Paenibacillus mucilaginosus, microbial inoculant thereof and application

Abstract

The present application relates to the technical field of microorganisms, and specifically discloses a Paenibacillus mucilaginosus, a microbial inoculum thereof and application. The Paenibacillus mucilaginosus N-002 has a preservation number of CGMCC No.32752. The Paenibacillus mucilaginosus N-002 provided by the present application can produce urease inhibitors and indole acetic acid in large amounts and has no denitrification effect. The urease inhibitors produced by the Paenibacillus mucilaginosus N-002 can reduce the urease activity of soil, significantly reduce the ammonia emission and nitrous oxide emission of soil, especially after nitrogen fertilizer is applied, are conducive to the nitrogen retention of soil, improve the fertilizer efficiency of nitrogen fertilizer, and reduce the influence of ammonia and nitrous oxide emission on the environment. The strain has no denitrification effect, can increase the proportion of non-denitrifying bacteria in soil after being applied to soil, reduce the denitrification effect of soil, thereby reducing the emission of nitrous oxide in soil, and is conducive to environmental protection.

Description

Technical Field

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

[0002] Urea dominates the global nitrogen fertilizer market, accounting for 60%–70% of all nitrogen fertilizers, due to its high nitrogen content, low price, wide applicability, and ease of use. However, the ammonia volatilization problem caused by urea application to farmland is becoming increasingly serious. Ammonia volatilization in farmland mainly originates from the application of nitrogen fertilizers such as urea, accounting for 50%–60% of global anthropogenic ammonia emissions. Urea rapidly decomposes into ammonia (NH3 or NH4OH) and CO2 under the action of urease in the soil. Because the rate of ammonia production is much higher than the rate at which crops utilize it, large amounts of ammonia volatilize from the soil. This not only causes nitrogen loss and reduces nitrogen fertilizer utilization, but the volatilized ammonia, as a precursor to PM2.5, also contributes to air pollution, leading to a series of environmental problems such as soil acidification, eutrophication, and biodiversity loss.

[0003] In addition, N2O emissions from farmland are another key pathway for nitrogen loss after the application of nitrogen fertilizers such as urea. N2O has an extremely strong greenhouse effect, with a warming potential 298 times that of CO2, significantly impacting climate change and ozone layer depletion. Atmospheric N2O mainly originates from emissions from agricultural land. After urea is applied to the soil, it is converted into ammonium nitrogen by urease. Some of the ammonium nitrogen undergoes nitrification in aerobic microzones to produce nitrate nitrogen. After diffusing into anaerobic areas, the nitrate nitrogen releases N2O through denitrification.

[0004] To address the aforementioned issues, applying urease-inhibiting microorganisms to the soil can reduce soil urease activity, inhibiting the conversion of urea to ammonium (ammonia), thereby reducing soil ammonia emissions. Simultaneously, the reduction in soil ammonia content can decrease nitrification and nitrite formation, reducing soil N2O emissions. Applying non-denitrifying microorganisms to the soil can increase the number of non-denitrifying microorganisms in crop roots, reducing soil denitrification and decreasing the conversion of nitrate and nitrite nitrogen to N2O, thus reducing N2O emissions.

[0005] Bacillus mucilaginosus, also known as Paenibacillus mucilaginosus, is a widely distributed bacterium in the rhizosphere soil of plants. Commonly called "silicate bacteria" or "potassium bacteria," it decomposes aluminosilicate minerals in the soil, converting insoluble elements such as phosphorus, potassium, and silicon into soluble substances for plant absorption and utilization. Simultaneously, it produces various bioactive substances that promote plant growth. It is an important functional strain widely used in microbial fertilizers and belongs to the "strains exempt from toxicology testing" permitted by the "NY / T1109-2017 General Technical Guidelines for Biosafety of Microbial Fertilizers." However, current research and applications have only focused on its functions and utilization value, without considering the impact of its application to the soil on the fertilizer efficiency of nitrogen-containing fertilizers such as urea, or its impact on air pollution caused by NH3 and N2O emissions.

[0006] Currently, there is a lack of urease inhibitor-producing microorganisms with practical application value, and there are no reports on using such microorganisms to make microbial fertilizers for field application to reduce soil urease activity, improve the efficiency of nitrogen fertilizers such as urea, reduce soil NH3 and N2O emissions, and reduce air pollution. Therefore, it is urgent to screen for mucilaginous Bacillus species that can produce urease inhibitors and indoleacetic acid without denitrification to develop novel microbial fertilizers and achieve multiple goals such as promoting crop growth, increasing yield, improving planting efficiency, and reducing environmental pollution. Summary of the Invention

[0007] To address the problem that existing technologies lack microorganisms capable of simultaneously producing urease inhibitors and indoleacetic acid without denitrification, thus failing to achieve the multiple benefits of promoting crop growth, increasing yield, and reducing environmental pollution, this invention provides a *Bacillus subtilis*, its microbial inoculant, and its applications. To address the aforementioned issues, this invention screened and obtained *Bacillus subtilis* that produces urease inhibitors and indoleacetic acid without denitrification function. Using this as a functional strain for microbial fertilizers, a microbial fertilizer was developed that can improve the efficiency of nitrogen fertilizers such as urea and reduce the emission of ammonia (NH3) and nitrous oxide (N2O) from the soil. After application, it can improve the utilization rate and efficiency of urea, promote crop growth, increase crop yield, and reduce the volatilization of NH3 and N2O after urea is applied to the soil, enhancing the soil's nitrogen retention capacity and reducing the environmental pollution caused by NH3 and N2O emissions.

