Methylomonas sp. rsm1 and application thereof

Abstract

This invention provides a strain of methylmona RSM1 and its applications. The methylmona provided by this invention ( Methylomonas The *Methylmonas* strain RSM1, with accession number CCTCC No: M 20242683, is provided in this invention. This strain was screened from saline-alkali paddy fields in Inner Mongolia. It can utilize methane as a carbon source for growth, reducing methane emissions during rice cultivation and increasing rice yield. This invention also provides a compound inoculant of *Methylmonas* RSM1 and nitrogen-fixing bacteria. This compound inoculant significantly enhances the effects of methane emission reduction and rice yield increase, reducing methane emissions by up to 79.8% and increasing rice yield by 84.1%. This invention is of great significance for environmental protection and agricultural development.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural microorganisms, in particular to a strain of methylobacterium RSM1 and its application. BACKGROUND

[0002] Paddy fields are one of the main sources of greenhouse gas methane, accounting for about 20% of total methane emissions. The greenhouse effect caused by these methane will lead to global warming, which has a huge impact on agricultural development and human survival. During the cultivation of rice, the paddy field is in a long-term flooded condition, which isolates the air and soil, creating a perfect anaerobic environment. In this environment, the organic matter in the soil is decomposed by methanogens to release methane, and the organic matter secreted by rice roots provides a source of nutrition for methanogens, further increasing methane production.

[0003] There are currently three main methods to reduce methane emissions in paddy fields. The first is to develop water-saving irrigation techniques for paddy fields, such as sprinkling irrigation and drip irrigation, to reduce soil moisture content, increase air permeability, and reduce anaerobic environments. However, this method requires strict agricultural management and has high costs. The second is to change the rice variety and plant low-methane-emission rice, but the overall effect is not obvious. The third is to use biological control to adjust the soil microbial structure and increase the content of methane-metabolizing bacteria to directly reduce or metabolize the produced methane.

[0004] Methane-metabolizing bacteria are microorganisms that use methane as a carbon source and energy, and they inhabit the root zone, which is crucial for reducing methane emissions in paddy fields (Shinoda et al., 2019; Bao et al., 2014). For example, the applicant isolated and identified the aerobic methylobacterium sp. PRM1 (Patent Application No. 202211083626.4), which can metabolize methane and promote rice growth. However, nitrogen-fixing bacteria are generally believed to significantly improve rice yield, but most of them have been found not to have methane reduction effects after verification, such as the nitrogen-fixing bacteria (Methylobacterium sp.) SGS-4 and other nitrogen-fixing bacteria isolated by the applicant in the early stage. Methylosinus Azotobacter salinestris

[0005] In order to further expand the advantages of biological control and enhance the biological control effect of methane emissions, it is necessary to explore more microorganisms that can improve methane emissions, and to achieve the purpose of reducing methane emissions and promoting crop growth through the combined application of multiple microorganisms, so as to provide more help for environmental protection and crop yield increase through the complementary advantages or synergistic effects of strains. SUMMARY

[0006] The present application provides a strain of methylobacterium RSM1 and its application. The methylobacterium (Methylobacterium sp.) RSM1 has the advantages of being able to metabolize methane and promote rice growth, and has a synergistic effect with nitrogen-fixing bacteria (Methylobacterium sp.) SGS-4 and other nitrogen-fixing bacteria. Methylomonas sp ​​RSM1 has the effect of reducing methane emission and increasing rice yield, and the present application also finds that by combining RSM1 with nitrogen-fixing bacteria (SGS-4) Methylomonas sp , the above effects can be greatly enhanced. Azotobacter salinestris

[0007] In a first aspect, the present application provides a methanogen (RSM1) strain, wherein the methanogen (RSM1) is deposited with the China Center for Type Culture Collection (CCTCC) on December 2, 2024, and the strain deposit number is CCTCC NO: M 20242683, and the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. Methylomonas sp Methylomonas sp In a second aspect, the present application provides a culture method of the above-mentioned methanogen (RSM1), specifically, inoculating the methanogen (RSM1) activated to the logarithmic phase into an inorganic nitrate medium at an inoculation amount of 1%-5%, growing at a temperature of 28-32℃, and introducing methane at a final concentration of 15%-25%.

