CN116064293B

CN116064293B - Methanogen and application thereof

Info

Publication number: CN116064293B
Application number: CN202211083626.4A
Authority: CN
Inventors: 包智华; 曹佳慧; 张萌; 曹伟伟
Original assignee: Inner Mongolia University
Current assignee: Inner Mongolia University
Priority date: 2022-09-06
Filing date: 2022-09-06
Publication date: 2024-09-17
Anticipated expiration: 2042-09-06
Also published as: CN116064293A

Abstract

The invention relates to a methanotrophic bacterium and application thereof. The strain name of the methanotrophic bacteria is methyl campylobacter PRM1 (Methylosinus sp.PRM1), and the preservation number is CCTCC NO: m20211475. The strain provided by the invention can promote the growth of rice and simultaneously reduce methane emission and nitrogen fertilizer application, and the rice growth promoter prepared by the strain PRM1 is injected into the rhizosphere water culture solution of rice seedlings, so that the plant type and plant height of the rice can be obviously increased, the growth of the rice seedlings is promoted, the emission of greenhouse gases is reduced, and meanwhile, the use of chemical fertilizers is reduced.

Description

Methanogen and application thereof

Technical Field

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a methanogen and application thereof.

Background

The Chinese is the largest grain producing country and consuming country in the world, the grain safety of the Chinese faces multiple challenges such as rapid increase of grain demands, increasingly narrow yield improving space, continuously reduced cultivated area, increasingly scarce fresh water resources, serious ecological environment pollution and the like, and the grain safety problem needs to be solved urgently. Rice is a staple food for 60% of the world population and more than 65% of Chinese people. The sowing area of the rice field accounts for 20 percent of the world, and the annual nitrogen fertilizer application amount exceeds 500 ten thousand tons, thereby causing the pollution of soil and groundwater. Paddy fields are also one of the sources of methane emission, accounting for about 10% of the total methane emission. Methane emissions may lead to an increase in air temperature, thereby reducing rice yield. The rice yield was reduced by 3.2% per 1℃increase in predicted temperature. Thus, according to the expected population growth, environmental problems caused by rice field greenhouse gases and nitrogen fertilizers will become more serious. The reduction of methane and nitrogenous fertilizer in paddy fields is significant for the realization of the green ecological and global low-carbon emission reduction targets advocated by China.

Methane-oxidizing bacteria are bacteria that use methane as the sole carbon source and play a key role in reducing methane emissions in paddy fields. Methane emissions from paddy fields are those produced in anaerobic layers that are released to the atmosphere primarily through the rice aeration tissues. In this process, the root canal methane-oxidizing bacteria oxidize about 90% of the methane.

Plant growth promoting bacteria (plant growth promoting rhizobacteria, PGPR) are beneficial bacteria that can promote plant growth and its absorption and utilization of mineral nutrition, either living freely in the soil or attached to the plant root system. Azotobacter is a plant growth-promoting bacterium which has been studied in many cases, and among them, azospirillum, azotobacter (Azorcus), pseudomonas (Pseudomonas sp.), bacillus sp and the like have been studied in many cases for promoting growth of crops.

However, no report on growth promotion and nitrogen fixation of rice by methane-oxidizing bacteria is found so far.

Disclosure of Invention

In view of the problems existing in the prior art, the invention provides a methanotrophic bacterium and application thereof, the strain and the microbial inoculum thereof provided by the invention have the effect of promoting growth, simultaneously reducing methane emission and nitrogen fertilizer application, and the growth promoter which can be prepared by using the strain PRM1 can obviously increase the plant type and the plant height, thereby promoting the growth of seedlings, reducing the emission of greenhouse gases and simultaneously reducing the use of chemical fertilizers.

The technical scheme for solving the technical problems is as follows:

The invention provides a methanotrophic bacterium, the name of which is methyl campylobacter PRM1 (Methylosinus sp.PRM1), the preservation number of which is CCTCC NO: m20211475. The compound is preserved in China Center for Type Culture Collection (CCTCC) at the year 11 and 22 of 2021, and the preservation addresses are as follows: chinese university of Wuhan and Wuhan.

