Growth-promoting methylobacterium R5018 and application thereof

Abstract

The invention discloses a growth-promoting methylobacterium R5018 strain and an application of the growth-promoting methylobacterium R5018 strain. The strain R5018 is obtained by separation from a rice phyllospheric microflora and methanol domestication and screening, and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M20252544. The rice growth-promoting microbial inoculum prepared from the strain can be stably colonized at phyllosphere and promote the growth of overground and underground parts of rice through seed soaking, foliage spraying or combined application of the seed soaking and the foliage spraying, and the ear number, the grain number and the yield are remarkably increased. Compared with the prior art, the strain disclosed by the invention is derived from a rice phyllospheric bacterial community, is high in adaptability, environment-friendly and efficient, provides a new microbial technical scheme for high-yield cultivation of rice, and has a wide popularization and application prospect.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a growth-promoting methyl bacillus R5018 and its applications. Background Technology

[0002] Methylotrophs are a class of microorganisms that can utilize single-carbon compounds such as methanol and methylamine as their sole carbon source, and are widely distributed in the phyllosphere environment of plants. Methylotrophs can utilize methanol, a byproduct of pectin metabolism in the cell walls of plant phyllospheres, as both a carbon and energy source for growth, and they colonize the phyllosphere of many plants and crops (such as rice, wheat, and corn). Methylotrophs (such as *Methylorubrum oryzae* and *M. extorquens*) not only exist stably in the phyllosphere but can also synthesize various plant growth-promoting substances, such as indoleacetic acid (IAA), gibberellins, ACC deaminase, siderophores, and soluble phosphorus. These substances promote crop growth and development by improving nutrient absorption, regulating endogenous hormone levels, and alleviating abiotic stress.

[0003] In rice production, screening for highly adaptable, growth-promoting, and stable Methylbacterium strains tailored to the rice foliage niche, and developing corresponding application systems, is crucial for increasing rice yield and reducing the use of chemical fertilizers and pesticides. However, there are few reports on the application of highly efficient growth-promoting Methylbacterium derived from the rice foliage and domesticated with methanol in rice cultivation, and insufficient colonization stability of these strains limits their widespread application in green and high-yield cultivation. There is an urgent need to develop highly efficient strains suitable for the rice niche and their application methods to increase yield and reduce chemical inputs. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a growth-promoting strain of Methylobacterium oryzae R5018 and its prepared inoculum.

[0005] Another object of the present invention is to provide the application of R5018 and the bacterial agents prepared therefrom.

[0006] Another object of the present invention is to provide a rice growth-promoting bacterial agent and its application method.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A strain of Methylobacterium oryzae R5018, classified as Methylobacterium oryzae R5018, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20252544 on November 13, 2025.

[0009] The Methylbacterium R5018 was derived from the rice leaf flora, using methanol as the sole carbon source, and was obtained as a Gram-negative bacterium through isolation and screening.

[0010] The 16S rRNA gene sequence of the Methylobacterium R5018 is shown in SEQ ID NO. 1.

[0011] The methylbacterium R5018 has functions such as producing ACC deaminase, indoleacetic acid, gibberellin, phosphate solubilization, and siderophore production.

[0012] The Methylobacterium R5018 culture medium is NMS1 medium, with 0.5% methanol as the sole carbon source, and the culture conditions are constant temperature shaking culture at 30℃ and 180 r / min.

[0013] The Methylobacterium R5018 can be cultured in liquid medium for 36 hours to obtain a pink liquid bacterial solution, which appears as pink colonies on solid medium.

[0014] A rice growth-promoting bacterial agent, comprising the above-mentioned Methylobacterium oryzae R5018.

[0015] After being sprayed on the leaves, the above-mentioned rice growth-promoting bacteria can utilize the trace amounts of methanol released from the leaves to promote foliar colonization, enhance the growth capacity of rice, and increase yield.

