A stress-resistant nitrogen-fixing strain NBL-B11002 and its application

Abstract

The present invention provides a strain of Bacillus amyloliquefaciens NBL‑B11002 and an application thereof, and belongs to the field of agricultural microbial technology. The strain is an endophytic nitrogen-fixing bacterium, isolated from the rhizosphere of corn, and belongs to non-leguminous plant-associated nitrogen-fixing bacteria. After verification, it was found that the strain has good stress resistance, can resist salt, alkali, and ultraviolet radiation of a certain intensity, and shows excellent nitrogen fixation ability, can reduce chemical nitrogen fertilizer by more than 30%, and is an important potential growth-promoting nitrogen-fixing bacterium with wide application; the strain can significantly promote the growth of wheat, corn, eggplant, etc., increase biomass, and show good growth-promoting effect. Therefore, the strain has good application value.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and specifically relates to a biological nitrogen fixation technology, in particular to a stress-resistant nitrogen fixation strain NBL-B11002 and an application thereof. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] The chemical methods currently used by humans to produce nitrogen fertilizers not only require very harsh conditions such as high temperature and high pressure, but also waste a lot of raw materials. They also lead to a series of problems such as low effective utilization rate of nitrogen molecules, environmental pollution, excessive use, nitrogen loss, and soil compaction, which pose a major threat to the sustainable development of agriculture and food security.

[0004] Biological nitrogen fixation is a special physiological function of nitrogen-fixing microorganisms. It awakens the nitrogen-fixing genes of microorganisms and allows them to actively participate in nitrogen fixation in the process of symbiosis with crops. Its nitrogen fixation amount can reach 3 times the industrial nitrogen fixation amount, accounting for about 2 / 3 of the total global nitrogen fixation. Compared with chemical fertilizers, biological nitrogen fixation is a new source of nitrogen fertilizer with great application potential, low cost, low pollution, and good application prospects.

[0005] Nitrogen-fixing bacteria can be divided into symbiotic nitrogen-fixing bacteria, associated nitrogen-fixing bacteria and autogenic nitrogen-fixing bacteria. All three types of nitrogen-fixing bacteria can convert elemental nitrogen in the atmosphere into inorganic nitrogen that can be absorbed and utilized by plants. Among them, autogenic nitrogen-fixing bacteria can independently fix nitrogen and have no dependence on plants, which plays a certain role in increasing crop yields. Research on nitrogen-fixing bacteria in the prior art mainly focuses on symbiotic nitrogen-fixing bacteria, such as Rhizobium, and the target of action is generally leguminous plants, and there are very few reports on autogenic nitrogen-fixing bacteria. Moreover, these few known autogenic nitrogen-fixing bacteria have requirements for environmental conditions such as ambient temperature and humidity, and have the defect of unsatisfactory nitrogen-fixing effect. The key to the research and development of biological nitrogen fixation technology is to screen out strains with high nitrogenase activity, efficient nitrogen fixation, and strong self-resistance, which can be applied in actual production to reduce the use of chemical nitrogen fertilizers. Summary of the invention

[0006] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a strain of Bacillus amyloliquefaciens NBL-B11002 and its application. The strain is an endophytic nitrogen-fixing bacterium isolated from the rhizosphere of corn and belongs to a non-leguminous plant-associated nitrogen-fixing bacterium. After verification, it was found that the strain has good stress resistance, can resist salt, alkali, and a certain intensity of ultraviolet radiation, and exhibits excellent nitrogen fixation ability, can reduce chemical nitrogen fertilizer by more than 30%, and is an important potential growth-promoting nitrogen-fixing bacterium with wide application; the strain can significantly promote the growth of wheat, corn, eggplant, etc., increase biomass, and exhibit good growth-promoting effects. Therefore, the strain has good application value.

[0007] To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions:

[0008] In a first aspect, a strain of Bacillus amyloliquefaciens NBL-B11002 is provided, and its deposit number is CCTCC NO: M 20232184.

[0009] This strain was deposited in the China Center for Type Culture Collection (address: Wuhan University Collection Center, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China) on November 10, 2023, and its biological deposit number is CCTCC NO:M 20232184.

[0010] The colony and bacterial characteristics of the Bacillus amyloliquefaciens NBL-B11002 are as follows: after being cultured on a NA solid plate, the colony is nearly circular, with irregular edges, off-white, small, rough and dull surface, with table-like protrusions and a ring-like protrusion in the middle. The bacterial body is rod-shaped, arranged in chains, with spores in the middle, and Gram-positive.

[0011] In a second aspect, a microbial agent is provided, comprising any of the following items and an acceptable adjuvant;

[0012] (I) Bacillus amyloliquefaciens NBL-B11002 as described in the first aspect; and / or

[0013] (II) the inactivated bacteria of Bacillus amyloliquefaciens NBL-B11002 as described in the first aspect;

[0014] and / or

[0015] (III) The fermentation broth, metabolites, culture, exosomes, lysate or extract of Bacillus amyloliquefaciens NBL-B11002 as described in the first aspect.

[0016] In a third aspect, a biofertilizer is provided, comprising any of the following items and an acceptable adjuvant;

[0017] (I) Bacillus amyloliquefaciens NBL-B11002 as described in the first aspect; and / or

[0018] (II) the inactivated bacteria of Bacillus amyloliquefaciens NBL-B11002 as described in the first aspect;

[0019] and / or

[0020] (III) The fermentation broth, metabolites, culture, exosomes, lysate or extract of Bacillus amyloliquefaciens NBL-B11002 as described in the first aspect.

[0021] In a fourth aspect, there is provided use of the Bacillus subtilisin NBL-B11002 or its fermentation liquid described in the first aspect, the microbial agent described in the second aspect, or the biofertilizer described in the third aspect in all or part of the following:

[0022] 1) Application in the production of indoleacetic acid;

[0023] 2) Application in the preparation of indoleacetic acid generating agents;

[0024] 3) Application in improving the nutritional status of plant rhizosphere soil;

[0025] 4) Application in the preparation of plant rhizosphere soil nutrient improver;

[0026] 5) Application in promoting plant growth;

[0027] 6) Application in the preparation of plant growth promoters;

[0028] 7) Application in reducing nitrogen fertilizer application in plants;

[0029] 8) Application in the preparation of nitrogen fertilizer for plants.