The methanotrophic bacteria provided by the invention are methyl campylobacter which can promote the growth of plants (such as rice), can reduce methane emission and nitrogen fertilizer application while promoting the growth, can be used for preparing a growth promoter, and can be injected into rhizosphere water culture liquid of seedlings to obviously increase plant type and plant height, so that the growth of the seedlings is promoted, and the use of chemical fertilizers is reduced while reducing the emission of greenhouse gases.

The research of the invention discovers that the methyl campylobacter PRM1 has good nitrogen fixation activity, can provide nitrogen for the attached plant root system, and the flagellum motility of the strain can find a proper living place for the plant root system, and is attached to the root surface or enters the root to enable the plant root system to obtain the energy required for nitrogen fixation through methane generated by the oxidized rice field. Therefore, the functional flora taking the rice rhizosphere methane oxidizing bacteria as the core can partially replace nitrogen fertilizer, promote rice growth to improve rice grain yield, solve the problem of serious grain shortage in China, reduce the use amount of chemical fertilizer, reduce the release of greenhouse gas, have important ecological environment significance, and also accord with the policy of ecological priority green development advocated by China.

The invention provides a microbial inoculum, which comprises the methanotrophic bacteria and/or fermentation liquor of the methanotrophic bacteria.

For example, the microbial inoculum can be used as a rice growth promoter, and the strain PRM1 or bacterial liquid thereof is used as an active ingredient. Other ingredients may be added in addition to the active ingredient. The concentration of the strain PRM1 bacterial liquid can be 1X 10 ⁸ CFU/mL.

The beneficial effects of adopting the technical scheme include: the growth promoter prepared from the strain PRM1 can obviously increase the plant type and the plant height, thereby promoting the growth of seedlings, reducing the emission of greenhouse gases and reducing the use of chemical fertilizers.

The invention provides a fermentation method of the methanotrophic bacteria, which comprises the following steps: inoculating the methanotrophic bacteria into a culture medium, and fermenting and culturing.

The invention provides the application of the methanotrophic bacteria in one or more of (1) to (6),

(1) Promoting plant growth;

(2) Preparing a plant growth promoter;

(3) Reducing methane emissions;

(4) The use amount of chemical fertilizers is reduced;

(5) Replacement or partial replacement of nitrogen fertilizer;

(6) The grain yield is improved.

Further, the plant is rice.

Further, the application in preparing greenhouse gas emission reduction rice growth promoting agent.

According to the invention, the rice seedlings are subjected to inoculation experiments, and the growth amount of rice inoculated with methane-oxidizing bacteria under the condition of applying nitrogen fertilizer with different contents is obviously higher than that of rice inoculated without methane, so that the rice seedlings are most obviously applied with a small amount of nitrogen fertilizer, and the fact that the methane-oxidizing bacteria plays a vital role in promoting the growth of rice is shown. Thus, the present invention provides the use of strain PRM1 in promoting rice growth. Can also be applied to reducing methane emission and increasing nitrogenous fertilizer in paddy fields.

The invention provides the application of the microbial inoculum in one or more of (1) to (6),

(1) Promoting plant growth;

(2) As plant growth promoters;

(3) Reducing methane emissions;

(4) The use amount of chemical fertilizers is reduced;

(5) Replacement or partial replacement of nitrogen fertilizer;

(6) The grain yield is improved.

Further, the plant is rice.

The invention provides a method for promoting rice growth, which comprises the following steps: the methanotrophic bacteria and/or the microbial inoculum are/is applied to rice.

Further, the bacterial suspension of the methanotrophic bacteria is injected into the water culture solution of the rhizosphere of the rice seedlings.

For example, the methanotrophic bacterium PRM1 can be prepared into a bacterial suspension with the final concentration of 1X 10 ⁸ CFU/mL, and the bacterial suspension is injected into the rhizosphere water culture solution of the rice seedlings, and 1mL of the rice salt-tolerant growth promoter (namely the bacterial preparation prepared by using the methanotrophic bacterium provided by the invention) can be injected into the rhizosphere of each rice seedling.

