CN111117929B

CN111117929B - Natural ammonium-resistant nitrogen-fixing paenibacillus AH-4 and application thereof

Info

Publication number: CN111117929B
Application number: CN202010060483.XA
Authority: CN
Inventors: 陈三凤; 李琴
Original assignee: China Agricultural University
Current assignee: Hubei Forbon Technology Co Ltd
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2022-02-01
Anticipated expiration: 2040-01-19
Also published as: CN111117929A

Abstract

The invention relates to the technical field of nitrogen-fixing microorganisms, in particular to a natural ammonium-resistant nitrogen-fixing paenibacillus AH-4 strain and application thereof. The invention provides a Paenibacillus sp AH-4 strain which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 18766. The bacterium contains a structural gene nifH of the azotobacter, can keep high azotobacter activity under the condition of high-concentration ammonium (50-300mM), breaks through the inhibiting effect of the high-concentration ammonium on biological azotobacter, can be popularized and used in agricultural production, and effectively reduces the using amount of fertilizer.

Description

Natural ammonium-resistant nitrogen-fixing paenibacillus AH-4 and application thereof

Technical Field

The invention relates to the technical field of nitrogen-fixing microorganisms, in particular to a natural ammonium-resistant nitrogen-fixing paenibacillus AH-4 strain and application thereof.

Background

Nitrogen is a major element essential for plant growth. The earth's surface is rich in nitrogen, but most of it exists in the atmosphere in the form of inert nitrogen gas and cannot be utilized by organisms. In order to ensure the high yield of grain crops, vegetables and fruits, a large amount of chemical nitrogen fertilizer is needed. However, the long-term excessive application of chemical nitrogen fertilizers can cause soil deterioration, environmental pollution and product quality reduction, and hinder the sustainable development of agricultural economy.

Biological nitrogen fixation refers to a process of reducing nitrogen in the air into ammonium by a few prokaryotic microorganisms under the action of in vivo nitrogen fixation enzymes. However, the biological nitrogen fixation efficiency is influenced by ammonium in the environment, and the high concentration of ammonium inhibits the nitrogen fixation effect. In other words, in poor soils, nitrogen fixing microorganisms can exert their nitrogen fixing effect sufficiently, whereas in fertile soils, nitrogen fixing microorganisms grow but not fix nitrogen. Generally, nitrogen-fixing microorganisms express nitrogenase in the presence of excess ammonium only if the ammonia assimilation mechanism is modified by chemical or genetic engineering. Almost all natural nitrogen-fixing bacteria can only fix nitrogen under low ammonium conditions. The nitrogen fixing activity of the currently reported nitrogen fixing microorganisms is low under the condition of high-concentration ammonium. The microorganism capable of efficiently fixing nitrogen under the condition of high-concentration ammonium is obtained, so that the nitrogen-fixing microorganism can fully play a nitrogen-fixing role in poor and fertile soil, and has important application value for reducing the using amount of chemical fertilizers in agricultural production.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide a natural ammonium-resistant Paenibacillus azotobacter AH-4 and application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides an ammonium-resistant nitrogen-fixing Paenibacillus (Paenibacillus sp.) AH-4 strain, which is obtained by separation from natural environment, is preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of West Lu No.1 of North Kyoho area of Tokyo, China academy of sciences, postal code 100101) in 2019, is named as Paenibacillus sp, and has a preservation number of CGMCC No.18766, within 10 months and 30 days.

The colony of the Paenibacillus sp (AH-4) on an LD solid culture medium is round, wet and white and has a complete edge.

The Paenibacillus sp AH-4 provided by the invention contains a structural gene nifH, and the nucleotide sequence for coding the gene is shown in SEQ ID NO. 1.

The 16S rDNA gene sequence of the Paenibacillus sp-4 is shown in SEQ ID NO. 2.

The invention also provides a microbial inoculum containing the ammonium-tolerant nitrogen-fixing bacillus (Paenibacillus sp.) AH-4.

In the invention, the microbial inoculum containing ammonium-tolerant nitrogen-fixing bacillus (Paenibacillus sp.) AH-4 can be a liquid microbial inoculum or a solid microbial inoculum. The microbial inoculum containing ammonium-resistant nitrogen-fixing bacillus (Paenibacillus sp.) AH-4 can be prepared by adding auxiliary materials allowed in the field of microbial preparations by adopting conventional technical means.

The azotobacter activity of the Paenibacillus (Paenibacillus sp.) AH-4 is obviously higher than that of the Paenibacillus strain Paenibacillus (Paenibacillus sp.) recognized in the field with the highest azotobacter activity in the Paenibacillus, namely the Paenibacillus durus ATCC35681, so that the Paenibacillus has higher application potential.