[0016] This invention provides a method for preparing and using the rice growth-promoting bacterial agent. Spraying method: The bacterial agent is fermented in a culture medium containing 0.5% methanol until the OD reaches [insert OD value here]. 600 = 1.0, after centrifugation, remove the supernatant, wash the bacterial cells twice with sterile water, resuspend the bacterial cells, and adjust the bacterial OD. 600 =1.0. Add 0.02% polyoxyethylene alkylphenyl ether as a surfactant and 0.1% carboxymethyl cellulose as a dispersant, then spray onto rice leaves to achieve a leaf colonization rate of 10 g fresh weight per plant per pot. 8 CFU. Soaking method: After rice seeds are disinfected, they are soaked in OD... 600 Soak rice seeds in a 1.0% microbial agent solution for 24 hours, then proceed with germination and normal sowing. Combined application method: Soak rice seeds in the microbial agent before sowing and transplanting; spray the seedlings with the microbial agent again when the plants reach the tillering, heading, and maturity stages.

[0017] The present invention has the following beneficial effects:

[0018] The rice growth-promoting microbial agent is derived from the foliage of rice plants. After methanol domestication and enrichment, it exhibits high stability and strong leaf surface colonization ability. The methyl-trophic bacterium R5018 utilizes methanol on the leaf surface for growth and possesses functions such as producing ACC deaminase, IAA, gibberellin, phosphate solubilizer, and siderophore, thus promoting rice growth and increasing yield. The microbial agent provided by this invention has advantages such as strong adaptability, high stability, and environmental friendliness, offering a microbial solution for promoting rice growth and increasing yield. Attached Figure Description

[0019] Figure 1 The colony morphology and phylogenetic diagram of strain R5018 provided by the present invention are shown below, wherein A: electron micrograph of strain R5018, B: solid culture colony morphology of strain R5018, C: liquid culture state of strain R5018, and D: phylogenetic tree of strain R5018.

[0020] Figure 2 The growth curve of strain R5018 provided by the present invention.

[0021] Figure 3 The graph shows the results of the growth-promoting performance test of strain R5018 provided by this invention.

[0022] Figure 4 The diagram shows the growth status of different rice varieties during the tillering stage, provided by this invention. 1: Foliar spraying of rice growth-promoting bacteria, 2: Foliar spraying of rice growth-promoting bacteria, 3: Soaking seeds in rice growth-promoting bacteria and combined treatment of soaking seeds in the same solution, 4: Control group.

[0023] Figure 5 This invention provides a statistical chart of biomass of different rice varieties.

[0024] Figure 6 The figure shows the yield measurement results of different rice varieties provided by this invention.

[0025] Information on the preservation of biological materials

[0026] Methylobacterium oryzae R5018 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on November 13, 2025, with accession number CCTCC NO: M20252544. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0028] Example 1: Isolation, screening, and identification of Methylobacterium oryzae R5018

[0029] 1. Obtaining the primary enrichment solution

[0030] Accurately weigh 10g of rice leaf sample and place it in a 250mL Erlenmeyer flask containing 100mL of sterile 1×PBS solution. Incubate at 30℃ and 180rpm for 4 hours using a shaker. Remove the rice leaves from the culture medium using sterile forceps and filter using a 0.22μm sterile filter membrane to trap microorganisms. Then, cut the filter membrane into small pieces and add it to 100mL of 1×NMS1 liquid culture medium, along with 0.5% methanol. Incubate at 30℃ and 180rpm for turbidity to obtain the primary enrichment solution.

[0031] 2. Domestication of Methyltrophic Bacteria

[0032] Five mL of primary enrichment solution was inoculated into 100 mL of 1×NMS1 liquid medium, with methanol added as the sole carbon source. The conical flask was then incubated in a shaker at 30°C and 180 rpm. Once the bacterial culture became turbid, it was subcultured into fresh medium with a higher methanol concentration using a smaller amount of transfer solution. This process was repeated for enrichment. The transfer volume gradients were: 5 mL, 3 mL, 1.5 mL, 1 mL, 0.5 mL; the methanol concentration gradients were: 0.3%, 0.5%, 0.8%, 1.0%, 1.5%. The final enrichment solution was used for PCR of the methanol dehydrogenase functional gene. The enrichment solution with the highest methanol utilization efficiency and no toxic effect on the bacteria was selected for stable subculturing.