[0008] Preferably, the methanogen (RSM1) activated to the logarithmic phase is inoculated into the inorganic nitrate medium at an inoculation amount of 2%, grown at a temperature of 30℃, and methane is introduced at a final concentration of 20%. Methylomonas sp Methylomonas sp

[0009] Preferably, the methanogen (RSM1) activated to the logarithmic phase is inoculated into the inorganic nitrate medium at an inoculation amount of 2%, grown at a temperature of 30℃, and methane is introduced at a final concentration of 20%. Methylomonas sp

[0010] In a third aspect, the present application provides a bacterial agent, which comprises the above-mentioned methanogen (RSM1) or its freeze-dried bacterial body, or the bacterial liquid obtained by the above-mentioned culture method or the supernatant after centrifugation of the bacterial liquid, or the resuspension of the precipitate after centrifugation of the bacterial liquid. Methylomonas sp Methylomonas sp

[0011] In a fourth aspect, the present application provides a composite bacterial agent, which contains the above-mentioned methanogen (RSM1) or its freeze-dried bacterial body, or the bacterial liquid obtained by the above-mentioned culture method or the supernatant after centrifugation of the bacterial liquid, or the resuspension of the precipitate after centrifugation of the bacterial liquid, and Methylomonas sp Azotobacter salinestris nitrogen-fixing bacteria or its freeze-dried bacterial body, or its bacterial liquid or the supernatant after centrifugation of the bacterial liquid, or the resuspension of the precipitate after centrifugation of the bacterial liquid.

[0012] The nitrogen-fixing bacteria are nitrogen-fixing bacteria (SGS-4) with a deposit number of CCTCC NO: M20232631.

[0013] Preferably, the methanogen (RSM1) in the bacterial agent is the methanogen (RSM1) deposited with the China Center for Type Culture Collection (CCTCC) on December 2, 2024, and the strain deposit number is CCTCC NO: M 20242683. Methylomonas sp

[0014] Preferably, the nitrogen-fixing bacteria in the composite bacterial agent are nitrogen-fixing bacteria (SGS-4) with a deposit number of CCTCC NO: M20232631. Azotobacter salinestris ​​​​​​​sp) RSM1 viable bacteria content ≥1.0×10 8 cfu / mL, nitrogen-fixing bacteria ( Methylomonas sp SGS-4 live bacteria content ≥1.0×10⁻⁶ 8 cfu / mL.

[0015] This invention further provides a method for preparing the above-mentioned compound bacterial agent, specifically comprising: [the following steps are described in the original text, which is not directly related to the preceding paragraph and can be omitted:] Methylmonas ([…]) Azotobacter salinestris sp) RSM1 and nitrogen-fixing bacteria (sp) RSM1 and nitrogen-fixing bacteria Methylomonas sp After centrifuging the SGS-4 bacterial suspension at 6500-7500 rpm for 5-15 minutes, discard the supernatant and add sterile water to allow the Methylmonas bacteria to settle. Azotobacter salinestris sp) RSM1 and nitrogen-fixing bacteria (sp) RSM1 and nitrogen-fixing bacteria Methylomonas sp The OD value of the SGS-4 resuspension was 1.0, and Methylmonas hydrophila (Methylmonas hydrophila) was also included. Azotobacter salinestris sp) RSM1 and nitrogen-fixing bacteria SGS-4 ( Methylomonas sp The resuspensions were mixed. In this embodiment of the invention, the two bacterial resuspensions were mixed at a volume ratio of 1:1.

[0016] Fifthly, the present invention provides a product containing the aforementioned methylmonas ( Methylomonas sp sp) RSM1 or its lyophilized cells, or the bacterial culture obtained by the above-described culture method, or the supernatant after centrifugation of the bacterial culture, or the precipitate resuspension after centrifugation of the bacterial culture; and / or

[0017] The compound microbial agent contains the above-mentioned compound microbial agent or the compound microbial agent prepared by the above-mentioned preparation method.

[0018] The product is a bio-fertilizer, additive, or environmental protection agent.

[0019] The bio-fertilizer or fertilizer additive contains Methylmonas ( Azotobacter salinestris sp) RSM1 viable bacteria content ≥1.0×10 8 cfu / mL, nitrogen-fixing bacteria ( Methylomonas sp SGS-4 live bacteria content ≥1.0×10 8 cfu / mL.

