Multifunctional rhizobium huanghuaiensis and application thereof

Abstract

The present application relates to a multifunctional Sinorhizobium meliloti and its application, and belongs to the technical field of microbial fertilizer. The Sinorhizobium meliloti SK1 is preserved in the China General Microbiological Culture Collection Center on October 21, 2024, and the address is No. 1, Beichen West Road, Haidian District, Beijing, and the preservation number is CGMCC NO. 32282. The bacteria have the abilities of degrading fat, dissolving potassium, fixing nitrogen, dissolving organic phosphorus, dissolving inorganic phosphorus, producing iron carrier, producing extracellular polysaccharide, producing auxin and improving sandy soil. The strain can produce good growth-promoting effect on wheat, corn and soybean planted in saline-alkali soil or sandy soil environment, and has a very broad application prospect.

Description

Technical Field

[0001] The present invention relates to a multifunctional Sinorhizobium meliloti and its application, belonging to the technical field of microbial fertilizers. Background Art

[0002] The reasons for soil desertification are complex, including not only natural causes such as wind erosion, drought, and climate change, but also related to human activities. With the acceleration of industrialization and urbanization, problems such as land degradation and pollution are becoming increasingly serious, and soil treatment and improvement have become the key to ensuring food security and the ecological environment. At present, relying solely on natural restoration cannot achieve the governance goal, and taking artificial means to transform sandy soil and restore its ecological environment is the most fundamental and effective means of land desertification treatment.

[0003] The essence of sandy soil improvement is to add various materials to sandy soil, improve the sandy soil structure, increase the water-holding and fertilizer-holding capacity of sandy soil, improve enzyme activity, optimize the microbial population structure, provide a suitable environment for plant growth, and gradually realize the restoration of the natural ecology of desertified soil through the combined action of microorganisms and plants. Plant growth-promoting rhizobacteria (PGPR) are common strain groups in microbial agents, including Bacillus sp., Sinorhizobium sq., Pseudomonas sp., Azotobacter sp., etc. They can directly or indirectly promote plant growth through various mechanisms such as phosphorus solubilization, potassium solubilization, nitrogen fixation, production of auxin, and production of extracellular polysaccharides.

[0004] In 1988, Chen Wenxin discovered a new genus, which is the fourth genus of rhizobia discovered by humans and the first genus of rhizobia discovered and named by Chinese scholars, namely "Sinorhizobium". Lajudie et al. mentioned in the International Journal of Systematic Bacteriology that Sinorhizobium meliloti is a rhizobium isolated from the root nodules of the leguminous plant Melilotus, and its specific epithet is derived from the Latinization of the genus name Melilotus of the host plant, and the Chinese translation is "Sinorhizobium meliloti". Sinorhizobium meliloti can not only nodulate and fix nitrogen with plants of the genus Melilotus, but also nodulate with plants of the related genus Medicago.

[0005] *Sinorhizobium meliloti*, a rhizobium strain for which the whole genome was sequenced earlier, is widely distributed in soil, on plant surfaces, and in the rhizosphere. It is frequently used as a representative strain in China to study its symbiotic relationships with legumes. As a free-living, soil-dwelling bacterium, *Sinorhizobium meliloti* also participates in the fixation symbiosis of legumes in the genera *Medicago*, *Melilotus*, and *Trigonella*, utilizing various forms of carbon, nitrogen, and phosphorus. The nitrogen-fixing capacity of *Sinorhizobium meliloti* is crucial for the growth of legumes, as it converts atmospheric nitrogen into a form usable by plants, thereby improving the yield and quality of legume crops. It plays an important role in agriculture, increasing soil fertility, reducing dependence on chemical nitrogen fertilizers, and contributing to environmental protection and sustainable agricultural development.

[0006] In recent years, research on the symbiotic nitrogen fixation and competitive nodulation of *Sinorhizobium* microorganisms and leguminous plants has been extensive and in-depth. Among these studies, *Sinomenium sinense* rhizobia has shown significant effects on promoting the growth of alfalfa. Yang Peizhi et al. found that *Sinomenium sinense* rhizobia has a good growth-promoting effect on alfalfa; Luo Ming et al. discovered a *Sinomenium sinense* rhizobia strain that not only has good salt-alkali resistance but also promotes alfalfa nodulation and increases alfalfa yield. Currently, there is no research on the effects of *Sinomenium sinense* rhizobia on promoting the growth of wheat, corn, and soybeans in sandy saline-alkali soils, or on improving sandy soil conditions. Therefore, research on multifunctional rhizobia suitable for promoting crop growth and improving sandy soils under sandy and saline-alkali conditions is of great significance and deserves further in-depth exploration. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a multifunctional rhizobium strain of Osmanthus fragrans and its applications. This strain has the ability to degrade fats, solubilize potassium, fix nitrogen, solubilize organic phosphorus, dissolve inorganic phosphorus, produce iron carriers, produce extracellular polysaccharides, produce auxin, and improve sandy soils. It can produce good growth-promoting effects on wheat, corn, and soybeans in saline-alkali soils and sandy infertile soils, and has a very broad application prospect.