[0030] In a specific embodiment of the present invention, the plant includes crops; preferably, the crops include grains or vegetables; more preferably, the grains or vegetables include radish, cabbage, wheat, corn, peanuts, soybeans, tomatoes, rapeseed, eggplant, cowpea, and cauliflower.

[0031] In a fifth aspect, a method for promoting plant growth is provided, comprising irrigating the roots and / or directly spraying and / or flushing the Bacillus amyloliquefaciens NBL-B11002 or its fermentation product described in the first aspect or the microbial nitrogen-fixing agent described in the second aspect or the microbial fertilizer described in the third aspect onto the plants / leaves and / or soil.

[0032] In a sixth aspect, a method for reducing nitrogen fertilizer application for plants is provided, comprising irrigating the roots and / or directly spraying and / or flushing the Bacillus amyloliquefaciens NBL-B11002 or its fermentation product described in the first aspect or the microbial nitrogen-fixing agent described in the second aspect or the microbial fertilizer described in the third aspect onto the plants / leaves and / or soil.

[0033] In a seventh aspect, a fermentation method of the Bacillus amyloliquefaciens NBL-B11002 of the first aspect is provided, wherein the strain NBL-B11002 is inoculated into a fermentation medium for fermentation culture.

[0034] The fermentation medium may be a conventional bacterial culture medium, which includes LB culture medium or NB culture medium.

[0035] In a specific embodiment of the present invention, the fermentation culture conditions are as follows: culture at 30-40°C for 15-30h, rotation speed: 150-200rpm;

[0036] Preferably, the fermentation culture conditions are as follows: culture at 35° C. for 24 hours, and a rotation speed of 160 rpm.

[0037] The culture conditions of the Bacillus amyloliquefaciens are as follows: the optimum pH value of the culture medium is 6.8.

[0038] In an eighth aspect, a method for producing a nitrogen-fixing bacterial agent is provided, wherein the bacterial powder of NBL-B11002 described in the first aspect, 30%-40% glucose, and Fumei powder are used to prepare a nitrogen-fixing bacterial agent; wherein the number of viable bacteria is 2×10 8 cfu / g—2×10 10 cfu / g;

[0039] Or, NBL-B11002 bacterial powder, 30%-40% glucose, and Fumei powder are used to prepare the nitrogen-fixing bacterial agent; wherein the number of viable bacteria is ≥1 billion CFU / g.

[0040] One or more of the above technical solutions have the following beneficial effects:

[0041] (1) The strain NBL-B11002 provided by the present invention has a stronger nitrogen fixation ability and can reduce the amount of chemical nitrogen fertilizer used by more than 30%. The strain of the present invention has both a nitrogen fixation gene cluster and a strong nitrogen fixation ability. The nitrogenase activity on the nitrogen-free medium reaches 486.11nmol C2H4 / (g·h), which is significantly higher than the control patented strain. The strain of the present invention is more adaptable to nitrogen-free environments and has better nitrogen fixation ability. It can fix nitrogen in the air and convert it into nitrogen that can be directly absorbed and utilized by crops, increase the nitrogen source, and at the same time reduce urease activity and prolong the action time of inorganic nitrogen sources. The prepared nitrogen-fixing bacterial agent is used in actual production, which improves the nitrogen levels of soil and plants and can reduce the use of chemical nitrogen fertilizers by more than 30%.

[0042] (2) Strong stress resistance and wide range of nitrogen fixation targets. The strain of the present invention is a Bacillus, which can produce dormant spores, has better stress resistance, high survival rate in adverse environments such as saline-alkali and ultraviolet radiation, is harmless to crops, has no crop variety specificity, can colonize not only in the root system and soil, but also in the plant body, and the nitrogen fixation targets are the entire soil and non-specific plants.

[0043] (3) It has a significant promoting effect on crop growth. The nitrogen-fixing strain of the present invention has the characteristics of solubilizing phosphorus and secreting IAA, etc., which can improve the nitrogen and phosphorus nutrition of crops and increase the nutrient utilization rate of fertilizers, thereby promoting crop growth and development and increasing production and income.

[0044] Advantages of additional aspects of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0046] Figure 1 This is the PCR amplification result of strain NBL-B11002 in Example 1 of the present invention.

[0047] Figure 2 This is a colony morphology diagram of strain NBL-B11002 in Example 3 of the present invention.

[0048] Figure 3 This is a bacterial morphology diagram of strain NBL-B11002 in Example 3 of the present invention.

[0049] Figure 4 The height of the hydroponic wheat plant after spraying the NBL-B11002 bacterial solution in Example 5 of the present invention.

[0050] Figure 5It is the fresh weight of hydroponic wheat after spraying NBL-B11002 bacterial solution in Example 5 of the present invention.

[0051] Figure 6 It is the dry weight of hydroponic wheat after spraying NBL-B11002 bacterial solution in Example 5 of the present invention.

[0052] Figure 7 The growth potential of corn plants in the nitrogen fertilizer reduction plot experiment in Example 6 of the present invention.

[0053] Figure 8 This is the growth trend of eggplant 15 days after the first application of the nitrogen-fixing bacteria agent in Example 7 of the present invention.