The invention has no special limitation on the rice varieties, and the rice varieties can be golden fragrance No.3 and No. 131, and can also be other varieties.

Drawings

FIG. 1 is a scanning electron microscope image of strain Methylosinus sp.PRM1.

FIG. 2 is a phylogenetic tree of strain Methylosinus sp.PRM1 and related bacteria based on the 16S rRNA gene sequence.

FIG. 3 is an optimum pH experiment for strain Methylosinus sp.PRM1.

FIG. 4 is an experiment of the optimal methane concentration of strain Methylosinus sp.PRM1.

FIG. 5 is an experiment of optimal salinity for strain Methylosinus sp.PRM1.

FIG. 6 shows the growth of golden yellow rice No. 3 under various treatments with different nitrogen contents.

FIG. 7 shows the growth of the empty 131 rice under different nitrogen contents.

FIG. 8 shows the experimental results of plant heights of golden fragrance No.3 (A) and empty cultivation No. 131 (B) rice under different nitrogen content conditions, wherein N+ represents inoculated bacteria and N-represents non-inoculated bacteria.

Detailed Description

The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.

Unless otherwise indicated, all reagents, methods and apparatus used in the present invention are those conventionally used in the art and the reagents and materials used in the present invention may be prepared by conventional methods or commercially available.

Liquid inorganic Nitrate Medium (NMS): the volume ratio of the solution 1 in the culture medium is as follows: solution 2: solution 3: solution 4=1000:0.1:1:1, ph=6.8 was adjusted, sterilization was performed at 121 ℃ for 20min, and solution 5 was added after the medium was returned to room temperature (10 ml of solution 5 was added per 1000ml solution 1).

Solid inorganic Nitrate Medium (NMS): 15g/L of Agar was added to the liquid inorganic nitrate medium.

NB medium: 10g/L peptone, 3g/L beef extract and 5g/L sodium chloride.

r-Taq mix：Takara，Japan。

DNA Marker 2000：Takara，Japan。

1 XTBE buffer: tris-base 54g/L, boric acid 27.5g/L, EDTA 0.5 mol/L20 mL pH=8.0.

10% Of carbon source nutrient solution: the original concentration of the carbon source was 10% (mass volume percentage).

Bacterial genome extraction kits were purchased from TIANGEN, beijing.

EXAMPLE 1 isolation and screening of strains

Collecting samples in the natural wetland of the inner Mongolian autonomous region and the Haote city Ha Suhai in the period of 4 months in 2015, digging all the whole reed roots, removing the soil which is outmost and has no root growth, collecting and exposing reed roots, rinsing off soil on the root surface by using sterile water, keeping the soil (rhizosphere soil) which is tightly attached to the roots, then placing the roots in a 50mL centrifuge tube, placing the centrifuge tube in a foam box filled with dry ice for fresh keeping, and taking the reed roots back to a laboratory in a short time.

The method for separating the rhizosphere soil from the root adopts an ultrasonic vibration cleaning-centrifugal removal method, about 40mL of deionized sterile water is added into a 50mL centrifuge tube filled with a root sample, ultrasonic vibration cleaning is carried out for 20min, then the centrifuge is carried out at 4000rpm on a high-speed centrifuge for 10min, the root is transferred to a new centrifuge tube, a precipitate is collected as a rhizosphere soil sample, the steps are repeated for more than 3 times until the soil outside the root is completely removed, and the obtained rhizosphere soil and root sample can be stored in the 50mL sterile centrifuge tube at the temperature of minus 8 ℃ for a long time for subsequent experiments.