[0008] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0009] In a first aspect, the present invention provides a Paenibacillus mucilaginosus N-002, which has the accession number CGMCC No.32752.

[0010] Paenibacillus mucilaginosus N-002 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 Paenibacillus mucilaginosus and deposited on November 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 32752. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] The biological characteristics of the gelatinous Bacillus N-002 provided by this invention are as follows: the colonies are round with neat edges, moist and smooth surface, viscous and elastic, colorless and transparent, raised like half a glass bead, and can be drawn into filaments; the bacterial cells are rod-shaped with capsules, and the size of the bacterial cells is about (5-6) μm × (7-10) μm; the spores are elliptical, central, and about 1.5 μm × 3.0 μm in size.

[0012] The described Bacillus spp. N-002 is a Gram-positive bacterium, and it is negative for glucose fermentation test, methyl red test, VP test, indole test, gelatin liquefaction test, casein test and urea hydrolysis test; it is positive for catalase test, citrate utilization test and starch hydrolysis test.

[0013] The Bacillus subtilis N-002 provided by this invention belongs to the "strain exempt from toxicological testing" as permitted by the "General Technical Guidelines for Biosafety of Microbial Fertilizers" (NY / T 1109-2017). It exhibits stable production performance and is a commonly used strain in microbial fertilizer production enterprises, suitable for producing microbial fertilizers. It possesses significantly different characteristics from existing microbial fertilizer production strains, namely, high production of urease inhibitors and indoleacetic acid, and no denitrification effect. This allows it to achieve multiple objectives, including promoting crop growth, increasing yield, enhancing planting efficiency, and reducing environmental pollution, making it widely applicable in agricultural planting and possessing high practical value.

[0014] Secondly, the present invention also provides the application of the above-mentioned Bacillus subtilis N-002 in reducing NH3 and N2O emissions in soil and improving nitrogen fertilizer utilization.

[0015] Furthermore, the present invention also provides the application of the above-mentioned Bacillus subtilis N-002 in promoting crop growth and increasing crop yield.

[0016] The Bacillus subtilis N-002 provided by this invention, in addition to its basic phosphorus and potassium solubilizing functions, is notably characterized by its ability to produce high levels of urease inhibitors and indoleacetic acid (IAA) during its growth, without denitrification. The urease inhibitors it produces can suppress urease activity in various soils, reducing the rate at which nitrogen fertilizers such as urea are converted into ammonia (ammonium), thereby reducing NH3 emissions from the soil. Simultaneously, the reduction in ammonia (ammonium) content in the soil also leads to reduced nitrification and NO3 emissions. - The supply of urea is limited, thereby reducing the rate of subsequent denitrification and decreasing N2O emissions from the soil caused by the application of nitrogen fertilizers such as urea. This protects the ecological environment while improving the utilization rate and fertilizer efficiency of urea. Simultaneously, non-denitrifying bacteria grow and reproduce in the crop root system, increasing the proportion of non-denitrifying bacteria and reducing soil denitrification, further lowering N2O emissions from farmland. In addition, this microorganism also produces high levels of indoleacetic acid, effectively promoting crop growth and increasing crop yield. Through synergistic effects, it further enhances soil improvement, crop growth promotion, and harmful gas emission reduction, providing strong support for the sustainable development of green agriculture and demonstrating significant economic, social, and environmental benefits.

[0017] Thirdly, the present invention also provides a microbial inoculant containing the aforementioned Bacillus subtilis N-002.

[0018] Furthermore, the microbial agent is a liquid agent or a powdered agent.

[0019] Bacillus subtilis N-002 can be made into a microbial agent, which is convenient for storage and transportation. It can be applied in a variety of ways, such as spraying, watering or as base fertilizer, depending on different crop types, planting patterns and soil conditions. It can be fully integrated into modern farming practices without increasing farmers' usage costs or operational difficulties.

[0020] Furthermore, the viable count of Bacillus subtilis N-002 in the liquid bacterial agent is ≥5.0 × 10⁻⁶. 8 CFU / mL; the viable count of Bacillus subtilis N-002 in the powdered bacterial agent is ≥1.0×10⁻⁶. 10 CFU / g.