[0054] Fig. 9 The flowering rate and fruiting rate of eggplants in different treatment groups in Example 7 of the present invention. DETAILED DESCRIPTION

[0055] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0056] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. Unless otherwise specified, the chemical reagents used in the examples are conventional commercial reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0057] Example 1 Isolation and Screening of Nitrogen-Fixing Bacteria

[0058] 1. Initial screening of nitrogen-fixing bacteria

[0059] (1) Experimental materials:

[0060] Culture medium: Ashby nitrogen-free culture medium: mannitol 10g, KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, K2SO4 0.3g, CaCO3 5g, distilled water 1000ml, sterilized at 121℃ for 30min

[0061] (2) Experimental methods:

[0062] Plant sample screening method: Wash the surface of the plant sample with corn root system with sterile water, cut it into appropriate size with scissors, soak it in 75% alcohol for 30 seconds, then soak it in sodium hypochlorite for 3-5 minutes, and finally wash it with sterile water for 3 times, 1 minute each time. After washing, put it on sterilized filter paper to dry, then grind it with a sterilized mortar and apply it on Ashby nitrogen-free medium, and culture it in a 30℃ incubator for 3-5 days. Pick single colonies with different shapes, colors and sizes, purify them in Ashby nitrogen-free medium, transfer the slant and examine it under a microscope, and store them at 4℃.

[0063] Soil sample screening method: add 10g sample into a triangular flask containing 90mL physiological saline and mix thoroughly, place at 30℃, shake at 180r / min for 30min, make suspension, and perform gradient dilution. -3 , 10 -4 , 10 -5 Dilute the concentration, pipette 0.1 mL of the sample suspension into a sterile culture dish containing Ashby nitrogen-free medium, spread evenly, invert and culture at 30°C for 3-5 days, pick a single colony, mark it, separate and purify it by streaking for multiple times, transfer it on a slant and examine it under a microscope, and store it at 4°C.

[0064] Bacterial spotting: Pipette 2.5 μl of bacterial solution from the glycerol tube and spot it on NA medium and Ashby nitrogen-free medium respectively. Place them in a 30°C incubator for 2 days and observe their growth.

[0065] (3) Results: A total of 7 soil and plant samples were isolated, and 78 nitrogen-fixing bacteria were screened.

[0066] 2. Determination of nitrogenase activity

[0067] The nitrogenase activity of the strain was determined by acetylene reduction method. The specific method is as follows:

[0068] Ashby medium was injected into a 10 mL bottle with a rubber stopper and made into a slope. The above bacteria grown on the Ashby medium were inoculated onto the slopes and cultured at 28°C for 24 h. 1 mL of air was extracted from the bottle with a syringe, and 1 mL of C2H2 was injected with a syringe. The reaction was carried out at 28°C for 12 h. Then 1 mL of gas was extracted from the bottle, and the peak areas of C2H4 and C2H2 were measured using a Shimadzu GC-14C gas chromatograph. At the same time, the peak areas of C2H4 and C2H2 with known concentrations were measured and a standard curve was prepared. The amount of ethylene generated was calculated from the standard curve. The nitrogenase activity was calculated according to the formula: nitrogenase activity = C2H4 mole (nmol) / [bacterial weight (g)*reaction time (h)].

[0069] The results showed that among the 78 bacterial strains, 24 had nitrogenase activity, ranging from 67.1875 to 486.11 nmol C2H4 / (g·h). Among them, NBL-B11002 had the highest nitrogenase activity, which was 486.11 nmol C2H4 / (g·h), and its nitrogenase activity was at a relatively high level.

[0070] Table 1 Nitrogenase activity of strains

[0071]

[0072]

[0073] 3. Detection of nitrogen-fixing gene nifH A. Primer design

[0074]

[0075] B. PCR reaction system - 25 μL

[0076]

[0077]

[0078] C. Reaction Conditions

[0079]

[0080] The nitrogen fixation gene nifH was detected in the above-mentioned bacteria with nitrogenase activity. The results were ( Figure 1 ) showed that the strain NBL-B11002 of the present invention had a target band at 894 bp, i.e., it had the nitrogen fixation gene nifH.

[0081] Example 2 Evaluation of nitrogen-fixing bacteria function

[0082] Control strains: N24 (deposit number CCTCC NO: M 2019281), GXGL-4A (deposit number CGMCC No. 12588), and oligotrophic nitrogen-fixing strain SI-NJAU-1 (CGMCC No: 17643).

[0083] The above strains were used as control strains to compare and analyze the functionality of the strains of the present invention, such as nitrogen fixation, phosphorus solubilization, and IAA secretion. The nitrogenase activity determination method was referred to Example 1, and the Salkowski colorimetric solution method was used to quantitatively determine the IAA production capacity of the strain, and the phosphorus platinum blue spectrophotometer method was used to determine the phosphorus solubilization activity. The specific determination method is as follows:

[0084] 1. IAA capability determination

[0085] (1) Qualitative determination of IAA-producing bacteria: The isolated and purified bacteria were inoculated into a liquid culture medium containing L-tryptophan (100 mg / L). After incubation in a shaker at 30°C and 180 r / min in the dark for 24 h, 50 μL of the bacterial suspension was dropped onto a white ceramic plate, and 50 μL of Salkowski colorimetric solution (50 mL 35% HClO4 + 1 mL 0.5 mol / L FeCl3) was added. 50 μL of uninoculated liquid culture medium and an equal volume of mixed solution were added as controls. The fully mixed white ceramic plate was placed at room temperature and protected from light. The results were observed after 30 min. Those that turned red indicated that IAA could be produced.

[0086] (2) Quantitative determination of IAA-producing bacteria: The IAA-producing bacteria obtained in the initial screening were quantitatively determined, and the culture conditions were the same as above. Each strain of bacteria was replicated three times. The OD600 value of the bacterial suspension after 24 hours of culture was first determined to calculate the growth of the bacteria. Then, the bacterial suspension was centrifuged at 10,000 r / min for 10 minutes, and the supernatant was added with an equal volume of Salkowski colorimetric solution. The suspension was kept in the dark for 30 minutes, and the OD530 value was immediately determined. The CK with the colorimetric solution was adjusted to zero to calculate the IAA production. The IAA content in the fermentation broth per unit volume was calculated when the bacterial concentration OD600 value was 1. The standard curve was prepared by gradient dilution of analytically pure IAA.

