Paenibacillus mucilaginosus as well as microbial agent and application thereof

Abstract

The invention relates to the technical field of microorganisms and particularly discloses paenibacillus mucilaginosus as well as a microbial agent and application thereof. The preservation number of the paenibacillus mucilaginosus N-002 is CGMCC (China General Microbiological Culture Collection Center) No.32752, and the preservation number of the paenibacillus mucilaginosus N-002 is CGMCC No.32752. The paenibacillus mucilaginosus N-002 provided by the invention can produce a urease inhibitor and indoleacetic acid at high yield and has no denitrification effect, the urease inhibitor produced by the paenibacillus mucilaginosus N-002 can reduce the urease activity of soil, significantly reduce the ammonia emission and nitrous oxide emission of the soil, especially after a nitrogen fertilizer is applied, is beneficial to soil nitrogen element maintenance, improves the fertilizer efficiency of the nitrogen fertilizer, and has a good application prospect. Meanwhile, the influence of emission of ammonia and nitrous oxide on the environment is reduced; the strain has no denitrification effect, and can increase the proportion of non-denitrifying bacteria in soil and reduce the denitrification effect of the soil after being applied to the soil, so that the emission of nitrous oxide in the soil is reduced, and environmental protection is facilitated.

Description

Technical Field

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

[0002] Urea dominates the global nitrogen fertilizer market, accounting for 60% to 70% of nitrogen fertilizers, thanks to its high nitrogen content, low price, wide applicability, and ease of use. However, the problem of ammonia volatilization caused by the application of urea to farmland is becoming increasingly serious. Ammonia volatilization from farmland mainly comes from the application of nitrogen fertilizers such as urea, and its emissions account for 50% to 60% of global anthropogenic ammonia emissions. Urea is rapidly decomposed into ammonia (NH3 or NH4OH) and CO2 by soil urease. Because the rate of ammonia production is much higher than the rate of its utilization by crops, large amounts of ammonia volatilize from the soil, causing not only nitrogen loss and reduced nitrogen fertilizer utilization, but also the volatilized ammonia, as a precursor to PM2.5, can cause air pollution, leading to a series of environmental problems such as soil acidification, eutrophication, and loss of biodiversity.

[0003] Furthermore, N2O emissions from farmland are another key pathway for nitrogen loss following the application of nitrogen fertilizers such as urea. N2O has a strong greenhouse effect, with a warming potential 298 times greater than that of CO2, significantly contributing to climate change and ozone layer depletion. Atmospheric N2O primarily originates from emissions from agricultural land. After urea is applied to the soil, it is converted to ammonium nitrogen by the action of urease. Some of this ammonium nitrogen undergoes nitrification in aerobic micro-zones to form nitrate nitrogen. This nitrate nitrogen diffuses into anaerobic zones and releases N2O through denitrification.

[0004] To address these issues, applying urease inhibitor-producing microorganisms to the soil can reduce soil urease activity, hindering the conversion of urea to ammonium (ammonia), and thus reducing soil ammonia emissions. This reduction in soil ammonia content can also reduce nitrification and nitrite, reducing soil N2O emissions. Applying non-denitrifying microorganisms to the soil can increase their numbers in crop roots, reducing soil denitrification and the conversion of nitrate and nitrite to N2O, thereby reducing N2O emissions.

[0005] Bacillus mucilaginosus, also known as Paenibacillus mucilaginosus, is a widely found bacterium in plant rhizosphere soils. Commonly known as "silicate bacteria" or "potassium bacteria," it decomposes aluminosilicate minerals in the soil, converting insoluble elements like phosphorus, potassium, and silicon into soluble substances for plant absorption and utilization. It also produces a variety of bioactive substances that promote plant growth. It is an important functional strain widely used in microbial fertilizers and is considered a "strain exempt from toxicology testing" as defined in the "NY / T1109-2017 General Technical Guidelines for Biosafety of Microbial Fertilizers." However, existing research and applications have focused solely on these functions and their potential, failing to consider the impact of soil application of microbial agents on the efficiency of nitrogen-containing fertilizers such as urea, or their impact on atmospheric pollution caused by NH3 and N2O emissions.

[0006] Currently, there is a lack of viable urease inhibitor-producing microorganisms, and there are no reports of using such microorganisms as microbial fertilizers for field applications to reduce soil urease activity, improve the efficiency of nitrogen fertilizers like urea, reduce soil NH3 and N2O emissions, and mitigate air pollution. Therefore, there is an urgent need to identify Paenibacillus species that can produce urease inhibitors and indoleacetic acid without denitrification, in order to develop new microbial fertilizers that can achieve the multiple goals of promoting crop growth, increasing yields, improving agricultural profitability, and reducing environmental pollution. Summary of the Invention

[0007] In view of the problem that the prior art lacks microorganisms that can simultaneously produce urease inhibitors and indoleacetic acid and have no denitrification function, and cannot achieve the multiple effects of promoting crop growth, increasing yields and reducing environmental pollution, the present invention provides a colloidal Bacillus, its microbial agent and application. In response to the above problems, the present invention screened and obtained a colloidal Bacillus that produces urease inhibitors and indoleacetic acid and has no denitrification function, and used it as a functional strain of microbial fertilizer to develop a microbial fertilizer that can improve the fertilizer efficiency of nitrogen fertilizers such as urea and reduce ammonia (NH3) and nitrous oxide (N2O) emissions in the soil. After application, the utilization rate and fertilizer efficiency of urea can be improved, crop growth can be promoted, and crop yield can be increased. It can also reduce the volatilization of NH3 and N2O after urea is applied to the soil, enhance the nitrogen conservation effect of the soil, and reduce the pollution of NH3 and N2O emissions to the environment.

[0008] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0009] In a first aspect, the present invention provides Paenibacillus mucilaginosus N-002, whose deposit number is CGMCC No.32752.

