonies are round with neat edges, and the edges become slightly irregular when the culture time is long. They are grayish-white, opaque, dry and rough on the surface, with a raised center that is slightly crater-like. The bacteria are straight rods, about 0.6 μm × (2.0–5.0) μm in size, and usually grow singly or in short chains. The spores are elliptical with blunt ends, located in the center, and the sporangia are slightly enlarged. The spores are about 0.8 μm × (1.2–1.5) μm in size.

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

[0012] The *Bacillus amyloliquefaciens* Y-102 provided by this invention belongs to the "strain exempt from toxicological testing" as permitted by the *NY / T 1109-2017 General Technical Guidelines for Biosafety of Microbial Fertilizers*. In actual production scenarios, this strain exhibits excellent stability, meeting the process requirements of microbial fertilizer production enterprises. Furthermore, compared to currently known microbial fertilizer production strains, *Bacillus amyloliquefaciens* Y-102 possesses unique functional characteristics. It can efficiently synthesize urease inhibitors and indoleacetic acid while lacking denitrification activity. This characteristic gives this strain multiple application values ​​in agricultural production: on the one hand, by inhibiting soil urease activity, it reduces ammonia and nitrous oxide emissions, thereby reducing nitrogen loss; combined with the plant growth-promoting effect of indoleacetic acid, it can significantly improve crop growth and yield; on the other hand, the absence of denitrification helps reduce greenhouse gas emissions and 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] Secondly, the present invention also provides the application of the above-mentioned Bacillus amyloliquefaciens Y-102 in reducing NH3 and N2O emissions in soil and improving nitrogen fertilizer utilization.

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

[0015] The present invention provides a high-yield urease inhibitor and indoleacetic acid from Bacillus amyloliquefaciens Y-102, which have no denitrification function and combine multiple beneficial functions. The urease inhibitor secreted by Bacillus amyloliquefaciens effectively inhibits the activity of various ureases in the soil, slows down the conversion of nitrogen fertilizers such as urea into ammonia (ammonium), thereby significantly reducing NH3 emissions; after the ammonia (ammonium) content is reduced, soil nitrification weakens, and NO3... - Limited supply further reduces denitrification, effectively decreasing N2O emissions. This not only improves urea utilization and fertilizer efficiency but also protects the ecological environment. Simultaneously, this strain lacks denitrification function; when applied to the soil, it grows and reproduces within the crop root system, increasing the proportion of non-denitrifying bacteria in the soil and further reducing soil denitrification, thus continuously lowering N2O emissions from farmland. Furthermore, Bacillus amyloliquefaciens Y-102's high production of indoleacetic acid effectively stimulates crop root development and above-ground growth. These functions work synergistically, not only improving soil quality and enhancing crop adaptability to the environment, increasing crop yields, but also reducing harmful gas emissions. This provides strong technical support for the sustainable development of green agriculture, demonstrating significant economic, social, and environmental benefits.

[0016] Thirdly, the present invention also provides a microbial inoculant comprising the aforementioned Bacillus amyloliquefaciens Y-102.

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

[0018] Preparing Bacillus amyloliquefaciens Y-102 into a microbial inoculant improves its storage stability and ease of transportation, facilitating its widespread application in various agricultural scenarios. This inoculant offers flexible application methods, allowing for foliar spraying, root application, or use as a base fertilizer, depending on the crop variety, planting pattern, and soil conditions. This reduces operational difficulties for farmers and avoids additional costs, thus meeting the diverse needs of modern agricultural production.

[0019] Furthermore, the viable count of Bacillus amyloliquefaciens Y-102 in the liquid bacterial agent is ≥1.0 × 10⁻⁶. 9 CFU / mL; the viable count of Bacillus amyloliquefaciens Y-102 in the powdered bacterial agent is ≥1.0×10⁻⁶. 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] Fermentation medium was loaded into a ventilated stirred fermenter with a loading coefficient of 0.65-0.75, sterilized at 121℃ for 30 min, and then cooled to 40℃ for inoculation. The seed culture of *Bacillus amyloliquefaciens* Y-102 was inoculated into the ventilated stirred fermenter at an inoculation rate of 5%-10%. Fermentation was carried out at 37℃, a rotation speed of 150-200 r / min, and an aeration rate of 1.0-2.0 VVM for 36-48 h, yielding a viable count ≥1.0 × 10⁻⁶. 9 Fermentation broth with a concentration of CFU / mL can be used directly as a liquid inoculum, 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] Fermentation medium was loaded into a ventilated stirred fermenter with a loading coefficient of 0.65-0.75, sterilized at 121℃ for 30 min, and then cooled to 40℃ for inoculation. The seed culture of *Bacillus amyloliquefaciens* Y-102 was inoculated into the ventilated stirred fermenter at an inoculation rate of 5%-10%. Fermentation was carried out at 37℃, a rotation speed of 150-200 r / min, and an aeration rate of 1.0-2.0 VVM for 36-48 h, yielding a viable count ≥1.0 × 10⁻⁶. 9 Fermentation broth with CFU / mL;

[0024] The fermentation broth was continuously centrifuged at 6000-7000 r / min using a disc centrifuge to concentrate it 5-10 times, obtaining bacterial sludge; the bacterial sludge was then added to auxiliary materials and spray-dried to obtain powdered bacterial agent.

