Methylomonas RSM1 and application thereof

Abstract

The invention provides a methylomonas sp RSM1 strain and application of the methylomonas sp RSM1 strain, and the preservation number of the methylomonas sp RSM1 strain provided by the invention is CCTCC (China Center for Type Culture Collection) No: M 20242683. The methymonas RSM1 strain provided by the invention is screened from an Inner Mongolia saline-alkali soil paddy field, and can grow by using methane as a carbon source, so that methane emission in a rice planting process is reduced, and the rice yield is increased. The invention further provides a complex microbial inoculant of the methymonas RSM1 and the nitrogen-fixing bacteria, the complex microbial inoculant can greatly enhance methane emission reduction and rice yield increase effects, methane emission is reduced by 79.8% at most, the rice yield is increased by 84.1% at most, and the complex microbial inoculant has important significance on environmental protection and agricultural development.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, and in particular to a strain of Methylomonas RSM1 and an application thereof. Background Art

[0002] Rice fields are one of the main sources of greenhouse gas methane emissions, accounting for about 20% of total methane emissions. The greenhouse effect caused by these methanes will lead to global warming, which has a huge impact on agricultural development and human survival. During the rice planting process, rice fields are flooded for a long time. This flooding isolates the air and soil, creating a perfect anaerobic environment. In such an environment, organic matter in the soil will be decomposed by methanogens and release methane. At the same time, the organic matter secreted by the rice roots provides a nutrient source for methanogens, further increasing the production of methane.

[0003] There are currently three main means to reduce methane emissions from rice fields. The first is to develop water-saving irrigation technology for rice fields, using sprinkler irrigation, drip irrigation and other means to reduce soil moisture content, increase air permeability, and reduce anaerobic environment, but this method has stricter agricultural management requirements and higher costs; the second is to change rice varieties and plant more low-methane emission rice, but the overall effect is not obvious; the third is to use biological control to adjust the soil microbial structure, increase the content of methane metabolizing bacteria, and directly reduce or metabolize the methane produced.

[0004] Methane metabolizing bacteria are microorganisms that use methane as a carbon source and energy source. They live in the rhizosphere and are crucial to reducing methane emissions from rice fields (Shinoda et al., 2019; Bao et al., 2014). For example, the applicant isolated and identified aerobic methylcampal bacteria Methylosinus sp. PRM1 (patent application number 202211083626.4), which can metabolize methane and promote rice growth. Nitrogen-fixing bacteria are generally believed to be able to significantly increase rice yields, but most of them have been found to have no methane emission reduction effect after verification, such as the nitrogen-fixing bacteria ( Azotobacter salinestris ) SGS-4 and other nitrogen-fixing bacteria.

[0005] In order to further expand the advantages of bioremediation and enhance the bioremediation effect of methane emissions, it is necessary to discover 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, in order to provide more support for environmental protection and crop yields through complementary advantages between strains or stimulating synergistic effects. Summary of the invention

[0006] The present invention provides a strain of Methylomonas RSM1 and an application thereof. The Methylomonas ( Methylomonas sp)RSM1 has the effect of reducing rice methane emission and increasing rice yield. The present invention also found that by introducing Methylomonas ( Methylomonas sp )RSM1 and nitrogen-fixing bacteria ( Azotobacter salinestris ) When used in combination with SGS-4, the above-mentioned effects can be greatly enhanced.

[0007] In a first aspect, the present invention provides a Methylomonas ( Methylomonas sp ) RSM1 strain, the Methylomonas ( Methylomonas sp )RSM1 was deposited in the China Center for Type Culture Collection (CCTCC) on December 2, 2024, with the strain deposit number CCTCC NO: M 20242683, and the deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] In a second aspect, the present invention provides the above-mentioned Methylomonas ( Methylomonas sp ) The method for culturing RSM1 is as follows: Methylomonas ( Methylomonas sp )RSM1 was inoculated into inorganic nitrate medium at an inoculation amount of 1%-5%, the growth temperature was 28-32°C, and methane was introduced at a final concentration of 15%-25%.