[0010] Paenibacillus mucilaginosus N-002 was screened out from the rhizosphere soil of corn in a well-growing corn field near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770). It was classified and named Paenibacillus mucilaginosus. It was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, abbreviated as CGMCC, on November 22, 2024. The strain deposit number is CGMCC No. 32752. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] The biological characteristics of the Bacillus subtilis N-002 provided by the present invention are as follows: the colonies are round, with neat edges, a moist and smooth surface, sticky and elastic, colorless and transparent, with ridges like half glass beads, and can be pulled into filaments; the bacteria are rod-shaped, have capsules, and are about (5-6) μm × (7-10) μm in size; the spores are oval, mesophytic, and about 1.5 μm × 3.0 μm in size.

[0012] The Paenibacillus subtilis N-002 is a Gram-positive bacterium, and its glucose fermentation test, methyl red test, VP test, indole test, gelatin liquefaction test, casein test and urea hydrolysis test are all negative; its catalase test, citrate utilization test and starch hydrolysis test are all positive.

[0013] The Paenibacillus mucilaginosus N-002 provided by the present invention is a "strain exempt from toxicology testing" as approved by the "NY / T 1109-2017 General Technical Guidelines for Biosafety of Microbial Fertilizers." It has stable production performance and is a commonly used strain in microbial fertilizer production. It can be used to produce microbial fertilizers. It has significant advantages that distinguish it from existing microbial fertilizer production strains: high production of urease inhibitors and indoleacetic acid, and no denitrification. It can achieve the multiple goals of promoting crop growth, increasing yields, boosting cultivation efficiency, and reducing environmental pollution. It has broad applications in agricultural cultivation and high practical value.

[0014] In a second aspect, the present invention also provides the use of the above-mentioned Paenibacillus subtilis N-002 in reducing NH3 and N2O emissions in soil and improving nitrogen fertilizer utilization rate.

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

[0016] The Paenibacillus subtilis N-002 provided by the present invention, in addition to its basic phosphorus and potassium solubilization functions, has the remarkable feature of being able to produce high levels of urease inhibitors and indoleacetic acid (IAA) during its growth process, and has no denitrification effect. The urease inhibitor it produces can inhibit urease activity in various soils, reducing the rate at which nitrogen fertilizers such as urea are converted to ammonia (ammonium) after application, thereby reducing NH3 emissions in the soil; at the same time, the reduction in ammonia (ammonium) content in the soil can also lead to nitrification and NO3 - The supply of nitrogen fertilizers is limited, which in turn reduces the rate of subsequent denitrification and reduces N2O emissions from the application of nitrogen fertilizers such as urea. This improves urea utilization and fertilizer efficiency while protecting the ecological environment. At the same time, bacteria without denitrifying functions grow and multiply within crop roots, increasing the proportion of these bacteria, reducing soil denitrification and further reducing N2O emissions from farmland. Furthermore, this microorganism also produces high levels of indoleacetic acid, effectively promoting crop growth and increasing crop yields. Through synergistic functional effects, it further enhances soil improvement, promotes crop growth, and reduces harmful gas emissions, providing strong support for the sustainable development of green agriculture and possessing significant economic, social, and environmental benefits.

[0017] In a third aspect, the present invention further provides a microbial agent comprising the above-mentioned Paenibacillus subtilis N-002.

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

[0019] Paenibacillus subtilis N-002 is made into a microbial agent, which is convenient for storage and transportation. It has flexible and diverse application methods. It can be sprayed, poured or used as base fertilizer according to different crop types, planting patterns and soil conditions. It is fully integrated into modern farming practices and will not increase farmers' usage costs or operational difficulties.

[0020] Furthermore, the number of viable bacteria of Paenibacillus subtilis N-002 in the liquid bacterial agent is ≥5.0×10 8 CFU / mL; the number of viable bacteria of Paenibacillus subtilis N-002 in the powdered bacterial agent is ≥1.0×10 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] The fermentation medium was filled into a ventilated stirred fermenter with a filling factor of 0.65-0.75, sterilized at 121°C for 30 minutes, and cooled to 35°C for inoculation. The seed liquid of Paenibacillus subtilis N-002 was inoculated into the ventilated stirred fermenter with an inoculation amount of 5%-10%, and fermented at 30°C, a speed of 150-200 r / min, and a ventilation volume of 0.5-1.2 VVM for 48-72 hours to obtain a viable cell count of ≥5.0×10 8 The fermentation broth with CFU / mL can be directly used as a liquid bacterial agent 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] The fermentation medium was filled into a ventilated stirred fermenter with a filling factor of 0.65-0.75, sterilized at 121°C for 30 minutes, and cooled to 35°C for inoculation. The seed liquid of Paenibacillus subtilis N-002 was inoculated into the ventilated stirred fermenter with an inoculation amount of 5%-10%, and fermented at 30°C, a speed of 150-200 r / min, and a ventilation volume of 0.5-1.2 VVM for 48-72 hours to obtain a viable cell count of ≥5.0×10 8 CFU / mL of fermentation broth;

[0025] The fermentation liquid is continuously centrifuged at 6000-7000 r / min in a disc centrifuge to be concentrated 5-10 times to obtain bacterial sludge. The bacterial sludge is added with auxiliary materials and spray-dried to obtain a powdered bacterial agent.

[0026] As a specific embodiment of the present invention, the preparation method of the above-mentioned Paenibacillus mucilaginosus N-002 seed solution comprises the following steps:

[0027] ① Preparation of eggplant bottle slant culture

[0028] Take several 500mL eggplant flasks and add 50mL of NA medium to each. Sterilize at 121°C for 30 minutes, arrange them into a slant, and allow to solidify before use. Pour 5mL of sterile water onto the activated test tube slant. Use a sterile bamboo stick to scrape off the bacterial moss and stir as much as possible. Pour the bacterial suspension onto the slant of the eggplant flask and shake gently to ensure even inoculation. Incubate the flask upside down at 30°C for 5-7 days to produce a large number of spores before use.

[0029] ②Seed liquid preparation

[0030] Fill the seed tank with culture medium at a filling factor of 0.65-0.75, sterilize at 121°C for 30 minutes, and inoculate after cooling to 35°C. Take the inclined surfaces of four eggplant bottles, pour 50 mL of sterile water into each, scrape the bacterial moss with a sterilized bamboo stick, and pour it into a 1000 mL inoculation bottle. Inoculate the seed tank using the pressure differential method. Incubate at 30°C, a rotation speed of 1500-2000 r / min, and a ventilation volume of 1.0-2.0 VVM for 12-14 hours to obtain the seed solution.