[0033] 3. Isolation of methyltrophic bacteria

[0034] Dilute the last generation of acclimatization culture medium with sterile water at a concentration of 10:10. 2 10 3 10 4 10 5 10 6 Each time, 100 μL of the diluted solution was pipetted onto NMS1 solid medium containing 0.5% sterile methanol. The medium was sealed tightly with breathable sealing film to prevent contamination and ensure respiration. The medium was then placed in a 30°C incubator for incubation. The growth of microorganisms on the plates was observed after several days.

[0035] Single colonies were picked from the NMS1 solid medium and inoculated into NMS1 liquid culture tubes with shaking. After the culture became turbid, it was streaked onto a plate for further purification. This process was repeated three times to obtain pure cultures of varying morphology and size. The methanol oxidation function of the pure cultures was then determined by PCR using the methanol dehydrogenase functional gene.

[0036] 4. Preparation of culture medium

[0037] 10×NMS1 stock solution (200mL): MgSO4·7H2O 0.4g, KNO3 2g, CaCl2·2H2O 0.028g, diluted to 200mL with ultrapure water.

[0038] 50× phosphate buffer (100mL, pH=6.8, final concentration 2.3mM): KH2PO4 0.544g, Na2HPO4·12H2O 2.701g, ultrapure water to a final volume of 100mL.

[0039] 5000×NMS Trance Elements (100mL): Na2EDTA 1g, FeSO4·H2O 2g, ZnSO4·7H2O 0.8g, H3BO3 0.03g, CuCl2·2H2O 0.6g, CoCl2·6H2O 0.2g, MnCl2·4H2O 0.03g, NiCl2·6H2O 0.02g, Na2MoO4·2H2O 0.05g. Adjust the volume to 100mL with ultrapure water, filter to sterilize, and store at 4℃.

[0040] To prepare 100 mL of 1×NMS1 liquid culture medium, add: 90 mL of ultrapure water, 10 mL of 10×NMS1 stock solution, and 2 mL of 50× phosphate buffer. Then autoclave at 121°C for 20 minutes. When using the culture medium, add 20 μL of filtered and sterilized 5000×NMS Trance Elements in a laminar flow hood. (Note: 5000×NMS Trance Elements must be filtered through a 0.22 μm sterile filter in a laminar flow hood. All stock solutions must be stored at 4°C protected from light.)

[0041] 5. Identification of Methyltrophic Bacteria

[0042] (1) Morphological identification

[0043] Through the above separation and screening process, a methyltrophic bacterium was finally obtained and named "R5018".

[0044] The strain R5018 obtained through the above screening was observed using a transmission electron microscope as shown in the following image. Figure 1 As shown; Figure 1 As shown in A, the bacteria are rod-shaped, measuring (0.8~1.0) μm × (2~4.0) μm, and can appear singly or in clusters. They move by lateral flagella and lack capsules, spores, and a mucus layer.

[0045] The strain R5018 obtained through the above screening stained red with Gram, indicating it is a Gram-negative bacterium. For example... Figure 1 As shown in Figure B, strain R5018 forms pink, round colonies on NMS1 solid medium. The surface is smooth, not very moist, with regular, slightly raised edges, opaque, and easily picked up. The colony diameter is approximately 0.1–0.2 mm. Figure 1 As shown in Figure C, liquid culture of strain R5018 also turns pink.

[0046] (2) Physiological and biochemical identification

[0047] The physiological and biochemical test results of the above-mentioned strain R5018 showed that strain R5018 was positive in glucose metabolism, urea metabolism, arginine decarboxylation and β-glucosidase reaction, indicating that the strain can ferment glucose to produce acid or gas, has the ability to form amines through arginine decarboxylation, and has the ability to produce urease to decompose urea to generate ammonia and synthesize β-glucosidase, as shown in Table 1.

