Bacillus NJAU-N6 with nitrogen fixation, low temperature resistance and drought resistance and application of bacillus NJAU-N6

Abstract

The invention discloses bacillus NJAU-N6 with nitrogen fixation, low temperature resistance and drought resistance and application of the bacillus NJAU-N6, the strain is classified and named as Paenibacillus illinoisis, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.35728. The bacillus NJAU-N6 has the advantages that the bacillus NJAU-N6 can be applied to nitrogen fixation, low temperature resistance and drought resistance; the invention discloses the capability of the compound in soil nitrogen fixation and soybean growth promotion. The invention also discloses a microbial inoculum prepared from the strain and application of the microbial inoculum in soil nitrogen fixation and soybean growth promotion. The strain disclosed by the invention has a relatively wide temperature adaptation range and relatively strong drought resistance, can stably play a nitrogen fixation role and a function of promoting soybean growth in a low-temperature and drought-resistant soil environment, and is particularly suitable for planting scenes of crops such as soybeans in semi-arid windy desert areas in northeast China; the method has good popularization and application values in soil fertility improvement and crop yield increase in the region.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural microorganisms, in particular to a bacillus NJAU-N6 with the abilities of nitrogen fixation, low temperature tolerance and drought resistance and its application. BACKGROUND

[0002] With the continuous growth of the world population and the increasing demand for food, chemical fertilizers are widely and massively used in agricultural production to ensure high crop yields. However, long-term over-reliance on chemical fertilizers has led to a series of serious ecological and environmental problems and challenges to agricultural sustainability: chemical fertilizers, especially nitrogen and phosphorus fertilizers, have generally low utilization rates, and the nutrients that are not absorbed enter water bodies through runoff and leaching, causing eutrophication of rivers, lakes and seas, or are converted into greenhouse gases (such as N2O) into the atmosphere, exacerbating environmental pollution and climate change. Excessive use of chemical fertilizers leads to soil compaction, decreased organic matter content, imbalanced pH, and simplified microbial community structure, disrupting the soil's healthy microecosystem and causing it to lose its natural fertility.

[0003] Microbial agents have attracted increasing attention due to their ability to improve crop nutrition, increase soil fertility, and promote the conversion of other life and matter in the soil, as well as their non-toxic, harmless, environmentally friendly, and low-cost advantages. Despite the wide variety of microbial agents on the market, the colonization rate and functional stability of microorganisms are easily affected by environmental factors such as soil moisture and temperature, and existing products have insufficient regional applicability, so there is an urgent need to develop microbial products that are more adaptable to regional environments and can improve soil fertility and drought resistance.

[0004] In addition, nitrogen-fixing bacteria can reduce N2 in the atmosphere, which most crops cannot directly utilize, to NH3 that can be absorbed and utilized by crops through the catalytic action of intracellular nitrogenase, providing a key reduced nitrogen source for crop growth and effectively promoting plant growth, thus playing an important role in agricultural production. In existing patents, CN202411305444.6 discloses a composite microbial agent that can significantly promote plant growth in alkaline soil; CN202410038998.8 discloses a salt-tolerant and alkali-tolerant nitrogen-fixing bacteria that not only has strong salt and alkali tolerance but also can secrete auxin IAA and improve plant chlorophyll content in a saline-alkali environment, with excellent growth-promoting effects; CN202311652849.2 discloses a Bacillus amyloliquefaciens with excellent nitrogen-fixing ability and resistance to salt, alkali, and a certain intensity of ultraviolet radiation, which can significantly promote the growth and biomass of crops such as wheat, corn, and eggplant. There are some nitrogen-fixing bacterial agents on the market. However, their environmental adaptability and functional stability are still relatively weak, and further exploration is needed. SUMMARY

[0005] The present application aims at the problem of microbial resources that can overcome the constraints of soil drought, low temperature and poor nutrition, and can stably colonize and function in the environment, thereby realizing soil nitrogen fixation and improving crop yield, and provides a bacillus NJAU-N6 and application thereof.