[0031] Specifically, the seed culture medium and fermentation culture medium include the following components: starch 0.7%, white sugar 0.2%, soybean cake 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%, and natural pH.

[0032] Specifically, the above-mentioned auxiliary materials can be the auxiliary materials commonly used in conventional microbial solid inoculants in the art, such as light calcium carbonate, starch, etc., which can be conventionally selected in the art and are not particularly limited in the present invention.

[0033] More specifically, the number of viable bacteria in the powdered bacterial agent is required to be ≥ 1.0 × 10 10 CFU / g, moisture content ≤10%, shelf life 18 months.

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

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

[0036] The mucilaginous Bacillus N-002 provided by the present invention can also be used in combination with other similar functional strains (such as Bacillus megaterium NPKM-001 and Bacillus amyloliquefaciens Y-102). Through functional synergy, it can further enhance the soil improvement effect and crop growth promotion effect, and show stronger advantages in reducing rhizosphere soil urease activity and reducing soil NH3 and N2O emissions. It not only helps to improve the yield and quality of crops 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 method of the microbial agent includes spraying, pouring or using as base fertilizer.

[0038] In a fourth aspect, the present invention further provides a microbial fertilizer comprising the above-mentioned Paenibacillus subtilis N-002.

[0039] The present invention provides a safe strain of Paenibacillus colloidus N-002 and a microbial agent thereof that simultaneously promotes crop growth, improves nitrogen fertilizer efficiency, and reduces ammonia and nitrous oxide emissions. This strain can produce high levels of urease inhibitors and indoleacetic acid and is non-denitrifying. The urease inhibitors it produces can reduce soil urease activity, significantly reducing ammonia emissions from the soil, especially after the application of nitrogen fertilizers such as urea, thereby facilitating soil nitrogen retention and improving nitrogen fertilizer efficiency while reducing the impact of ammonia emissions on the atmosphere, soil, water bodies, and other environmental factors. This strain has no denitrifying effect and, after application to the soil, can increase the proportion of bacteria without denitrifying function in the soil, reduce soil denitrification, and thus reduce nitrous oxide emissions in the soil, thereby benefiting environmental protection. The microbial agent produced from this strain is primarily used in crop cultivation, with optimal application during the nitrogen fertilizer application stage. The widespread application of this agent is conducive to the upgrading of microbial fertilizer production strains and has great potential for promoting the sustainable development of green agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The colony morphology of the present invention's Paenibacillus subtilis N-002 on a silicate bacteria screening medium;

[0041] Figure 2 The bacterial and spore morphologies of Paenibacillus subtilis N-002 of the present invention;

[0042] Figure 3 is the phylogenetic tree of Paenibacillus mucilaginosus N-002 of the present invention;

[0043] Figure 4 The growth status of strain N-002 was obtained by cross-inoculation on a culture medium containing strain NPKM-001.

[0044] Figure 5 The figure shows the growth status of the NPKM-001 strain inoculated with the cross method on the culture medium containing the N-002 strain. DETAILED DESCRIPTION

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

[0046] Unless otherwise specified, analytically pure reagents that meet national standards were used in the examples, and the analytical water was grade 3 water as specified in GB / T6682.

[0047] Culture medium used in the examples:

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

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

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

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

[0052] Potassium-solubilizing fermentation medium: sucrose 5.0 g, MgSO4·7H2O 0.5 g, Na2HPO4 2 g, FeCl3 0.005 g, K2HPO4 0.2 g, CaCO3 0.1 g, potassium feldspar 5.0 g, distilled water 1000 mL, pH 7.0-7.5. Sterilize at 121°C for 30 min.

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

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

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

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

[0058] Organophosphate medium: glucose 10 g, (NH4)2SO4 0.5 g, MgSO4·7H2O 0.3 g, NaCl 0.3 g, KCl 0.3 g, FeSO4·7H2O 0.03 g, MnSO4·7H2O 0.03 g, lecithin 2 g, CaCO3 5 g, distilled water 1 L, pH 7.0. Sterilize at 121°C for 30 min.

[0059] Example 1

[0060] Strain screening

[0061] 1. Enrichment of Bacillus potassium-solubilizing

[0062] 10.0 g of soil was collected from the rhizosphere of thriving corn in a farmland near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770). The soil was added to a flask containing 90 mL of sterile water and glass beads. The suspension was shaken at 200 rpm for 10 minutes, followed by an 80°C water bath for 15 minutes to isolate bacteria in the spore state. After a short period of stagnation, 5 mL of this soil suspension was inoculated into Alexandrov liquid medium and incubated at 30°C with shaking for 4 days. After stagnation, 5 mL of the suspension was again inoculated into Alexandrov liquid medium. The above steps were repeated to complete the secondary enrichment, and the secondary enrichment solution was reserved.

[0063] 2. Isolation of Bacillus potassium-solubilizing

[0064] Use a sterile pipette to take 1.0 mL of the secondary enrichment solution and dilute it to 10 -6Pipette 0.1 mL of each gradient onto an Alexandrov plate. Spread evenly with a sterile spreader. Incubate inverted in a 30°C incubator for 3–7 days. Observe for the formation of clear zones around the colonies. If clear zones appear, the strain is a potassium-solubilizing Bacillus with some potassium-solubilizing ability.

[0065] The strain with transparent circle was transferred to NA slant medium and agar truffle cake with diameter of 8mm was prepared (area s = r 2 π=4 2 ×3.14=50.24mm 2 ) and inoculated onto Alexandrov plates; five replicates were grown for each strain. After 3-7 days of culture, the clear zone area (S) was measured and the potassium-solubilizing capacity of the strain was determined as S / s.