[0020] Sixthly, the present invention provides the above-mentioned methylmonas ( Methylomonas sp The application of sp) RSM1 or its freeze-dried cells, or the bacterial solution obtained by the above culture method, or the supernatant after centrifugation of the bacterial solution, or the precipitate resuspension after centrifugation of the bacterial solution, or the above-mentioned bacterial agent or the above-mentioned product in reducing methane emissions during rice cultivation.

[0021] In a seventh aspect, the present invention provides the application of the above-mentioned compound microbial agent or the compound microbial agent or the above-mentioned product prepared by the above-mentioned preparation method in reducing methane emissions, increasing the number of rice tillers and / or increasing rice yield during rice cultivation.

[0022] In an eighth aspect, the present application provides a method for reducing methane emission during rice planting, specifically, inoculating the above-mentioned methylobacterium (Methylobacterium radiotolerans RSM1) or its freeze-dried bacterial body, or the bacterial liquid obtained by the above-mentioned culture method or the supernatant after centrifugation of the bacterial liquid, or the resuspension of the precipitate after centrifugation of the bacterial liquid, or the above-mentioned bacterial agent or the above-mentioned product to the rhizosphere of rice. Methylomonas sp In the examples of the present application, the above-mentioned inoculation is performed during the rice seedling raising period.

[0023] The present application further provides a method for simultaneously reducing methane emission during rice planting and increasing rice yield, specifically, inoculating the above-mentioned composite bacterial agent or the composite bacterial agent prepared by the above-mentioned preparation method or the above-mentioned product to the rhizosphere of rice.

[0024] The present application has the following beneficial effects:

[0025] (1) The present application discovers a methylobacterium (Methylobacterium radiotolerans RSM1), and application of the strain can reduce methane emission during rice planting and increase rice yield, and reduce methane emission by 12.1% and 15.2% respectively between the 65th day and the 79th day of rice planting, and increase the per mu yield of rice by 15.9%. Azotobacter salinestris (2) The present application mixes methylobacterium (Methylobacterium radiotolerans RSM1) and nitrogen-fixing bacteria (Azospirillum brasilense SGS-4) to prepare a bacterial agent, and application of the bacterial agent can produce significant synergistic effect, and reduce methane emission by 65.6% and 79.8% respectively between the 65th day and the 79th day of rice planting, increase the tiller number by 20.0 at the 79th day, and increase the final per mu yield of rice by 84.1%.

[0026] Figure 1 Figure 2 Figure 3 The present application further provides a method for simultaneously reducing methane emission during rice planting and increasing rice yield, specifically, inoculating the above-mentioned composite bacterial agent or the composite bacterial agent prepared by the above-mentioned preparation method or the above-mentioned product to the rhizosphere of rice. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the examples or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0028] Figure 4 is the 16S rRNA gene phylogenetic tree of the methanotrophic bacteria separation strain RSM1 provided by the present application.

[0029] Figure 5 ​​is the influence result figure of the complex microbial agent treatment of the methane-oxidizing bacteria RSM1 and the nitrogen-fixing bacteria SGS-4 provided by the present application on the methane emission of a rice field.

[0030] Figure 6 is the influence result figure of the complex microbial agent treatment of the methane-oxidizing bacteria RSM1 and the nitrogen-fixing bacteria SGS-4 provided by the present application on the methane emission reduction rate of a rice field.

[0031] Methylomonas sp is the influence result figure of the complex microbial agent treatment of the methane-oxidizing bacteria RSM1 and the nitrogen-fixing bacteria SGS-4 provided by the present application on the ear number per plant of a field rice.

[0032] Figure 1 is the influence result figure of the complex microbial agent treatment of the methane-oxidizing bacteria RSM1 and the nitrogen-fixing bacteria SGS-4 provided by the present application on the yield per plant of a field rice.

[0033] Methylomonas rhizoryzae is the influence result figure of the complex microbial agent treatment of the methane-oxidizing bacteria RSM1 and the nitrogen-fixing bacteria SGS-4 provided by the present application on the total yield per mu of a field rice. DETAILED DESCRIPTION

[0034] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0035] The inorganic nitrate medium (NMS) and the SSM medium used in the following embodiments have the formulations shown in Tables 1 and 2.