[0006] Another object of the present application is to provide a microbial inoculant prepared from the bacillus NJAU-N6 and application thereof.

[0007] The above-mentioned objects of the present application can be achieved by the following technical solutions.

[0008] The bacillus NJAU-N6 has the ability of nitrogen fixation, low temperature resistance and drought resistance, is classified as Paenibacillus illinoisensis, is preserved in the China General Microbiological Culture Collection Center (CGMCC), and has a preservation date of August 23, 2025 and a preservation number of CGMCC No. 35728.

[0009] The culture characteristics and identification basis of the strain NJAU-N6 are as follows: after being cultured at 25℃ for 2d on 1 / 10 TSB medium, the colony is round, gray, and the bacterial body surface is smooth and translucent; the 16s rRNA gene sequence of the strain is compared with similar sequences, and a phylogenetic tree is constructed, and the result shows that the homology of the strain with Paenibacillus illinoisensis is 96.73%; combined with the colony morphology and 16s rRNA phylogenetic tree analysis, the strain is identified as Paenibacillus illinoisensis.

[0010] The bacillus NJAU-N6 is applied to promoting soil nitrogen fixation and / or promoting plant growth.

[0011] The bacillus NJAU-N6 is applied to preparing a microbial inoculant for promoting soil nitrogen fixation and / or promoting plant growth.

[0012] The bacillus NJAU-N6 is applied to preparing a microbial inoculant for promoting soil nitrogen fixation and / or promoting plant growth.

[0013] The preparation method of the microbial inoculant.

[0014] The microbial inoculant is preferably prepared mainly by the following method: the bacillus NJAU-N6 is inoculated into TSB liquid medium, and cultured at 25-28℃ and 170-190r / min for 2-3d to obtain a fermentation broth; the fermentation broth is subjected to centrifugal treatment, and the precipitate is collected and adjusted to a bacterial body concentration of 1×10 9 CFU / mL or more, i.e. the microbial inoculant is obtained.

[0015] The application of the bacillus NJAU-N6 microbial agent in promoting soil nitrogen fixation and / or promoting soybean growth.

[0016] Advantages of the present application:

[0017] The bacillus NJAU-N6 is screened from a soil sample in a semi-arid sandy area, and compared with conventional nitrogen-fixing and growth-promoting bacteria, the strain has stronger low-temperature tolerance, drought resistance, and stronger environmental adaptability; in practical application, it can effectively increase the soil nitrogen content and promote the growth of soybeans, and has good popularization value in the black soil fertilization and crop yield increase in the semi-arid sandy area of northeast China. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 OD values of different strains after 15 DEG C low-temperature culture and 28 DEG C normal culture for 48h 600 Note: Different letters above the column chart are significant (P<0.05) differences defined by one-way ANOVA, and significant difference comparison is performed among the five different treatments at the same temperature.

[0019] Figure 2 OD values of different strains after 48h culture in medium with PEG600 concentration of 0, 10%, 20%, and 30% 600

[0020] Figure 3 Influence of different microbial agent treatments on the 30d soybean height

[0021] Figure 4 Influence of different microbial agent treatments on the 30d soybean aboveground and underground fresh weight

[0022] Figure 5 Phylogenetic tree constructed based on the 16S rRNA gene sequence of the strain NJAU-N6

[0023] BIOLOGICAL MATERIAL PRESERVATION INFORMATION

[0024] The strain NJAU-N6, classified as Paenibacillus illinoisensis, is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology of Chinese Academy of Sciences, on August 25, 2025, with the preservation number of CGMCC No. 35728.​ Detailed Implementation

[0025] The following embodiments and accompanying drawings are intended to help in a better understanding of the present invention, but do not constitute a limitation thereof. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods; and the experimental materials used, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0026] The culture medium formulations involved in the following examples are as follows:

[0027] 1 / 10 TSA medium (1L): 1.5g tryptone, 0.5g soybean peptone, 0.5g sodium chloride, 15g agar powder.

