Azotobacter HWN14 with activity promoting function and application of azotobacter HWN14

Abstract

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to nitrogen-fixing bacteria HWN14 with activity promotion and application of the nitrogen-fixing bacteria HWN14. The nitrogen-fixing bacterium (Azobacter sp.) HWN14 is preserved in the Guangdong Microbial Culture Collection Center on December 26, 2024, and the preservation number of the nitrogen-fixing bacterium is GDMCC No: 65669. The strain HWN14 is a new species of nitrogen-fixing bacteria, and has various plant living promoting properties such as nitrogen fixation, phosphorus solubilization, potassium solubilization, siderophore production, indole-3-acetic acid (auxin, IAA) production and the like; the strain has the characteristics of no pollution, biological environmental protection and the like in the application process, and is a plant growth promoting strain with a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microorganisms, and particularly relates to a nitrogen-fixing bacterium HWN14 with plant growth-promoting activity and its application. Background Art

[0002] Nitrogen, phosphorus, and potassium are the three most important nutrient elements during the growth process of plants, and the three affect the growth, development, yield, and quality of crops. However, most farmland soils in China are nitrogen-deficient soils, and almost all need to be supplemented with nitrogen. More than 90% of the potassium in the soil exists in the form of mineral potassium, which is difficult for plants to directly absorb and utilize. In modern agricultural production, the demand for soil nutrient elements is generally solved by applying chemical fertilizers. However, the abuse of chemical fertilizers has also led to environmental pollution, soil compaction, etc., seriously hindering the sustainable development of the ecosystem.

[0003] Plant growth-promoting rhizobacteria (PGPR) are one of the raw materials of bio-agricultural preparations that are currently expected to replace or partially replace chemical fertilizers. Plant growth-promoting rhizobacteria can, through their own life activities, release nutrients existing in the form of compounds in the soil into the environment, increase the content of available nutrients such as nitrogen, phosphorus, and potassium in the soil, and have the function of promoting plant growth, including but not limited to decomposing organic phosphorus, decomposing inorganic phosphorus, nitrogen fixation, decomposing potassium, producing siderophores, degrading phenolic acids, producing IAA, producing ACC deaminase, etc. Nitrogen fixation can convert atmospheric N 2 into nitrogen-containing compounds that plants can absorb and utilize, such as ammonium salts, nitrates, etc., and can also produce substances such as organic acids during the nitrogen fixation process, improve the soil structure, and promote the growth of plant roots; Phosphorus solubilization can convert organic phosphorus compounds in the soil into phosphates, or convert inorganic insoluble phosphorus into soluble phosphorus, thereby promoting the dissolution and utilization of ineffective phosphorus in the soil by plants. Most of the potassium in the soil is bound in minerals and cannot be absorbed by plants; Potassium-solubilizing bacteria can decompose aluminosilicate minerals and convert mineral potassium in the soil into soil solution potassium, thereby promoting plant growth and development; Fe in the environment 2+ is easily oxidized into insoluble Fe 3+ , resulting in a lack of soluble iron and reducing the resistance of plants; Siderophores produced by microorganisms can bind to trivalent iron ions, convert insoluble iron into soluble iron that can be utilized by microorganisms, and provide the iron element required for microbial metabolism. Auxin IAA is a main regulator of plant growth, development, and stress response, and plant growth-promoting bacteria can promote plant growth by producing IAA.

[0004] Therefore, screening plant growth-promoting bacteria with functions such as nitrogen fixation, phosphorus solubilization, potassium solubilization, producing siderophores, and producing IAA has important research significance for aspects such as plant growth and development and environmental protection. Summary of the Invention

[0005] In view of the above-mentioned prior art, the first object of the present invention is to provide a nitrogen-fixing bacterium (Azotobacter sp.) HWN14 having multiple plant life-promoting activities such as nitrogen fixation, phosphorus solubilization, potassium solubilization, siderophore production and IAA production.

[0006] The second object of the present invention is to provide a fermentation culture of nitrogen-fixing bacteria HWN14.

[0007] The third object of the present invention is to provide applications of the above-mentioned nitrogen-fixing bacteria HWN14 and the above-mentioned fermentation culture.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a new species of nitrogen-fixing bacteria HWN14. The nitrogen-fixing bacteria (Azotobacter sp.) HWN14 has been deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC for short; address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Postal Code: 510070) on December 26, 2024, and its deposit number is GDMCC No: 65669.

[0010] Furthermore, the present invention provides a fermentation culture of the above-mentioned nitrogen-fixing bacteria HWN14.