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

[0026] ①Preparation of slant culture in eggplant flasks

[0027] Take several 500mL eggplant flasks, add 50mL of NA medium to each flask, sterilize at 121℃ for 30min, arrange them into slant agar, and let them solidify before use. Pour 5mL of sterile water into the activated test tube slant agar, scrape off the bacterial growth with a sterile bamboo stick and stir as evenly as possible, pour the bacterial suspension into the eggplant flask slant agar, and gently shake to ensure even inoculation. Incubate upside down at 37℃ for 5-7 days to produce a large number of spores for later use.

[0028] ② Seed liquid preparation

[0029] Fill the seed tank with culture medium at a filling coefficient of 0.65–0.75, sterilize at 121℃ for 30 min, and inoculate after cooling to 40℃. Take four slant cultures from the above-mentioned eggplant flasks, pour 50 mL of sterile water into each, scrape off the mycelial growth with a sterile bamboo stick, and pour it into a 1000 mL inoculation bottle. Inoculate the seed tank using the pressure difference method, and incubate at 37℃, a rotation speed of 1500–2000 r / min, and an aeration rate of 1.0–2.0 VVM for 10–12 h to obtain the seed culture.

[0030] Specifically, the seed culture medium and fermentation culture medium consist of 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, pH at rest, sterilized at 121℃ for 30 min.

[0031] Specifically, the above-mentioned excipients can be commonly used excipients in conventional microbial solid inoculants in the field, such as light calcium carbonate, starch, etc. Conventional selections are possible in the field, and the present invention does not impose any special limitations.

[0032] More specifically, the above-mentioned powdered bacterial agent must contain ≥1.0 × 10⁻⁶ viable bacteria. 10 CFU / g, moisture content ≤10%, shelf life 18 months.

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

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

[0035] The *Bacillus amyloliquefaciens* Y-102 provided by this invention can be used in combination with functional strains such as *Bacillus mucilaginosus* N-002 and *Primulina megaterium* NPKM-001. Through functional synergy among the strains, the soil improvement effect and crop growth promotion capacity are significantly enhanced. This synergy among strains can more effectively inhibit rhizosphere soil urease activity, significantly reduce NH3 volatilization and N2O emissions, effectively reduce nitrogen loss, and improve fertilizer utilization efficiency. This not only helps create a better rhizosphere microenvironment for crop growth, promoting robust plant development and significantly increasing crop yield and quality, but also effectively reduces the emission of harmful gases from farmland, alleviating ecological pressure on the atmosphere, soil, and water bodies, and providing strong support for the sustainable development of ecological agriculture.

[0036] Specifically, the application methods of the microbial agent include spraying, watering, or using it as a base fertilizer.

[0037] Fourthly, the present invention also provides a microbial fertilizer comprising the aforementioned Bacillus amyloliquefaciens Y-102.

[0038] The *Bacillus amyloliquefaciens* Y-102 and its formulation provided by this invention possess multiple functional characteristics, demonstrating significant advantages in crop cultivation. This strain not only exhibits outstanding phosphorus and potassium solubilizing abilities but also produces urease inhibitors and indoleacetic acid, while lacking denitrification capabilities. These characteristics work together to achieve a win-win situation for both ecological and economic benefits. Functionally, the indoleacetic acid produced effectively promotes crop growth and significantly increases crop yield; the urease inhibitor reduces soil urease activity, especially after urea application, significantly reducing ammonia and nitrous oxide emissions. This helps maintain soil nitrogen levels, improves the utilization efficiency of nitrogen fertilizers such as urea, and reduces ammonia pollution to the atmosphere, soil, and water. Furthermore, since the strain itself lacks denitrification capabilities, after being applied to the soil, it will grow and multiply extensively in the crop rhizosphere, increasing the proportion of non-denitrifying bacteria in the soil, further reducing soil denitrification and nitrous oxide emissions, which is of great significance for environmental protection. Attached Figure Description

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

[0040] Figure 2 The cell and spore morphology of Bacillus amyloliquefaciens Y-102 of this invention;

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

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0043] Unless otherwise stated, all examples used analytical grade reagents that meet national standards, and the water used for analysis was grade III water as specified in GB / T6682.

[0044] Culture medium used in the examples:

[0045] Phosphate-solubilizing liquid culture medium: 10g glucose, 5g Ca3(PO4)2, 5g MgCl2, 0.25g MgSO4·7H2O, 0.2g KCl, 0.1g (NH4)2SO4, 1000mL distilled water, pH 7.0~7.5, sterilized at 115℃ for 30min.

[0046] Phosphate-solubilizing plate medium: glucose 10g, Ca3(PO4)2 5g, MgCl2 5g, MgSO4·7H2O 0.25g, KCl 0.2g, (NH4)2SO4 0.1g, distilled water 1000mL, agar 15.0g, pH 7.0~7.5, sterilized at 115℃ for 30min.

[0047] NA slant culture medium: 10.0g peptone, 3.0g beef extract powder, 5.0g sodium chloride, 15.0g agar, 1000mL distilled water, adjust pH to 7.0, and sterilize at 121℃ for 30min.

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

[0049] Phosphate-solubilizing fermentation medium: 10g glucose, 5g Ca3(PO4)2, 5g MgCl2, 0.25g MgSO4·7H2O, 0.2g KCl, 0.1g (NH4)2SO4, 1000mL distilled water, pH 7.0~7.5, sterilized at 115℃ for 30min.

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

[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, sterilized at 121℃ for 30 min.

[0052] NB medium: 10.0g peptone, 3.0g beef extract powder, 5.0g sodium chloride, 1000mL distilled water, adjust pH to 7.0, and sterilize at 121℃ for 30min.