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

[0067] 3. Screening of Bacillus with strong potassium and phosphorus solubilizing abilities

[0068] Each of the 176 strains screened above was inoculated into 50 mL of LB medium and incubated at 30°C and 180 rpm for 12 hours, at which point the culture reached its logarithmic growth phase and served as the seed culture. One mL of the seed culture was added to 100 mL of potassium-dissolving fermentation medium. A blank control, containing no inoculated potassium-dissolving fermentation medium, served as the blank control. Each treatment was replicated three times, and the culture was incubated at 30°C and 150 rpm for 7 days. After incubation, the fermentation broth was centrifuged at 6000 rpm for 10 minutes to obtain the supernatant. 10 mL of the supernatant was added to a 50 mL volumetric flask, diluted to the mark with distilled water, and, after digestion, the potassium ion content was determined using a flame photometer. The available potassium (K₂O) concentrations in each flask were compared with those in the blank control, and the degree of increase in available potassium (K₂O) concentration corresponding to each strain was calculated.

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

[0070] Results: From the 176 strains with S / s values greater than 4.0, a total of 59 strains were screened out 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 the concentration of available phosphorus (calculated as P2O5) by 21.38 times.

[0071] 4. Screening of Bacillus that do not produce urease

[0072] Using sterile bamboo sticks, isolate the 59 strains of Bacillus strains with strong potassium and phosphate solubilization abilities screened above. These strains were then inoculated in a cross pattern onto urea-phenol red bacterial plates. After incubation at 30°C for 24 hours, the color of the cross surrounding the plate was observed. Colonies with yellow surrounding areas (not red) were selected and inoculated onto NA slant medium. The plates were incubated at 30°C for 24–48 hours before being stored for future use.

[0073] Principle of the test: Phenol red is an acid-base indicator that turns yellow in acidic conditions, orange in neutral conditions, and red in alkaline conditions. Its color change range is from pH 6.8 (yellow) to 8.4 (red). If the area around the bacterial lawn remains yellow, it indicates that the strain may not produce urease, is not breaking down urea in the culture medium around the lawn, and has not significantly changed the pH. Conversely, if the area around the bacterial lawn turns red, the strain may produce urease, breaking down urea in the culture medium to produce ammonia, which increases the pH and turns red.

[0074] Results: Twenty-one non-urease-producing Bacillus strains were screened from 59 Bacillus strains with strong potassium and phosphate solubilization abilities, including strain N-002.

[0075] 5. Screening of Bacillus spp. producing urease inhibitors

[0076] The 21 non-urease-producing strains obtained in the previous step were inoculated into a 250 mL Erlenmeyer flask containing 50 mL of NB medium and cultured in a shaking incubator at 30°C and 150 rpm for 48 h. The fermentation broth was then centrifuged at 6000 rpm for 10 min to obtain the supernatant, which was the test solution for the urease inhibitor.

[0077] Soil was excavated from a well-growing cornfield near Bajiazi Village, Changtu County, Tieling City, Liaoning Province (longitude 123.965, latitude 42.770). Urease activity in the cornfield soil was determined using the sodium phenol-sodium hypochlorite colorimetric method. The principle is that urea is hydrolyzed by soil urease to produce ammonia, which reacts with phenol-sodium hypochlorite at room temperature to form blue indigophenol. The color depth of indigophenol is proportional to the amount of ammonia generated. Therefore, the amount of ammonia can be measured colorimetrically, indicating urease activity. The inhibition rate of urease activity in the cornfield soil can then be calculated using the test solution. The specific test method is as follows.

[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 hours, and dissolve in water. The volume is adjusted to 1000 mL to obtain a stock solution containing 0.1 mg of ammonia per mL. Before use, dilute this solution 10-fold with water to a working solution of 0.01 mg / mL.

[0080] Sodium phenolate solution (1.35 mol / L): Solution A: Weigh 62.50 g of phenol (accurate to 0.01 g) and dissolve it in a small amount of ethanol. Add 2 mL of methanol and 18.5 mL of acetone, and dilute to 100 mL with ethanol. Solution B: Weigh 27.00 g of sodium hydroxide (accurate to 0.01 g), dissolve it in water, and dilute to 100 mL. Store solutions A and B in a refrigerator at 4°C. Before use, mix 20 mL of each solution A and solution B and dilute to 100 mL with water.

[0081] Sodium hypochlorite solution: Dilute the reagent according to the concentration of commercially available sodium hypochlorite solution to an active chlorine concentration of 0.9%.

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

[0083] Citrate buffer (pH 6.7): Weigh 184.00 g of citric acid (accurate to 0.01 g) and 147.50 g of potassium hydroxide (accurate to 0.01 g) and dissolve them separately in water. Combine the two solutions, adjust the pH to 6.7 with 1 mol / L sodium hydroxide, and dilute to 1000 mL with water.

[0084] (2) Preparation of standard curve

[0085] Before measuring sample absorbance, pipette 0.00mL, 1.00mL, 3.00mL, 5.00mL, 7.00mL, 9.00mL, 11.00mL, and 13.00mL of the ammonia working solution into a 50mL volumetric flask. Then, add 20mL of water, followed by 4mL of sodium phenolate solution and 3mL of sodium hypochlorite solution, shaking thoroughly. After 20 minutes, color development will occur. Adjust the volume to a set of standard ammonia concentrations of 0.0μg / mL, 0.2μg / mL, 0.6μg / mL, 1.0μg / mL, 1.4μg / mL, 1.8μg / mL, 2.2μg / mL, and 2.6μg / mL. Compare the colors at a wavelength of 578nm on a spectrophotometer within 1 hour (the blue color remains stable for 1 hour). Plot a standard curve with ammonia concentration as the horizontal axis and absorbance as the vertical axis.

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

[0087] ① Determination of soil urease activity

[0088] Take 5 g of soil sample (accurate to 0.0001 g; if the content is high, the sample weight can be appropriately reduced) and 5.0 mL of redistilled water in a 100 mL stoppered Erlenmeyer flask, add 1 mL of toluene, and shake evenly. After 15 minutes, add 10 mL of 100 g / L urea solution and 20 mL of pH 6.7 citrate buffer solution, shake well, and place in a constant temperature box at 37℃±1℃ for 24 hours.