[0087] 2. Determination of phosphate-solubilizing activity of strains - phosphoplatinum blue spectrophotometer method

[0088] (1) Drawing of the standard curve of phosphomolybdenum blue

[0089] Accurately pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 ml of phosphorus standard solution into 10 ml stoppered test tubes respectively, add 5 ml of key blue colorimetric mixture, add water to the scale and mix well. After placing in a 45°C water bath for 25 min, use tube No. 0 as a blank, measure the absorbance at a wavelength of 660 nm, and draw a phosphorus standard curve with the measured absorbance as the ordinate and the soluble phosphorus content in the solution as the abscissa.

[0090] (2) Determination of phosphate solubilization effect

[0091] Take 1 mL of seed solution and centrifuge at 5000 x g for 5 min. Wash the bacteria three times with sterile water, resuspend the bacteria, and adjust the concentration of the bacteria to about 1X 10 8cfu / mL. Inoculate into a conical flask containing NBRIP medium at a 1% inoculation rate, sample 2 mL every 24 hours after inoculation, centrifuge at 10000xg for 10 minutes, accurately aspirate 1 ml of supernatant and place in a 10 ml EP tube, add 1 ml of colorimetric mixture, dilute to the appropriate multiple in a 45°C water bath for 25 minutes, measure the absorbance at a wavelength of 660 nm, and calculate the phosphate solubilization effect of the strain according to the standard curve.

[0092] The results are shown in Table 2. The results show that the nitrogen fixation, phosphorus solubilization ability and IAA secretion of the strain NBL-B11002 of the present invention are significantly higher than those of the control strain.

[0093] Table 2 Comparison of nitrogen-fixing bacteria functions

[0094]

[0095]

[0096] Example 3 Identification and safety evaluation of strains

[0097] 1. Morphological characteristics of strains

[0098] Colony morphology: Inoculate a single colony onto a NA solid plate medium, place the plate upside down in a constant temperature incubator, and culture at 30°C for 24 hours. The colony is nearly circular, with irregular edges, off-white, small, rough and matte surface, with a table-like protrusion and a ring-shaped protrusion in the middle. Figure 2 .

[0099] Bacterial morphology: Gram staining was used to observe that the bacterial cells of strain NBL-B11002 were rod-shaped, arranged in chains, with spores in the spores, and Gram staining was positive. Figure 3 .

[0100] 2. Physiological and biochemical characteristics

[0101] The physiological and biochemical characteristics of the strain were identified using Haibo Biotech HBI Bacillus biochemical identification strips, see Table 3.

[0102] Table 3 Physiological and biochemical reactions of Bacillus amyloliquefaciens NBL-B11002

[0103]

[0104] Bacillus amyloliquefaciens NBL-B11002 can utilize glucose and various sugars (D-xylose, L-arabinose and starch), can utilize citrate, cannot utilize propionate and D-mannitol, can decompose animal protein into small molecules for utilization, can grow in 7% sodium chloride, and can reduce nitrate. This indicates that strain NBL-B11002 has strong salt tolerance and can be used in saline-alkali adverse environments.

[0105] 3. Molecular identification

[0106] The 16S rDNA gene sequence analysis method was used to identify the genus and species of the isolated and screened strain NBL-B11002. The 16s rDNA gene sequence (SEQ ID NO.1) has a homology of 99.86% with the Bacillus amyloliquefaciens sequence in the NCBI database, and the strain NBL-B11002 was identified as Bacillus amyloliquefaciens. The strain NBL-B11002 was deposited in the China Center for Type Culture Collection (Address: Wuhan University Collection Center, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China) on November 10, 2023, and its biological collection number is CCTCC NO: M 20232184, and its classification name is Bacillus amyloliquefaciens.

[0107] NBL-B11002 16S rRNA sequence:

[0108] >

[0109]

[0110] 4. Safety evaluation of bacterial strains - hemolytic test

[0111] The strain NBL-B11002 was streaked in a "Z" shape on Columbia blood agar (Qingdao Haibo), cultured for 24 hours, and observed to see if there was a hemolytic ring. The results showed that no hemolytic ring appeared after the strain was cultured, proving that the strain NBL-B11002 was non-hemolytic and had high safety during production and use.

[0112] Example 4 Stress resistance of bacterial strains and their colonization ability in soil

[0113] 1. Salt tolerance:

[0114] Using NB (Qingdao Haibo Biological) as the basic culture medium, the amount of sodium chloride added was adjusted to make the concentration of sodium chloride in the culture medium 1%, 3%, 5%, 8%, and 10%, respectively, and NBL-B11002 bacteria were inoculated. NB culture medium was used as a control, and the number of viable bacteria in the control was set to 100%. The number of viable bacteria was determined by the hemacytometer.

[0115] Table 4 Effects of different concentrations of sodium chloride in the culture medium on bacterial growth

[0116] Salt content of culture medium Number of viable bacteria% CK 100 1％ 98.45 3％ 93.28 5％ 81.25 8％ 52.43 10％ 23.15

[0117] The results showed that the strain still maintained a certain activity under high salt concentration, indicating that it could tolerate high salt adverse environment.

[0118] 2. Acid and alkali resistance research

[0119] The NBL-B11002 strain was inoculated into NB (Qingdao Haibo Biological) culture medium and cultured at 30°C and 180r / min for 24h. The seed solution was inoculated into a conical flask containing 100mL NB (Qingdao Haibo Biological) culture medium at a 1% inoculation rate, and the initial pH value of the fermentation liquid was adjusted to 2.0, 4.0, 6.0, 8.0, 10.0, and 12.0, respectively, and the natural pH value was used as a control, and the number of viable bacteria was determined by the hemocytometer.

[0120] Table 5 Effect of different pH on bacterial growth

[0121] PH Number of viable bacteria% 2 20.14 4 52.78 6 96.78 CK 100 8 90.08 10 48.12 12 17.20

[0122] The results showed that the strain NBL-B11002 still maintained good activity under strong acid and strong alkaline conditions, indicating that the strain of the present invention has good stability to pH.