[0053] Giltay liquid medium: Solution A: 1.0 g KNO3, 1.0 g asparagine, 5 mL of 1% (w / v) bromothymol blue (BTB) ethanol solution, 500 mL 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, add distilled water to 500 mL; Mix solutions A and B, adjust pH to 7.0–7.2, sterilize at 121 °C for 30 min before use.

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

[0055] Example 1

[0056] strain screening

[0057] 1. Enrichment of Bacillus phosphate-solubilizing

[0058] 10.0g of soil was collected from the rhizosphere of vigorously growing corn in a farmland near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770). This soil was added to an Erlenmeyer flask containing 90mL of sterile water and glass beads. The mixture was shaken on a shaker at 200 rpm for 10 minutes, followed by a water bath at 80℃ for 15 minutes to obtain bacteria in spore form. After a short period of settling, 5mL of this soil suspension was inoculated into phosphate-solubilizing liquid medium and cultured at 37℃ with shaking for 3 days. After settling, 5mL of this solution was inoculated again into phosphate-solubilizing liquid medium, and the above operation was repeated to complete the secondary enrichment. The secondary enriched solution was then used for further processing.

[0059] 2. Isolation of Bacillus phosphate-solubilizing

[0060] Take 1.0 mL of the above secondary enrichment solution using a sterile pipette and serially dilute it to 10⁻⁶. -6 Take 0.1 mL of each gradient and inoculate it onto a phosphate-solubilizing plate. Spread it evenly using a sterile spreader and incubate upside down in a 37°C incubator for 2–4 days. Observe whether a clear zone forms around the colony. If a clear zone appears, it indicates that the strain is a phosphate-solubilizing Bacillus.

[0061] The strains with clear zones were transferred to NA slant agar medium, and agar blocks with a diameter of 8 mm (area s = r) were prepared. 2 π = 4 2 ×3.14=50.24mm 2 The strains were inoculated into phosphate-solubilizing agar plates; five replicates were made for each strain. After culturing for 2–4 days, the area of ​​the clear zone (S) was measured, and the S / s ratio was used to determine the phosphate-solubilizing ability of the strains.

[0062] Results: 525 strains of Bacillus with a clear zone were obtained through screening, of which 113 strains had an S / s value greater than 5.0, with strain Y-102 having the highest S / s value of 6.9.

[0063] 3. Screening for Bacillus species with strong phosphorus-solubilizing and potassium-solubilizing abilities.

[0064] The 113 strains selected above were inoculated into 50 mL of LB medium and cultured at 37℃ and 180 r / min for 12 h. At this point, the cultures were in the logarithmic growth phase and were used as seed cultures. 1 mL of the seed culture was added to 100 mL of phosphate-solubilizing fermentation medium, with the uninoculated phosphate-solubilizing fermentation medium serving as a blank control. Each treatment was repeated three times, and cultured at 37℃ and 150 r / min for 7 days. After culture, the fermentation broth was centrifuged at 6000 r / min for 10 min to obtain the supernatant. 10 mL of the supernatant was added to a 50 mL volumetric flask, and the volume was adjusted to the mark with distilled water. After digestion, the available phosphorus content was determined by the molybdenum-phosphorus colorimetric method. The concentration of available phosphorus (calculated as P2O5) for each strain was compared with that of the blank control group to calculate the increase in available phosphorus (calculated as P2O5) concentration.

[0065] In the potassium solubilization experiment, the procedures were the same as above, except that potassium-soothing fermentation medium was used instead of phosphorus-soothing fermentation medium and the available potassium content was measured with a flame photometer after digestion. The concentration of available potassium (calculated as K2O) for each strain was compared with that of the blank control group, and the degree of increase in available potassium (calculated as K2O) concentration for each strain was calculated.

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

[0067] 4. Screening for non-urease-producing Bacillus species

[0068] Using sterilized bamboo sticks, 51 strains of Bacillus with strong phosphorus-solubilizing and potassium-solubilizing abilities were selected and inoculated in a cross pattern on urea phenol red bacterial culture medium plates. The plates were incubated upside down at 37°C for 24 hours. The color change around the cross pattern was observed. Colonies that remained yellow (not turning red) were selected and inoculated into NA slant medium. After incubation at 37°C for 24–48 hours, the colonies were stored for later use.

[0069] Experimental principle: Phenol red is an acid-base indicator. It is yellow in acidic conditions, orange in neutral conditions, and red in alkaline conditions, with a color change range of pH 6.8 (yellow) to 8.4 (red). If the area around the bacterial growth remains yellow, it indicates that the strain may not produce urease, has not decomposed the urea in the culture medium surrounding the bacterial growth, and has not caused a significant change in pH. Conversely, if the area around the bacterial growth turns red, it indicates that the strain may produce urease, decompose the urea in the culture medium to produce ammonia, and thus increase the pH and turn red.

[0070] Results: 23 non-urease-producing Bacillus strains were screened from 51 Bacillus strains with strong phosphorus-solubilizing and potassium-solubilizing abilities, including strain Y-102.