[0009] Preferably, Methylomonas ( Methylomonas sp )RSM1 was inoculated into inorganic nitrate medium with an inoculum size of 2%, a growth temperature of 30°C, and a final concentration of 20% methane was introduced.

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

[0011] In a fourth aspect, the present invention provides a composite bacterial agent comprising the above-mentioned Methylomonas ( Methylomonas sp ) RSM1 or its freeze-dried bacterial cells, or the bacterial solution obtained by the above-mentioned culture method, or the supernatant after centrifugation of the bacterial solution, or the precipitate resuspension after centrifugation of the bacterial solution, and Azotobacter or its freeze-dried bacterial bodies, or its bacterial solution or the supernatant after centrifugation of the bacterial solution, or the precipitate resuspension after centrifugation of the bacterial solution.

[0012] The nitrogen-fixing bacteria are nitrogen-fixing bacteria ( Azotobacter salinestris )SGS-4, deposit number CCTCC NO: M20232631.

[0013] Preferably, the Methylomonas ( Methylomonassp) RSM1 live bacteria content ≥ 1.0×10 8 cfu / mL, nitrogen-fixing bacteria ( Azotobacter salinestris ) SGS-4 live bacteria content ≥1.0×10 8 cfu / mL.

[0014] The present invention further provides a method for preparing the composite bacterial agent, which comprises: Methylomonas sp)RSM1 and nitrogen-fixing bacteria ( Azotobacter salinestris ) SGS-4 bacterial solution was centrifuged at 6500-7500rpm for 5-15 minutes, the supernatant was discarded, and sterile water was added to make Methylomonas ( Methylomonas sp)RSM1 and nitrogen-fixing bacteria ( Azotobacter salinestris ) The OD value of SGS-4 resuspension was 1.0, and Methylomonas ( Methylomonas sp)RSM1 and nitrogen-fixing bacteria SGS-4 ( Azotobacter salinestris In the embodiment of the present invention, the two bacterial resuspensions are mixed in a volume ratio of 1:1.

[0015] In a fifth aspect, the present invention provides a product comprising the above-mentioned Methylomonas ( Methylomonas sp) RSM1 or its freeze-dried bacterial cells, or the bacterial solution obtained by the above-mentioned culture method, or the supernatant after centrifugation of the bacterial solution, or the precipitate resuspension after centrifugation of the bacterial solution; and / or Contains the above-mentioned composite bacterial agent or the composite bacterial agent prepared by the above-mentioned preparation method.

[0016] The product is a biological fertilizer, an additive or an environmental protection agent.

[0017] The biological fertilizer or fertilizer additive contains Methylomonas ( Methylomonas sp) RSM1 live bacteria content ≥ 1.0×10 8 cfu / mL, nitrogen-fixing bacteria ( Azotobacter salinestris )SGS-4 live bacteria content ≥1.0×10 8 cfu / mL.

[0018] In a sixth aspect, the present invention provides the above-mentioned Methylomonas ( Methylomonas sp) Use of RSM1 or its freeze-dried bacteria, or the bacterial solution obtained by the above-mentioned 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.

[0019] In a seventh aspect, the present invention provides the use of the above-mentioned composite bacterial agent or the composite bacterial agent prepared by the above-mentioned preparation method or the above-mentioned product in reducing methane emissions during rice cultivation, increasing the number of rice tillers and / or improving rice yield.

[0020] In an eighth aspect, the present invention provides a method for reducing methane emissions during rice planting, specifically, inoculating the above-mentioned Methylomonas ( Methylomonas sp ) RSM1 or its freeze-dried bacterial cells, or the bacterial solution obtained by the above-mentioned culture method, or the supernatant after the bacterial solution is centrifuged, or the precipitate resuspension after the bacterial solution is centrifuged, or the above-mentioned bacterial agent or the above-mentioned product. In the embodiment of the present application, the above-mentioned inoculation is carried out during the rice seedling raising period.

[0021] The present invention further provides a method for simultaneously reducing methane emissions during rice cultivation 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 into the rice root circle. In the embodiment of the present application, the above-mentioned inoculation is carried out during the rice seedling raising period.