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

[0022] Fermentation medium was loaded into a ventilated stirred fermenter with a loading coefficient of 0.65–0.75, sterilized at 121°C for 30 min, and then cooled to 35°C for inoculation. The *Bacillus subtilis* N-002 seed culture was inoculated into the ventilated stirred fermenter at an inoculation rate of 5%–10%. Fermentation was carried out at 30°C, a rotation speed of 150–200 r / min, and an aeration rate of 0.5–1.2 VVM for 48–72 h to obtain a viable count ≥5.0 × 10⁻⁶. 8 Fermentation broth with a concentration of CFU / mL can be used directly as a liquid inoculum, with a shelf life of 6 months.

[0023] As a specific embodiment of the present invention, the preparation method of the powdered bacterial agent includes the following steps:

[0024] Fermentation medium was loaded into a ventilated stirred fermenter with a loading coefficient of 0.65–0.75, sterilized at 121°C for 30 min, and then cooled to 35°C for inoculation. The *Bacillus subtilis* N-002 seed culture was inoculated into the ventilated stirred fermenter at an inoculation rate of 5%–10%. Fermentation was carried out at 30°C, a rotation speed of 150–200 r / min, and an aeration rate of 0.5–1.2 VVM for 48–72 h to obtain a viable count ≥5.0 × 10⁻⁶. 8 Fermentation broth with CFU / mL;

[0025] The fermentation broth was continuously centrifuged at 6000–7000 r / min using a disc centrifuge to concentrate it 5–10 times, obtaining a bacterial sludge. The bacterial sludge was then mixed with auxiliary materials and spray-dried to obtain a powdered bacterial agent.

[0026] As a specific embodiment of the present invention, the preparation method of the above-mentioned Bacillus subtilis N-002 seed liquid includes the following steps:

[0027] ①Preparation of slant culture in eggplant flasks

[0028] Take several 500mL eggplant flasks, add 50mL of NA medium to each flask, sterilize at 121℃ for 30min, arrange them as 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 30℃ for 5-7 days to produce a large number of spores for later use.

[0029] ② Seed liquid preparation

[0030] 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 35℃. 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 30℃, a rotation speed of 1500–2000 r / min, and an aeration rate of 1.0–2.0 VVM for 12–14 h to obtain the seed culture.

[0031] Specifically, the seed culture medium and fermentation culture medium include the following components: starch 0.7%, white sugar 0.2%, soybean meal 0.08%, yeast extract 0.08%, MgSO4 0.3%, K2HPO4 0.2%, ferric chloride 0.01%, (NH4)2SO4 0.05%, calcium carbonate 0.15%, and natural pH.

[0032] 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.

[0033] 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.

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

[0035] Furthermore, the microbial agent also includes Bacillus amyloliquefaciens Y-102, the preservation number of which is CGMCC No. 33966.

[0036] The Bacillus spp. N-002 provided by this invention can also be used in combination with other similar functional strains (such as Bacillus megaterium NPKM-001 and Bacillus amyloliquefaciens Y-102). Through synergistic effects, it can further enhance soil improvement and crop growth promotion. It shows stronger advantages in reducing rhizosphere soil urease activity and reducing soil NH3 and N2O emissions. It not only helps to improve crop yield and quality and ensure food security, but also reduces the negative impact of agricultural production on the environment and contributes to the construction of ecological agriculture.

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

[0038] Fourthly, the present invention also provides a microbial fertilizer comprising the aforementioned Bacillus subtilis N-002.

[0039] This invention provides a safe Bacillus subtilis N-002 strain and its microbial inoculant that simultaneously promotes crop growth, improves nitrogen fertilizer efficiency, and reduces ammonia and nitrous oxide emissions. This strain produces high levels of urease inhibitors and indoleacetic acid without denitrification. The urease inhibitors it produces reduce soil urease activity, significantly decreasing ammonia emissions from the soil, especially after the application of nitrogen fertilizers such as urea. This is beneficial for soil nitrogen retention, improving nitrogen fertilizer efficiency, and reducing the environmental impact of ammonia emissions on the atmosphere, soil, and water. Since this strain does not denitrify, its application to the soil increases the proportion of non-denitrifying bacteria, reducing soil denitrification and thus decreasing nitrous oxide emissions, which is beneficial for environmental protection. The microbial inoculant made from this strain is mainly used in crop cultivation, with better results when applied during the nitrogen fertilizer application stage. The widespread application of this inoculant is conducive to the upgrading of microbial fertilizer production strains and has great application potential in promoting the sustainable development of green agriculture. Attached Figure Description

[0040] Figure 1 This is the colony morphology of Bacillus spp. N-002 of the present invention on a silicate bacterial screening medium;

[0041] Figure 2 The cell and spore morphology of Bacillus subtilis N-002 of this invention;

[0042] Figure 3 This is the phylogenetic tree of Bacillus lentigines N-002 of the present invention;

[0043] Figure 4 To observe the growth of strain N-002 when inoculated onto a medium containing strain NPKM-001 using the cross-inoculation method;

[0044] Figure 5 To observe the growth of NPKM-001 strain when inoculated onto a medium containing N-002 strain using the cross-inoculation method. Detailed Implementation

[0045] 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.