[0011] Preferably, the fermentation culture is obtained by inoculating the nitrogen-fixing bacteria HWN14 into a fermentation medium and fermenting it.

[0012] Preferably, the fermentation medium is nitrogen-free Ashby medium or R2A medium.

[0013] Preferably, the culture conditions are: 30°C, 150-200rpm / min.

[0014] The present invention also provides an application of the above-mentioned nitrogen-fixing bacteria HWN14 or the above-mentioned fermentation culture, which is at least one of the following (1)-(17):

[0015] (1) Application in promoting plant growth;

[0016] (2) Application in the preparation of products that promote plant growth;

[0017] (3) Application in the production of siderophores;

[0018] (4) Application in the preparation of products for producing siderophores;

[0019] (5) Application in the preparation of products containing iron carriers;

[0020] (6) Application in increasing soil iron carrier content;

[0021] (7) Use in nitrogen fixation;

[0022] (8) Use in the preparation of products with nitrogen fixation ability;

[0023] (9) Use in phosphorus solubilization;

[0024] (10) Use in the preparation of products with phosphorus solubilization ability;

[0025] (11) Use in increasing the available phosphorus content in soil;

[0026] (12) Use in potassium solubilization;

[0027] (13) Use in the preparation of products with potassium solubilization ability;

[0028] (14) Use in increasing the potassium content in soil;

[0029] (15) Use in IAA production;

[0030] (16) Use in the preparation of products with IAA production ability;

[0031] (17) Use in increasing the IAA content in soil.

[0032] Preferably, in the above applications, the product is a bacterial agent, a microbial preparation or a bio-fertilizer.

[0033] Preferably, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0034] The present invention also provides a composition, comprising the above-mentioned nitrogen-fixing bacterium HWN14 or the above-mentioned fermentation culture.

[0035] The present invention has the following beneficial effects:

[0036] A nitrogen-fixing bacterium HWN14 was isolated and screened in the present invention. After identification, it has the highest homology with Azotobacter nigricans subsp. nigricans IAM 15005 T The similarity of the 16S rRNA gene sequence is 97.7%, which is less than the species boundary value of 98.65%, indicating that strain HWN14 is a new species of the genus Azotobacter.

[0037] The strain HWN14 provided by the present invention has various plant growth-promoting activities such as nitrogen fixation, phosphorus solubilization, potassium solubilization, siderophore production, and IAA production; this strain has the characteristics of pollution-free and biological environmental protection during the application process, and is a plant growth-promoting strain with good application prospects; this strain enriches the resource library of growth-promoting bacterial strains and has important research significance in the development of microbial fertilizers, reducing the use of chemical fertilizers, and promoting plant growth. Brief Description of the Drawings

[0038] Figure 1 : A: The cell morphology of HWN14 on Ashby medium; B: The individual morphology of HWN14;

[0039] Figure 2 : The maximum likelihood (ML) phylogenetic tree constructed based on the 16S rRNA gene;

[0040] Figure 3 : Study on the plant growth-promoting characteristics of strain HWN14: A: Results of phosphorus solubilization experiment; B: Results of potassium solubilization experiment; C: Results of siderophore production experiment; D: Results of nitrogen fixation experiment; E: Results of IAA production experiment. Detailed Embodiments

[0041] To make the technical problems, technical solutions, and technical advantages to be solved by the present invention clearer, the following will be described in detail with specific examples. However, the protection scope of the present invention is not limited to the following specific embodiments. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments, and even less a limitation of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention. Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0042] The nitrogen-fixing bacterium (Azotobacter sp.) HWN14 provided by the present invention was deposited at the Guangdong Provincial Microbial Culture Collection Center (abbreviation: GDMCC; address: 5th Floor, Building 59, 100th Yard, Xianlie Middle Road, Guangzhou; postal code: 510070) on December 26, 2024, and its deposit number is GDMCC No: 65669.

[0043] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0044] The reagents and media used in the following examples are as follows:

[0045] Nitrogen-free Ashby solid medium: 10 g glucose, 0.2 g KH 2 PO 4 4 4 ·7H 2 O, 0.2 g NaCl, 0.2 gCaSO 4 ·2H 2 O, 5 g CaCO3 , 18 g of agar, made up to 1000 mL with deionized water; pH = 7.2, sterilized at 121 °C for 20 min.