[0089] After the incubation period, filter the filtrate. Pipette 1.00 mL of the filtrate into a 50 mL volumetric flask. Add 4 mL of sodium phenolate solution and 3 mL of sodium hypochlorite solution, shaking thoroughly. After 20 minutes, color will develop and the volume will be adjusted to 50 mL with water. Repeat the above steps using 10 mL of redistilled water instead of 10 mL of 100 g / L urea solution as a control. Measure the color within 1 hour using a spectrophotometer at a wavelength of 578 nm.

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

[0091] Except replacing "5.0 mL of redistilled water" in ① with "5.0 mL of the test solution", the other steps are the same as above ①.

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

[0093] ① Calculation of soil urease activity

[0094] Urease activity is expressed as the amount of ammonia produced by hydrolyzing the substrate (urea) in 1 g of air-dried soil in 24 hours, and is calculated as follows:

[0095]

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

[0097] c1 is the amount of ammonia (μg / mL) obtained from the standard curve using the absorbance value of the sample plus urea;

[0098] c2 is the amount of ammonia (μg / mL) obtained from the standard curve using the absorbance value of the sample without urea added;

[0099] V is the color development volume (mL), V = 50mL.

[0100] N is the fractionation multiple, N = leachate volume (mL) / filtrate volume (mL) = 41mL / 1mL = 41.

[0101] V0 is the amount of soil sampled (g), V0 = 5 g.

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

[0103] f is the dry matter content of the soil sample, %. When the sample is the supernatant of the fermentation liquid, f=1.

[0104] ②Calculation of urease activity inhibition rate

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

[0106] Results: Among 21 non-urease-producing Bacillus strains, five strains were found to have inhibition rates greater than 50% on urease activity in cornfield soil. The fermentation supernatant of strain N-002 exhibited an inhibition rate of 81.53% on urease activity in cornfield soil. Using the same method, the fermentation supernatant of strain N-002 exhibited inhibition rates of 71.85%, 73.61%, and 44.29% on urease activity in fluvo-aquic soil (from the western Mancheng District, Baoding City), cinnamon soil (from the western Mancheng District, Baoding City), and black soil (from the northern suburbs of Shenyang City), respectively.

[0107] 6. Screening of Bacillus sp. that produces high indoleacetic acid (IAA)

[0108] The five strains with urease inhibition rates greater than 50% obtained in the previous step were inoculated into 50 mL of NB medium in a 250 mL Erlenmeyer flask. The culture was shaken at 30°C and 150 rpm for 48 hours, and then centrifuged at 6000 rpm for 10 minutes to obtain the supernatant. The supernatant was used as the IAA test solution. IAA content was determined using the Salksowski colorimetric method as follows.

[0109] (1) Drawing of standard curve

[0110] Prepare IAA standard solutions of 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, and 35 mg / L in distilled water. Mix 2.0 mL of each IAA standard solution with 2.0 mL of Salksowski colorimetric reagent and incubate in a dark water bath at 40°C for 30 minutes. Measure the absorbance at 530 nm using a spectrophotometer. Plot a standard curve with IAA concentration as the horizontal axis and absorbance as the vertical axis.

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

[0112] Pipette 2.0 mL of the test solution and mix with 2.0 mL of Salksowski colorimetric reagent. Incubate in a dark water bath at 40°C for 30 min. Measure the absorbance at 530 nm using a spectrophotometer. Calculate the IAA content in the fermentation broth using the standard curve.

[0113] Salksowski color developing reagent: 1 mL of 0.5 mol / L FeCl3 solution was mixed with 50 mL of 35% HClO4 solution.

[0114] Results: Four of the five strains produced IAA, among which the fermentation broth of strain N-002 had the highest IAA content, reaching 113.5 mg / L.

[0115] 7. Screening of Bacillus without denitrification function

[0116] Use a sterile bamboo stick to transfer the above four Bacillus strains with strong IAA production ability to NA slant culture medium and culture at 30°C for 24 hours for activation.

[0117] In a clean bench, add 10 mL of Giltay liquid medium to a large test tube (20 mm x 200 mm). Place a small Dulbecco's tube (5 mm x 20 mm) upside down into the large test tube to allow the gas in the small test tube to escape. Four activated Bacillus strains with strong IAA production abilities were then inoculated into the tubes. Culture at 30°C for 5-7 days and observe whether bubbles are generated in the tubes. If gas is generated, the strain has denitrification; if not, it does not.

[0118] The test showed that two of the four strains mentioned above had no denitrification function, including strain N-002.

[0119] Identification of strain species

[0120] 1. Morphological Observation

[0121] Use a sterile bamboo stick to pick a slant of strain N-002 and inoculate it into NB medium. After incubation at 30°C, 150 rpm, and shaking for 24 hours, take 1.0 mL of the culture medium and dilute it appropriately. Then spread it onto a silicate bacterial screening medium plate. Incubate it inverted at 30°C for 24-48 hours and observe the colony morphology. Take an appropriate amount of the culture medium for smearing and stain with crystal violet to observe the morphology of the bacteria and spores.

[0122] Results: On silicate bacterial screening medium plates, the colonies were round, with neat edges, a moist and smooth surface, viscous and elastic, colorless and transparent, with ridges resembling half glass beads that could be pulled into filaments. Gram staining was positive; the bacteria were rod-shaped, with capsules, and were approximately (5-6) μm × (7-10) μm in size; the spores were oval, mesophytic, and approximately 1.5 μm × 3.0 μm in size. Figures 1 and 2 .

[0123] 2. Physiological and biochemical identification

[0124] Following the experimental methods in Bergey's Manual of Systematic Bacteriology, strain N-002 was subjected to physiological and biochemical tests, including 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.