[0123] 3. Anti-ultraviolet research

[0124] The strain NB-B11002 was inoculated on a NA culture dish by the plate streak method and irradiated under UV light for 0, 1, 2, 3 and 4 min, respectively, and then cultured in a 37°C incubator for 24 h. The culture medium without UV light was used as a control. The number of viable bacteria was determined by the hemocytometer.

[0125] Table 6 Effects of different UV irradiation times on bacterial growth

[0126] Irradiation time min Number of viable bacteria% 0 100 1 80.39 2 52.22 3 24.54 4 10.67

[0127] The results showed that strain NBL-B11002 remained active under ultraviolet irradiation, indicating that it had strong tolerance and could adapt well to adverse environments.

[0128] 4. Colonization ability of strains in saline-alkali soil

[0129] Experimental method: Add 50 μg / mL of rifampicin to the culture medium, and domesticate the NBL-B11002 strain that can grow stably on 50 ng / μl rifampicin LB plates. Use an inoculation needle to pick the domesticated strain and inoculate it into the LB liquid culture medium containing rifampicin. Shake and culture for about 24 hours until the bacterial suspension OD600 = 0.8. Put the saline-alkali soil (PH7.85, salt 2‰) collected from Dongying and sterilized at 121℃ for 3 hours into pots, and irrigate the cultured bacterial suspension, 20 mL per pot. The same amount of culture solution without bacteria was used as the control CK. Cultivate corn plants, with 5 replicates for each treatment. Take rhizosphere soil samples from each treatment at 2, 5, 10, 15, 20 and 30 days of culture, and spread them on LB plates containing 50 ng / μl rifampicin after gradient dilution. Count the colonies after culture.

[0130] Table 7 The number of colonies of the collected samples in the medium containing rifampicin

[0131]

[0132] The results show that the strain of the present invention can be colonized and survive in saline-alkali soil and has strong environmental adaptability potential.

[0133] Example 5 Wheat Growth Promotion Experiment-Hydroponics

[0134] 1. Test materials

[0135] Test strains: GN-26, NBL-B17001, GN-16, NBL-B11002 Bacillus amyloliquefaciens, BE416

[0136] Test crops: wheat;

[0137] Nutrient solution: Hoagland nutrient solution (nitrogen-free), Hoagland nutrient solution (nitrogen-containing)

[0138] NB liquid culture medium: 18 g of this product, 1 L of distilled water, autoclave at 121°C for 20 min.

[0139] Preparation of bacterial solution: The above strains were activated by NA slant transfer and inoculated into NB medium respectively, and cultured at pH 7.0, 35°C, and 160 r / min with shaking for 24 h to obtain fermentation solution for later use.

[0140] 2. Experimental treatment

[0141] A total of 3 control groups and 5 treatment groups were set up, with 5 replicates for each treatment. The experimental settings are shown in Table 8. The bacterial solution was diluted to 10 7 CFU / ml, spray on the wheat leaves until the leaves are covered with water droplets, spray once every 7 days, and spray 3 times in total.

[0142] Table 8 Hydroponic wheat experimental treatments

[0143]

[0144] 3. Results

[0145] 3.1 Wheat plant height

[0146] Wheat plant height after spraying nitrogen-fixing bacteria solution Figure 4 The results showed that the plant heights of all treatments were higher than those of CK1 (nitrogen-containing nutrient solution) and CK2 (NB), among which treatments T4 and T5 were significantly higher than CK1 (nitrogen-containing nutrient solution), and T4 (NBL-B11002) was significantly higher than CK2 (NB). The plant height of T4 treatment (NBL-B11002) was the highest, significantly higher than that of CK2 (NB) by 19.47%.

[0147] 3.2 Wheat biomass

[0148] Fresh weight and dry weight of wheat plants after spraying nitrogen-fixing bacteria solution Figure 4 and Figure 5 The results showed that the fresh weight and dry weight of plants in all treatments were higher than those in CK2(NB), among which the fresh weight and dry weight of treatment T4(NBL-B11002) were the highest, which were 53.52% and 26.32% higher than those in CK2(NB), respectively.

[0149] Example 6 Experiment on reducing nitrogen fertilizer application in maize

[0150] 1. Experimental materials:

[0151] Test crops: Summer corn Zhengdan 958.

[0152] Test fertilizers: Nitrogen fertilizer was urea (containing 46% N), phosphate fertilizer was diammonium phosphate (containing 18% N, 46% P2O5) and superphosphate (containing 12% P2O5), and potash fertilizer was potassium sulfate (containing 52% K2O).

[0153] Preparation of bacterial liquid: LB medium - glucose 0.2%, peptone 1.0%, sodium chloride 0.5%, yeast extract 0.5%, the balance is water; рH6.8; temperature 35°С, rotation speed 160r / min, culture time 24h, obtain Bacillus amyloliquefaciens NBL-B11002 fermentation liquid, the number of viable bacteria is 1.8 billion / ml.

[0154] 2. Experimental methods:

[0155] The experiment was conducted in the new industrial park of Shandong Baolaililai Bioengineering Co., Ltd. (Taishan District, Tai'an City, Shandong Province). The soil type is sandy loam with a sandy texture. The experiment was divided into 4 groups: CK1-NB + 20% nitrogen reduction, CK2-NB + 40% nitrogen reduction, T1-bacteria solution + 20% nitrogen reduction and T2-bacteria solution + 40% nitrogen reduction. Each group was fertilized according to the conventional recommended fertilizer amount N 180kg / hm 2 、P2O590kg / hm 2 、K2O 90kg / hm 2 (ASI method) was used to conduct a nitrogen fertilizer reduction experiment. The bacterial solution was diluted 200 times and sprayed on the corn leaves at the seedling stage until the leaves were covered with water droplets. It was applied every 7-14 days and sprayed 2-3 times. The corn plant height, stem diameter, dry weight and total nitrogen content of the leaves were measured about 45 days after corn sowing.