[0022] The beneficial effects of the present invention are: (1) The present invention discovered a strain of Methylomonas ( Methylomonas sp )RSM1, the application of this strain can reduce methane emissions during rice planting and increase rice yield, reducing methane emissions by 12.1% and 15.2% between the 65th and 79th days of rice planting, and increasing rice yield per mu by 15.9%.

[0023] (2) The present invention uses Methylomonas ( Methylomonas sp )RSM1 and nitrogen-fixing bacteria ( Azotobacter salinestris The application of this microbial agent can produce significant synergistic effects, reducing methane emissions by 65.6% and 79.8% between the 65th and 79th days of rice planting, respectively, increasing the number of tillers by 20.0 on the 79th day, and increasing the final per-acre yield of rice by 84.1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The present invention discloses a 16S rRNA gene phylogenetic tree of the methanotrophic bacteria isolated strain RSM1.

[0026] Figure 2 This is a graph showing the effect of the composite bacterial agent treatment of the methane oxidizing bacteria RSM1 and the nitrogen fixing bacteria SGS-4 provided by the present invention on the methane emission from the paddy field.

[0027] Figure 3 This is a graph showing the results of reducing methane emissions from paddy fields by treating with a composite bacterial agent of the methane oxidizing bacteria RSM1 and the nitrogen fixing bacteria SGS-4 provided by the present invention.

[0028] Figure 4 This is a graph showing the effect of the composite bacterial agent treatment of methanotrophic bacteria RSM1 and nitrogen-fixing bacteria SGS-4 provided by the present invention on the number of panicles per plant of rice in the field.

[0029] Figure 5 This is a graph showing the effect of the composite bacterial agent treatment of methanotrophic bacteria RSM1 and nitrogen-fixing bacteria SGS-4 provided by the present invention on the yield of single rice plant in the field.

[0030] Figure 6 This is a graph showing the effect of the composite bacterial agent treatment of methane oxidizing bacteria RSM1 and nitrogen fixing bacteria SGS-4 provided by the present invention on the total yield per mu of field rice. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The inorganic nitrate medium (NMS) and SSM medium recipes used in the following examples are shown in Tables 1 and 2.

[0033] Table 1 NMS medium formula The preparation method of liquid inorganic nitrate medium (NMS) medium is as follows: the volume ratio of solution 1: solution 2: solution 3: solution 4 = 1000: 0.1:1:1 in the medium, adjust the pH to 6.8, sterilize at 121°C for 20 minutes, and add solution 5 after the temperature of the medium is below 40°C (add 10 ml solution 5 for every 1000 ml solution 1).

[0034] Table 2 SSM medium formula The preparation method of SSM medium is as follows: first prepare 1g / 50ml biotin stock solution and filter sterilize. Add components other than biotin in proportion, adjust pH to 8.5, sterilize at 121℃ for 20 min, and add an appropriate amount of 1g / 50ml biotin stock solution after the culture medium temperature is below 30℃.

[0035] Example 1 Methylomonas ( Methylomonas Isolation, screening and identification of sp)RSM1 The strain was obtained in August 2021 through screening, separation and purification from the rhizosphere soil of saline-alkali rice in Tuzuo Banner, Hohhot City, Inner Mongolia Autonomous Region. When sampling, the whole rice plant with roots was dug up, and the exposed rice roots (including rhizosphere soil) were collected and placed in a 50mL centrifuge tube containing 40mL sterile water, placed in a foam box filled with dry ice for preservation, and brought back to the laboratory within a short time.

[0036] The separation of rhizosphere soil from roots was carried out by ultrasonic vibration cleaning-centrifugal removal. The centrifuge tube containing the root sample was ultrasonically cleaned for 20 minutes, and then centrifuged at 4000rpm for 10 minutes in a high-speed centrifuge to remove the roots and supernatant to obtain the rhizosphere soil. The obtained rhizosphere soil was used for the subsequent separation experiment of Methylomonas and nitrogen-fixing bacteria.