[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 from strain N-002 was extracted using a bacterial genomic DNA extraction kit (Biomiga, catalog number BW-GD2411-01) according to the instructions. PCR amplification was performed using universal primers for the prokaryotic 16S rDNA gene sequence (upstream 5′-ACTGGAGGAAGGTGGGGA-3′, downstream 5′-AGGAGGTGATCCAACCGCA-3′) to obtain the amplified product. Sequencing of the amplified product was commissioned to BGI. The 16S rDNA sequencing results were as follows:

[0130] AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCGTAATACATGCAAGTCGAGCGGAGCACTTCGGTGCTTAGCGGCGGACGGGTGAGTAACAGGTAGGCAACCTGCCTGTAAGATCGGGATAACTACCGGAAACGGTAGCTAAGACCGGATAGCTGGTTTCGGTGCATGCCGGAATCATGAAACACGGGGCAACCTGTGGCTTACGGATGGGCCTGCGGCGCATTAGCTAGTTGGCGGGGTAATGGCCCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGCAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAATGTCGTGGAGAGTAACTGCTCTGCGAATGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCTTTTAAGTCTGGTGTTTAAGCCCGGGGCTCAACCCCGGTTCGCACCGGAAACTGGAAGACTTGAGTGCAGGAGAGGAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTAAACACAATAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACG.

[0131] The sequencing results were aligned at NCBI, and a phylogenetic tree was constructed using MEGA 11.0 based on the Neighbor-Joining method. Figure 3 Based on 16S rDNA sequence similarity analysis, and referring to the results of physiological and biochemical tests and colony and bacterial morphology, the N-002 strain was determined to be Paenibacillus mucilaginosus, also known as Bacillus mucilaginosus.

[0132] The Paenibacillus mucilaginosus was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, abbreviated as CGMCC, on November 22, 2024. The strain deposit number is CGMCC No. 32752, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

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

[0134] A sterile bamboo skewer was used to pick a slant of N-002 strain culture and inoculated into 50 mL of LB medium. The culture was shaken at 30°C and 180 rpm for 12 hours to obtain a seed solution. In the experimental group, the seed solution was inoculated into an organophosphate medium at a 3% inoculum concentration. In the control group, the seed solution was first inactivated by heating at 121°C for 20 minutes. All other procedures were the same as for the experimental group, with three replicates per group. After inoculation, the culture was shaken at 30°C and 180 rpm for 5 days. The culture was then centrifuged at 5000 rpm for 10 minutes. The supernatant was then decanted and the available phosphorus content 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, 35.15 times that of the control group. This suggests that strain N-002 can decompose and release phosphorus from some lecithin (organic phosphorus) in the culture medium, demonstrating a significant dephosphorylation effect on organic phosphorus.

[0136] Example 2

[0137] This embodiment provides a preparation of a microbial fertilizer:

[0138] (1) Fermentation medium: starch 0.7%, 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 and add 50mL of NA medium to each. Sterilize at 121°C for 30 minutes, arrange them into a slope, and allow to solidify before use. Pour 5mL of sterile water into the slope of the activated N-002 culture tube. Use a sterile bamboo stick to scrape off the bacterial moss and stir as much as possible. Pour the bacterial suspension into the slope of the eggplant flask and shake slowly to ensure even inoculation. Incubate the flask upside down at 30°C for 5-7 days to produce a large number of spores before use.

[0141] Fill a 100L ventilated agitated seed tank with fermentation medium at a charge factor of 0.65-0.75, sterilize at 121°C for 30 minutes, and inoculate after cooling to 35°C. Take the slopes of four eggplant bottles and pour 50mL of sterile water into each. Use a sterile bamboo stick to scrape off the bacterial moss and pour it into a 1000mL inoculation bottle. Inoculate the seed tank using the pressure differential method. Incubate at 30°C, a rotation speed of 1500 r / min, and an air volume of 1.0-2.0 VVM for 12-14 hours to obtain the seed solution.

[0142] (3) Add 1M 3 In a ventilated agitator fermenter, sterilize at 121°C for 30 minutes at a charge factor of 0.65-0.75. After cooling to 35°C, inoculate the above-mentioned seed solution at a 5%-10% inoculum level. Ferment for 48-72 hours at 30°C, 150 rpm, and an aeration rate of 0.5-1.2 VVM to obtain a fermentation broth. The viable bacterial content in the fermentation broth was determined by the gradient dilution plate spread method, and the spore count was calculated by microscopic observation using a smear staining method.

[0143] Results: The spore content in the fermentation broth was (5.0~8.0)×10 8 CFU / mL, so the content of viable bacteria (spores) ≥5.0×10 8 CFU / mL liquid bacterial agent, that is, liquid microbial fertilizer (liquid bacterial agent), has a shelf life of 6 months.

[0144] (4) The fermentation broth was continuously centrifuged at 6000 r / min in a disc centrifuge to concentrate 5 to 10 times to obtain bacterial sludge. After adding auxiliary materials to the bacterial sludge, the mixture was spray-dried to obtain bacterial powder.

[0145] Results: The spore content in the sludge can reach (2.0~8.0)×10 9 CFU / mL. The spore content in bacterial powder can reach (1.0~3.0)×10 11 CFU / g, can be used to prepare spore (live bacteria) content ≥1.0×10 10 CFU / g powdered microbial fertilizer (powdered bacterial agent), moisture content ≤ 10%, shelf life 18 months.

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

[0147] Table 2 uses 1M 3 Technical parameters of N-002 inoculant preparation in fermentation tank

[0148]

[0149]

[0150] Example 3

[0151] 1. Experimental study on the effect of liquid bacterial agent of Paenibacillus subtilis N-002 on reducing ammonia emissions in potted plants

[0152] Twenty-eight plastic flower pots (15 cm inner diameter x height x 18 cm) were filled with soil (from farmland at a depth of 5 to 15 cm) to a depth of approximately 15 cm. Twenty-eight pepper seedlings, approximately uniform in size, thickness, and leaf count, were selected and potted one week after seedling cultivation. Each seedling had its own pot. These seedlings were divided into four groups: CK, Experiment I, Experiment II, and Experiment III, each containing seven pots. Fertilization was initiated at the start of potting, and the experimental period lasted 15 days. The experimental plan is shown in Table 3.