[0156] Plant height: Use a tape measure to measure the straightened height (cm) from the substrate surface to the top growth point of the plant.

[0157] Stem diameter: measured using a digital vernier caliper.

[0158] Determination of SPAD value: Use handheld SPAD-502 (produced by Minolta, Japan) to measure the wider middle part of each leaf 5 times, and calculate the average value as the SPAD value of the leaf. The average value of the SPAD value of each layer of leaves is used as the SPAD value of that layer of leaves.

[0159] Determination of nitrogen content in plant leaves: Wash and dry fresh leaves, put them in an oven, fix them at 105°C for 30 minutes, and then dry them at 65°C for 24 hours. The baked samples are crushed and sieved, digested using the H2SO4–H2O2 method, and the nitrogen content of the plants is determined using the Kjeldahl method.

[0160] 3. Results

[0161] The plant traits and biomass of corn in different treatment groups are shown in Table 9 below.

[0162] Table 9 Maize plant traits and biomass

[0163]

[0164] The results showed that the growth of corn in the T1 and T2 treatment groups inoculated with nitrogen-fixing bacteria was significantly better than that of the uninoculated CK1 and CK2. The plant height of the bacterial solution + 20% nitrogen reduction and bacterial solution + 40% nitrogen reduction treatment groups increased by 28.3% and 67.6% respectively compared with the CK1 and CK2 groups, and the biomass increased significantly. The dry weight increased by 54.5% and 44.4% respectively compared with the control groups CK1 and CK2, and the leaf nitrogen content increased by 52.4% and 80% respectively compared with the control groups CK1 and CK2, indicating that the inoculation of nitrogen-fixing bacteria increased the nitrogen level of the plant, promoted the growth of crops, and reduced the use of some nitrogen fertilizers.

[0165] Growth trend of corn plants in the small plot experiment of nitrogen fertilizer reduction Figure 7 .

[0166] Example 7 Application experiment of nitrogen-fixing bacteria agent on eggplant

[0167] 1. Test materials

[0168] Eggplant seedling variety: "Da Hong Pao" long eggplant

[0169] Nitrogen-fixing bacteria agent: NBL-B11002 bacterial powder, 30%-40% glucose, and Fumei powder to make up the nitrogen-fixing bacteria agent, with a viable bacterial count of 2×10 9 cfu / g.

[0170] 2. Test methods

[0171] The experiment was conducted at the Shengfeng Base in Tai'an, Shandong Province. It was divided into three treatment groups. The control group CK was the conventional fertilization group, T1 was the conventional fertilization + nitrogen-fixing bacteria, and T2 was the group that reduced the chemical nitrogen fertilizer by 30% + nitrogen-fixing bacteria. The nitrogen-fixing bacteria was diluted about 200 times and used for root irrigation. The experimental area was 132m 2 The nitrogen-fixing agent was first applied on May 3, 30 days after the eggplant seedlings were transplanted, and then applied once every 12-14 days for a total of 3 times. The growth indicators of eggplants, soil and leaf nitrogen levels were measured on May 15 (12 days after the first application of the agent) and May 30 (15 days after the second application of the agent).

[0172] Soil sample collection and determination: The soil samples at a depth of 0-20 cm from the rhizosphere of each treatment plant were collected using the multi-point sampling method and placed in sterile sealed bags for testing and analysis. The total nitrogen content of the soil was determined by the Kjeldahl method; the ammonium nitrogen, available phosphorus, and available potassium content of the HM-TYD high-precision full-project soil nutrient detector were used; the soil urease activity was determined by the Nessler colorimetric method, and the soil alkaline nitrogen content was determined by the alkaline diffusion method.

[0173] Plant height: Use a tape measure to measure the straightened height (cm) from the substrate surface to the top growth point of the eggplant plant; calculate its growth rate (AGR): AGR = ΔH / Δt, ΔH is the difference in plant height between two measurements, and Δt is the number of days between the two measurements.

[0174] Blade: Measure blade length and blade width.

[0175] Flowering rate (%): Number of flowers on eggplant plants / Total number of eggplant plants × 100

[0176] Fruiting rate (%): Number of fruits per eggplant plant / Total number of eggplant plants × 100

[0177] Leaf nitrogen level: Wash and dry fresh plant leaves, put them in an oven, and kill them at 105°C for 30 minutes. Then, bake them at 65°C for 24 hours. The baked samples are crushed and sieved, and the nitrogen content in the plant leaves is determined by the Kjeldahl method.

[0178] Stem diameter: The diameter of the eggplant stem was measured using a digital vernier caliper (mm).

[0179] Single eggplant fruit weight: Use an electronic balance to measure the single fruit weight of 15 eggplants and calculate the yield.

[0180] 3. Results and Analysis

[0181] 3.1 Soil physical and chemical properties

[0182] The effects of nitrogen-fixing bacteria on soil physical and chemical indicators are shown in Table 10.

[0183] Table 10 Changes in soil physical and chemical indicators under different treatments

[0184]

[0185] Twelve days after the first application of the product, the total nitrogen content of T1 and T2 treatments increased by 25.9% and 14.3% respectively compared with CK, the ammonium nitrogen content increased by 29.2% and 6.9% respectively, and the nitrate nitrogen and urease activity also increased to varying degrees.

[0186] Fifteen days after the second application of the product, the eggplant was in the fruiting period. At this time, the nutrient content of different treatments was quite different. The total nitrogen, ammonium nitrogen and nitrate nitrogen contents of the T1 treatment were still higher than those of the CK, but the increase was not large. The available phosphorus content and urease activity of T1 were lower than those of the CK treatment. The total nitrogen and ammonium nitrogen contents of the T2 treatment were not much different from those of the CK. It is speculated that this may be due to the fact that the eggplant was in the fruiting period and the plants absorbed a large amount of nutrients from the soil, resulting in differences in soil nutrients. The decrease in urease is conducive to the slow release of nitrogen in the soil and prolongs the action time of nitrogen.