[0046] 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.

[0047] Culture medium used in the examples:

[0048] Alexandrov agar plate medium: sucrose 5.0g, Na₂HPO₄ 2.0g, MgSO₄·7H₂O 0.5g, FeCl₃ 0.005g, bromothymol blue 0.1g, CaCO₃ 0.1g, agar 19.0g, potassium feldspar powder 1.0g, distilled water 1000mL, adjust pH to 7.0. Sterilize at 121℃ for 30min.

[0049] Alexandrov broth: 5.0 g sucrose, 2.0 g Na₂HPO₄, 0.5 g MgSO₄·7H₂O, 0.005 g FeCl₃, 0.1 g bromothymol blue, 0.1 g CaCO₃, 1.0 g potassium feldspar powder, 1000 mL distilled water, adjust pH to 7.0. Sterilize at 121℃ for 30 min.

[0050] 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. Sterilize at 121℃ for 30min.

[0051] LB medium: 10g tryptone, 5g yeast extract, 10g NaCl, 1000mL distilled water, pH 7.4. Sterilize at 121℃ for 30min.

[0052] 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. Sterilize at 121℃ for 30min.

[0053] Phosphate-solubilizing fermentation medium (NBRIP): glucose 10g, Ca3(PO4)2 5g, MgCl2 5g, MgSO4·7H2O 0.25g, KCl 0.2g, (NH4)2SO4 0.1g, distilled water 1000mL, pH 7.0~7.5. Sterilize at 115℃ for 30min.

[0054] 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. Sterilize at 121℃ for 30 min.

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

[0056] 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.

[0057] Silicate bacteria selection medium: 10g sucrose, 0.5g yeast extract, 1.0g (NH4)2SO4, 2.0g Na2HPO4, 0.5g MgSO4·7H2O, 1.0g CaCO3, 1.0g potassium feldspar, 15g agar, 1000mL distilled water, adjust pH to 7.0. Sterilize at 121℃ for 30min.

[0058] 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. Sterilize at 121℃ for 30min.

[0059] Example 1

[0060] strain screening

[0061] 1. Enrichment of potassium-solubilizing Bacillus

[0062] 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 Alexandrov Liquid Culture Medium and cultured at 30℃ with shaking for 4 days. After settling, 5mL of this medium was inoculated again into Alexandrov Liquid Culture Medium, and the above operation was repeated to complete the secondary enrichment. The secondary enriched solution was then used for further processing.

[0063] 2. Isolation of potassium-solubilizing Bacillus

[0064] Take 1.0 mL of the above secondary enrichment solution using a sterile pipette and serially dilute it to 10⁻⁶. -6Take 0.1 mL of each gradient and inoculate it onto Alexandrov agar plates. Spread the plates evenly using a sterile spreader and incubate upside down in a 30°C incubator for 3–7 days. Observe whether a clear zone forms around the colonies. If a clear zone appears, it indicates that the strain is a potassium-solubilizing Bacillus.

[0065] 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 Alexandrov agar plates; five replicates were made for each strain. After 3-7 days of culture, the area of ​​the clear zone (S) was measured, and the potassium-solubilizing ability of the strain was determined by the S / s ratio.

[0066] Results: 382 strains of Bacillus with a clear zone were screened, of which 176 strains had an S / s value greater than 4.0, with strain N-002 having the highest S / s value of 8.6.

[0067] 3. Screening for Bacillus species with strong potassium- and phosphorus-solubilizing abilities.

[0068] The 176 strains selected above were inoculated into 50 mL of LB medium and cultured at 30 °C and 180 rpm 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 potassium-solubilizing fermentation medium, with the uninoculated potassium-solubilizing fermentation medium serving as a blank control. Each treatment was repeated three times, and cultured at 30 °C and 150 rpm for 7 days. After culture, the fermentation broth was centrifuged at 6000 rpm 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 potassium ion content was measured using a flame photometer. 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 was calculated.

[0069] In the phosphorus solubilization experiment, the procedures were the same as above, except that the phosphorus solubilization fermentation medium was used instead of the potassium solubilization fermentation medium and the available phosphorus content was determined by molybdenum-phosphorus colorimetric method after digestion. The available phosphorus (calculated as P2O5) concentration of each strain was compared with that of the blank control group, and the degree of increase in available phosphorus (calculated as P2O5) concentration was calculated.

[0070] Results: Among the 176 strains with an S / s value greater than 4.0, a total of 59 strains were screened that could increase the concentration of available potassium (calculated as K2O) by more than 10% and the concentration of available phosphorus (calculated as P2O5) by more than 15 times. Among them, strain N-002 could increase the concentration of available potassium (calculated as K2O) in the fermentation broth by 18.64% and increase the concentration of available phosphorus (calculated as P2O5) by 21.38 times.