[0153] Table 3N-002 fungal agent pepper pot test plan

[0154]

[0155] The flower pots from each of the four groups were placed in a transparent glass jar measuring 0.5 m (length × width × height) × 0.5 m (height × height). Daily watering was provided through water supply to the bottom of the jar and through drainage holes in the bottom of the pots. All other management procedures remained consistent across the groups, including maintaining soil water retention at 40%–60%, an average temperature of 25°C, and 8–10 hours of sunlight per day. The jars were sealed with a lid to create an airtight static chamber. Three PVC plastic tubes with valves were attached to the lid. One PVC tube was used to supply water to the bottom of the jar, another to provide airflow at a controlled rate of approximately 60 L / h (using a small blower to continuously supply air). The third PVC plastic tube was a tee-shaped pipe. One end of the pipe allowed the exhaust gas to be introduced into 200 mL of 0.01 mol / L sulfuric acid absorption solution. The other end could be inserted with a 20 cm long needle to aspirate the gas inside the jar for sampling and measurement of N2O emissions.

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

[0157] Table 4 Effect of applying N-002 liquid microbial agent on ammonia emission reduction in potted pepper soil

[0158]

[0159] The experimental results show that: from the data, it can be seen that urea application can significantly increase the amount of ammonia volatilization in the soil during the experimental period; compared with group I (urea application), group III applied the same amount of urea (15g / m 2) under the condition of irrigating with N-002 liquid microbial agent, the ammonia emission of soil was reduced by 65.82% (P<0.01); compared with the CK group (no urea application), the ammonia emission of group II was reduced by 43.46% (P<0.01) when irrigating with N-002 liquid microbial agent without urea application. Therefore, the liquid microbial agent can significantly reduce the ammonia emission of soil, especially the soil irrigated with urea. The reason for the reduction of ammonia emission is that the N-002 strain can produce urease inhibitors, which can inhibit the conversion of urea to ammonium or ammonia (NH4 + / NH3) conversion process, so that urea in the soil mainly exists in the form of amide nitrogen, reducing the NH4 + / NH3 concentration, thereby reducing ammonia emissions.

[0160] 2. Effect of Paenibacillus subtilis N-002 liquid inoculant on urease activity in potted pepper soil

[0161] At the completion of the potted plant experiment (day 15), the urease activity of the soil in each pot was measured using the "T / NAIA 011-2020 Soil Urease Activity Determination - Sodium Phenol-Sodium Hypochlorite Colorimetric Method" method. The urease activity in the soil of the pots treated with the N-002 inoculant was compared with that in the pots not treated with the N-002 inoculant. For the specific testing method, see Example 1. The specific test results are shown in Table 5.

[0162] Table 5 Effects of applying N-002 liquid microbial agent on urease activity in soil of potted peppers

[0163]

[0164] Note: The mean in the table is the average of 7 corresponding values after removing the highest and lowest values; different capital letters after the mean indicate differences at the P<0.01 level.

[0165] The test results showed that compared with group I (urea application), group III had the same urea application rate (15g / m 2 ), application of N-002 liquid inoculant reduced soil urease activity by 67.15% (P < 0.01). Compared with the CK group (no urea application), application of N-002 liquid inoculant in Group II, without urea application, reduced soil urease activity by 71.96% (P < 0.01). Therefore, regardless of urea application, this liquid inoculant significantly reduced soil urease activity, thereby significantly reducing fertilizer losses caused by ammonia volatilization after urea fertilization and improving urea utilization and efficiency.

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

[0167] On the 0th, 3rd, 6th, 9th, 12th, and 15th day after fertilization, air intake and exhaust were stopped for 4 hours between 8:00 AM and 12:00 PM. Each time, at 12:00 PM, 50 mL of air was drawn from the glass cylinder using a syringe and injected into a 12 mL headspace tube evacuated with a vacuum pump. N₂O content was determined using an Agilent 7890A gas chromatograph with an ECD detector, a detection temperature of 300°C, a column temperature of 60°C, and a carrier gas of 95% argon and 5% methane at a flow rate of 40 mL / min. A standard gas with a concentration of 5 mg / L was selected based on the N₂O sample concentration to establish a calibration curve. Manual injection was used: 40 μL of sample was drawn using a 100 μL gas-tight syringe and injected into the analyzer within 1 second.

[0168] To maintain the growth of the pepper seedlings, the glass jars required a constant supply of air. However, during the four hours when air intake and exhaust were stopped, the jars became a confined space, confining N₂O emitted from the soil in the flowerpots to the jars. Therefore, by comparing the N₂O concentrations within each group of glass jars, we could assess the N₂O reduction effectiveness of the N-002 liquid inoculant (microbial fertilizer). The results are shown in Table 6.

[0169] Table 6 Effect of applying N-002 liquid microbial agent on N2O emission reduction in potted pepper soil

[0170]

[0171] Results: Urea application significantly increased soil nitrogen content in Groups I and III, leading to a significant increase in N2O emissions through nitrification and denitrification, with peak emissions occurring around the third day. However, Group III showed significant differences compared to Group I: on days 0, 3, 6, 9, 12, and 15 after fertilization, N-002 liquid inoculant application reduced soil N2O emissions by 4.3%, 30.4%, 27.7%, 20.9%, 17.6%, and 13.3%, respectively, compared to Group I (urea application). The average N2O emission reduction from day 3 to day 15 was 22.0%. (In addition to the urease inhibitor contained in the liquid inoculant, which has a N2O emission-reducing effect, spores are also an active ingredient in the liquid inoculant. However, the spores that reach the pepper roots have not yet germinated by day 0 and therefore have no N2O emission-reducing effect, so calculations were performed starting from day 3.)

[0172] In addition, compared with the CK group (no urea application), in the group II without urea application, the N-002 liquid bacterial agent can reduce the soil N2O emissions by 1.2%, 12.6%, 8.1%, 9.3%, 9.2% and 7.0% respectively, and the average N2O emission reduction from the 3rd to the 15th day is 9.2%. Therefore, applying N-002 bacterial agent (microbial fertilizer) at the same time as urea application can significantly reduce soil N2O emissions, and during the experimental period, the emission reduction effect on the 3rd day was the best, reaching more than 30%. Applying N-002 bacterial agent without urea application also has a certain emission reduction effect. There are two main reasons for this: First, the N-002 strain can produce urease inhibitors, which inhibit the rate at which urea decomposes into ammonia (ammonium) and reduces the emission of NH3. At the same time, it also leads to nitrification and NO3 - The supply of N2O is limited, which in turn reduces the subsequent denitrification rate, thereby reducing the emission of N2O; 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 bacteria without denitrification ability in the soil, especially the root soil, reducing the denitrification of the soil, and hindering the conversion of nitrate, nitrite, ammonium salts, etc. to N2O, ultimately reducing the emission of N2O.