[0036] Table 1 NMS medium formulation

[0037]

[0038] The preparation method of the liquid inorganic nitrate medium (NMS) is as follows: after the medium is adjusted to pH = 6.8, sterilized at 121 ℃ for 20 min, and the medium temperature is below 40 ℃, solution 5 is added (10 ml of solution 5 is added to 1000 ml of solution 1).

[0039] Table 2 SSM medium formulation

[0040]

[0041] The preparation method of SSM medium is as follows: First, prepare 1g / 50ml of biotin stock solution, filter and sterilize. Add the components other than biotin according to the ratio, adjust the pH to 8.5, sterilize at 121 ℃ for 20 min, and add an appropriate amount of the filtered and sterilized 1g / 50ml biotin stock solution after the medium temperature has dropped below 30℃.

[0042] Example 1 Methylmonas ( Methylomonas sp Isolation, screening and identification of sp) RSM1

[0043] This strain was obtained in August 2021 from the rhizosphere soil of saline-alkali rice in Tuozuo Banner, Hohhot City, Inner Mongolia Autonomous Region, through screening, isolation, and purification. During sampling, the entire rice plant was dug up along with its roots, and the exposed rice roots (including the rhizosphere soil) were collected and placed in a 50 mL centrifuge tube containing 40 mL of sterile water. The tube was then placed in a foam box filled with dry ice for preservation and brought back to the laboratory within a short period of time.

[0044] The rhizosphere soil was separated from the roots using an ultrasonic washing-centrifugation method. Centrifuge tubes containing root samples were ultrasonically washed for 20 min, followed by centrifugation at 4000 rpm for 10 min to remove the roots and supernatant, thus obtaining the rhizosphere soil. This rhizosphere soil was used for subsequent isolation experiments of methylmonas and nitrogen-fixing bacteria.

[0045] Methylmonas hydrophila were cultured using NMS medium. Initially, 1 g of rhizosphere soil was placed in a 70 ml serum bottle containing 20 ml of NMS liquid medium. Then, 8 ml of air was removed using a sterile disposable syringe, and an equal volume of high-purity CH4 (99.9%) was injected to adjust the methane concentration in the serum bottle to 20%. The serum bottle was then placed at 30°C and cultured with shaking (200 rpm) for 10 days. Afterward, the enriched stock solution obtained from the above experimental process was subjected to extreme dilution, i.e., the bacterial concentration was reduced from 10... -1 Up to 10 -9 The gradient dilution process was performed. 200 μl of the enrichment stock solution was added to a serum bottle containing 20 ml of NMS liquid culture medium, resulting in a dilution of 10⁻⁶. -2 The culture medium was further diluted 10-fold serially to a final concentration. -9 For each concentration, the serum bottle opening was sealed with a rubber stopper and then secured with an aluminum cap. 20% of the air inside the serum bottle was replaced with methane. Samples of each dilution gradient were incubated at 30°C and 200 rpm in a shaker. Once the culture medium at the highest dilution became turbid, a second round of gradient dilutions was rapidly performed, and this process was repeated multiple times for subculturing.

[0046] During the process of microbial dilution subculture, take an appropriate amount of enriched stock solution (about 100 μl) and inoculate on solid NMS medium, place in airtight incubator filled with 40% CH4 gas to culture methanopilus, and the temperature is set to 30°C. After colonies grow on the culture dish, single colonies are picked and separated and purified by repeated streaking. Strains that grow well on solid NMS medium in the presence of CH4 and cannot grow on NB medium (without CH4) are selected, and finally strain RSM1 is obtained, which is preliminarily determined as a methanotroph.

[0047] The isolated strain RSM1 is inoculated into NMS medium and placed in a methane tank, and cultured at 30°C and 40% methane concentration for 3-5 days. The bacterial genome DNA is extracted using TIANamp Bacteria DNA Kit, and the extracted DNA is used as a template for PCR amplification of the 16S rRNA gene sequence with bacterial universal primers.