[0071] 4. Screening for non-urease-producing Bacillus species

[0072] Using sterilized bamboo skewers, bacterial colonies of the 59 Bacillus strains with strong potassium- and phosphorus-solubilizing abilities selected above were picked up and cross-inoculated on urea phenol red bacterial agar plates. After incubation at 30°C upside down for 24 hours, the color change around the cross-shaped bacterial colonies was observed. Colonies that remained yellow around the edges (without turning red) were selected and inoculated on NA slant agar plates. After incubation at 30°C for 24–48 hours, they were stored for later use.

[0073] 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.

[0074] Results: 21 non-urease-producing Bacillus strains were screened from 59 strains of Bacillus with strong potassium- and phosphorus-solubilizing abilities, including strain N-002.

[0075] 5. Screening for Bacillus strains that produce urease inhibitors

[0076] The 21 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 30 °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.

[0077] 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.

[0078] (1) Reagents and solutions

[0079] 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.

[0080] 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.

[0081] 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%.

[0082] Urea solution (100g / L): Weigh 10.00g of urea (accurate to 0.01g), dissolve in water, and bring the volume up to 100mL.

[0083] 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.

[0084] (2) Standard Curve Construction

[0085] 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 into 50 mL volumetric flasks. 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.

[0086] (3) Determination of urease inhibition rate of the test solution

[0087] ① Soil urease activity assay

[0088] 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 place in a constant temperature incubator at 37℃±1℃ for 24h.

[0089] 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.

[0090] ② After adding the test solution, soil urease activity was measured.

[0091] 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.

[0092] (4) Calculation of urease activity inhibition rate

[0093] ① Calculation of soil urease activity

[0094] 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:

[0095]

[0096] In the formula, X is the content of urease in the soil sample (mg / (g·24h));

[0097] c1 is the ammonia content (μg / mL) obtained from the standard curve when the sample is treated with urea;

[0098] c2 is the ammonia content (μg / mL) obtained from the standard curve for the absorbance of the sample without urea.

[0099] V is the volume of the colorimetric final volume (mL), V = 50 mL.

[0100] N is the fractional amount, N = volume of leachate (mL) / volume of filtrate taken (mL) = 41mL / 1mL = 41.

[0101] V0 is the soil sample size (g), V0 = 5g.

[0102] t is the sample incubation time, t = 1 unit (unit is 24h).

[0103] f represents the dry matter content of the soil sample, in percentages (%). When the sample is the supernatant of the fermentation broth, f = 1.

[0104] ② Calculation of urease activity inhibition rate

[0105] Urease activity inhibition rate (%) = (Soil urease activity - Soil urease activity after adding test solution) / Soil urease activity × 100%

[0106] Results: Among 21 non-urease-producing Bacillus strains, 5 strains showed an inhibition rate of more than 50% on urease activity in maize soil. The supernatant of the fermentation broth from strain N-002 showed an inhibition rate of 81.53% on urease activity in maize soil. Using the same method, the supernatant of the fermentation broth from strain N-002 showed inhibition rates of 71.85%, 73.61%, and 44.29% on urease activity in alluvial soil (from western Mancheng District, Baoding City), brown soil (from western Mancheng District, Baoding City), and black soil (from northern suburbs of Shenyang City), respectively.

[0107] 6. Screening for Bacillus strains that produce high levels of indoleacetic acid (IAA)

[0108] Five strains with urease inhibition rates greater than 50% obtained in the previous step were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of NB medium and cultured at 30 °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.

[0109] (1) Plotting the standard curve

[0110] 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.

[0111] (2) Determination of IAA content in fermentation broth

[0112] 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.

[0113] Salksowski colorimetric reagent: Mix 1 mL of 0.5 mol / L FeCl3 solution with 50 mL of 35% HClO4 solution.

[0114] Results: Four of the five strains produced IAA, with strain N-002 having the highest IAA content in its fermentation broth, reaching 113.5 mg / L.

[0115] 7. Screening for Bacillus strains without denitrification function

[0116] The four strains of Bacillus with strong IAA-producing ability were transferred to NA slant culture medium using sterile bamboo sticks and activated by incubation at 30°C for 24 hours.

[0117] 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 four activated Bacillus strains with strong IAA-producing capacity into the small tube. Incubate at 30°C for 5–7 days and observe whether 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.

[0118] The experiment showed that two of the above four strains lacked denitrification function, including strain N-002.

[0119] Strain species identification

[0120] 1. Morphological observation

[0121] Using a sterile bamboo stick, pick up slant culture of strain N-002 and inoculate it into NB medium. After incubating at 30℃ and 150 rpm for 24 h on a shaker, take 1.0 mL of culture medium, dilute appropriately, and spread it onto a silicate bacteria selection medium plate. Incubate upside down at 30℃ for 24–48 h to observe colony morphology. After smearing an appropriate amount of culture medium, stain with crystal violet to observe the morphology of bacterial cells and spores.