[0048] Table 1. Physiological and biochemical assays of strain R5018

[0049]

[0050] (3) Molecular biological identification

[0051] The 16S rRNA sequence of strain R5018 was sequenced by Beijing Qingke Biotechnology Co., Ltd., and the sequencing results are shown in SEQ ID NO. 1. The obtained 16S rRNA sequence was compared with existing sequences in the NCBI database using BLAST analysis, and strains with similar homology were selected to construct a phylogenetic tree using MEGA 7.0 software.

[0052] The results are as follows Figure 1 As shown in D, the results showed that the strain R5018 obtained by the above screening had 100% similarity to Methylobacterium oryzae CBMB20 and was relatively close in evolutionary distance. Based on the physiological and biochemical characteristics of the strain, it was identified as Methylobacterium oryzae and named Methylobacterium oryzae R5018.

[0053] The aforementioned Methylobacterium oryzae R5018 was deposited at the China Center for Type Culture Collection on November 13, 2025, with accession number CCTCC NO: M20252544.

[0054] Example 2: Antibiotic resistance test and growth curve of strain R5018

[0055] To determine the antibiotic resistance of R5018 in the complex stress environment of the leaf margin, the bacterial suspension was spread on NMS1 plates, and then drug susceptibility test strips were attached to the plates. The plates were then incubated at 30°C for 36 hours, and the presence of inhibition zones was observed.

[0056] The results of the antibiotic resistance test of the above-mentioned strain R5018 are shown in Table 2. Strain R5018 has good resistance to ampicillin, ciprofloxacin, compound sulfamethoxazole and lincomycin. Therefore, in the subsequent fermentation production process of the strain, the above-mentioned antibiotics can be added in appropriate amounts to the culture medium to effectively prevent contamination by other microorganisms.

[0057] Table 2 Antibiotic resistance of strain R5018

[0058]

[0059] To determine the optimal methanol concentration for strain R5018, pure strains were cultured with 0.5%, 1.0%, and 1.5% methanol, and bacterial growth in NMS1 medium was observed. The growth curves of strain R5018 are shown below. Figure 2 As shown, methanol concentration has a significant impact on the growth of strain R5018. A suitable methanol concentration promotes strain growth, with the fastest growth rate and highest final biomass observed at a methanol concentration of 0.5%. Increasing the methanol concentration to 1.5% significantly inhibits growth, manifested as a decrease in growth rate and a substantial reduction in maximum biomass. Therefore, a methanol concentration of 0.5% represents the optimal growth condition for strain R5018 under methanol-induced conditions.

[0060] Example 3: Determination of growth-promoting characteristics of strain R5018

[0061] (1) Determination of indoleacetic acid (IAA) secretion

[0062] To detect the ability of strain R5018 to produce indoleacetic acid (IAA), the isolated and purified strain was inoculated into NMS1 liquid medium containing 100 mg / L L-tryptophan and cultured in a shaker at 30 ℃ and 180 rpm in the dark for 24 h until the logarithmic growth phase was reached. 1 mL of the culture medium was centrifuged at 8000 rpm for 5 min to collect the supernatant. 50 μL of the supernatant was mixed with 50 μL of Salkowski chromogenic reagent on a white ceramic plate. After developing the color in the dark at room temperature for 30 min, the absorbance was measured at 530 nm. The IAA yield was calculated according to the standard curve. The result was the average of three replicates.

[0063] (2) Measurement of gibberellin secretion

[0064] To detect the gibberellin production capacity of strain R5018, 0.5 mL of the bacterial culture in the logarithmic growth phase was centrifuged at 8000 rpm for 5 min, the supernatant was collected, and 4.5 mL of concentrated sulfuric acid was added and mixed well. The absorbance was measured at 412 nm using a spectrophotometer. The gibberellin concentration in the bacterial culture was calculated based on the standard curve equation.