Experiments prove that the Paenibacillus sp AH-4 can tolerate50 to 300mM of high concentration ammonium, NH in the environment₄ ⁺When the concentration of the azotobacter reaches 50-300mM, the azotobacter can still keep higher azotobacter activity and carry out high-efficiency biological nitrogen fixation.

The invention provides application of the Paenibacillus sp AH-4 or a microbial inoculum containing the Paenibacillus sp AH-4 in biological nitrogen fixation.

In one embodiment of the present invention, the biological nitrogen fixation is performed under a low ammonium concentration condition in which the ammonium ion concentration is 0 to 5 mM. Preferably, the biological nitrogen fixation is performed under the condition that the concentration of ammonium ions is 0-4 mM. More preferably, the concentration of ammonium ion is 0-1 mM.

As another embodiment of the present invention, the biological nitrogen fixation is biological nitrogen fixation under a high ammonium concentration condition that the ammonium ion concentration is more than 20 mM.

The ammonium ion concentration in the high ammonium concentration condition where the ammonium ion concentration is more than 20mM may be 30 to 450 mM. Preferably 50 to 400 mM. More preferably 50 to 300 mM.

The invention provides application of Paenibacillus sp AH-4 or a microbial inoculum containing the Paenibacillus AH-4 in fertilizer preparation.

The invention provides a biological fertilizer which comprises the Paenibacillus (Paenibacillus sp.) AH-4 or a microbial inoculum containing the Paenibacillus AH-4.

The invention provides application of the Paenibacillus sp AH-4 or a microbial inoculum containing the Paenibacillus AH-4 or the biological fertilizer in plant cultivation.

The invention provides application of the Paenibacillus sp AH-4 or a microbial inoculum containing the Paenibacillus sp AH-4 in breeding nitrogen-fixing microorganisms.

The application in breeding the nitrogen-fixing microorganisms can be breeding the nitrogen-fixing microorganisms by utilizing Paenibacillus sp (AH-4) through breeding methods such as mutagenesis, genetic engineering transformation and the like.

The invention provides application of the paenibacillus AH-4 or a microbial inoculum containing the paenibacillus AH-4 or the biological fertilizer in promoting plant growth or improving plant yield.

Preferably, the above application is the administration of Paenibacillus sp (Paenibacillus sp.) AH-4 to the plant.

The present invention also provides a method for promoting plant growth or increasing plant yield, comprising: promoting plant growth or increasing plant yield by applying the Paenibacillus sp AH-4 or a fungicide comprising the Paenibacillus sp AH-4 to the plant.

Preferably, the Paenibacillus sp AH-4 is applied by soaking seeds with a suspension of the Paenibacillus sp AH-4 or irrigating the plant with a suspension of the Paenibacillus sp AH-4; or applying a fertilizer to the plant in combination with a suspension of Paenibacillus AH-4.

The invention has the beneficial effects that: the invention provides a natural ammonium-resistant nitrogen-fixing Paenibacillus AH-4 strain, which can keep higher nitrogen-fixing activity under the condition of high-concentration ammonium and carry out high-efficiency biological nitrogen fixation (the nitrogen-fixing activity of the strain under the ammonium-free condition is 7689.2nmol C)₂H₄Permg protein h, nitrogenase activity in 100mM ammonium enrichment of 4124.59nmol C₂H₄Permg protein h, while the prior art natural nitrogen-fixing bacteria can only be generally 0-5 mM NH₄ ⁺Has azotobacter activity in the range), breaks the inhibiting effect of high ammonium condition on biological nitrogen fixation, ensures that the azotobacter can fully play the nitrogen fixation effect in poor and fertile soil, and has wide application prospect in agricultural production.

Drawings

FIG. 1 shows the colony morphology of the strain Bacillus Paenibacillus sp (Paenibacillus sp.) AH-4 of example 1 of the present invention on nitrogen-free plates.

FIG. 2 is an electrophoretogram of amplified products of nifH and 16S rDNA of the strain Bacillus sphaericus sp AH-4 in example 1 of the present invention;

FIG. 3 shows the nitrogenase activity of the strain Paenibacillus sp (Paenibacillus sp.) AH-4 in example 2 of the present invention at different ammonium concentrations.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 isolation, culture and characterization of Paenibacillus AH-4

1. Paenibacillus AH-4 isolation

Paenibacillus AH-4 was isolated from a sample of rhizosphere soil from trees in Hefei city, Anhui province.

2. Cultivation of Paenibacillus AH-4

Preparation of a culture medium: nitrogen-free liquid medium (1L): 20g of sucrose; 12.06g K₂HPO₄；3.4g KH₂PO₄；0.2g MgSO₄·7H₂O；0.01g NaCl；0.01g FeCl₃；0.002g NaMoO₄·2H₂O; adding H₂And O is metered to 1L. Sterilizing at 115 deg.C for 30 min. The solid medium was prepared by adding 1.5g of agar per 100ml of liquid medium.