The culture of methane-oxidizing bacteria usually uses an inorganic nitrate culture medium (NMS), the enrichment culture is initiated, reed roots are cut into short sections of about 1cm along the cross section under the aseptic operation condition, each section is longitudinally cut along the center to prepare root slices, the inside of the roots is directly contacted with the NMS solid culture medium, a culture dish cover is covered, the culture is placed in an airtight type drying incubator, air in the incubator is pumped out to half the pressure in the incubator by a water circulation type air pump, a methane gas tank (99.9% high-purity CH ₄) is connected, methane is introduced into the incubator to normal atmospheric pressure, at the moment, the volume fraction of the methane in the incubator is just 50%, the reed roots are placed in a biochemical incubator for constant-temperature enrichment culture for 2 weeks at 30 ℃, at the moment, a pressure gauge on the drying incubator is observed to be obvious negative pressure, and the primary presumption that a part CH ₄ is consumed by the growth of the methane-oxidizing bacteria is carried out. The root slices in the solid NMS culture medium and colonies growing around the root slices are all transferred into a 2ml sterile centrifuge tube by using a sterile toothpick, and 1ml liquid NMS culture medium is added to prepare an enrichment stock solution. And carrying out limiting dilution on the enriched stock solution obtained in the experimental process, namely carrying out gradient dilution on the concentration of the thalli from 10 ^-1 to 10 ^-9. Adding 200 μl of the enriched stock solution into a serum bottle (total volume 60 ml) filled with 20ml of NMS liquid culture solution to obtain culture solution with dilution degree of 10 ^-2, sequentially diluting to 10 ^-9 concentration by 10 times serial dilution method, sealing the bottle mouth of the serum bottle with a rubber plug, fixing with an aluminum cover, pumping 8ml of air with a disposable sterile syringe, injecting an equal volume of high-purity CH ₄, adjusting to 20% of final methane concentration in the serum bottle, and culturing samples with each dilution gradient in a shaking table at 30 ℃ and 200 rpm. And (3) rapidly carrying out gradient dilution on the culture solution with the highest dilution multiple for the second round after the culture solution is turbid, and repeating subculture for a plurality of times.

In the process of microbial dilution subculture, a proper amount of enrichment stock solution (about 10 μl) is coated and inoculated on solid NMS, and the solid NMS is placed in an airtight incubator filled with 50% CH ₄ gas to culture methane-oxidizing bacteria, and the temperature is set at 30deg.C. After bacterial colonies grow out on the culture dish, single bacterial colonies are picked, and strains with different bacterial colony forms are separated and purified by adopting a repeated streak transfer mode. Strains which grew well in solid NMS medium with CH ₄ but failed to grow in NB medium (without CH ₄) were selected, and strain PRM1 was finally obtained and initially identified as methane-oxidizing bacteria.

Example 2

1. Identification of Strain PRM1

General morphological characteristics of strain PRM 1: bacterial strain PRM1 has white colony on NMS solid culture medium, large colony, wet softness, round protrusion, regular edge, opacity and smooth surface.

2. Strain flagellum analysis

The strain PRM1 was cultured in NMS liquid medium at 30℃under 20% (v/v) methane at 150rpm for 2 days, and cells of the strain PRM1 were extracted and observed by using a field emission scanning electron microscope. Before using a scanning electron microscope, firstly, the silicon wafer is ultrasonically treated with water, then is ultrasonically treated with ethanol, and is dried by nitrogen. And (3) dripping the bacteria on a silicon wafer, then placing the silicon wafer in a dryer, placing the silicon wafer in a refrigerator at 4 ℃ until the silicon wafer is half-dried, dripping 2.5% glutaraldehyde solution for fixing for one hour, sucking residual liquid, and placing the silicon wafer in the dryer, and placing the silicon wafer in the refrigerator at 4 ℃. Sequentially dripping 30%, 50%, 75%, 95% and 100% ethanol for gradient elution, eluting for 10 min each gradient, and eluting twice with 100% ethanol. Cells grown in NMS broth were fixed in 0.1M phosphate buffer containing 2% (v/v) glutaraldehyde and washed in 0.1M cacodylate buffer when viewed by transmission electron microscopy. Thereafter, the bacterial cells were stained with 1% (w/v) uranyl acetate and observed with a transmission electron microscope at 100 kV.

The scanning electron microscope result is shown in figure 1, the strain PRM1 is rod-shaped, has the length of about 1.5-2.0 mu m, has flagella, and has the movement capability, so that favorable conditions are provided for the strain to find a proper methane living environment and communicate with plant root system information and enter plant roots.