[0071] 5. Screening for Bacillus strains that produce urease inhibitors

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

[0073] Soil samples were collected from a well-grown cornfield near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770), at depths of 5–20 cm below the surface. The urease activity in the cornfield soil was detected using the sodium phenolate-sodium hypochlorite colorimetric method. The principle is as follows: urea is hydrolyzed by soil urease to produce ammonia. Ammonia reacts with phenol-sodium hypochlorite under normal temperature conditions to produce blue indophenol. The color depth is directly proportional to the amount of ammonia produced. Therefore, the amount of ammonia can be determined colorimetrically to represent urease activity, and the inhibition rate of the test solution on urease activity in the cornfield soil can then be calculated. The specific detection 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 h, dissolve it in water and dilute to 1000 mL to obtain a stock solution containing 0.1 mg of ammonia per mL. Before use, dilute the above solution 10 times with water to prepare a working solution with a concentration 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), dissolve 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 in water, and dilute to 100 mL. Store solutions A and B in a refrigerator at 4°C. Before use, mix 20 mL of solution A and 20 mL of 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 until the concentration of active chlorine is 0.9%.

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

[0079] Citrate buffer (pH 6.7): Weigh 184.00 g citric acid (accurate to 0.01 g) and 147.50 g 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 bring the volume to 1000 mL with water.

[0080] (2) Standard Curve Construction

[0081] Before measuring the absorbance of the samples, pipette 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 ammonia working solution (0.1 mg / mL) into 50 mL volumetric flasks, respectively. Add 20 mL of water, then add 4 mL of sodium phenolate solution and 3 mL of sodium hypochlorite solution sequentially, shaking well while adding. After 20 min, the color develops, and the solution is diluted to volume to prepare 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. Measure the absorbance at 578 nm using a spectrophotometer within 1 hour (the blue color remains stable within 1 hour). Plot a standard curve with ammonia concentration on the x-axis and absorbance on the y-axis.

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

[0083] ① Soil urease activity assay

[0084] Take 5g of soil sample (accurate to 0.0001g; if the content is high, the sample weight can be reduced appropriately) and 5.0mL of redistilled water into a 100mL Erlenmeyer flask with a stopper. Add 1mL of toluene, shake well, and after 15min, add 10mL of 100g / L urea solution and 20mL of pH 6.7 citrate buffer solution. Shake well and incubate in a constant temperature incubator at 37℃±1℃ for 24h.

[0085] After incubation, filter to obtain the filtrate. Pipette 1.00 mL of the filtrate into a 50 mL volumetric flask, then add 4 mL of sodium phenolate solution and 3 mL of sodium hypochlorite solution sequentially, shaking constantly. After 20 min, color development will occur, and the solution will be diluted to 50 mL with water. A control was prepared by repeating the above procedure using 10 mL of redistilled water instead of 10 mL of 100 g / L urea solution. Colorimetric measurements were taken at 578 nm using a spectrophotometer within 1 hour.

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

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

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

[0089] ① Calculation of soil urease activity

[0090] Urease activity is expressed in mg as the amount of ammonia produced by the hydrolysis of 1g of air-dried soil substrate (urea) over 24 hours, and is calculated using the following formula:

[0091]

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

[0093] c1 is the ammonia content (μg / mL) obtained from the standard curve when the sample is treated with urea;

[0094] c2 is the ammonia content (μg / mL) obtained from the standard curve for the absorbance of the sample without urea.

[0095] V is the volume of the colorimetric final volume (mL), V = 50 mL.

[0096] N is the fractional amount, N = volume of leachate (mL) / volume of filtrate taken (mL) = 41mL / 1mL = 41.

[0097] V0 is the soil sample size (g), V0 = 5g.

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

[0099] f represents the dry matter content of the soil sample, in percentages.

[0100] ② Calculation of urease activity inhibition rate

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

[0102] Results: Five strains of Bacillus that do not produce urease were found to inhibit urease activity in maize soil by more than 60% from 23 non-urease-producing Bacillus strains. Among them, the supernatant of fermentation broth of strain Y-102 inhibited urease activity in maize soil by 89.22% (urease activity in maize soil was 0.529±0.064 mg / (g·24h), and urease activity after adding fermentation supernatant was 0.057±0.011 mg / (g·24h).

[0103] Using the same method, the supernatant of the fermentation broth of strain Y-102 showed inhibition rates of 90.16%, 87.84%, and 83.99% on urease activity in soil samples from alluvial soil (from the western part of Mancheng District, Baoding City), brown soil (from the western part of Mancheng District, Baoding City), and black soil (from the northern suburbs of Shenyang City), respectively.

[0104] 6. Screening for Bacillus strains that produce high levels of indoleacetic acid (IAA)

[0105] Five strains with urease activity inhibition rates greater than 60% obtained in the previous step were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of NB medium and cultured at 37 °C and 150 rpm for 48 h on a shaker. The flasks were then centrifuged at 6000 rpm for 10 min to obtain the supernatant. The supernatant was the test solution for IAA. The IAA content was determined using the Salksowski colorimetric method, the specific procedure of which is as follows.

[0106] (1) Plotting the standard curve

[0107] 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 using distilled water. 2.0 mL of each IAA standard solution was mixed with 2.0 mL of Salksowski reagent and incubated at 40°C in the dark for 30 min. The absorbance was measured at 530 nm using a spectrophotometer. A standard curve was plotted with IAA concentration on the x-axis and absorbance on the y-axis.

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

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

[0110] Salksowski colorimetric reagent: Mix 1 mL of 0.5 mol / L FeCl3 solution with 50 mL of 35% HClO4 solution.

[0111] Results: Among the above 5 strains, 3 strains produced IAA, with strain Y-102 having the highest IAA content in its fermentation broth, reaching 135.8 mg / L.