[0065] (3) ACC deaminase activity assay

[0066] Strain R5018 was inoculated into NMS1 liquid medium containing 5 mmol / L ACC substrate and cultured at 30°C and 180 r / min for 24 h to induce ACC deaminase production. The bacterial suspension was centrifuged to collect the cell pellet, which was then washed twice with Tris-HCl buffer (0.1 mol / L, pH=7.6). The collected cell pellet was then resuspended in 600 μL Tris-HCl buffer (0.1 mol / L, pH=8.5), and 30 μL of toluene was added followed by rapid shaking for 30 s to lyse the cells and obtain ACC deaminase. 20 μL of ACC solution (0.5 mol / L) was added to 200 μL of the cell extract, mixed thoroughly, and incubated at 30°C for 24 h. A solution without added ACC served as a control. Then, add 1 mL of 0.56 mol / L HCl, centrifuge at 11000 r / min for 5 min at room temperature, take 1 mL of supernatant, add an equal volume of 0.56 mol / L HCl and mix well. Then add 300 μL of 2,4-dinitrophenylhydrazine (0.2%, 2,4-dinitrophenylhydrazine dissolved in 2 mol / L HCl), and place in a 30℃ water bath for 30 min. Finally, add 2 mL of 2 mol / L NaOH for color development, and measure its absorbance at 540 nm. Calculate the α-butanone content (μmol) according to the standard curve. Take 50 μL of cell extract to determine the bacterial protein content according to the Bradford method. ACC deaminase activity is expressed as the amount of ACC deaminization to α-butanone produced per milligram of bacterial protease per hour in the reaction system, and its enzyme activity unit is μmol·μg. -1 ·h -1 The results were the average of three replicates.

[0067] (4) Ability to decompose inorganic phosphorus

[0068] Strain R5018 was cultured in NMS1 liquid medium at 30℃ and 180 r / min for 24 h to prepare a bacterial suspension. Four sterile filter paper discs (6 mm in diameter) were placed at four inoculation points arranged in a cross shape on an inorganic phosphate agar plate. 10 μL of the bacterial suspension was inoculated into the center of each filter paper disc. The plate was sealed and incubated at 30℃. Each treatment was repeated three times. After incubation at 30℃ for 3–4 days, the presence of phosphate-solubilizing zones around the colonies was observed.

[0069] (5) Iron production capacity

[0070] Strain R5018 was cultured in NMS1 liquid medium at 30℃ and 180 rpm for 12 h to prepare a bacterial suspension. Four sterile filter paper discs (8 mm in diameter) were placed at four inoculation points arranged in a cross shape on a CAS medium plate. 10 μL of the bacterial suspension was inoculated into the center of each filter paper disc. The plate was sealed and incubated at 30℃. Each treatment was repeated three times. After incubation at 30℃ for 3–4 days, the presence of an orange-yellow halo around the colonies was observed.

[0071] Experimental results are as follows Figure 3 As shown in Table 3, strain R5018 has functions such as producing indoleacetic acid, gibberellin, ACC deaminase, phosphate solubilizer, and siderophore.

[0072] Table 3. Production of auxin and related bioactive substances by strain R5018

[0073]

[0074] Example 4: Rice pot experiment

[0075] Rice variety selection: Nanjing 5718 (Su-approved rice 20190004), a conventional late-maturing medium-grained japonica rice variety bred by the Institute of Food Crops, Jiangsu Academy of Agricultural Sciences; Changnongjing 8 (Su-approved rice 201412), a conventional early-maturing late-grained japonica rice variety bred by the Changshu Municipal Institute of Agricultural Sciences.

[0076] 1. Rice seedling cultivation

[0077] Sterilization: Select plump and healthy rice seeds and wash them with sterile water. Soak the washed seeds in 70% ethanol for 1 minute and in 0.1% sodium hypochlorite solution for 30 minutes, then rinse them with sterile water.

[0078] Soaking: Soak the sterilized seeds in sterile water at 30℃ for 24 hours, changing the water halfway through to maintain water quality and oxygen supply. Soaking is complete when the seeds have absorbed enough water (the seeds are swollen and plump, and soft to the touch).

[0079] Germination: Wrap the soaked rice seeds in a damp gauze in a sterile petri dish, leaving some space between the seeds. Place in a 30℃ incubator for germination, changing the air and water every 12 hours to ensure seed respiration and prevent mold. Sowing is suitable when the rice seed embryos are about 1-2 mm in length and uniform in size.