[0046] Inorganic phosphorus medium: 5.0 g of Ca 3 (PO 4 ), 2 , 10.0 g of glucose, 0.5 g of (NH 4 ), 2 SO 4 , 0.3 g of NaCl, 0.3 g of KCl, 0.3 g of MgSO 4 ·7H 2 O, 0.03 g of MnSO 4 , 0.03 g of FeSO 4 ·7H 2 O, 0.5 g of yeast extract powder, 17.0 g of agar, made up to 1000 mL with deionized water; pH = 7.2, sterilized at 121 °C for 20 min.

[0047] Potassium medium: 10.0 g of sucrose, 0.5 g of yeast extract, 1.0 g of ammonium sulfate, 2.0 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 1.0 g of calcium carbonate, 1.0 g of potassium feldspar powder, made up to 1000 mL with deionized water; pH = 7.2, sterilized at 121 °C for 20 min.

[0048] CAS detection medium:

[0049] (1) CAS medium (g / L): glucose 2.0, casein 3.0, CaCl 2 0.1, MgSO 4 4.9, agar 17.0, sterilized at 121 °C for 20 min;

[0050] (2) CAS stain solution (g / L): resazurin 0.3, FeCl 3 0.1, cetyltrimethylammonium bromide 1.5, sterilized at 115 °C for 20 min;

[0051] (3) When cooled to 60 °C, slowly add 5 mL each of 0.1 mol / L phosphate buffer solution and 5% (v / v) CAS stain solution to the CAS medium, which is the CAS detection medium.

[0052] PC colorimetric solution: 12 g of FeCl 3 , 429.7 mL of 98% concentrated H 2 SO 4 4, made up to 1 L.

[0053] Example 1: Isolation and purification of strains

[0054] (1) Sample collection and pretreatment

[0055] Approximately 200 g of soil from a 10 - 20 cm soil layer was collected from the campus of Huizhou University in Huicheng District, Huizhou, Guangdong (23°2'31”N; 114°24'40”E). After removing visible roots and other debris, it was passed through a sieve with a 2 - mm pore size. The filtered soil was mixed evenly and left to air - dry naturally at room temperature.

[0056] (2) Isolation and purification of soil bacteria

[0057] Take 1 g of air - dried soil and put it into a 50 - mL centrifuge tube containing 9 mL of sterilized normal saline. Place the centrifuge tube on a shaker (oscillator) and shake it at 120 - 160 rpm for 2 h. In a laminar flow hood, use a pipette to suck 0.1 mL of the mixed soil suspension and put it into a centrifuge tube containing 0.9 mL of sterilized normal saline. After mixing, it is the 10 -1 dilution. According to the 1:10 ratio, prepare 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 gradient dilutions respectively. Use a pipette to suck 0.2 mL of different dilutions respectively and spread them evenly on the surface of nitrogen - free Ashby solid medium. Invert and place it in a 30℃ constant - temperature incubator and observe after culturing for different times. After colonies grow, pick single colonies with different morphological characteristics and streak - purify them on Ashby medium; preserve the purified strains in 30% glycerol and store them in a - 80℃ refrigerator.

[0058] Example 2: Identification of strain HWN14

[0059] (1) Morphological identification of strain HWN14

[0060] Figure 1 shows the morphological characteristics of the isolated strain HWN14, where Figure 1 A is the colony morphology of the strain cultured on nitrogen - free Ashby solid medium at 30℃ for 3 d. The cells are milky white, viscous, secrete a large amount of mucus, and can form an obvious transparent circle on the nitrogen - free medium; Figure 1 B is the cell morphological characteristics observed under a scanning electron microscope. The cells are oval, and the cell size: length is 1.3 - 1.6 μm, width is 0.9 - 1.0 μm.

[0061] (2) Physiological and biochemical characteristics of strain HWN14

[0062] The physiological and biochemical characteristics of strain HWN14 are shown in Table 1; the oxidase of strain HWN14 is negative, the reductase is positive, Gram - staining is negative, the growth temperature range is 15 - 37℃, it is intolerant to NaCl, grows on R2A medium, produces acid, and does not produce gas.

[0063] Table 1:

[0064]

[0065]

[0066] (3) Molecular identification of strain HWN14

[0067] Scrape a small amount of bacterial cells from the nitrogen-free Ashby solid medium, add 15 μL of sterile water, and heat-treat in a boiling water bath for 10 min to use this as the template for colony PCR reaction. Use the universal bacterial 16S rDNA 27F / 1492R primer pair to perform PCR amplification of the 16S rDNA of the strain.

[0068] PCR reaction system (20 μL): 2×Taq enzyme buffer: 10 μL, 27F: 0.5 μL, 1492R: 0.5 μL, template: 1 μL, ddH 2 O: 8 μL. PCR reaction conditions: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 1 min, 30 cycles; extension at 72 °C for 10 min.