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

[0174] After the experiment, the pepper seedling pots were removed from the glass jars. Most of the soil surrounding the roots was carefully removed. The roots were then washed in a basin of tap water and placed on absorbent paper to dry. Plant height was measured with a ruler (accurate to mm). The roots and aboveground parts of the pepper seedlings were cut with scissors at the boundary between the aboveground and underground parts. Stem diameter was measured with a vernier caliper. Root and aboveground fresh weights were measured using a balance (accurate to 0.01 g). The results are shown in Table 7.

[0175] Table 7 Effect of applying N-002 liquid microbial agent on the growth promotion of potted pepper seedlings

[0176]

[0177] Note: The mean in the table is the average of 7 corresponding values after removing the highest and lowest values; different capital letters indicate differences at the P < 0.01 level, and different lowercase letters indicate differences at the P < 0.05 level.

[0178] Results: Measurements of plant height, stem diameter, leaf number, root fresh weight, and aboveground fresh weight of peppers in the four groups showed that the liquid inoculant significantly promoted pepper growth. Compared with group CK (no urea application), group II increased the average plant height, stem diameter, leaf number, root fresh weight, and aboveground fresh weight of peppers by 27.65%, 20.14%, 5.21%, 73.61%, and 41.26%, respectively, without urea application. Compared with group I (urea application), group III increased the average plant height, stem diameter, leaf number, root fresh weight, and aboveground fresh weight of peppers by 21.91%, 21.66%, 6.55%, 45.87%, and 52.41%, respectively, when the same amount of urea was applied. In summary, regardless of whether urea was applied, the N-002 inoculant significantly increased plant height, stem diameter, root fresh weight, and aboveground fresh weight of peppers, promoting crop growth. The main reason for this is that urea is a high-quality nitrogen fertilizer that provides nutrition to peppers and promotes their growth. In addition to containing natural plant growth hormones such as IAA, the liquid inoculant also produces IAA after its spores germinate and multiply in the pepper root system. Therefore, applying this inoculant can significantly promote the growth of pepper seedlings.

[0179] Example 4

[0180] This embodiment provides a composite microbial agent, including Paenibacillus mucilaginosus N-002 and Priestia megaterium NPKM-001, wherein the deposit number of Priestia megaterium NPKM-001 is CGMCC No. 32751.

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

[0182] Using the plate standoff test, it can be proved that there is no mutual inhibition between the two test strains N-002 and NPKM-001. The specific test steps are as follows: Pour 5mL of sterile water into the inclined surface of a test strain, scrape the bacterial lawn with a bamboo stick, transfer the bacterial suspension into a sterile empty test tube, and oscillate it with a vortex shaker for 2 minutes to form a uniform bacterial suspension. Immediately pour it into 100mL of sterilized NA culture medium that has not yet solidified (50-60℃), mix it and immediately pour the plate. After solidification, place the plate in a refrigerator at 4℃ overnight. The next day, inoculate the bacterial lawn of another test strain on the plate using the cross method, culture it at 37℃ for 24-48 hours, and observe whether there is an inhibition zone around the cross bacterial lawn. If the cross bacterial lawn grows well and there is no inhibition zone around it, it indicates that the two strains do not inhibit each other.

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

[0184] (2) N-002 and NPKM-001 strains have synergistic effects

[0185] The combined application of N-002 and NPKM-001 liquid microbial agents in the same potted plant experiment as in Example 3 above showed that the two microbial agents had a synergistic or enhancing effect 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 potted plants, urea application with 0.13 mL of the liquid inoculant Paenibacillus mucilaginosus N-002 reduced soil urease activity by 67.15%, NH3 emissions by 65.82%, and average N2O emissions by 22.0% from the 3rd to the 15th day. Similarly, urea application with 0.13 mL of the liquid inoculant NPKM-001 reduced soil urease activity by 64.09%, NH3 emissions by 71.32%, and average N2O emissions by 21.4% from the 3rd to the 15th day.

[0187] In the pot experiment, when 0.26 g of urea was applied to each pot, and 0.065 mL of N-002 liquid agent and 0.065 mL of NPKM-001 liquid agent were applied at the same time, the soil urease activity was reduced by 77.13%, and the soil NH3 emissions and average N2O emissions were reduced by 75.82% and 29.86%, respectively, which were 9.98 percentage points, 10.00 percentage points and 7.86 percentage points higher than the effect of applying N-002 alone; and 13.04 percentage points, 4.50 percentage points and 8.46 percentage points higher than the effect of applying NPKM-001 alone.

[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 in the scope of protection of the present invention.

Claims

1. A Paenibacillus mucilaginosus N-002, characterized in that: Its deposit number is CGMCC No.32752.

2. Use of the Paenibacillus subtilis N-002 according to claim 1 in reducing NH3 and N2O emissions in soil and improving nitrogen fertilizer utilization efficiency.

3. Use of the Paenibacillus subtilis N-002 according to claim 1 in promoting crop growth and increasing crop yield.

4. A microbial agent, characterized in that: The invention comprises the Paenibacillus mucilaginosus N-002 according to claim 1.

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

6. The microbial agent according to claim 5, wherein The number of viable bacteria of Paenibacillus subtilis N-002 in the liquid bacterial agent is ≥5.0×10 8 CFU / mL; the number of viable bacteria of Paenibacillus subtilis N-002 in the powdered bacterial agent is ≥1.0×10 10 CFU / g.

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

8. The microbial agent according to claim 4 or 7, wherein Also included is Bacillus amyloliquefaciens Y-102, and the deposit number of Bacillus amyloliquefaciens Y-102 is CGMCC No.33966.

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

10. A microbial fertilizer, characterized in that: The invention comprises the Paenibacillus mucilaginosus N-002 according to claim 1.

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