[0112] 7. Screening for Bacillus strains without denitrification function

[0113] The three strains of Bacillus with strong IAA-producing ability were transferred to NA slant culture medium using sterile bamboo sticks and activated by incubation at 37°C for 24 hours.

[0114] In a clean bench, add 10 mL of Giltay liquid culture medium to a large test tube (20 mm × 200 mm). Invert a small DuPont test tube (5 mm × 20 mm) into the large test tube to purge the gas from the small tube. Inoculate each of the three activated Bacillus strains with strong IAA-producing capacity with the small tube. Incubate at 37°C for 5–7 days and observe whether gas bubbles are produced in the small tube. If gas is produced, the strain has denitrification ability; if no gas is produced, the strain does not have denitrification ability.

[0115] Experiments showed that two of the three strains lacked denitrification function, including strain Y-102.

[0116] Strains and species identification

[0117] 1. Morphological observation

[0118] Using a sterile bamboo stick, collect slant culture of strain Y-102 and inoculate it into NB medium. After incubating at 37℃ and 150 rpm for 24 h on a shaker, take 1.0 mL of culture medium, dilute appropriately, and spread it onto NA medium plates. Incubate at 37℃ upside down for 24–48 h to observe colony morphology. Take an appropriate amount of culture medium from 24–48 h, smear it, and stain it with crystal violet to observe the morphology of bacterial cells and spores.

[0119] Results: On NA agar plates, colonies were round with neat edges, though slightly irregular edges appeared with prolonged incubation. They were grayish-white, opaque, dry, and somewhat rough, with a raised center resembling a crater. The bacteria were rod-shaped, approximately 0.6 μm × (2.0–5.0) μm in size, usually solitary or arranged in short chains, and Gram-positive. Spores were elliptical, with blunt ends, centrally located, and the sporangia were slightly enlarged, with spores approximately 0.8 μm × (1.2–1.5) μm in size. See [link to relevant documentation]. Figures 1-2 .

[0120] 2. Physiological and biochemical identification

[0121] Following the experimental methods outlined in Bergey's Manual of Systematic Bacteriology, physiological and biochemical tests were conducted on strain Y-102, including tests for oxidase, peroxidase, 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 was extracted from strain Y-102 using a bacterial genomic DNA extraction kit (Biomiga, catalog number BW-GD2411-01) following the instructions. PCR amplification was performed using universal primers for prokaryotic 16S rDNA gene sequencing (upstream 5′-ACTGGAGGAAGGTGGGGA-3′, downstream 5′-AGGAGGTGATCCAACCGCA-3′), yielding the amplified products. Sequencing of the amplified products was performed by BGI Genomics. The 16S rDNA sequencing results are as follows:

[0127]

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

[0129] This Bacillus amyloliquefaciens was deposited on March 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33966. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

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

[0131] The mycelial growth of strain Y-102 was picked up with a sterile bamboo stick and inoculated into 50 mL of LB medium. The culture was then incubated at 37℃ and 180 rpm for 12 h with shaking to obtain the seed culture. In the experimental group, the seed culture was inoculated into organophosphate medium at a 3% inoculation rate. In the control group, the seed culture was first inactivated by heating at 121℃ for 20 min, and the remaining procedures were the same as in the experimental group. Each group had three replicates. After inoculation, the culture was incubated at 37℃ and 180 rpm for 5 days with shaking. After centrifugation at 5000 rpm for 10 min, the supernatant was collected, and the available phosphorus content in the supernatant 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; the available phosphorus content in the experimental group was (1.936±0.105) mg / L, which was 27.27 times that of the control group. This indicates that strain Y-102 can decompose and release some of the phosphorus in the lecithin (organic phosphorus) in the culture medium.

[0133] Example 2

[0134] This embodiment provides a method for preparing 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 30min.

[0136] (2) Preparation of seed solution:

[0137] Take several 500mL eggplant flasks, add 50mL of NA medium to each flask, sterilize at 121℃ for 30min, arrange them into slant agar, and let them solidify before use. Pour 5mL of sterile water into the activated Y-102 bacterial culture slant tube, scrape off the bacterial growth with a sterile bamboo stick and stir as evenly as possible, pour the bacterial suspension into the eggplant flask slant, and gently shake to ensure even inoculation. Incubate upside down at 37℃ for 5-7 days to produce a large number of spores for later use.

[0138] Fermentation medium was loaded into a 100L aerated stirred seed tank with a loading coefficient of 0.65–0.75. The tank was sterilized at 121℃ for 30 min and cooled to 40℃ before inoculation. Four slant cultures of the above-mentioned eggplant flasks were taken, and 50 mL of sterile water was poured into each. The mycelial growth was scraped off with a sterile bamboo stick and poured into a 1000 mL inoculation bottle. The seed tank was inoculated using the pressure difference method. The culture was carried out at 37℃, 1500 r / min, and 1.0–2.0 VVM aeration for 10–12 h to obtain the seed culture.

[0139] (3) Add the above fermentation medium to 1M 3 In a ventilated stirred fermenter with a loading coefficient of 0.65–0.75, sterilization was performed at 121℃ for 30 min. After cooling to 40℃, the above seed culture was inoculated at an inoculation rate of 5%–10%. Fermentation was carried out at 37℃, 150 r / min, and an aeration rate of 1.0–2.0 VVM for 36–48 h to obtain the fermentation broth. The viable cell content in the fermentation broth was detected by the serial dilution plate 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%–95%, and the spore content was (2.0–5.0) × 10⁻⁶. 9 The concentration of CFU / mL is between 1.0 and 10, therefore it is possible to prepare bacteria with a viable bacterial (spore) content ≥ 1.0 × 10⁻⁶. 9 Liquid microbial agent with a concentration of CFU / mL, also known as liquid microbial fertilizer (liquid microbial agent), has a shelf life of 6 months.