[0069] Take 5 μL of the PCR product for agarose gel electrophoresis. After confirmation, the remaining samples are sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequenced sequences are spliced by SeqMan software to obtain the 16S rRNA gene sequence. Upload the sequence to the EzBiocloud (https: / / www.ezbiocloud.net / ) database and perform homologous sequence alignment with the known 16S rRNA genes. Select the standard strains with higher homology and construct a maximum likelihood method (ML) phylogenetic evolution tree.

[0070] The sequence alignment results show that HWN14 is a member of the genus Azotobacter, and has the highest sequence similarity with the standard strain Azotobacter nigricans subsp. nigricans IAM 15005 T at 97.7%, followed by Azotobacter armeniacus DSM 2284 T and Azotobacter salinestris ATCC 49674 T , with sequence similarities of 97.4% and 97.3% respectively.

[0071] The results of the ML phylogenetic tree are as Figure 2As shown, strain HWN14 and A. nigricans subsp. nigricans IAM15005 T , A. armeniacus DSM 2284 T and A. salinestris ATCC 49674 T cluster on a large branch.

[0072] Strain HWN14 was sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for whole-genome sequencing on the Illumina Hiseq platform. After filtering the raw genomic data for Reads, high-quality paired Reads were assembled into Scaffold data. In addition, the published genomic sequences of type species were downloaded from NCBI, and the average nucleotide identity (ANI) of the genomes was calculated using the online website ANIcalculator (https: / / www.ezbiocloud.net / tools / ani); the digital DNA-DNA hybridization value (dDDH) of the genomes was calculated through the online tool Genome-to-Genome Calculator GGDC (http: / / ggdc.dsmz.de / home.php). The ANI and dDDH values of nitrogen-fixing bacterium HWN14 and some related type species of nitrogen-fixing bacteria are shown in Table 2.

[0073] Table 2:

[0074]

[0075]

[0076] Table 2 shows that the ANI values of the genomes of HWN14 and some published type species are 82.5% - 88.9%, and the dDDH values are 26.0% - 36.5%. These ANI values and dDDH values are all lower than the standard values of 95% - 96% and 70% for identifying new species, further indicating that strain HWN14 is a new species of nitrogen-fixing bacteria.

[0077] Based on the comprehensive analysis of the morphological characteristics, physiological and biochemical characteristics, 16S rRNA gene sequence similarity, ML phylogenetic tree analysis, and the alignment results of genomic ANI and dDDH of the strain, it is shown that strain HWN14 of the present invention belongs to a new species of the genus Azotobacter, and its 16S rRNA gene sequence is as shown in SEQ ID NO.1 (Azotobacter sp. HWN14 16S rRNA gene sequence).

[0078] Example 3: Study on the growth-promoting activities of strain HWN14

[0079] (1) Qualitative study on growth-promoting activities

[0080] Strain HWN14 was inoculated onto nitrogen-free Ashby solid medium, inorganic phosphorus medium, potassium medium, and CAS detection medium respectively, and cultured at 30 °C for 2 - 5 d under constant temperature. Observe whether there is a clear zone formed, or whether the medium around the bacteria on the CAS detection medium turns red, to determine whether the strain has the abilities of dissolving inorganic phosphorus, decomposing potassium, nitrogen fixation, and producing siderophores.

[0081] In addition, strain HWN14 was inoculated into Ashby liquid medium supplemented with 100 mg / L L-tryptophan, and cultured at 30 °C and 150 r / min for 3 d, with Ashby liquid medium without bacteria as the control. Take 1 mL of the bacterial suspension and centrifuge it at 10000 r / min for 5 min. Mix the supernatant with PC colorimetric solution in a ratio of 1:1, and let it stand at room temperature for 15 min. Observe the color change. If it turns red, it is positive for IAA production; if it does not change color, it is negative.

[0082] The qualitative results showed that HWN14 could form obvious clear zones on inorganic phosphorus medium ( Figure 3 A), potassium medium ( Figure 3 B), and nitrogen-free Ashby solid medium ( Figure 3 D). On the CAS detection medium ( Figure 3 C), the medium around the bacteria turned red. Compared with the control in the IAA production experiment, the solution turned red ( Figure 3 D). The above results indicated that HWN14 had the abilities of dissolving phosphorus, decomposing potassium, producing siderophores, nitrogen fixation, and producing IAA.