[0008] The technical solution of this invention is as follows:

[0009] A strain of *Sinorhizobium meliloti* SK1 was deposited on October 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.32282.

[0010] The 16S rRNA gene sequence of *Rhizobium sinense* SK1 is shown in SEQ ID NO.1.

[0011] The above-mentioned method for culturing Rhizobium sinense SK1 includes the following steps:

[0012] The Rhizobium sinense SK1 strain was inoculated into a liquid culture medium to activate and culture it to obtain an activated bacterial solution; the activated bacterial solution was then inoculated into a liquid culture medium at a volume ratio of 1%-2% to expand the culture, thus obtaining the Rhizobium sinense SK1 bacterial solution.

[0013] According to a preferred embodiment of the present invention, the liquid culture medium is LB liquid culture medium.

[0014] According to a preferred embodiment of the present invention, the activation culture conditions are: 120-180 rpm, 36-38℃ for 12 h.

[0015] According to a preferred embodiment of the present invention, the conditions for the expanded culture are: 120-180 rpm, 36-38℃ for 12 h.

[0016] A microbial inoculant, comprising the aforementioned Rhizobium sinense SK1.

[0017] The above-mentioned Rhizobium sinense SK1 or its inoculum agent are used in one or more of the following applications: degradation of fats, potassium solubilization, nitrogen fixation, organic phosphorus solubilization, inorganic phosphorus solubilization, iron carrier production, extracellular polysaccharide production, auxin production, and improvement of sandy soil.

[0018] The above-mentioned application of Rhizobium sinense SK1 or its inoculum in promoting the growth of wheat, corn and soybean.

[0019] According to a preferred embodiment of the present invention, the application method involves preparing *Rhizobium sinense* SK1 with a live bacteria content of 2.0–3.0 × 10⁻⁶. 8 The live bacteria preparation at CFU / mL should be diluted more than 100 times and applied to the soil at a rate of 2-5 L / acre.

[0020] According to a preferred embodiment of the present invention, the soil is saline-alkali soil with a salt content ≤10g / Kg and pH ≤9, or sandy soil with a salt content ≤2g / Kg and pH ≤9.

[0021] The beneficial effects of this invention are:

[0022] The Rhizobium sinense SK1 provided by this invention has the ability to degrade fats, solubilize potassium, fix nitrogen, solubilize organic phosphorus, dissolve inorganic phosphorus, produce iron carriers, produce extracellular polysaccharides, produce auxin, and improve sandy soil. Moreover, this strain can produce good growth-promoting effects on wheat, corn, and soybeans planted in saline-alkali soil or sandy soil environments, and has a very broad application prospect. Attached Figure Description

[0023] Figure 1The images are of strain SK1; (A) shows the colony morphology of strain SK1, and (B) shows strain SK1 under a microscope after Gram staining.

[0024] Figure 2 Phylogenetic tree of strain SK1.

[0025] Figure 3 This is a diagram showing the growth of strain SK1 in glycerol tretinoin medium.

[0026] Figure 4 This is a diagram showing the growth of strain SK1 in a silicate bacteria culture medium.

[0027] Figure 5 This is a diagram showing the growth of strain SK1 in Assumption medium.

[0028] Figure 6 This is a diagram showing the growth of strain SK1 in Monkina organophosphate medium.

[0029] Figure 7 This is a diagram showing the growth of strain SK1 in Monkina inorganic phosphorus medium.

[0030] Figure 8 This is a diagram showing the growth of strain SK1 in CAS medium.

[0031] Figure 9 These are experimental images of strain SK1 producing extracellular polysaccharides; (A) shows an image of extracellular polysaccharides being produced in an Erlenmeyer flask, and (B) shows an image of extracellular polysaccharides placed on filter paper.

[0032] Figure 10 Experimental images showing the application of SK1 bacterial solution to sandy soil followed by drying.

[0033] Figure 11 Experimental images showing the production of auxin by strain SK1.

[0034] Figure 12 The images show the morphological characteristics of wheat plants in different treatment groups after 10 days of cultivation in saline-alkali soil.

[0035] Figure 13 The images show the morphological characteristics of wheat plants in different treatment groups after 14 days of cultivation in saline-alkali soil.