[0028] TSA medium (1L): 15g tryptone, 5g soybean peptone, 5g sodium chloride, 15g agar powder.

[0029] TSB medium (1L): 15g tryptone, 5g soybean peptone, 5g sodium chloride.

[0030] Nitrogen-fixing medium (1L): Potassium dihydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, calcium carbonate 5g, mannitol 10g, calcium sulfate 0.1g

[0031] Example 1: Isolation, Screening and Identification of Functional Bacteria

[0032] 1. Preparation of gradient dilution solutions for soil samples

[0033] Soil samples were collected from the semi-arid sandy area of ​​Zhangwu County, Fuxin City, Liaoning Province. 10g of soil sample was weighed and placed in a sterile Erlenmeyer flask, along with 90mL of sterile water and 4-5 glass beads (6mm in diameter). After mixing, the sample was incubated at 25℃ and 170rpm for 30min. Dilution gradients of 10 were then prepared sequentially. -3 10 -4 10 -5 and 10 -6 A suspension.

[0034] 2. Plate coating and culture

[0035] A dilution gradient of 10 was selected. -3 10 -4 and 10 -5 The suspensions were plate-spread. After vortexing each dilution gradient, 100 μL of the suspension was spread evenly onto 1 / 10 TSA agar plates, with three replicates for each dilution gradient. After spreading, the plates were incubated at 30°C, and colony growth was observed and recorded every 24 hours.

[0036] 3. Separation, purification and preservation

[0037] The characteristics (color, shape, texture) of the colonies on the culture medium were observed, and single colonies with different morphological characteristics were selected and streaked onto nitrogen fixation medium and incubated in a 30°C incubator. The growth of the colonies was observed and recorded every 24 hours. The strains that could grow normally in the nitrogen fixation medium were preliminarily determined to have nitrogen fixation potential.

[0038] The strains with nitrogen fixation potential described above were each streaked onto a new 1 / 10 TSA medium plate, incubated at 30°C for 48 hours, and single colonies were then selected and streaked again onto a new 1 / 10 TSA medium plate for purification. The purified strains (bacteria) were inoculated into TSB medium, incubated at 30°C and 170 rpm until the logarithmic growth phase, and then mixed with 40% (v / v) glycerol at a volume ratio of 1:1, aliquoted into 2 mL sterile strain preservation tubes, and stored in a -80°C ultra-low temperature refrigerator for standby use. A total of 53 nitrogen-fixing bacteria were isolated from the soil in nine areas in the semi-arid sandy region.

[0039] 4. Screening of high-efficiency stress-resistant nitrogen-fixing bacteria

[0040] The nitrogen fixation capacity and low-temperature and drought tolerance of the nitrogen-fixing bacteria isolated above were determined, and a strain NJAU-N6 with excellent comprehensive performance was selected. The specific operation is as follows:

[0041] (1) Determination of nitrogen fixation capacity of strains

[0042] The strains were inoculated on nitrogen fixation medium plates and incubated at 20°C for 5 days. Then, 500 μL of 1 mg mL-1 methylene blue solution was added to each plate, and the plates were incubated at 30°C for 24 hours. The plates were then observed for the formation of transparent zones around the colonies. -1 Congo red solution was added to the plates, and after standing for 1 hour, the dye solution was discarded. Then, 500 μL of 1 mol L-1 NaCl solution was added, and the plates were placed for 1 hour. The formation of transparent zones around the colonies was observed. The diameters (mm) of the transparent zones and the colonies were measured, and the Hc value was calculated according to the following formula: -1 Hc value = transparent zone diameter / colony diameter

[0043] Hc value = transparent zone diameter / colony diameter

[0044] Five bacterial strains (strains NJAU-N6, FS-N4, FS-N5, ZW-N11, and XL-N22) with an Hc value (ratio of hydrolysis zone to colony diameter) greater than 1.7, as shown in Table 1, were selected for subsequent tests.