[0080] Sowing: Mix organic cultivation substrate and vermiculite in a 3:1 volume ratio and fill the mixture into seedling trays. Select seeds with good germination and uniformity, bury them in the substrate, and cover with a shallow layer of cultivation substrate, keeping the surface moist. Water daily after sowing to ensure the soil remains moist. Transplant seedlings when they reach the 3-3.5 leaf stage.

[0081] 2. Transplanting

[0082] After drying, crushing, and sifting the paddy soil collected from the field, transfer it to rice pots and soak it in water to make the soil moist and loose. Select rice seedlings that are growing well and uniformly for transplanting, two holes per pot.

[0083] 3. Water and fertilizer management

[0084] During the seedling establishment period, maintain shallow water while providing shade and cooling to help seedlings recover and grow. During the tillering stage, maintain a water layer of 3-5 cm to promote tillering, and apply 0.5g of urea per pot as tillering fertilizer. During the jointing and booting stage, apply 0.4g of urea and 0.6g of compound fertilizer per pot as panicle fertilizer. Maintain a stable water supply during the heading and flowering stage. During the grain-filling and ripening stage, maintain shallow water, reduce watering, and alternate between dry and wet conditions to facilitate grain filling. At harvest time, stop watering when most of the grains turn yellow, keep the potting soil dry, and harvest after the grains are fully ripe. During rice cultivation, it is important to promptly remove weeds and control diseases and pests to maintain the health and good growth of rice plants.

[0085] (1) Experimental Example 1: Soaking rice seeds with growth-promoting bacteria

[0086] Select plump and intact seeds, disinfect them, and then immerse them in water during the logarithmic growth phase (OD). 600 =0.8~1.2, viable count ≥1×10 8 The bacteria were soaked in the fermentation broth of strain R5018 (CFU / mL) at 30°C for 24 hours. Then, germination, sowing, and transplanting were carried out according to the above method.

[0087] (2) Experimental Example 2: Foliar spraying of rice growth-promoting bacteria

[0088] Strain R5018 was fermented with 0.5% methanol until OD was reached. 600 = 1.0, after centrifugation, remove the supernatant, wash the bacterial cells twice with sterile water, resuspend the bacterial cells, and adjust the bacterial OD. 600=1.0, then add 0.02% polyoxyethylene alkylphenyl ether as a surfactant and 0.1% carboxymethyl cellulose as a dispersant, and then spray it on the rice leaves to achieve a leaf colonization rate of 10 g fresh weight per plant per pot. 8 CFU. The application period is during the tillering, heading, and maturity stages of rice.

[0089] (3) Experimental Example 3: Combined Administration

[0090] The rice growth-promoting bacteria agent is applied by soaking rice seeds in water and spraying the leaves. The rice seeds are soaked in the rice growth-promoting bacteria agent before sowing and transplanting. The rice growth-promoting bacteria agent is then sprayed again when the rice plants grow to the tillering, heading and maturity stages.

[0091] Compare with Example 1

[0092] Equal volumes of sterile water were sprayed on the leaves of rice plants during the tillering, heading, and maturity stages as a control group.

[0093] In the above pot experiment, each treatment was set up with 9 replicates. Sampling was carried out at the tillering and heading stages to measure indicators such as plant height, root length, above-ground fresh weight, underground fresh weight, and number of tillers. At the maturity stage, indicators such as total number of grains, thousand-grain weight, and number of ears were measured, with 3 replicates for each stage.