3. Bacterial DNA extraction and PCR amplification of 16S rRNA gene sequence

The strain PRM1 isolated in example 1 was inoculated into NMS medium and placed in a methane tank, cultured at 30℃under 40% methane concentration for 3-5 days, the cells were collected, genomic DNA was extracted using a bacterial genome extraction kit of Takara Bio Inc., and PCR amplification of the 16SrRNA gene sequence was performed using the extracted DNA as a template, and the amplification primers were bacterial universal primers (manufactured by Biotechnology (Shanghai) Co., ltd.) and forward primer 27 (F): 5'-AGAGTTTGATCMTGGCTCAG-3'; reverse primer 1492 (R) 5'-GGTTACCTTGTTACGACTT-3'.

50. Mu.L PCR amplification system was used: 21. Mu.L of sterile water, 1. Mu.L of forward and reverse primers, 25. Mu. L r-Taq mix, 2. Mu.L of target microorganism genomic DNA.

Amplification reaction procedure: ① Pre-denaturation at 94℃for 2min, denaturation at ② 94 ℃for 30s, annealing at ③ 52 ℃for 30s, extension at ④ 72 ℃for 30s, and after 30 cycles of steps ② to ④, the system was continued to extend at 72℃for 10min and the amplified product was stored at 4 ℃.

PCR amplified products were detected by 1% agarose gel electrophoresis using DNA MARKER 2000,2000 as a reference and subjected to 100V constant pressure electrophoresis in 1 XTBE buffer for 20min. The PCR amplification product bands were then observed under a gel imaging system.

4. Strain 16S rRNA gene sequence sequencing and phylogenetic analysis

The PCR amplified products were sent to general biosystems for sequencing, and the obtained 16S rRNA gene sequences were aligned with BLASTN from NCBI (strain PRM1 has two different copies of the 16S rRNA gene, designated PRM1-1 and PRM1-2, respectively), and closely related species were selected to construct phylogenetic trees of the 16S rRNA gene sequences, as shown in FIG. 2, with a similarity of 99.1% between the 16S rRNA gene sequences of strain PRM1 and Methylococcus Methylosinus sporium NCIMB, belonging to Methylococcus. The strain is preserved in China Center for Type Culture Collection (CCTCC) in the year 2021, 11 and 22, and the strain is preserved with the number of CCTCC NO: m20211475, the preservation address is: the specific address of the university of Wuhan, wuhan is No. 299 in Wuhan, hubei province.

Example 3

1. Pre-cultivation of the strain PRM1

20ML NMS broth was added to a 60mL serum bottle, and the strain turbidity was added to give an initial turbidity (OD ₆₀₀) of 0.02. The rubber plug is clamped by the tweezers and put into the bottle mouth, and the bottle mouth is clamped by the clamp after the aluminum cover is covered, so that a closed space is ensured, and gas dissipation is prevented. After 10ml of air was drawn in by syringe, a corresponding volume of methane gas (99.0%) which had been sterilized by filtration through a 0.22 μm filter was flushed. The serum bottles were placed in a shaker and shake-cultured at 30℃and 150 rpm. After 2 days of culture the strain enters the logarithmic growth phase.

2. Experiment of carbon source utilization of Strain

The strain PRM1 was cultured in 32 carbon source nutrient solutions, respectively. A carbon source nutrient solution having an original concentration of 10% was prepared and sterilized by filtration using a 0.22 μm filter. The carbon source type is ethanol, formic acid, formamide, urea, methanol, dimethylamine, trimethylamine N-oxide, trimethylamine N-thio iodide, tetramethyl ammonium chloride, dimethyl carbonate, D-arabinose, D-xylose, D-fructose, D-glucose, sodium acetate, DL-lactic acid, dioctyl sebacate, DL-potassium sodium tartrate, sodium citrate, L-aspartic acid, L-glutamic acid, betaine, sodium oxalate, D-maltose, succinic acid, sucrose, sodium salicylate, DL-malic acid, malonic acid, D+ glucose, and arabinose. The final concentration of the carbon source in the culture solution is controlled to be 0.1%, and three parallel experiments are set for each carbon source. Adding the pre-cultured bacterial turbid liquid, and keeping the final turbidity (OD ₆₀₀) of the solution to be about 0.02. The culture was performed according to the strain preculture conditions described above.