[0045] Table 1 Ratio of transparent zone to colony diameter of screened bacterial strains

[0046] Strain No. Colony growth diameter (d) Transparent circle diameter (D) Hc value (D / d) NJAU-N6 2.48 4.69 1.89 FS-N5 2.17 4.08 1.88 FS-N4 2.54 4.64 1.83 XL-N22 5.76 10.31 1.79 ZW-N11 4.12 7.29 1.77 ZW-N23 5.54 9.20 1.66 FS-N26 5.78 9.54 1.65 XL-N32 2.45 3.99 1.63 ZW-N51 2.48 4.02 1.62

[0047] (2) Determination of low-temperature tolerance of strains

[0048] Preparation of inoculant:

[0049] Five strains of nitrogen-fixing bacteria (strains NJAU-N6, FS-N4, FS-N5, ZW-N11, XL-N22) stored in glycerol tubes at -20°C were activated by streaking on TSA medium plates and incubated at 30°C for 24 h. Single colonies were inoculated into 3 mL TSB medium and incubated at 28°C, 160 rpm and 15°C, 160 rpm for 48 h, respectively. The growth of the strains was evaluated by measuring the OD 600 values of the culture solutions.

[0050] The results are shown in Table 1. Figure 1 After incubation at 15°C, 160 rpm for 48 h, the OD 600 value of strain NJAU-N6 was the highest (1.6), followed by strain FS-N4 (1.17), indicating that the growth of these two strains was better than that of the other strains under low-temperature conditions. Compared with the incubation conditions at 28°C, 160 rpm, the OD 600 values of the five strains decreased by 19% (NJAU-N6), 26% (FS-N4), 40% (FS-N5), 57% (ZW-N11), and 34% (XL-N22), respectively, among which strains NJAU-N6 and FS-N4 had the lowest decrease, indicating that they were less affected by low temperature and were more likely to stably colonize and function under low-temperature conditions.

[0051] (3) Determination of the drought tolerance of the strains

[0052] PEG6000 was used to simulate osmotic stress to construct an artificial drought environment to screen drought-tolerant bacteria. The activated five nitrogen-fixing strains described above were inoculated into TSB medium and incubated at 28°C, 150 r / min for 12-16 h. 50 μL of the bacterial solution was taken and inoculated into a shaking tube containing 3 mL of drought stress medium (TSB medium containing 0, 10%, 20%, or 30% PEG6000) and incubated at 28°C, 150 r / min for 48 h. The OD 600 value at 600 nm was measured to determine the growth of the strains and draw a growth curve.

[0053] The results are shown in Table 2. Figure 2 When the concentration of PEG6000 was 20%, the OD 600 values of strains NJAU-N6 and FS-N4 were significantly higher than those of the other strains (0.32 and 0.31, respectively), and even when the concentration of PEG6000 was increased to 30%, the OD 600 values of these two strains were still higher than those of the other strains (0.16 and 0.11, respectively). This indicates that strains NJAU-N6 and FS-N4 have better drought tolerance and are more likely to stably colonize and function under drought conditions.

[0054] Based on the above test results of (1), (2), and (3), strains NJAU-N6 and FS-N4 were selected for subsequent tests. After strain NJAU-N6 was cultured in 1 / 10 TSB medium at 25°C for 2 days, the colonies were round, gray, smooth, and translucent. Homology comparison of the 16s rRNA gene sequence and construction of a phylogenetic tree (results shown in FIG. 1) showed that the strain was identified as Paenibacillus illinoisensis. The strain was preserved in the China General Microbiological Culture Collection Center on August 23, 2025, and the preservation number was CGMCC No. 35728. Figure 5

[0055] Example 2 Effect of nitrogen-fixing bacterial strain NJAU-N6 inoculant treatment on soybean growth for 30 days

[0056] The nitrogen-fixing bacterial strain NJAU-N6 obtained in Example 1 was prepared into an inoculant as the experimental group, and sterile water was used as the blank control. A potting test was carried out, and 3 replicates were set for each treatment. The specific test steps were as follows:

[0057] Potting soil treatment: The sterile soil (pH 5.9) used in the potting test was collected from Zhangwu County, Fuxin City, Liaoning Province. After the soil was dried, it was sieved through a 10-mesh sieve and treated with gamma ray sterilization for standby use.