[0122] Results: On silicate bacteria selection agar plates, colonies were round with neat edges, moist and smooth surfaces, viscous and elastic, colorless and transparent, raised like half a glass bead, and could be drawn into filaments. Gram staining was positive; the bacteria were rod-shaped, with capsules, and approximately (5–6) μm × (7–10) μm in size; spores were oval, centrally located, and approximately 1.5 μm × 3.0 μm in size. See Figures 1-2 .

[0123] 2. Physiological and biochemical identification

[0124] Following the experimental methods outlined in Bergey's Manual of Systematic Bacteriology, physiological and biochemical tests were conducted on strain N-002, including peroxidase assay, glucose fermentation, citrate utilization assay, methyl red assay, VP assay, starch hydrolysis assay, indole assay, gelatin liquefaction assay, casein assay, and urea hydrolysis assay. The results are shown in Table 1.

[0125] Table 1 Physiological and biochemical characteristics of strain N-002

[0126]

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

[0128] 3. Molecular biological identification

[0129] Total DNA was extracted from strain N-002 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:

[0130] AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCGTAATACATGCAAGTCGAGCGGAGCACTTCGGTGCTTAGCGGCGGACGGGTGAGTAACAGGTAGGCAACCTGCCTGTAAGATCGGGATAACTACCGGAAACGGTAGCTAAGACCGGATAGCTGGTTTCGGTGCATGCCGGAATCATGAAACACGGGGCAACCTGTGGCTTACGGATGGGCCTGCGGCGCATTAGCTAGTTGGCGGGGTAATGGCCCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGCAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAATGTCGTGGAGAGTAACTGCTCTGCGAATGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCTTTTAAGTCTGGTGTTTAAGCCCGGGGCTCAACCCCGGTTCGCACCGGAAACTGGAAGACTTGAGTGCAGGAGAGGAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTAAACACAATAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACG.

[0131] 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 N-002 was identified as Paenibacillus mucilaginosus, also known as Bacillus mucilaginosus.

[0132] The mucilaginosus species was deposited on November 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 32752. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0133] Phosphate solubilization effect of strain N-002 on organophosphates

[0134] The mycelial growth of strain N-002 was picked up with a sterile bamboo stick and inoculated into 50 mL of LB medium. The culture was then incubated at 30℃ 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 30℃ 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.

[0135] Results: The available phosphorus content in the control group was (0.065±0.010) mg / L; the available phosphorus content in the experimental group was (2.285±0.114) mg / L, which was 35.15 times that of the control group. This indicates that strain N-002 can decompose and release some of the phosphorus in lecithin (organic phosphorus) in the culture medium, and has a significant phosphorus-releasing effect on organic phosphorus.

[0136] Example 2

[0137] This embodiment provides a method for preparing a microbial fertilizer:

[0138] (1) Fermentation medium: starch 0.7%, white sugar 0.2%, soybean meal powder 0.08%, yeast extract 0.08%, MgSO4 0.3%, K2HPO4 0.2%, ferric chloride 0.01%, (NH4)2SO4 0.05%, calcium carbonate 0.15%, natural pH.

[0139] (2) Preparation of seed solution:

[0140] 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 N-002 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 30℃ for 5-7 days to produce a large number of spores for later use.

[0141] 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, cooled to 35℃, and then inoculated. 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 then incubated at 30℃, a rotation speed of 1500 r / min, and an aeration rate of 1.0–2.0 VVM for 12–14 h to obtain the seed culture.

[0142] (3) Add the above fermentation medium to 1M 3 In a ventilated stirred fermenter with a loading coefficient of 0.65–0.75, the mixture was sterilized at 121°C for 30 min, cooled to 35°C, and then inoculated with the above seed culture at an inoculation rate of 5%–10%. Fermentation was carried out at 30°C, 150 r / min, and an aeration rate of 0.5–1.2 VVM for 48–72 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 observation under a microscope using the smear staining method.

[0143] Results: The spore content in the fermentation broth ranged from (5.0 to 8.0) × 10⁻⁶. 8 The concentration of CFU / mL is between 5.0 and 10⁻⁶, therefore it is possible to prepare bacteria with a viable bacterial (spore) content ≥ 5.0 × 10⁻⁶. 8 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.

[0144] (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.

[0145] Results: The spore content in the fungal substrate could reach (2.0–8.0) × 10⁻⁶. 9 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 Powdered microbial fertilizer (powdered inoculant) with CFU / g, moisture content ≤10%, shelf life 18 months.

[0146] The technical parameters for preparing microbial fertilizer from strain N-002 are shown in Table 2.

[0147] Table 2 uses 1M 3 Technical parameters for preparing N-002 microbial agent in fermenter

[0148]

[0149]

[0150] Example 3

[0151] 1. Pot experiment on the ammonia emission reduction effect of Bacillus subtilis N-002 liquid inoculant.

[0152] 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.

[0153] Table 3. Experimental Scheme for Potted Pepper Cultivation with Inoculant N-002

[0154]

[0155] 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.