[0037] Methylomonas was cultured using NMS medium. At the beginning of enrichment culture, 1g of rhizosphere soil was placed in a 70ml serum bottle containing 20ml NMS liquid medium. Then, 8ml of air was extracted with a disposable sterile syringe, and an equal volume of high-purity CH4 (99.9%) was injected to adjust the final methane concentration in the serum bottle to 20%. The serum bottle was placed at 30℃ and shaken (200 rpm) for enrichment culture for 10 days. After that, the enrichment stock solution obtained in the above experimental process was diluted to the limit, that is, the bacterial concentration was increased from 10 -1 to 10 -9 200 μl of the enriched stock solution was added to a serum bottle containing 20 ml of NMS liquid culture medium to obtain a dilution of 10 -2 The culture medium was diluted to 10 by 10-fold serial dilution method. -9 Concentration, the mouth of the serum bottle is sealed with a rubber stopper and then fixed with an aluminum cap, 20% of the air in the serum bottle is replaced with methane, and each dilution gradient sample is placed in a shaker at 30°C and 200 rpm for incubation. When the culture medium with the highest dilution multiple becomes turbid, a second round of gradient dilution is quickly performed on it, and the subculture is repeated multiple times.

[0038] During the dilution and subculture of microorganisms, take an appropriate amount of enrichment stock solution (about 100 μl) and spread it on the solid NMS medium, place it in an airtight incubator filled with 40% CH4 gas to culture Methylomonas, and the temperature is set to 30°C. After the colonies grow on the culture dish, pick a single colony, and use repeated streaking and transfer to separate and purify the strains with different colony morphologies. Select the strains that grow well in the solid NMS medium with CH4, but cannot grow in the NB medium (without CH4), and finally obtain the strain RSM1, which is preliminarily determined to be a methane oxidizing bacterium.

[0039] The isolated strain RSM1 was inoculated into NMS medium and placed in a methane box. It was cultured at 30°C and 40% methane concentration for 3-5 days. The bacteria were collected and the genomic DNA was extracted using the TIANamp Bacteria DNA Kit. The extracted DNA was used as a template and PCR amplification of the 16S rRNA gene sequence was performed using bacterial universal primers.

[0040] The above PCR amplification products were sent to General Biotech for sequencing, and the obtained 16S rRNA gene sequences were compared with strain RSM1 using NCBI's BLASTN. Species with close genetic relationships were screened to construct a phylogenetic tree of 16S rRNA gene sequences, as shown in Figure 1 As shown, strain RSM1 and Methylomonas spp. Methylomonas rhizoryzae The 16S rRNA gene sequence of GJ1 has a similarity of 99.8% and belongs to the genus Methylomonas ( Methylomonas sp ). The strain was deposited in the China Center for Type Culture Collection (CCTCC) on December 2, 2024, with the strain deposit number of CCTCC NO: M 20242683. The deposit address is: Wuhan University, Wuhan, China, and the specific address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0041] Example 2 Methylomonas ( Methylomonas sp )RSM1 and nitrogen-fixing bacteria ( Azotobacter salinestris ) Preparation of SGS-4 composite bacterial agent Primary seed culture of Methylomonas RSM1: Take 20 μL of Methylomonas strain from the bacterial preservation tube and inoculate it into a 70 ml serum bottle containing 20 ml NMS liquid culture medium, fill it with 20% methane gas with a purity of 99.9%, and culture it in a 30°C incubator with shaking (150 rpm) until the logarithmic growth phase.

[0042] Secondary seed culture of Methylomonas RSM1: Take 5 ml of the primary Methylomonas RSM1 seed solution in the logarithmic growth phase with a sterile syringe and inject it into a 500 ml serum bottle containing 250 ml NMS liquid medium, flush with 20% methane gas and culture with shaking (150rpm), for a total of 4-8 bottles. Centrifuge the above bacterial solution at 7000rpm for 10 minutes and remove the supernatant. Add sterile water to the remaining bacteria to adjust the suspension of Methylomonas RSM1 to OD1.0.