[0083] (2) Quantitative detection of phosphorus-dissolving ability

[0084] The phosphorus-dissolving ability of HWN14 was determined by the molybdenum-antimony anti-colorimetric method. Strain HWN14 was inoculated into a conical flask containing 100 mL of PKO liquid medium at a ratio of 1%, with PKO medium without inoculation as the control. Cultured at 30 °C and 150 r / min for 3 d, centrifuged at 10000 r / min for 10 min at 4 °C, and the supernatant was taken. The molybdenum-antimony anti-colorimetric method was used to determine the increase in available phosphorus in the culture solution. The measurement results were the replicates of three experiments. The results showed that the phosphorus-dissolving amount after 3 d of culture was (25.5 ± 0.3) U / mL.

[0085] (3) Quantitative detection of siderophore production ability

[0086] Inoculate strain HWN14 into a nitrogen-free liquid medium at a ratio of 1%, culture it at 30 °C and 150 r / min for 3 days, take 1 mL of the bacterial cells, centrifuge at 10000 r / min for 2 min, and then take the supernatant. Mix the supernatant with the CAS detection solution at a ratio of 1:1 in a 96-well plate, and react for 1 h to measure the absorbance at OD 630 at 630 nm. Use the uninoculated liquid medium as a control. Calculation of the siderophore content: %Siderophore = (Ar - As) / Ar × 100%. Ar is the absorbance of the control, and As is the absorbance of the sample at OD

[0087] (4) Quantitative detection of the ability to produce IAA

[0088] Preparation of the standard curve: Prepare IAA solutions with concentrations of 0, 3, 6, 9, 12, 15, and 18 mg / L. Mix the IAA solution with the PC colorimetric solution at a ratio of 1:1, let it stand in the dark for 15 min, and then measure the OD 530 at

[0089] Inoculate strain HWN14 into Ashby liquid medium supplemented with 100 mg / L L-tryptophan, culture it at 30 °C and 150 r / min for 3 days, and use the Ashby liquid medium without bacteria as a control. Take 1 mL of the bacterial suspension, centrifuge at 10000 r / min for 5 min, mix the supernatant with the PC colorimetric solution at a ratio of 1:1, let it stand at room temperature for 15 min, and measure the OD 530 at

[0090] nm, substitute the measured absorbance value into the regression equation to calculate the IAA concentration produced by HWN14.

[0091] It can be understood that the above specific embodiments are further descriptions of the present invention and are not used to limit the protection scope of the present invention. For those skilled in the art, all other retouches and modifications obtained without creative labor fall within the protection scope of the present invention.

Claims

1. A nitrogen-fixing bacterium (Azotobacter sp.) HWN14, whose deposit number is GDMCC No: 65669.

2. The fermentation culture of the nitrogen-fixing bacterium HWN14 according to claim 1.

3. The fermentation culture of the nitrogen-fixing bacteria HWN14 according to claim 2, characterized in that: The fermentation culture is obtained by inoculating the nitrogen-fixing bacteria HWN14 into a fermentation medium and fermenting and culturing it.

4. The fermentation culture of the nitrogen-fixing bacterium HWN14 according to claim 3, characterized in that The fermentation medium is nitrogen-free Ashby medium or R2A medium.

5. The fermentation culture of the nitrogen-fixing bacterium HWN14 according to claim 4, characterized in that The culture conditions are: 30°C, 150-200rpm / min.

6. The use of the nitrogen-fixing bacteria HWN14 according to claim 1 or the fermentation culture according to any one of claims 2 to 5, which is at least one of the following (1) to (17): (1) Application in promoting plant growth; (2) Application in the preparation of products that promote plant growth; (3) Application in the production of siderophores; (4) Application in the preparation of products for producing siderophores; (5) Application in the preparation of products containing iron carriers; (6) Application in increasing soil iron carrier content; (7) Application in nitrogen fixation; (8) Application in the preparation of products with nitrogen fixation capability; (9) Application in phosphorus dissolution; (10) Application in the preparation of products with phosphorus dissolving ability; (11) Application in increasing the available phosphorus content in soil; (12) Application in potassium solution; (13) Application in the preparation of products with potassium dissolving ability; (14) Application in increasing soil potassium content; (15) Application in IAA production; (16) Application in the preparation of products capable of producing IAA; (17) Application in increasing soil IAA content.

7. The use according to claim 7, characterized in that: The product is a bacterial agent, a microbial preparation or a biological fertilizer.

8. A composition, characterized in that It comprises the nitrogen-fixing bacteria HWN14 described in claim 1 or the fermentation culture described in any one of claims 2-5.

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