[0141] (4) The above fermentation liquid is continuously centrifuged at 6000r / min using a disc centrifuge to concentrate it by 5 to 10 times to obtain bacterial mud. After adding auxiliary materials to the bacterial mud, it is spray-dried to obtain bacterial powder.

[0142] Results: The spore content in the fungal substrate could reach (1.0–5.0) × 10⁻⁶. 10 CFU / mL. The spore content in the mycelium powder can reach (1.0~3.0)×10⁻¹⁰. 11 CFU / g can be used to prepare spores (live bacteria) with a content ≥1.0×10⁻⁶. 10 CFU / g or ≥1.0×10 11Powdered microbial fertilizer (powdered inoculant) with CFU / g, moisture content ≤10%, shelf life 18 months.

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

[0144] Table 2 uses 1M 3 Technical parameters for preparing Y-102 microbial agent in fermenter

[0145]

[0146] Example 3

[0147] 1. Pot experiment on the ammonia emission reduction effect of Bacillus amyloliquefaciens Y-102 liquid inoculant.

[0148] Twenty-eight plastic flowerpots with an inner diameter of 15cm and a height of 18cm were prepared and filled with soil (soil from farmland with a depth of 5-15cm) to a depth of approximately 15cm. Twenty-eight chili seedlings, approximately one week old and of similar size, thickness, and number of leaves, were selected and planted in separate flowerpots, one seedling per pot. These were divided into four groups: CK group, Experiment I group, Experiment II group, and Experiment III group, with seven pots in each group. Fertilizer was applied at the beginning of the potting process, and the experiment lasted for 15 days. The experimental protocol is shown in Table 3.

[0149] Table 3 Y-102 inoculant pepper pot experiment scheme

[0150]

[0151]

[0152] Four groups of flowerpots were placed in transparent glass jars measuring 0.5m x 0.5m x 0.5m. Water was supplied to the bottom of the jars and the pots were irrigated daily through drainage holes at the bottom. Other management practices remained consistent across groups, including maintaining soil moisture content at 40%–60%, an average air temperature of 25°C, and 8–10 hours of sunlight per day. The glass jars were sealed with caps to form a closed, static chamber. Three PVC pipes with valves were installed on the caps. One pipe supplied water to the bottom of the jar, another pipe provided air intake at a controlled rate of approximately 60L / h (using a small fan to continuously supply air into the jar), and the third pipe was a T-junction. One end of the T-junction allowed the exhaust gas to be passed into 200mL of 0.01mol / L sulfuric acid absorbent, while the other end allowed the insertion of a 20cm needle to sample the gas inside the jar and determine the N2O emissions.

[0153] The ammonia absorbed by the absorbent was determined using the method described in "HJ533-2009 Determination of Ammonia in Ambient Air and Exhaust Gas - 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 directly proportional to the ammonia concentration. The absorbance was measured at a wavelength of 420 nm, and the ammonia concentration in the air was calculated based on the absorbance. The experimental results are shown in Table 4.

[0154] Table 4. Effect of Y-102 liquid bacterial agent on ammonia emission reduction in potted chili soil.

[0155]

[0156]

[0157] The experimental results show that, as shown in Table 4, irrigating with urea significantly increases the ammonia volatilization rate in the soil during the experimental period; compared with Group I (irrigated with urea), Group III, after irrigating with the same amount of urea (15 g / m³), showed a significant increase in ammonia volatilization. 2 Under certain conditions, the application of Y-102 liquid microbial agent reduced soil ammonia emissions by 75.43%; compared with the CK group (without urea application), the application of Y-102 liquid microbial agent in group II reduced soil ammonia emissions by 56.48% even without urea application. Therefore, this liquid microbial agent can significantly reduce ammonia emissions from soil, especially soil treated with urea. The reason for the ammonia reduction is that the Y-102 strain can produce urease inhibitors, which can inhibit the conversion of urea to ammonium (ammonia), ensuring that urea exists mainly in the form of amide nitrogen in the soil, thus reducing NH4+ in the soil solution. + The concentration of NH3 was reduced, thereby lowering ammonia emissions.

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

[0159] At the end of the above potted plant experiment (day 15), the urease activity of the soil in each pot was detected using the method of "Determination of Soil Urease Activity - Sodium Phenol-Sodium Hypochlorite Colorimetric Method" (T / NAIA 011-2020). The urease activity of the soil in pots treated with Bacillus amyloliquefaciens Y-102 inoculant and those not treated with Bacillus amyloliquefaciens Y-102 inoculant were compared. For specific detection methods, please refer to Example 1. Specific experimental results are shown in Table 5.

[0160] Table 5. Effects of Y-102 liquid bacterial agent application on soil urease activity in potted peppers.

[0161]

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

[0163] The experimental results showed that compared with Group I (urea application), Group III, when applying the same amount of urea (15g / m³), showed better results. 2 Under the condition of no urea application, the application of Y-102 liquid microbial agent reduced soil urease activity by 67.45% (P<0.01); compared with the CK group (no urea application), the application of Y-102 liquid microbial agent in group II reduced soil urease activity by 63.87% (P<0.01) even without urea application. Therefore, regardless of whether urea is applied, this liquid microbial agent can significantly reduce soil urease activity, thereby significantly reducing fertilizer efficiency loss caused by ammonia volatilization after urea fertilization and improving urea utilization rate and fertilizer efficiency.