[0187] 3.2 Growth characteristics of eggplant

[0188] The effects of nitrogen-fixing bacteria on the growth characteristics of eggplant are shown in Table 11. The growth of eggplant after 15 days of application of nitrogen-fixing bacteria is shown in Table 11. Figure 8 .

[0189] Table 11 Growth characteristics of eggplant in different treatment groups

[0190]

[0191] The overall growth of eggplant in the nitrogen-fixing bacteria group was better than that in the control group. Twelve days after the first application of the bacteria, the plant height of the T1 and T2 treatments increased by 30% and 19.2% respectively compared with the CK; the leaf length increased by 25% and 6% respectively; the leaf width increased by 13.2% and 4% respectively; and the stem diameter increased by 21% and 14.3% respectively.

[0192] Fifteen days after the second application of the fungicide, the plant height of the T1 and T2 treatments increased by 12.8% and 7.2% respectively, the leaf length increased by 6.2% and 3% respectively, the leaf width increased by 18.1% and 5% respectively, and the stem diameter increased by 13% and 8% respectively compared with CK. The growth rate of eggplant in the nitrogen-fixing bacteria group was significantly higher than that in CK. In summary, the application of nitrogen-fixing bacteria can significantly promote the growth of eggplant plants.

[0193] The flowering rate and fruiting rate of eggplant in different treatment groups are as follows Fig. 9 . The results showed that the eggplants flowered and set fruits earlier after the application of nitrogen-fixing bacteria, and could be put on the market earlier. The flowering rates of the T1 and T2 treatments were increased by 81.2% and 77.3% respectively compared with CK; the first batch fruiting rate of eggplants was increased by 153.2% and 40% respectively in the T1 and T2 treatments compared with CK. This shows that nitrogen-fixing bacteria can effectively increase the number of flowers and fruits of eggplants, while promoting the rapid expansion and growth of fruits, thereby increasing yields. Therefore, the application of nitrogen-fixing bacteria can reduce the use of chemical nitrogen fertilizers by more than 30%.

[0194] 3.3 Eggplant fruit weight and yield

[0195] The yield was collected and counted on June 15, and only the yield of one harvest was calculated and converted into per-acre yield. The average single fruit weight and yield of eggplants in different treatments are shown in Table 12. The results showed that the eggplant yield of T1 and T2 treatments increased by 6.1% and 2.9% compared with CK. The single fruit weight of T1 and T2 increased by 9.3% and 3% respectively compared with CK. Compared with the normal level, the yield of eggplant was slightly increased when the nitrogen application rate was reduced by 30%. This shows that the reduction of chemical nitrogen fertilizer and the application of nitrogen-fixing bacteria have improved the nitrogen supply in the soil and are conducive to reducing the application of nitrogen fertilizer.

[0196] In summary, the nitrogen-fixing bacteria agent can reduce the application amount of nitrogen fertilizer by more than 30%.

[0197] Table 12 Eggplant yield and average single fruit weight under different treatments (single crop)

[0198]

[0199] Example 8 Field application effect experiment of nitrogen-fixing bacteria agent on corn

[0200] 1. Experimental Materials

[0201] Crop: The corn variety is Zhengdan 958.

[0202] Inorganic fertilizers: urea (N content 46%), superphosphate (P2O5 content 12%) and potassium sulfate (K2O content 50%).

[0203] Nitrogen-fixing bacteria agent: NBL-B11002 bacterial powder, 30%-40% glucose, and Fumei powder are used to complete the preparation of nitrogen-fixing bacteria agent, with the number of live bacteria ≥1 billion CFU / g.

[0204] 2. Experimental Methods

[0205] The experiment was conducted in a corn field in Jiyang City. The experiment adopted a randomized block design and was divided into four treatments: clean water irrigation treatment CK1 (no industrial nitrogen fertilizer), CK2 (industrial nitrogen fertilizer-urea (containing pure nitrogen 46) conventional fertilization treatment (150kgN / hm 2 ), T1 was urea treatment + nitrogen-fixing bacteria 2kg / mu with water flushing or / and foliar spraying, T2 was urea reduction of 15% + nitrogen-fixing bacteria treatment 2kg / mu with water flushing or / and foliar spraying, T3 was urea reduction of 30% + nitrogen-fixing bacteria treatment 2kg / mu with water flushing or / and foliar spraying. Each treatment group was set up with 3 replicates (3 plots), and the area of ​​each plot was set to 100m 2 The treatment group used nitrogen-fixing bacteria (10 days after urea application) 2-3 times at the jointing stage and booting stage on the basis of farmers' usual fertilization, and the application amount of phosphorus and potassium fertilizers was the same in each group. Each plot had a clear boundary separation and was randomly arranged.

[0206] Sample collection and determination method: During the growth period and harvest period, 10 plants with the same growth in each treatment were selected and dug out. The aboveground parts of the plants were weighed, sterilized (sterilized at 105℃ for 30 minutes), dried (dried at 75℃ to constant weight), weighed and crushed, and then the nitrogen content of the aboveground parts was determined. After the plant samples were digested and alkalized with H2SO4-H2O2, the total nitrogen content of the plants was determined by the Kjeldahl method; (or the total nitrogen content of stems, leaves, and ears) to calculate the nitrogen absorption of the aboveground parts and the nitrogen fertilizer absorption and utilization rate. The calculation formula is as follows:

[0207] Amount of nitrogen absorbed by aboveground parts (kg / ha) = aboveground biomass × nitrogen content in aboveground parts

[0208] Nitrogen fertilizer absorption and utilization rate (%) = (total nitrogen absorption of the aboveground parts of plants in the nitrogen application treatment - nitrogen absorption of the aboveground parts of the control area) × 100 / nitrogen application amount

[0209] Yield determination method: The yield is measured by the actual yield of the whole plot and converted into hectare yield. The output effect, agronomic utilization rate of nitrogen fertilizer and partial productivity of nitrogen fertilizer are calculated. The calculation formula is as follows:

[0210] Nitrogen partial productivity (NPEP, kg / kg) = crop yield in nitrogen application area / nitrogen application amount

[0211] 4. Results and Analysis

[0212] The effects of nitrogen-fixing bacteria on yield and nitrogen efficiency are shown in Table 13.