[0058] Inoculant preparation: The strain NJAU-N6 with the preservation number of CGMCC No. 35728 was inoculated into TSB liquid medium and cultured at 28°C and 180 r / min for 2 days to obtain a fermentation broth. After centrifugation at 6000 r / min for 10 min, the bacterial concentration was adjusted to 1×10 9 CFU / mL or more with sterile water to obtain the inoculant, which was used for standby.

[0059] Soybean seed treatment and seedling raising: The soybean variety "Dayu Lüxin" was selected. The soybean seeds were disinfected by soaking in a 5% sodium hypochlorite solution for 3 min, and then washed with deionized water for 2-3 times to remove the residual disinfectant on the seed surface. The disinfected seeds were sown in 50-hole seedling trays filled with sterilized seedling substrate, and cultivated until the soybean seedlings grew to the "two-leaf-one-heart" stage. Then, the soybean seedlings were transplanted into the pots (each pot was filled with 600 g of the above-mentioned sterile soil), and 1 soybean seedling was transplanted into each pot. After transplantation, the soil was watered thoroughly until it was moist. After the soybean seedlings grew stably for 1 week, the inoculation treatment was carried out at a ratio of 10%, and the control group was watered with an equal amount of sterile water. After the inoculation was completed, the soil was watered regularly to maintain the soil moisture, and the soybean was cultured in a 25°C greenhouse for about 3 weeks. The growth indicators of the soybean were measured.

[0060] (1) Measurement of soybean plant height

[0061] ​After 30d, the soybean plant height was determined, and the specific determination method was as follows: the height from the root of soybean to the highest leaf tip was measured with a ruler. The results are shown in Table 1. Figure 3 As shown in Table 1: the plant height of the control group (CK) soybean was 80.6 cm, and the plant height of the experimental group (NJAU-N6) soybean was 103.2 cm, which was 28% higher than that of the control group, and the plant height of soybean was significantly improved.

[0062] (2) Determination of fresh weight of aboveground and underground parts of soybean

[0063] The whole soybean plant was collected, washed and dried with absorbent paper, and the aboveground part (stem and leaf) and underground part (root) were separated with scissors, and the fresh weight of the two parts was determined.

[0064] The results are shown in Table 2. Figure 4 As shown in Table 2: the fresh weight of the aboveground part of the control group (CK) soybean was 5.54 g, and the fresh weight of the underground part was 2.23 g; the fresh weight of the aboveground part of the experimental group (NJAU-N6) soybean was 7.83 g, and the fresh weight of the underground part was 3.23 g, and the fresh weight of the aboveground and underground parts of soybean was significantly improved.

[0065] Example 3 Influence of microbial agent treatment on soil physical and chemical properties

[0066] The detection of sample physical and chemical properties was performed according to the standard of "Organic Fertilizer" (NY525-2012), and the specific determination indexes, methods and results are as follows:

[0067] (1) Determination of alkaline hydrolysis nitrogen content

[0068] The soil sample was dried and passed through a 100-mesh sieve, and the alkaline hydrolysis nitrogen content of the sample was determined by a flow analyzer (Vario EL, Germany).

[0069] The results are shown in Table 2. The alkaline hydrolysis nitrogen content of the control group (CK) soil was 70.3 mg / kg, and the alkaline hydrolysis nitrogen content of the treatment group inoculated with NJAU-N6 microbial agent was 90.9 mg / kg, which was 30% higher than that of the control group. Inoculation of NJAU-N6 microbial agent can significantly improve the alkaline hydrolysis nitrogen content in the soil.