Paenibacillus AH-4 is inoculated into a solid or liquid culture medium and cultured in an incubator at 30 ℃ for 2 days. The colony morphology of Paenibacillus AH-4 on nitrogen-free medium is shown in FIG. 1.

3. Identification of Paenibacillus AH-4

(1) Extraction of total DNA of bacteria: genomic DNA was extracted from a pure culture of the strain Paenibacillus AH-4 using a bacterial genome extraction kit (Tiangen corporation) and was performed according to the kit instructions.

(2) Identification of strains

Taking the bacterial genome DNA extracted in the step (1) as a template, and taking nifH F: GGCTGCGATCCVAAGGCCGAYTCVACCCG and nifH R: CTGVGCCTTGTTYTCGCGGATSGGCATGGC is a primer (wherein V represents A, G, C; Y represents C, T; and S represents G, C), the NIfH structural gene of the azotobacter is amplified by PCR, and a band with the size of about 295bp is obtained. The band was recovered and purified, and then sent to a company for sequencing. The nucleotide sequence of the gene nifH is shown in SEQ ID NO. 1. The sequencing results were aligned by on-line BLAST in the GenBank database and showed 98.26% homology to Paenibacillus zanthoxyli JH 29.

Taking the bacterial genome DNA extracted in the step (1) as a template, and designing a primer 16S F by using a 16S rDNA sequence of a model strain escherichia coli of prokaryotes: AGAGTTTGATCCTGGCTCAGAACGAACGCT and 16SR: TACGGCTACCTTGTTACGACTTCACCC, PCR amplified the 16S rDNA sequence. As a result, a band of about 1.5kb was obtained, and this band was recovered, purified, and subjected to sequencing. The 16S rDNA sequence is shown in SEQ ID NO.2, and the sequencing result is subjected to online BLAST comparison, so that the strain belongs to Paenibacillus (Paenibacillus).

The amplification system of the PCR is as follows: 10 × PCR buffer: 5 μ L, dNTP: 1.25 μ L, forward primer: 1.25 μ L, reverse primer: 1.25. mu.L, template DNA: 5 μ L, Taq DNA polymerase: 0.75. mu.L.

The amplification conditions for the above PCR were as follows:

amplification of nifH gene:

amplification of 16S rDNA:

the result of the electrophoresis detection of the PCR amplification product of the structural gene nifH of the azotobacter and the 16S rDNA is shown in figure 2.

Paenibacillus (Paenibacillus sp.) AH-4 is stored in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of West Siro 1 of Beijing Korean district, Microbiol institute of Chinese academy of sciences, postal code 100101) in 2019, 10 months and 30 days, and is named as Paenibacillus sp with the storage number of CGMCC No. 18766.

EXAMPLE 2 determination of the Nitrogen-fixing enzyme Activity of Paenibacillus AH-4 under different ammonium concentrations

1. Determination of nitrogenase Activity

Inoculating Paenibacillus AH-4 into 5mL LD culture medium, culturing at 30 deg.C overnight, transferring into 500mL triangular flask according to 1% inoculum size, culturing at 30 deg.C for 8 hr, collecting thallus, and adding appropriate amount of culture medium with different ammonium concentrations (basic culture medium for measuring enzyme activity and adding NH with different concentrations)₄Cl) suspension of the cells, adjustment of OD₆₀₀To 0.4. Inoculating 4mL of bacterial liquid into an anaerobic culture tube, pumping out air by using an air extractor, introducing argon, injecting 10% acetylene into the anaerobic tube, culturing at 30 ℃, and injecting 100 mu L of gas into a gas chromatograph every 2 hours to measure the ethylene content.

The minimal medium (1L) for measuring the enzyme activity is as follows: 26.3g Na₂HPO₄·12H₂O；3.4g KH₂PO₄(ii) a 10 μ g biotin; 26mg of CaCl₂·2H₂O；30mg MgSO₄；0.33mg MnSO₄·H₂O; 36mg ferric citrate; 7.6mg Na₂MoO₄·2H₂O; 10 μ g of p-aminobenzoic acid; 0.3g glutamic acid; 4g glucose. Sterilizing glutamic acid and glucose at 115 deg.C for 30min, adding into the mixture, and sterilizing other reagents at 121 deg.C for 20 min.

2. Method for determining protein content (Bradford,1976)

(1) Preparation of solutions

Bradford stock: 100mL of 95% ethanol, 200mL of 88% phosphoric acid, 350mg of Coomassie Brilliant blue G250;

bradford working solution: 425mL of double distilled water, 15mL of 95% ethanol, 30mL of 88% phosphoric acid, 30mL of Bradford stock; filter paper, store in brown bottle, and stand at room temperature. Can be stored for several weeks, but is filtered before use.