After observation and growth amount (OD ₆₀₀) measurement for 9 days, when methane and methanol are the only carbon sources, the culture solution of the strain PRM1 has turbidity, which indicates that the strain can use methane and methanol as self-growth carbon sources. Under the culture conditions of other carbon sources, the strain culture solution is clear, the turbidity of the strain culture solution and the initial turbidity are not obviously changed, and the strain cannot utilize the carbon sources to maintain the self-growth, and the strain PRM1 is a proprietary methane-oxidizing bacterium.

3. Bacterial strain nitrogen source utilization experiment

The strain PRM1 was cultured and detected in 27 nitrogen source nutrient solutions, respectively. The 27 nitrogen sources are nitrogen-free, sodium nitrite, ammonium sulfate, hydroxylamine, L-alanine, DL-2-aminobutyric acid, DL-4-aminobutyric acid, DL-aminopentanoic acid, DL-5-aminopentanoic acid, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamine, hippurate, L-histidine, L-lysine, L-methionine, L-ornithine, L-proline, histamine, putrescine, L-tryptophan, ethanolamine, dimethylamine, and formamide, respectively.

A nitrogen source nutrient solution having an original concentration of 10% (i.e., an original concentration of the nitrogen source in the nitrogen source nutrient solution was 10%) was prepared and filtered and sterilized using a 0.22 μm filter membrane. To a 60mL serum bottle was added 20mL of N-free NMS broth (NMS broth without KNO ₃). Adding 10% of the original nitrogen source nutrient solution to make the final concentration of the nitrogen source be 0.1%. Three parallel experiments were set up for each nitrogen source. Adding the pre-cultured bacterial turbid liquid, and keeping the initial turbidity of the solution at about 0.02. After 10mL of air was withdrawn by syringe, a corresponding volume of methane gas (methane gas was filtered and sterilized with a 0.22 μm filter) was flushed. The culture was performed according to the strain preculture conditions described above.

Through observation and OD600 measurement, the strain PRM1 can grow under the condition that L-alanine, L-arginine, L-asparagine, L-citrulline, putrescine, histamine, L-glutamine, L-lysine, L-proline, L-tryptophan and the like are taken as nitrogen sources, and the strain can grow under the condition of no nitrogen, so that the strain has the nitrogen fixation capability.

4. Optimum pH test

NMS culture solutions were prepared at pH 4,5, 6, 7, 8, 9, and 10, respectively. NMS liquid medium of different pH was added to a 60mL serum bottle at 20mL, and three parallel experiments were performed for each pH gradient. Adding the pre-cultured bacterial turbid liquid, and keeping the initial turbidity of the solution at about 0.02. Methane concentration was 20%, and culture was performed according to the strain preculture conditions described above. Absorbance measurements were performed using Nano Photometer P-Class. The results showed that the strain grew the highest at pH 7 (FIG. 3).

5. Optimum methane concentration test

The bacterial turbid liquid which is pre-cultured is added into a 20mL NMS serum bottle, and the initial turbidity of the solution is kept to be about 0.02. And (3) respectively filling different volumes of methane to form a mixed gas system with the final concentration (v/v) of 10%, 20%, 40%, 60% and 90% of methane. The culture was performed according to the strain preculture conditions described above. Absorbance was measured using Nano Photometer P-Class. The results showed that strain PRM1 grew the highest under 20% CH ₄ and had an OD of 0.4 (FIG. 4).

6. Salinity test of strains

NMS liquid culture media with 8 salinity gradients were prepared separately. The NaCl concentration (w/v) was 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 5% culture broth, pH 7, three parallel experiments were performed for each salinity gradient. Adding the pre-cultured bacterial turbid liquid, and keeping the initial turbidity of the solution at about 0.02. Methane concentration was 20%, and culture was performed according to the strain preculture conditions described above. Absorbance measurements were performed using Nano Photometer P-Class. The results show that strain PRM1 grew best at a salinity of 0.1%, strain PRM1 grew well at a salinity of 0.5%, and growth was almost completely inhibited at a salinity of 1%.