[0070] (2) Determination of available phosphorus content

[0071] The dried soil sample was passed through a 20-mesh sieve, and concentrated sulfuric acid and hydrogen peroxide were added for digestion until it was nearly colorless and transparent. The digestion solution was then diluted to constant volume, and after standing, it was filtered to obtain the test solution. The test solution was taken, and the phosphorus content in the test solution was determined by vanadium molybdenum ammonium colorimetry with an enzyme marker, and each sample was set for 3 times of repetition.

[0072] The results are shown in Table 2: the available phosphorus content in the control group (CK) soil is 50.5 mg / kg, and the available phosphorus content in the experimental group (NJAU-N6) is 53.2 mg / kg, which is higher than that of the control group, indicating that inoculation of NJAU-N6 strain can also have a certain promoting effect on the available phosphorus content in the soil.

[0073] (3) Available potassium content determination

[0074] The air-dried soil sample was passed through a 20-mesh sieve, concentrated sulfuric acid and hydrogen peroxide were added for digestion, until it was nearly colorless and transparent, the digestion solution was removed, and after standing, it was filtered and diluted. The potassium content in the diluted solution was determined by flame photometry, and each sample was set for 3 replicates.

[0075] The results are shown in Table 2: the available potassium content in the control group (CK) soil is 98 mg / kg, and the available potassium content in the experimental group (NJAU-N6) is 121 mg / kg, which is 23.5% higher than that of the control group, indicating that NJAU-N6 bacterial agent can significantly increase the available potassium content in the soil.

[0076] Table 2: Physicochemical properties of soil in each treatment

[0077]

[0078] In summary, the bacterial agent prepared by strain NJAU-N6 can not only significantly increase the content of soil alkali-hydrolyzed nitrogen, available potassium and other nutrients, but also effectively promote the growth index of soybeans. At the same time, relying on the excellent low-temperature tolerance and drought resistance of the strain, it has a positive effect on enhancing the stress resistance of soybeans, and the overall application effect is outstanding.

Claims

1. A strain of Paenibacillus illinoisensis NJAU-N6, deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 25, 2025, with accession number CGMCC No. 35728.

2. The application of Paenibacillus illinoisensis NJAU-N6 as described in claim 1 in promoting soil nitrogen fixation and / or promoting plant growth.

3. The application according to claim 2, characterized in that, The plant in question is soybean.

4. The use of Paenibacillus illinoisensis NJAU-N6 as described in claim 1 in the preparation of microbial agents that promote soil nitrogen fixation and / or promote plant growth.

5. The microbial inoculant prepared from Paenibacillus illinoisensis NJAU-N6 as described in claim 1.

6. The microbial agent according to claim 5, characterized in that, The bacterial agent is mainly prepared by the following method: Paenibacillus illinoisensis NJAU-N6 (CGMCC No. 35728) is inoculated into TSB liquid medium and cultured at 25-28℃ and 170-190 r / min for 2-3 days to obtain the fermentation broth. After centrifugation, the bacterial concentration of the fermentation broth is adjusted to 1×10⁻⁶ with sterile water. 9 The microbial inoculant was obtained when the concentration of CFU / mL was above a certain level.

7. The method for preparing the microbial inoculant according to claim 5, characterized in that, The process includes the following steps: Paenibacillus illinoisensis NJAU-N6 (CGMCC No. 35728) is inoculated into TSB liquid medium and cultured at 25-28℃ and 170-190 r / min for 2-3 days to obtain the fermentation broth. After centrifugation, the bacterial concentration of the fermentation broth is adjusted to 1×10⁻⁶ cells / mL with sterile water. 9 The bacterial inoculum was obtained by obtaining a concentration of CFU / mL or higher.

8. The use of the microbial agent according to any one of claims 4-7 in promoting soil nitrogen fixation and / or promoting plant growth.

9. The application according to claim 8, characterized in that... The plant in question is soybean.

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