[0048] The above PCR amplification product is sent to General Biosystems for sequencing, and the obtained 16S rRNA gene sequence is compared with strain RSM1 using BLASTN of NCBI to screen species with close genetic relationship, and a phylogenetic tree of 16S rRNA gene sequence is constructed as shown in Methylomonas sp , the 16S rRNA gene sequence similarity of strain RSM1 and methanopilus Azotobacter salinestris GJ1 is 99.8%, belonging to the genus Methanopilus Figure 2 . The strain was deposited at China Center for Type Culture Collection (CCTCC) on December 02, 2024, with the strain deposit number CCTCC NO: M 20242683, and the deposit address is Wuhan University, Wuhan, China, the specific address is No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, China.

[0049] Example 2 Preparation of methanopilus (Methanopilus sp.) Figure 3 ) RSM1 and azotobacter (Azotobacter sp.) Bradyrhizobium, Rhizobium Figure 4 Preparation of composite microbial inoculant of methanopilus (Methanopilus sp.)

[0050] Primary seed culture of methanopilus RSM1: 20 μL of methanopilus strain was inoculated into a 70 ml serum bottle containing 20 ml of NMS liquid medium and cultured, and 20% methane gas with a purity of 99.9% was filled. In a 30°C incubator, the culture was shaken (150 rpm) to the logarithmic growth phase.

[0051] Methylobacterium RSM1 secondary seed culture: 5 ml of the first methylobacterium RSM1 seed liquid in the logarithmic growth phase was injected into a 500 ml serum bottle containing 250 ml of NMS liquid medium using a sterile syringe, and was cultured by oscillation (150 rpm) in 20% methane gas. A total of 4-8 bottles were used. The supernatant was removed after centrifugation of the above-mentioned bacterial liquid at 7000 rpm for 10 minutes. Sterile water was added to the remaining bacterial bodies to obtain a methylobacterium RSM1 suspension with an OD value of 1.0.

[0052] Azotobacter SGS-4 primary seed culture: 20 μL of azotobacter SGS-4 was inoculated into a 200 ml triangular flask containing 50 ml of nitrogen-free SSM liquid medium, and was cultured by oscillation (150 rpm) at 30°C until the logarithmic growth phase.

[0053] Azotobacter SGS-4 secondary seed culture: 5 ml of the first azotobacter liquid seed liquid was injected into a 500 ml serum bottle containing 300 ml of SSM liquid medium for culture. A total of 4-8 bottles were used. The supernatant was removed after centrifugation of the above-mentioned bacterial liquid at 7000 rpm for 10 minutes. Sterile water was added to the remaining bacterial bodies to obtain an azotobacter SGS-4 suspension with an OD value of 1.0.

[0054] Finally, the methylobacterium RSM1 and azotobacter SGS-4 suspensions were mixed at a ratio of 1:1 to obtain a liquid compound microbial agent.

[0055] Example 3: Experiment of compound microbial agent for reducing methane emission of rice

[0056] The compound microbial agent was poured into the roots of the seedlings in the greenhouse. Each dish (about 2000 plants) of the microbial agent group was poured with 1000 ml of the compound microbial agent (RSM1 and SGS-4), the RSM1 group and the SGS-4 group were poured with 1000 ml of the respective microbial suspension with an OD value of 1.0, and the blank control group was poured with an equal amount of water. After 5 days, the seedlings were transplanted to the field.

[0057] Methane emission determination method: The methane concentration was determined using a rectangular, transparent acrylic plate to seal the top chamber (30 cm long, 30 cm wide, 100 cm high). When the field was flooded, the chamber was gently placed on the four steel pipe bases fixed around a rice plant in advance, because the steel pipe bases were located 10 cm below the water surface, so the static chamber covered the rice to form a closed system. Each time, the methane flux sample was collected and measured at 10:00-12:00 am, and 20 ml of gas was taken from the headspace of the static chamber at 0, 10 and 20 minutes, respectively. Then the bottles were taken back to the laboratory, and the methane concentration was measured using a 2014 GC Shimadzu gas chromatograph with a flame ionization detector. The methane emission flux was calculated based on the increase in the concentration of the gas in the chamber per square meter and per hour.