(2) Preparation of Standard Curve

1mL of Bradford working solution was put in a test tube, and 8. mu.L of 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, and 1.0mg/mL BSA solutions were added, mixed well, left to stand for 3 to 5min, and developed into blue. Determination of OD₅₉₅The value is obtained. As a control, 1mL of Bradford working solution to which 8. mu.L of water was added was used. By OD₅₉₅Values are plotted on the ordinate and BSA solution concentration is plotted on the abscissa. The standard curve equation y is obtained as 0.6.5853x-0.1067, R²＝0.9986。

(3) Protein content detection

The cells were collected by centrifugation, 200. mu.L of 0.5M NaOH was added and boiled for 5min, 200. mu.L of L0.5M HCl was added and mixed well, 8. mu.L of supernatant was centrifuged and extracted, and added to 1mL of Bradford working solution, mixed well, left to stand for 5min and developed. Determination of OD₅₉₅The value is obtained. Will OD₅₉₅Values were taken into the standard curve equation and protein concentrations were calculated.

3. Calibration of 1nmol ethylene

(1) Preparing 2 120mL serum bottles marked as 1# and 2# bottles;

(2) filling a serum bottle with water, plugging the serum bottle with a rubber plug inserted with a needle head to prevent bubbles from generating, taking down the rubber plug, pouring out 100mL of water (measured by a volumetric flask), and replacing with a new rubber plug, wherein the volume of air in the bottle is 100 mL;

(3) 2.24mL (2.24 mL in the standard case, and the injection amount is calculated according to the formula: PV ═ nRT in the non-standard case) of ethylene was injected into the 1# bottle, 1mL of gas was taken out from the 1# bottle and injected into the 2# bottle, 100. mu.L of gas was taken out from the 2# bottle and introduced into a gas chromatograph (model HP6890), and the peak area indicated was the amount of 1nmol of ethylene, from which it was possible to calculate how much nmol of ethylene was represented per unit peak area on the recording sheet.

(4) The calculation formula of the azotase activity is as follows:

the results of the azotobacter activity test of the Paenibacillus AH-4 strain under different ammonium concentration conditions are shown in FIG. 3, and the AH-4 strain has the highest azotobacter activity under the ammonium-free (0mM) condition, and the azotobacter activity thereofThe activity was 7689.2nmol C₂H₄/mg protein h; the azotase activity is very low under the condition of 5-20mM ammonium concentration; but the nitrogen-fixing enzyme activity is recovered with the continuous increase of the ammonium concentration, and higher nitrogen-fixing activity occurs under the condition of 100mM ammonium ion concentration, and the nitrogen-fixing enzyme activity is 4124.59nmol C₂H₄/mg protein h; and can still maintain higher nitrogen fixation activity under the condition of 200mM ammonium ion concentration, and the nitrogen fixation activity of the nitrogen fixation enzyme is 2336.7nmol C₂H₄/mg protein h; the azotase activity under the condition of 300mM ammonium ion concentration is 1087.87nmol C₂H₄/mg protein h。

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims

1. An ammonium-resistant nitrogen-fixing bacillus (Paenibacillus sp.) AH-4 strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 18766.

2. A microbial agent comprising the Paenibacillus sp (Paenibacillus sp.) AH-4 of claim 1.

3. Use of the Paenibacillus sp AH-4 of claim 1 or the microbial inoculum of claim 2 for biological nitrogen fixation.

4. The use according to claim 3, wherein the biological nitrogen fixation is biological nitrogen fixation under the condition of low ammonium concentration with ammonium ion concentration of 0-5 mM.

5. The use according to claim 3, wherein the biological nitrogen fixation is biological nitrogen fixation at high ammonium concentrations greater than 20mM ammonium ion concentration.

6. The use according to claim 5, wherein the high ammonium concentration condition has an ammonium ion concentration of 30 to 450 mM.

7. The use according to claim 6, wherein the high ammonium concentration condition has an ammonium ion concentration of 50 to 400 mM.

8. Use of a Paenibacillus sp AH-4 according to claim 1 or a bacterial agent according to claim 2 in the preparation of a fertilizer.

9. A biofertilizer characterized by comprising the Paenibacillus sp (Paenibacillus sp.) AH-4 according to claim 1 or the microbial agent according to claim 2.

10. Use of a Paenibacillus sp (AH sp) AH-4 according to claim 1 or a bacterial agent according to claim 2 or a biological fertilizer according to claim 9 for plant cultivation.

11. Use of the Paenibacillus sp (Paenibacillus sp.) AH-4 of claim 1 or the microbial inoculum of claim 2 for breeding nitrogen-fixing microorganisms.