Example 4 growth-promoting test of PRM1 Strain on Rice

Selecting rice seeds golden fragrance No. 3 and empty culture No. 131 which are consistent in size, respectively placing the rice seeds golden fragrance No. 3 and the empty culture No. 131 into a centrifuge tube filled with sterile water, and placing the centrifuge tube into a water bath kettle at 55 ℃ for water bath for 20min; pouring out water in the tube, adding 2.4% NaClO solution to soak the surface for sterilization for 15min, repeatedly washing the seeds with deionized water, soaking the seeds in the deionized water for 24h, and then placing the seeds on a culture dish paved with wet gauze to accelerate germination at 27 ℃ for 2d. Seeds with consistent buds are selected to be cultivated in the rice water culture solution with different nitrogen contents. Before the experiment, water culture solutions with different nitrogen contents (namely NMS liquid culture media with different nitrogen contents) are needed to be prepared for standby. The water culture liquid contains N, P, K, ca, mg, fe major elements, B, mn, cu, zn, mo trace elements and the like, the content of KNO ₃ in the high-nitrogen water culture liquid is 10% on the basis of NMS liquid culture medium, the content of KNO ₃ in the low-nitrogen water culture liquid is 1% on the basis of NMS liquid culture medium, and the nitrogen-free water culture liquid does not contain N on the basis of NMS liquid culture medium.

Preparing a rice growth promoting agent: inoculating the strain PRM1 to NMS liquid culture medium, charging 20% methane gas, and culturing at 180rpm and 30deg.C until OD ₆₀₀ is 1 to obtain bacterial liquid with concentration of 1.0X10- ⁸ CFU/ml; centrifuging the bacterial liquid at 5000rpm for 5min, discarding the supernatant, injecting an equal volume of 0.01M PBS buffer, oscillating and re-suspending, centrifuging at 5000rpm for 5min, discarding the supernatant, repeating for 2 times, and re-suspending to obtain the rice growth promoter.

Seeds with consistent sprouting are selected to be cultivated in rice water planting solutions with different nitrogen contents, 1ml of rice growth promoter is added into each 100ml of water planting solution, a group of 4 seedlings are arranged in experimental rice, and the samples are sampled after 7d treatment for index measurement. The whole culture process is carried out in an artificial intelligence climate incubator, and the temperature is as follows: day 26+ -5deg.C and night 21+ -5deg.C, relative humidity: 50% ± 10% in daytime and 60% ± 6% in night; illumination is carried out for 16 hours in the culture process dark for 8h.

As shown in FIG. 6, the growth of golden yellow rice No. 3 is obviously different under different nitrogen contents. Under the condition of different nitrogen content application, the plant height of the rice seedlings inoculated with the PRM1 growth promoting agent is obviously better than that of rice seedlings not inoculated with the growth promoting agent under the condition of methane existence, especially under the condition of low nitrogen, the difference is more obvious, and the two groups have no obvious difference under the condition of no methane existence. Fig. 8 (a) and table 1 show plant height data and growth promotion rate of golden fragrance No. 3 under various treatment conditions under different nitrogen contents after water culture for 7 days, and can be intuitively seen: under the condition of High Nitrogen (HN), in the presence of methane, the plants after inoculation grow from 17.9cm to 24cm, and the growth promotion rate of the treated group is 34% compared with that of the control group; the situation is more remarkable under the condition of Low Nitrogen (LN), the plant after inoculation grows from 12.2cm to 17.8cm, and the growth promotion rate is up to 46%; under the condition of no nitrogen, the plants after inoculation grow from 7.5cm to 10cm, and the growth promotion rate also reaches 33%. In contrast, the plant heights of the control group and the treatment group are almost the same under the condition of different nitrogen contents of the control group without methane, and the growth promotion rate is not obvious. Taken together, the data reveal that methane-oxidizing bacteria PRM1 plays a vital role in promoting the growth of golden yellow rice No. 3.