[0043] Primary seed culture of nitrogen-fixing bacteria SGS-4: Take 20 μL of nitrogen-fixing strain SGS-4 from the bacterial preservation tube and inoculate it into a 200 ml Erlenmeyer flask containing 50 ml of nitrogen-free SSM liquid culture medium, and culture it at 30°C and 150 rpm with shaking (150 rpm) until the logarithmic growth phase.

[0044] Secondary seed culture of nitrogen-fixing bacteria SGS-4: Take 5 ml of the first-level seed solution of nitrogen-fixing bacteria and inject it into a 500 ml serum bottle containing 300 ml of SSM liquid medium for culture, a total of 4-8 bottles. Centrifuge the above bacterial solution at 7000rpm for 10 minutes and remove the supernatant. Add sterile water to the remaining bacteria to adjust the nitrogen-fixing bacteria SGS-4 suspension to OD1.0.

[0045] Finally, the suspensions of Methylomonas RSM1 and nitrogen-fixing bacteria SGS-4 were mixed in a ratio of 1:1 to obtain a liquid composite bacterial agent.

[0046] Example 3 Experiment on reducing rice methane emission by composite bacterial agent The compound bacterial agent was poured onto the roots of the seedlings in the greenhouse. Each tray (about 2000 plants) of seedlings in the bacterial agent group was poured with 1000ml of the compound bacteria (RSM1 and SGS-4). The RSM1 group and the SGS-4 group were poured with 1000ml of their respective bacterial suspensions with an OD value of 1.0. The blank control group was poured with an equal amount of clean water. The seedlings were transplanted to the field after 5 days.

[0047] Methane emission measurement method: Methane concentration was measured using a rectangular, transparent acrylic plate enclosed top chamber (30 cm long, 30 cm wide, 100 cm high). When the field was flooded, the chamber was gently placed on four steel pipe bases fixed in advance around a rice plant. Because the steel pipe base was located 10 cm below the water surface, the static box covered the rice to form a closed system. Methane flux samples were collected and measured every time from 10:00 to 12:00 in the morning. 20 ml of gas was taken from the top space of the static box at 0 time, 10 minutes, and 20 minutes, and then the bottle was brought back to the laboratory, where the methane concentration was measured using a 2014GC Shimadzu gas chromatograph flame ionization detector. Methane emission flux was calculated based on the increase in indoor gas concentration per square meter and hour.

[0048] The methane emission flux and reduction rate are as follows: Figure 2 and Figure 3 As shown, the methane emission flux (mg C / h / m 2 ) were 4.61, 5.02, 4.00, 2.13 on the 65th day after planting, 7.98, 7.85, 3.27, 1.59 on the 72nd day, 5.27, 5.87, 2.99, 1.32 on the 79th day, and 4.18, 4.00, 3.41, 3.44 on the 100th day. The methane emission flux of the rice rhizosphere inoculated with the composite bacteria (SGS-4+RSM1) was significantly lower than that of the single inoculation of Methylomonas RSM1 and nitrogen-fixing bacteria SGS-4. The inoculation of nitrogen-fixing bacteria SGS-4 in the rice rhizosphere alone had basically no effect on methane emission reduction, while the single inoculation of Methylomonas RSM1 and the inoculation of composite bacteria (SGS-4+RSM1) reduced methane emission by 12.1%-65.6% (2.99 mgC / h / m 2 - 4.00mgC / h / m 2 ) and 15.2%-79.8% (1.32 mgC / h / m 2 - 2.13 mgC / h / m 2 ) of the 1,000 methane emission fluxes, of which the composite agent had the highest reduction rate of 79.8%. Overall, the composite agent (SGS-4+RSM1) had a better methane emission reduction effect than the single Methylomonas RSM1 and nitrogen-fixing bacteria SGS-4. The applicant also compared Methylomonas RSM1 and nitrogen-fixing bacteria Bradyrhizobium, Rhizobium When used in combination, it was found that the methane emission reduction effect was significantly inferior to the composite bacterial agent (SGS-4+RSM1).