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

[0165] At 8:00-12:00 on days 0, 3, 6, 9, 12, and 15 after fertilization, gas intake and exhaust were accurately stopped for 4 hours. At 12:00 each time, 50 mL of air was drawn from the glass cylinder using a syringe and injected into a 12 mL headspace tube that had been evacuated by a vacuum pump. The N2O content was determined using an Agilent 7890A gas chromatograph with an ECD detector at 300℃, a column temperature of 60℃, and a carrier gas composition of 95% argon and 5% methane at a flow rate of 40 mL / min. A standard curve was established using a standard gas with a concentration of 5 mg / L based on the N2O concentration in the sample. 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] For the chili seedlings to grow, air needs to be continuously supplied to the glass container. However, during the 4 hours when air intake and exhaust are stopped, the glass container becomes a closed space, and the N2O emitted from the soil in the flowerpot can only accumulate in the glass container. Therefore, by measuring and comparing the N2O concentration in each group of glass containers, the emission reduction effect of Y-102 liquid inoculant (microbial fertilizer) can be obtained. The results are shown in Table 6.

[0167] Table 6. Effect of Y-102 liquid bacterial agent on soil N2O reduction in potted chili peppers.

[0168]

[0169] Results: Groups I and III shared a common pattern: urea application significantly increased soil nitrogen content, leading to a significant increase in N2O emissions through nitrification and denitrification. The emission peak occurred around day 3, gradually decreasing after that, which may be related to the gradual utilization of urea. However, significant differences also existed between Groups I and III: at days 0, 3, 6, 9, 12, and 15 after fertilization, compared to Group I (urea application), Group III, with the same amount of urea applied, showed that the application of Y-102 liquid microbial agent reduced soil N2O emissions by 1.9%, 34.9%, 29.6%, 22.8%, 21.6%, and [missing data], respectively. The average emission reduction from day 3 to day 15 was 25.4% (besides the urease inhibitor in the liquid inoculant having an N2O reduction effect, spores are also an active ingredient in the liquid inoculant; however, the spores reaching the pepper roots had not yet germinated by day 0 and therefore did not have the corresponding N2O reduction effect, so the calculation started from day 3). Compared with the CK group (no urea application), the Y-102 liquid inoculant in group II, without urea application, reduced soil N2O emissions by 1.2%, 14.4%, 10.4%, 9.1%, 8.3%, and 7.4%, respectively, with an average emission reduction of 9.9% from day 3 to day 15. Therefore, applying Y-102 liquid inoculant (microbial fertilizer) at the same time as urea application can significantly reduce soil N2O emissions, and the emission reduction effect is best on day 3 of the experiment, reaching more than 30%. Applying Y-102 bacterial agent without urea application also has a certain emission reduction effect. The reasons for this are mainly twofold. First, the Y-102 strain can produce urease inhibitors, which inhibit the rate at which urea decomposes into ammonia (ammonium), reducing NH3 generation and emissions. It also promotes nitrification and NO3 production. - The supply of nitrogen is limited, which reduces the rate of subsequent denitrification and thus reduces N2O emissions. Secondly, because strain Y-102 has no denitrification ability, after being applied to the soil, it grows and reproduces in the plant root system, thereby increasing the number of non-denitrifying bacteria in the soil, especially the root soil, reducing the denitrification process in the soil, hindering the conversion of nitrates, nitrites, ammonium salts, etc. into N2O, and ultimately reducing N2O emissions.

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

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

[0172] Table 7. Effects of Y-102 liquid inoculant on available phosphorus content in soil of potted chili peppers.

[0173]

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

[0175] Results: Compared with Group I (urea application), Group III, under the condition of applying the same amount of urea, showed a 62.94% increase in available phosphorus content in the soil (P<0.01) after applying Y-102 liquid inoculant, and a 53.76% increase in available phosphorus content compared to before potting (P<0.01). Compared with Group CK (no urea application), Group II, without urea application, showed a 59.12% increase in available phosphorus content in the soil (P<0.01) after applying Y-102 liquid inoculant, and a 48.37% increase in available phosphorus content compared to before potting (P<0.01). This indicates that regardless of whether urea is applied, the application of Y-102 liquid inoculant can significantly increase the available phosphorus content in the soil, providing sufficient phosphorus for the growth of crops and even the next crop.

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

[0177] After the experiment, the flowerpots containing the chili seedlings were removed from the glass jar. Most of the soil around the roots was carefully removed, and the roots were washed in a basin of tap water and dried on absorbent paper. The height of the seedlings was measured using a ruler (accurate to mm). The roots and above-ground parts were cut at the dividing line between the above-ground and underground sections using scissors. The stem diameter was measured using calipers, and the fresh weight of the roots and above-ground parts was measured using a balance (accurate to 0.01 g). The results are shown in Table 8.

[0178] Table 8. Effects of Y-102 liquid bacterial agent on the growth promotion of potted pepper seedlings.