TABLE 1 plant height and growth promoting ratio of golden fragrance No.3 Rice under various treatments with different nitrogen contents

Similarly, the growth promoter has the same growth promoting effect on the rice of the No. 131 (figure 7), and the growth of the rice of the No. 131 is obviously different under different nitrogen contents. Under the condition of different nitrogen content application, the plant height of the empty-cultured rice No. 131 inoculated with the methane-oxidizing bacteria growth promoting agent in the presence of methane is obviously higher than that of the rice inoculated without methane, and the obvious difference exists. The results of fig. 8 (B) and table 2 show that: under the condition of High Nitrogen (HN), the plants after inoculation grow from 12.3cm to 16cm in the presence of methane, and the growth promotion rate of the treated group is 31% compared with that of the control group; the situation is more remarkable under the Low Nitrogen (LN) condition, the plants after inoculation grow from 10cm to 14cm, and the growth promotion rate reaches 40%; under the condition of No Nitrogen (NN), the plants after inoculation grow from 7.5cm to 9.1cm, and the growth promotion rate also reaches 21 percent. Namely, the methane-oxidizing bacteria PRM1 plays a vital role in promoting the growth of the empty-bred No. 131 rice.

TABLE 2 plant height and growth promoting ratio of empty 131 rice under various treatments with different nitrogen contents

According to the inoculation experiment of rice seedlings, the growth amount of rice inoculated with methane-oxidizing bacteria under the condition of applying nitrogen fertilizer with different contents is obviously higher than that of rice inoculated without methane, and the most obvious effect of the methane-oxidizing bacteria under the condition of applying a small amount of nitrogen fertilizer is suggested to play a vital role in promoting the growth of rice.

In conclusion, the inventor conjectures that the separated methane-oxidizing bacteria PRM1 strain has flagella and other motion chemotactic elements, so that the strain can be attached to the root surface or enter the root, and a good foundation is laid for information communication between the strain and plants and establishment of symbiotic relation. In addition, under the condition of applying nitrogen fertilizer with different contents, the growth amount of the rice inoculated with methane-oxidizing bacteria in the presence of methane is obviously higher than that of the rice inoculated without methane. Therefore, the functional flora part taking the rice rhizosphere methane oxidizing bacteria as the core is used for replacing nitrogen fertilizer, so that the growth of rice is promoted, the yield of rice grains is improved, the problem of shortage of serious grains in China is solved, meanwhile, the use amount of chemical fertilizer can be reduced, the release of greenhouse gases can be reduced, the method has important ecological environment significance, and the method also accords with the policy of ecological priority green development advocated by China.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form or details of construction and operation disclosed herein, and is intended to cover various modifications, equivalents, alternatives, variations, modifications, alternatives, combinations, and simplifications as may be included within the spirit and scope of the invention.

Claims

1. The methanotrophic bacterium is characterized by having a strain name of Methylotrichum PRM1 (Methylosinus sp.prm1) and a preservation number of CCTCC NO: m20211475.

2. A microbial inoculum comprising the methanotrophic bacterium of claim 1.

3. The method for fermenting methanotrophic bacteria of claim 1, comprising the steps of: inoculating the methanotrophic bacterium of claim 1 to a culture medium, and fermenting and culturing.

4. The use of the methanotrophic bacterium of claim 1 in one or more of (1) to (3),

(1) Promoting plant growth;

(2) Preparing a plant growth promoter;

(3) Instead of or in part of the nitrogen fertilizer.

5. The use according to claim 4, wherein the plant is rice.

6. The use of the microbial inoculum according to claim 2 in one or more of (1) to (3),

(1) Promoting plant growth;

(2) As plant growth promoters;

(3) Instead of or in part of the nitrogen fertilizer.

7. The use according to claim 6, wherein the plant is rice.

8. A method for promoting rice growth, comprising the steps of: applying the methanotrophic bacterium of claim 1 and/or the microbial inoculum of claim 2 to rice.

9. The method of claim 8, wherein the bacterial suspension of methanotrophic bacteria of claim 1 is injected into a rhizosphere hydroponic solution of rice seedlings.