[0156] 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.

[0157] Table 4. Effect of N-002 liquid bacterial agent on ammonia emission reduction in potted chili soil.

[0158]

[0159] The experimental results show that applying urea significantly increases the ammonia volatilization rate in the soil during the experimental period. Compared with Group I (applied urea), Group III, after applying the same amount of urea (15 g / m³), showed a higher ammonia volatilization rate. 2Under the condition of urea application, drenching with N-002 liquid microbial agent reduced soil ammonia emissions by 65.82% (P<0.01); compared with the CK group (no urea application), group II, without urea application, showed a 43.46% reduction in soil ammonia emissions after drenching with N-002 liquid microbial agent (P<0.01). Therefore, this liquid microbial agent can significantly reduce soil ammonia emissions, especially in soils drenched with urea. The reason for the ammonia emission reduction is that the N-002 strain can produce urease inhibitors, which can inhibit the conversion of urea into ammonium or ammonia (NH4+). + The transformation process of NH3 causes urea to exist mainly in the form of amide nitrogen in the soil, thus reducing the NH4+ in the soil solution. + The concentration of NH3 was reduced, thereby lowering ammonia emissions.

[0160] 2. Effects of Bacillus subtilis N-002 liquid inoculant on soil urease activity in potted peppers

[0161] 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 "T / NAIA 011-2020 Determination of Soil Urease Activity - Sodium Phenol-Sodium Hypochlorite Colorimetric Method". The urease activity of the soil in pots treated with N-002 inoculant and those not treated with N-002 inoculant was compared. Specific detection methods are detailed in Example 1. Specific experimental results are shown in Table 5.

[0162] Table 5. Effects of N-002 liquid bacterial agent application on soil urease activity in potted peppers.

[0163]

[0164] 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.

[0165] The experimental results showed that, compared with Group I (urea application), Group III, at the same urea application rate (15g / m³), showed better performance. 2 Under the condition of urea application, the application of N-002 liquid microbial agent reduced soil urease activity by 67.15% (P<0.01); compared with the CK group (without urea application), the application of N-002 liquid microbial agent in group II reduced soil urease activity by 71.96% (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.

[0166] 3. Effect of Bacillus subtilis N-002 liquid inoculant on N2O emission reduction in potted chili soil.

[0167] 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.

[0168] 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 N-002 liquid inoculant (microbial fertilizer) can be obtained. The results are shown in Table 6.

[0169] Table 6. Effect of applying N-002 liquid bacterial agent on soil N2O reduction in potted chili peppers.

[0170]

[0171] Results: After applying urea, both Group I and Group III showed a significant increase in soil nitrogen content, which in turn significantly increased N2O emissions through nitrification and denitrification, with the emission peak occurring around day 3. However, Group III showed a significant difference compared to Group I: on days 0, 3, 6, 9, 12, and 15 after fertilization, compared to Group I (applied urea), Group III, with the same amount of urea applied, showed that the application of N-002 liquid inoculant reduced soil N2O emissions by 4.3%, 30.4%, 27.7%, 20.9%, 17.6%, and 13.3%, respectively. The average reduction in N2O emissions from day 3 to day 15 was 22.0% (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 on day 0 and therefore did not have the corresponding N2O reduction effect, so the calculation started from day 3).

[0172] Furthermore, compared to the control group (no urea application), in group II (without urea application), the N-002 liquid microbial agent reduced soil N2O emissions by 1.2%, 12.6%, 8.1%, 9.3%, 9.2%, and 7.0%, respectively, with an average reduction of 9.2% from day 3 to day 15. Therefore, applying N-002 microbial agent (microbial fertilizer) simultaneously with urea application can significantly reduce soil N2O emissions, with the best reduction effect (over 30%) observed on day 3 of the experiment. Applying N-002 microbial agent without urea application also showed some emission reduction effect. The reasons for this are mainly twofold: firstly, the N-002 strain can produce urease inhibitors, inhibiting the rate of urea decomposition into ammonia (ammonium), thus reducing NH3 emissions. Simultaneously, it also leads to nitrification and NO3- reduction. - The supply of N2O is limited, which reduces the rate of subsequent denitrification and thus reduces N2O emissions. Secondly, because the N-002 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 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.

[0173] 4. Effects of N-002 liquid bacterial agent on the growth performance of potted pepper seedlings

[0174] 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 7.

[0175] Table 7. Effects of N-002 liquid bacterial agent on the growth promotion of potted pepper seedlings.

[0176]

[0177] 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.