[0058] Methane emission flux and reduction rate are shown in FIGS. 1-3, respectively. Figure 5 and Figure 6 The average methane emission flux (mg C / h / m 2 ) of the control group SGS-4, RSM1 and the complex microbial agent (RSM1+SGS-4) treated groups were 4.61, 5.02, 4.00, 2.13, respectively on the 65th day of planting, 7.98, 7.85, 3.27, 1.59, respectively on the 72nd day, 5.27, 5.87, 2.99, 1.32, respectively on the 79th day, and 4.18, 4.00, 3.41, 3.44, respectively on the 100th day. The methane emission flux of the rice rhizosphere inoculated with the complex microbial agent (SGS-4+RSM1) was significantly lower than that inoculated with the methanogen RSM1 and the nitrogen-fixing bacteria SGS-4 alone. Inoculation of the nitrogen-fixing bacteria SGS-4 alone had little effect on methane emission reduction, while inoculation of the methanogen RSM1 alone and the complex microbial agent (SGS-4+RSM1) reduced the methane emission flux by 12.1%-65.6% (2.99 mg C / h / m 2 - 4.00 mg C / h / m 2 ) and 15.2%-79.8% (1.32 mg C / h / m 2 - 2.13 mg C / h / m 2 ), respectively, during the rice heading stage, especially between the 65th and 79th day of planting, with the complex microbial agent achieving the highest reduction rate of 79.8%. Overall, the methane emission reduction effect of the complex microbial agent (SGS-4+RSM1) was better than that of the methanogen RSM1 and the nitrogen-fixing bacteria SGS-4 alone. The applicant also found that the methane emission reduction effect of the methanogen RSM1 and the nitrogen-fixing bacteria ​ used in combination was significantly worse than that of the complex microbial agent (SGS-4+RSM1).

[0059] Example 4: Experiment on the improvement of rice yield by the complex microbial agent

[0060] The complex microbial agent was poured into the roots of the greenhouse seedlings, 1000 ml of the complex microbial agent (RSM1 and SGS-4) was poured for each tray (about 2000 plants) of the microbial agent group, 1000 ml of the microbial suspension with an OD value of 1.0 was poured for each tray of the RSM1 group and the SGS-4 group, and an equal amount of water was poured for the blank control group. After 5 days, the seedlings were transplanted to the field, and the growth indicators such as tiller number and plant height were measured during different growth stages of the rice, and the yield was measured when the rice matured.

[0061] Rice growth index determination: On the 79th day, 10-12 rice plants were randomly sampled to determine plant height and tiller number. The average tiller number of the control, complex microbial agent (RSM1+SGS-4), nitrogen-fixing bacteria SGS-4 and methanotrophic bacteria RSM1 treatments were 18.6, 38.6, 36.8 and 19, respectively. Compared with the methanotrophic bacteria RSM1 treatment alone, the tiller number of the complex microbial agent (RSM1+SGS-4) and nitrogen-fixing bacteria SGS-4 treatments increased by 20.0 and 18.2, respectively. There was no significant difference in rice plant height among the four groups, indicating that the combined use of methanotrophic bacteria RSM1 and nitrogen-fixing bacteria SGS-4 not only did not reduce the growth-promoting effect of nitrogen-fixing bacteria SGS-4, but also improved the growth-promoting effect of nitrogen-fixing bacteria SGS-4.

[0062] Rice yield determination method: After the rice was basically mature, random sampling was used for sampling to determine the average single plant yield and single spike grain number of rice. In mid-October, a full-automatic harvester (PRO988Q, Kubota Corporation) was used for overall harvesting, and three plots were separately harvested and labeled for weighing.

[0063] The results of rice single plant spike number are shown in ​ The average single plant spike number of the control, nitrogen-fixing bacteria SGS-4, methanotrophic bacteria RSM1 and complex microbial agent (RSM1+SGS-4) treatments were 30, 40, 24 and 42, respectively. Nitrogen-fixing bacteria SGS-4 and complex microbial agent (RSM1+SGS-4) treatments significantly increased the single plant spike number of rice by 31.9% and 38.5%, respectively. Methanotrophic bacteria RSM1 treatment had no effect on the single plant spike number of rice, indicating that the combined use of methanotrophic bacteria RSM1 and nitrogen-fixing bacteria SGS-4 not only did not reduce the effect of nitrogen-fixing bacteria SGS-4 on the increase of single plant spike number of rice, but also improved the growth-promoting effect of nitrogen-fixing bacteria SGS-4.