[0094] Experimental results

[0095] On the 68th day after transplanting, during the rice tillering stage, the growth of plants in each treatment group was observed and measured. The results are as follows: Figure 4 As shown in the figure. Compared with the control group, all three treatments using rice growth-promoting bacteria (foliar spraying, seed soaking, and combined treatment) resulted in better growth vigor and significantly increased plant height for both Nanjing 5718 and Changnongjing 8 varieties. Figure 5 As shown, at the tillering stage of Nanjing 5718, the aboveground fresh weight and number of tillers in the combined treatment group were significantly higher than those in the control group (p < 0.05). At the heading stage, the underground fresh weight of the combined treatment group of Nanjing 5718 was significantly higher than that in the control group (p < 0.05). For Changnongjing 8, at the tillering stage, the aboveground fresh weight of all three treatment groups was significantly higher than that in the control group, and the plant height of the combined treatment group was significantly higher than that in the control group; at the heading stage, the underground fresh weight of the combined treatment group was significantly higher than that in the control group (p < 0.05). Comprehensive analysis shows that different treatments all resulted in a significant increase in plant height, root length, aboveground / underground fresh weight, and panicle number compared to the control group, with the combined treatment of rice growth-promoting bacteria agents showing better results.

[0096] Rice yield measurement results as follows Figure 6As shown in the figure, compared with the control group, foliar spraying and the combined treatment significantly increased the total number of grains in Nanjing 5718, and the combined treatment significantly increased the total number of grains in Changnongjing 8 (p < 0.05), with the number of panicles also showing an increasing trend. Among the three treatments, the combined treatment had the most significant effect on increasing the yield of both rice varieties. The experimental results indicate that rice growth-promoting bacteria can significantly promote rice growth, improve plant height, root system and aboveground biomass, and effectively increase the number of panicles, grains, and yield, showing good application prospects for high-yield rice cultivation.

Claims

1. A strain of Methylobacterium extorquens R5018, characterized in that, The classification name is Methylobacterium oryzae R5018, which is preserved in China Center for Type Culture Collection, with the preservation number CCTCC NO: M20252544, and the preservation time is November 13, 2025.

2. The use of Methylobacterium R5018 in promoting the growth and / or increasing the yield of rice according to claim 1.

3. The use of Methylobacterium R5018 in the preparation of a product for promoting the growth and / or increasing the yield of rice according to claim 1.

4. A bacterial agent prepared from Methylobacterium R5018 according to claim 1.

5. The bacterial agent of claim 4, characterized in that, The bacterial agent is obtained by fermenting the growth-promoting Methylobacterium R5018 in NMS1 medium containing 0.5% methanol at 30°C, 180 r / min-200 r / min to OD 600 = 1.

0.

6. The use of the bacterial agent according to claim 4 or 5 in promoting the growth and / or increasing the yield of rice.

7. The use of the bacterial agent according to claim 4 or 5 in the preparation of a product for promoting the growth and / or increasing the yield of rice.

8. A rice growth promoting bacterial agent, characterized by, The bacterial agent of claim 4 or 5 is centrifuged to remove supernatant, the bacterial body is washed twice with sterile water and resuspended, the OD of the bacterial body is adjusted to 1.0, then 0.01%-0.04% polyoxyethylene alkyl phenyl ether is added as a surfactant and 0.05%-0.2% carboxymethyl cellulose is added as a dispersing agent, to obtain the bacterial agent for promoting the growth of rice. 600 =1.0, then 0.01%-0.04% polyoxyethylene alkyl phenyl ether is added as a surfactant and 0.05%-0.2% carboxymethyl cellulose is added as a dispersing agent, to obtain the bacterial agent for promoting the growth of rice.

9. The bio-fertilizer of Oryza sativa as claimed in claim 8, wherein, The concentration of polyoxyethylene alkyl phenyl ether in the rice growth-promoting bacterial agent is 0.02%, and the concentration of carboxymethyl cellulose is 0.1%.

10. The method for using the growth promoting bacterial agent of rice according to claim 8 or 9, characterized by, Any one selected from the following: I: soaking rice seeds with the rice growth-promoting bacterial agent according to claim 8 or 9 for 20-28 hours, followed by germination and normal sowing; II: when the rice plants grow to the tillering stage, the heading stage and the mature stage, the rice growth promoting bacterial agent of claim 8 or 9 is sprayed on the leaves, so that the colonization amount of each pot plant reaches 10 8 CFU per gram of fresh weight; III: treating by the method combining I and II.

Images