[0179]

[0180] Note: The mean in the table is the average of the seven corresponding values ​​after removing the highest and lowest values; different uppercase 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, number of leaves, and total fresh weight of chili peppers in four groups showed that the liquid microbial agent significantly promoted chili pepper growth. Compared with the control group (no urea application), group II (without urea application) showed increases in plant height, stem diameter, number of leaves, and total fresh weight of chili peppers by 25.04%, 25.17%, 3.12%, and 49.13%, respectively. Compared with group I (urea application), group III (with the same amount of urea application) showed increases in plant height, stem diameter, number of leaves, and total fresh weight of chili peppers by 22.99%, 17.40%, 4.14%, and 44.62%, respectively. In conclusion, regardless of whether urea is applied, Y-102 microbial agent significantly increases plant height, stem diameter, and total fresh weight of chili peppers (but has no significant effect on the number of leaves), promoting crop growth. The main reason is that urea, being a nitrogen fertilizer, provides nutrients to chili peppers and promotes their growth; while the liquid microbial agent, in addition to containing natural plant growth hormones such as IAA, also produces IAA through the growth and reproduction of its spores after germination of the chili pepper roots. Therefore, applying this microbial agent can significantly promote the growth of chili pepper seedlings.

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

[0183] To verify the growth-promoting effect of Y-102 liquid inoculant on gramineous crops, this study conducted a pot experiment on maize.

[0184] The tested maize variety was Zhengdan 958. Twenty-eight plastic flowerpots (with several drainage holes at the bottom) measuring 15cm x 18cm (inner diameter x height) were used. Soil (5-15cm deep from farmland) was filled to a depth of approximately 15cm, and 500mL of tap water was added to each pot. After soaking overnight, five maize seeds were sown in each pot. One week after emergence, the seedlings were thinned to two plants per pot. The 28 pots were then randomly divided into four groups: a control group (CK), experimental group I, experimental group II, and experimental group III, with seven pots in each group. After fertilizing according to Table 9, all potted plants were immediately moved to a leveled bed in a greenhouse. Water was applied weekly to the bed, supplying water to the seedlings through the drainage holes at the bottom of the pots. The greenhouse temperature was controlled at 25-30℃. After 20 days of growth in the greenhouse, the seedling height, stem diameter, and total fresh weight of the maize seedlings in each group were measured. After removing the corn seedlings from the flowerpots, carefully wash the roots with tap water, place them on absorbent paper to dry the surface moisture, measure the length of the above-ground part (seedling height) with a ruler, measure the stem diameter at the base with calipers, and weigh the entire plant using a balance. The experimental results are shown in Table 10.

[0185] Table 9. Experimental scheme for potted maize treated with Y-102 liquid inoculant.

[0186]

[0187] Table 10 Results of the experiment on the growth promotion effect of Y-102 liquid bacterial agent on potted maize seedlings

[0188] Group Fertilization details for each pot (see Table 9) Plant height (cm) Stem diameter (mm) Fresh weight of the whole plant (g) CK Do not fertilize, water with 200mL of water. 38.65±3.04b 8.92±0.61b 116.24±6.14Bb Ⅰ Apply 200mL of urea solution 44.82±3.25ab 10.54±0.73ab 135.03±8.05Bab Ⅱ Apply 200mL of diluted bacterial agent solution. 45.01±4.06ab 10.63±0.64ab 138.06±9.11Bab Ⅲ Apply 200mL of urea-bacterial agent 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 two highest and two lowest values; different uppercase 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: Measurements of plant height, stem diameter, and total fresh weight of maize in four groups showed that Y-102 liquid microbial agent significantly promoted maize growth. Compared with the control group (no urea application), group II, without urea application, showed increases in plant height, stem diameter, and total fresh weight of maize by 16.46%, 19.17%, and 18.77%, respectively. Compared with group I (urea application), group III, with the same amount of urea applied and simultaneously treated with Y-102 liquid microbial agent, showed increases in plant height, stem diameter, and total fresh weight of maize by 16.42%, 15.37%, and 32.82%, respectively. In conclusion, regardless of whether urea is applied, Y-102 liquid microbial agent significantly increases maize plant height, stem diameter, and total fresh weight, promoting crop growth. The main reason for this is that urea, being a nitrogen fertilizer, provides nutrients to maize and promotes growth. Y-102 liquid microbial agent contains natural plant growth regulators such as IAA, and the live bacteria it contains can also produce IAA by growing and multiplying in the corn root system. At the same time, this liquid microbial agent contains (or produces) urease inhibitors in the roots, which improves the fertilizer efficiency of urea. Therefore, applying this microbial agent 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 within the protection scope of the present invention.

Claims

1. A type of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Y-102, characterized in that, Its accession number is CGMCC No. 33966.

2. The application of Bacillus amyloliquefaciens Y-102 as described in claim 1 in reducing NH3 and N2O emissions in soil and improving nitrogen fertilizer utilization.

3. The application of Bacillus amyloliquefaciens Y-102 as described in claim 1 in promoting crop growth and increasing crop yield.

4. A microbial inoculant, characterized in that, It includes Bacillus amyloliquefaciens Y-102 as described in claim 1.

5. The microbial agent as described in claim 4, characterized in that, The microbial agent is either a liquid agent or a powdered agent.

6. The microbial agent as described in claim 5, characterized in that, The viable count of Bacillus amyloliquefaciens Y-102 in the liquid bacterial agent is ≥1.0×10⁻⁶. 9 CFU / mL; the viable count of Bacillus amyloliquefaciens Y-102 in the powdered bacterial agent is ≥1.0×10⁻⁶. 10 CFU / g.

7. A microbial fertilizer, characterized in that, It includes Bacillus amyloliquefaciens Y-102 as described in claim 1.

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