[0178] Results: Measurements of plant height, stem diameter, number of leaves, root fresh weight, and above-ground fresh weight in four groups of chili peppers showed that the liquid microbial agent significantly promoted chili pepper growth. Compared with the control group (no urea application), the average values ​​of plant height, stem diameter, number of leaves, root fresh weight, and above-ground fresh weight in group II (without urea application) increased by 27.65%, 20.14%, 5.21%, 73.61%, and 41.26%, respectively. Compared with group I (urea application), the average values ​​of plant height, stem diameter, number of leaves, root fresh weight, and above-ground fresh weight in group III (with the same amount of urea application) increased by 21.91%, 21.66%, 6.55%, 45.87%, and 52.41%, respectively. In conclusion, regardless of whether urea is applied, the N-002 microbial agent can significantly increase the plant height, stem diameter, root fresh weight, and above-ground fresh weight of chili peppers, promoting crop growth. The main reason is that urea is a high-quality nitrogen fertilizer that can provide nutrients for chili peppers and promote 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 the chili pepper roots germinate. Therefore, applying this microbial agent can significantly promote the growth of chili pepper seedlings.

[0179] Example 4

[0180] This embodiment provides a compound microbial agent, including Bacillus subtilis N-002 and Priestia megaterium NPKM-001, wherein the preservation number of Priestia megaterium NPKM-001 is CGMCC No. 32751.

[0181] (1) There is no mutual inhibition between strains N-002 and NPKM-001.

[0182] The plate confrontation test can demonstrate that there is no mutual inhibition between the two tested strains, N-002 and NPKM-001. The specific experimental procedure is as follows: Pour 5 mL of sterile water into the slant of one tested strain, scrape off the bacterial growth with a bamboo skewer, transfer the bacterial suspension to a sterile empty test tube, and vortex for 2 minutes to form a homogeneous bacterial suspension. Immediately pour this suspension into 100 mL of sterile, still-liquid (50-60℃) NA medium, mix well, and immediately pour onto a plate. After solidification, refrigerate the plate overnight at 4℃. The next day, inoculate the plate with the bacterial growth of the other tested strain using the cross-hatching method, and incubate at 37℃ for 24–48 hours. Observe whether an inhibition zone appears around the cross-hatching bacterial growth. If the cross-hatching bacterial growth is good and no inhibition zone appears around it, it indicates that the two strains do not inhibit each other.

[0183] The growth status of strains N-002 and NPKM-001 on the same plate is shown in the figure. Figures 4-5 As shown in the figure, the two tested strains, N-002 and NPKM-001, do not inhibit each other.

[0184] (2) Strains N-002 and NPKM-001 have a synergistic effect

[0185] The combined application of two liquid microbial agents, N-002 and NPKM-001, in the same pot experiment as in Example 3 above showed that the two microbial agents have synergistic or synergistic effects in reducing rhizosphere soil urease activity, reducing soil NH3 and N2O emissions, promoting plant growth, and improving the fertilizer efficiency of nitrogen fertilizers such as urea.

[0186] In pot experiments, when urea was applied, drenching with 0.13 mL of Bacillus subtilis N-002 liquid inoculant reduced soil urease activity by 67.15%, NH3 emissions by 65.82%, and the average N2O emissions from day 3 to day 15 by 22.0%. When urea was applied, drenching with 0.13 mL of NPKM-001 liquid inoculant reduced soil urease activity by 64.09%, NH3 emissions by 71.32%, and the average N2O emissions from day 3 to day 15 by 21.4%.

[0187] In pot experiments, when each pot was irrigated with 0.26g of urea, and simultaneously irrigated with 0.065mL of N-002 liquid bacterial agent and 0.065mL of NPKM-001 liquid bacterial agent, the soil urease activity decreased by up to 77.13%, and the average soil NH3 and N2O emissions decreased by 75.82% and 29.86%, respectively. Compared with irrigating with N-002 alone, the effects were improved by 9.98 percentage points, 10.00 percentage points, and 7.86 percentage points, respectively; and compared with irrigating with NPKM-001 alone, the effects were improved by 13.04 percentage points, 4.50 percentage points, and 8.46 percentage points, respectively.

[0188] 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 Bacillus mucilaginosus (B. mucilaginosus) strain N-002, characterized by, Paenibacillus mucilaginosus and has a preservation number of CGMCC No. 32752. ​ 2. The application of the Paenibacillus mucilaginosus N-002 in reducing NH3 and N2O emissions in soil, improving nitrogen fertilizer utilization efficiency according to claim 1.

3. The application of the Paenibacillus mucilaginosus N-002 in promoting crop growth, improving crop yield according to claim 1.

4. A microbial inoculant, characterized in that, The Paenibacillus mucilaginosus N-002 according to claim 1.

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

6. The microbial inoculant of claim 5, wherein, The viable cell number of Bacillus mucilaginosus N-002 in the liquid inoculant is ≥5.0×10 8 CFU / mL; and the viable cell number of Bacillus mucilaginosus N-002 in the powder inoculant is ≥1.0×10 10 CFU / g.

7. The microbial inoculant of claim 4, wherein The application method of the microbial agent includes spraying, pouring or using as a base fertilizer.

8. A microbial fertilizer, characterized by, The Paenibacillus mucilaginosus N-002 according to claim 1. The Paenibacillus mucilaginosus N-002 according to claim 1.

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