[0049] Example 4 Experiment on improving rice yield with composite bacterial agent The compound bacterial agent was poured onto the roots of the seedlings in the greenhouse. Each tray (about 2000 plants) of seedlings in the bacterial agent group was poured with 1000ml of the compound bacteria (RSM1 and SGS-4), the RSM1 group and the SGS-4 group were poured with 1000ml of their respective bacterial suspensions with an OD value of 1.0, and the blank control group was poured with an equal amount of clean water. The rice seedlings were transplanted to the field after 5 days, and the growth indicators such as the tiller number and plant height of rice were measured at different rice growth stages. The yield was measured when the rice matured.

[0050] Determination of rice growth index: On the 79th day, the plant height and tiller number of 10-12 rice plants were measured by random sampling. The mean tiller numbers of the control, composite bacteria (RSM1+SGS-4), nitrogen-fixing bacteria SGS-4 and Methylomonas RSM1 treatments were 18.6, 38.6, 36.8 and 19, respectively. Compared with the treatment of Methylomonas RSM1 alone, the tiller numbers of the composite bacteria (RSM1+SGS-4) and nitrogen-fixing bacteria SGS-4 treatments increased significantly by 20.0 and 18.2, respectively. There was no significant change in the rice plant height among the four groups, indicating that the combined use of Methylomonas RSM1 and nitrogen-fixing bacteria SGS-4 will not only not reduce the growth-promoting effect of nitrogen-fixing bacteria SGS-4 itself, but also enhance the growth-promoting effect of nitrogen-fixing bacteria SGS-4.

[0051] Rice yield determination method: After the rice is basically mature, random sampling is used to determine the average yield per plant and the number of grains per panicle. In mid-October, a fully automatic harvester (PRO988Q, Kubota Corporation) is used for overall harvesting. The three plots are harvested separately, marked and weighed.

[0052] The results of the number of panicles per rice plant are as follows Figure 4 As shown in the figure, the mean number of rice panicles per plant in the control, nitrogen-fixing bacteria SGS-4, methanotrophic bacteria RSM1 and composite bacteria (RSM1+SGS-4) treatments were 30, 40, 24 and 42, respectively. The treatments with nitrogen-fixing bacteria SGS-4 and composite bacteria (RSM1+SGS-4) significantly increased the number of rice panicles per plant by 31.9% and 38.5%, respectively. However, the treatment with Methylomonas RSM1 had no effect on the number of rice panicles per plant, indicating that the combined use of Methylomonas RSM1 and nitrogen-fixing bacteria SGS-4 will not only not reduce the effect of nitrogen-fixing bacteria SGS-4 on the increase of rice panicles per plant, but also enhance the growth-promoting effect of nitrogen-fixing bacteria SGS-4.

[0053] According to the yield data, compared with the single Methylomonas RSM1 treatment, the composite bacteria (RSM1+SGS-4) and nitrogen-fixing bacteria SGS-4 treatment significantly increased the average single plant yield and total yield ( Figure 5 , Figure 6), while the effect of Methylomonas RSM1 treatment was not obvious. The average yield per plant of the control, composite agent (RSM1+SGS-4) and nitrogen-fixing bacteria SGS-4 treatments were 41.6g, 67.6g and 60.2g, respectively. The composite agent (RSM1+SGS-4) and nitrogen-fixing bacteria SGS-4 treatments increased the average yield per plant by 62.5% and 44.9%, respectively; the total yield of the control, composite agent (RSM1+SGS-4), nitrogen-fixing bacteria SGS-4 and Methylomonas RSM1 treatments were 284.1 kg / mu, 522.9 kg / mu, 448.8 kg / mu and 329.4 kg / mu, respectively, increasing the total yield by 84.1% and 58.1%, respectively, while the Methylomonas RSM1 treatment increased the total yield by 15.9%. Overall, the composite agent was significantly better than the single use of Methylomonas RSM1 and the single use of nitrogen-fixing bacteria SGS-4 in increasing rice yield.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Methylomonas Methylomonas sp) RSM1 strain, characterized in that The Methylomonas Methylomonas sp)RSM1 deposit number is CCTCC NO: M20242683.