[0213] Table 13 Yield and nitrogen efficiency of different treatment groups

[0214]

[0215] The results showed that the yield of each treatment group was 17.15%, 15.44% and 8.05% higher than that of the conventional nitrogen application level CK2 group, respectively. The combination of nitrogen-fixing bacteria and nitrogen fertilizer reduction can significantly improve the nitrogen fertilizer absorption and utilization rate and nitrogen fertilizer partial productivity. Nitrogen-fixing bacteria can provide more nitrogen nutrition for plants and is more conducive to plant growth and development. It can be used as a substitute for inorganic nitrogen sources, help coordinate the abundance of bacteria in various links of the nitrogen cycle, form a good soil nitrogen cycle, promote efficient nitrogen utilization, and improve the nitrogen fertilizer absorption and utilization rate. It can be promoted and applied as a recommended nitrogen fertilizer reduction program.

[0216] Example 9 Stability Verification of Nitrogen-Fixing Bacteria Product

[0217] 1. Experimental Materials

[0218] Nitrogen-fixing bacteria agent: NBL-B11002 bacterial powder, 30%-40% glucose, and Fumei powder are used to complete the preparation of nitrogen-fixing bacteria agent, with the number of live bacteria ≥1 billion CFU / g.

[0219] 2. Experimental methods

[0220] (1) Constant temperature accelerated test: Take 20 bags of samples and place them in an incubator at a temperature of 40℃±2℃ and a humidity of 75%±5% for 3 months. Take out 3 bags of samples on the 0th, 10th, 20th, 30th, 60th and 90th day respectively, and detect the number of live bacteria in the samples.

[0221] (2) Room temperature storage test: Take 20 bags of samples and place them in a sample storage cabinet. Place them naturally at room temperature for 12 months. Take out 3 bags of samples at the 0th, 2nd, 4th, 6th, 8th and 12th month respectively and test the number of live bacteria in the samples.

[0222] 3. Results and analysis

[0223] The stability of the nitrogen-fixing bacteria in the constant temperature accelerated test and the room temperature placement test is shown in Table 14 and Table 15. The results show that the total number of viable bacteria of the nitrogen-fixing bacteria can still be maintained at 10 after 90 days of constant temperature acceleration and 12 months of room temperature placement. 9 The CFU / g level is above 1.0, which indicates that the nitrogen-fixing bacteria agent has good stability.

[0224] Table 14 Stability of nitrogen-fixing bacteria in the constant temperature accelerated test

[0225]

[0226] Table 15 Stability of nitrogen-fixing bacteria in room temperature test

[0227]

[0228] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens NBL-B11002, whose deposit number is CCTCC NO: M 20232184.

2. A microbial agent, characterized in that: Includes any of the following and acceptable adjuvants; (I), Bacillus amyloliquefaciens NBL-B11002 as claimed in claim 1; and / or (II) A fermentation broth comprising the Bacillus amyloliquefaciens NBL-B11002 according to claim 1.

3. A biological fertilizer, characterized in that: Includes any of the following and acceptable adjuvants; (I), Bacillus amyloliquefaciens NBL-B11002 as claimed in claim 1; and / or (II) A fermentation broth comprising the Bacillus amyloliquefaciens NBL-B11002 according to claim 1.

4. Use of the Bacillus subtilisin NBL-B11002 according to claim 1 or the microbial agent according to claim 2 or the biological fertilizer according to claim 3 in all or part of the following: 1) Application in the production of indoleacetic acid; 2) Application in the preparation of indoleacetic acid generating agents; 3) Application in improving the nutritional status of plant rhizosphere soil; 4) Application in the preparation of plant rhizosphere soil nutrient improver; 5) Application in promoting plant growth; 6) Application in the preparation of plant growth promoters; 7) Application in reducing nitrogen fertilizer application in plants; 8) Application in the preparation of nitrogen fertilizer for plants.

5. The use according to claim 4, characterized in that The plants include grains or vegetables.

6. The use according to claim 5, characterized in that The grains or vegetables include radish, cabbage, wheat, corn, peanut, soybean, tomato, rape, eggplant, cowpea and cauliflower.

7. A method for promoting plant growth, characterized in that: The method comprises irrigating the roots and / or directly spraying and / or flushing the Bacillus amyloliquefaciens NBL-B11002 according to claim 1 or the microbial agent according to claim 2 or the microbial fertilizer according to claim 3 onto the plants / leaves and / or soil.

8. A method for reducing nitrogen fertilizer application in plants, characterized in that: The method comprises irrigating the roots and / or directly spraying and / or flushing the Bacillus amyloliquefaciens NBL-B11002 according to claim 1 or the microbial agent according to claim 2 or the microbial fertilizer according to claim 3 onto the plants / leaves and / or soil.

9. The fermentation method of Bacillus amyloliquefaciens NBL-B11002 according to claim 1, characterized in that: The Bacillus amyloliquefaciens NBL-B11002 is inoculated into a fermentation medium for fermentation culture.

10. The fermentation method according to claim 9, characterized in that The fermentation culture conditions are: culture at 30-40°C for 15-30 hours, rotation speed: 150-200 rpm.

11. The fermentation method according to claim 9, characterized in that The fermentation conditions were as follows: 35°C for 24 h, rotation speed: 160 rpm.

12. A method for producing a nitrogen-fixing bacterial agent, characterized in that: The Bacillus amyloliquefaciens NBL-B11002 described in claim 1 is prepared into bacterial powder, and the powder is mixed with 30wt%-40wt% of glucose and Fumei powder to prepare a nitrogen-fixing bacterial agent.

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