[0064] According to the yield data, compared with the methanotrophic bacteria RSM1 treatment alone, the complex microbial agent (RSM1+SGS-4) and nitrogen-fixing bacteria SGS-4 treatments significantly increased the average single plant yield and total yield ​ , ​), while the effect of methanotrophic bacteria RSM1 was not obvious. The average yield per plant of the control, the complex microbial inoculant (RSM1+SGS-4), and the nitrogen-fixing bacteria SGS-4 was 41.6 g, 67.6 g, and 60.2 g, respectively, and the complex microbial inoculant (RSM1+SGS-4) and the nitrogen-fixing bacteria SGS-4 increased the average yield per plant by 62.5% and 44.9%, respectively; the total yield of the control, the complex microbial inoculant (RSM1+SGS-4), the nitrogen-fixing bacteria SGS-4, and the methanotrophic bacteria RSM1 was 284.1 kg / ha, 522.9 kg / ha, 448.8 kg / ha, and 329.4 kg / ha, respectively, and the complex microbial inoculant (RSM1+SGS-4) and the nitrogen-fixing bacteria SGS-4 increased the total yield by 84.1% and 58.1%, respectively, while the methanotrophic bacteria RSM1 increased the total yield by 15.9%. Overall, the complex microbial inoculant was significantly better than the methanotrophic bacteria RSM1 and the nitrogen-fixing bacteria SGS-4 in increasing the yield of rice.

[0065] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Methylmonas ( Methylomonas strain RSM1 (sp.) is characterized by, The methylomonas RSM1 has a preservation number CCTCC NO: M20242683.

2. The culture method of the methylobacterium RSM1 according to claim 1, characterized in that, The methylomonas RSM1 activated to logarithmic phase is inoculated into inorganic nitrate medium at an inoculation amount of 1%-5% and a growth temperature of 28-32℃.

3. The culture method of the Methylobacterium RSM1 according to claim 2, characterized in that, The methylomonas RSM1 activated to logarithmic phase is inoculated into inorganic nitrate medium at an inoculation amount of 2% and a growth temperature of 30℃.

4. An inoculant characterized in that, The bacterial agent comprises the methylomonas RSM1 of claim 1 or the freeze-dried bacterial body thereof, or the bacterial liquid obtained by the culture method of claim 2.

5. A complex microbial agent, characterized in that, The bacterial agent comprises the methylomonas RSM1 of claim 1 or the freeze-dried bacterial body thereof, or the bacterial liquid obtained by the culture method of claim 2. The bacterial agent comprises the methylomonas RSM1 of claim 1 or the freeze-dried bacterial body thereof, or the bacterial liquid obtained by the culture method of claim 2. The nitrogen-fixing bacteria is Azotobacter chroococcum (A. chroococcum) Azotobacter salinestris ) SGS-4.

6. The preparation method of the complex bacterial agent of claim 5, characterized in that, The methylomonas RSM1 and the azotobacter SGS-4 bacterial liquid are centrifuged at 6500-7500 rpm for 5-15 minutes, the supernatant is discarded, sterile water is added to make the OD value of the methylomonas RSM1 and the azotobacter SGS-4 resuspension liquid 1.0, and the methylomonas RSM1 and the azotobacter SGS-4 resuspension liquid are mixed.

7. A product characterized by, The bacterial agent comprises the methylomonas RSM1 of claim 1 or the freeze-dried bacterial body thereof, or the bacterial liquid obtained by the culture method of claim 2. The product is a biological fertilizer or an environmental protection agent.

8. The methylomonas RSM1 of claim 1 or the freeze-dried bacterial body thereof, or the bacterial liquid obtained by the culture method of claim 2 or the bacterial agent of claim 4 in reducing methane emission and increasing rice yield in the process of rice planting.

9. The application of the composite bacterial agent of claim 5 or the composite bacterial agent prepared by the preparation method of claim 6 in reducing methane emission, increasing the number of tillers, and increasing rice yield in the process of rice planting.

10. A method of simultaneously reducing methane emission and increasing rice yield in rice cultivation, characterized by, The composite bacterial agent of claim 5 or the composite bacterial agent prepared by the preparation method of claim 6 or the product of claim 7 is inoculated into the rhizosphere of rice.

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