2. The Methylomonas according to claim 1 ( Methylomonas sp) A method for culturing RSM1, characterized in that: Methylomonas ( Methylomonas sp) RSM1 was inoculated into inorganic nitrate medium at an inoculum of 1%-5% and a growth temperature of 28-32°C; Preferably, Methylomonas ( Methylomonas sp)RSM1 was inoculated into inorganic nitrate medium with an inoculum size of 2% and a growth temperature of 30°C.

3. A bacterial agent, characterized in that: The bacterial agent includes the Methylomonas described in claim 1 ( Methylomonas sp) RSM1 or freeze-dried bacterial cells thereof, or a bacterial solution obtained by the culture method according to claim 2, or a supernatant after centrifugation of the bacterial solution, or a resuspension of a precipitate after centrifugation of the bacterial solution.

4. A composite bacterial agent, characterized in that: Containing the Methylomonas according to claim 1 ( Methylomonas sp) RSM1 or its freeze-dried bacterial cells, or the bacterial solution obtained by the culture method according to claim 2, or the supernatant after centrifugation of the bacterial solution, or the precipitate resuspension after centrifugation of the bacterial solution, and Azotobacter or its freeze-dried bacterial cells, or its bacterial solution or the supernatant after centrifugation of the bacterial solution, or the precipitate resuspension after centrifugation of the bacterial solution; The nitrogen-fixing bacteria are nitrogen-fixing bacteria ( Azotobacter salinestris )SGS-4, deposit number CCTCC NO: M20232631.

5. The method for preparing the composite bacterial agent according to claim 4, characterized in that: Including Methylomonas Methylomonas sp)RSM1 and nitrogen-fixing bacteria ( Azotobacter salinestris ) SGS-4 bacterial solution was centrifuged at 6500-7500rpm for 5-15 minutes, the supernatant was discarded, and sterile water was added to make Methylomonas ( Methylomonas sp)RSM1 and nitrogen-fixing bacteria ( Azotobacter salinestris ) The OD value of SGS-4 resuspension was 1.0, and Methylomonas ( Methylomonas sp)RSM1 and nitrogen-fixing bacteria SGS-4 ( Azotobacter salinestris ) resuspension mix.

6. A product, characterized in that Containing the Methylomonas according to claim 1 ( Methylomonas sp) RSM1 or its freeze-dried bacterial cells, or the bacterial solution obtained by the culture method according to claim 2, or the supernatant after centrifugation of the bacterial solution, or the precipitate resuspension after centrifugation of the bacterial solution; and / or Containing the composite bacterial agent according to claim 4 or the composite bacterial agent prepared by the preparation method according to claim 5; The product is a biological fertilizer, an additive or an environmental protection agent.

7. The Methylomonas according to claim 1 ( Methylomonas sp) Use of RSM1 or its freeze-dried bacteria, or the bacterial solution obtained by the culture method of claim 2, or the supernatant after centrifugation of the bacterial solution, or the precipitate resuspension after centrifugation of the bacterial solution 3, or the product of claim 6 in reducing methane emissions during rice cultivation.

8. Use of the composite bacterial agent according to claim 4 or the composite bacterial agent prepared by the preparation method according to claim 5 or the product according to claim 6 in reducing methane emissions during rice cultivation, increasing the number of rice tillers and / or improving rice yield.

9. A method for reducing methane emissions during rice planting, characterized in that: The rice rhizosphere is inoculated with the Methylomonas described in claim 1 ( Methylomonas sp) RSM1 or its freeze-dried bacteria, or the bacterial solution obtained by the culture method according to claim 2, or the supernatant after centrifugation of the bacterial solution, or the precipitate resuspension after centrifugation of the bacterial solution, or the bacterial agent according to claim 3, or the product according to claim 6.

10. A method for simultaneously reducing methane emissions during rice cultivation and increasing rice yield, characterized in that: The composite bacterial agent according to claim 4, the composite bacterial agent prepared by the preparation method according to claim 5, or the product according to claim 6 is inoculated into the rice rhizosphere.

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