[0036] Figure 14 Figure 1 shows the results of the growth-promoting effect of strain SK1 on wheat under different treatment groups in saline-alkali soil. Figure 2 shows the physiological plant height of each treatment group after 10 and 14 days of wheat cultivation. Figure 3 shows the aboveground fresh weight and underground fresh weight of each treatment group after 14 days of wheat cultivation. Figure 4 shows the aboveground dry weight and underground dry weight of each treatment group after 14 days of wheat cultivation.

[0037] Figure 15 The image shows the morphological characteristics of wheat plants in different treatment groups after 10 days of cultivation in sandy soil.

[0038] Figure 16 The images show the morphological characteristics of wheat plants in different treatment groups after 14 days of cultivation in sandy soil.

[0039] Figure 17 The results show the growth-promoting effects of strain SK1 on wheat under different treatment groups in sandy soil. Among them, (A) is a bar graph of physiological plant height of each treatment group after 10 and 14 days of wheat cultivation, (B) is a bar graph of aboveground fresh weight and belowground fresh weight of each treatment group after 14 days of wheat cultivation, and (C) is a bar graph of aboveground dry weight and belowground dry weight of each treatment group after 14 days of wheat cultivation.

[0040] Figure 18 The images show the morphological characteristics of soybean plants in different treatment groups after 12 days of cultivation in saline-alkali soil.

[0041] Figure 19 Morphological images of soybean plants in different treatment groups after 16 days of cultivation in saline-alkali soil.

[0042] Figure 20 The results show the growth-promoting effects of strain SK1 on soybeans under different treatment groups in saline-alkali soil. Among them, (A) is a bar graph of physiological plant height of each treatment group after 12 and 16 days of soybean culture, (B) is a bar graph of stem diameter of each treatment group after 12 and 16 days of soybean culture, (C) is a bar graph of aboveground fresh weight and belowground fresh weight of soybeans after 16 days of culture, and (D) is a bar graph of aboveground dry weight and belowground dry weight of soybeans after 16 days of culture.

[0043] Figure 21 The images show the morphological characteristics of soybean plants in different treatment groups after 14 days of cultivation in sandy soil.

[0044] Figure 22 The images show the morphological characteristics of soybean plants in different treatment groups after 21 days of cultivation in sandy soil.

[0045] Figure 23 The results show the growth-promoting effects of strain SK1 on soybeans under different treatment groups in sandy soil. Among them, (A) is a bar graph of physiological plant height of each treatment group after 14 and 21 days of soybean culture, (B) is a bar graph of stem diameter of each treatment group after 14 and 21 days of soybean culture, (C) is a bar graph of aboveground fresh weight and belowground fresh weight of soybeans after 21 days of culture, and (D) is a bar graph of aboveground dry weight and belowground dry weight of soybeans after 21 days of culture.

[0046] Figure 24 The image shows the morphological characteristics of maize plants in different treatment groups after 12 days of cultivation in saline-alkali soil.

[0047] Figure 25 The results show the growth-promoting effect of strain SK1 on maize under different treatment groups in saline-alkali soil. Among them, (A) is a bar graph of physiological plant height of each treatment group after 9 and 12 days of maize cultivation, (B) is a bar graph of aboveground fresh weight and belowground fresh weight of each treatment group after 12 days of maize cultivation, and (C) is a bar graph of aboveground dry weight and belowground dry weight of each treatment group after 12 days of maize cultivation.

[0048] Figure 26 The image shows the morphological characteristics of maize plants in different treatment groups after 14 days of cultivation in sandy soil.

[0049] Figure 27 The results show the growth-promoting effect of strain SK1 on maize under different treatment groups in sandy soil. Among them, (A) is a bar graph of physiological plant height of each treatment group after 14 days of maize cultivation, (B) is a bar graph of aboveground fresh weight and belowground fresh weight of each treatment group after 14 days of maize cultivation, and (C) is a bar graph of aboveground dry weight and belowground dry weight of each treatment group after 14 days of maize cultivation. Detailed Implementation

[0050] The following description is based on specific embodiments. Any substitutions or modifications made based on this invention without departing from the scope of this invention are within the protection scope of this invention.

[0051] Explanation of the source of experimental materials:

[0052] Wheat seeds: Jinchun No. 6, purchased from Fuyichun Seed Company.

[0053] Soybean seeds: Zhonghuang 57, purchased from Henan Yellow River Beach Source Seed Industry Co., Ltd.

[0054] Corn seeds: Zhengdan 958, purchased from Gansu State Farms Seed Co., Ltd.

[0055] Example 1

[0056] Isolation, screening and identification of Rhizobium SK1 from Osmanthus fragrans

[0057] Soil was collected from sandy soil in Yanggu County, Liaocheng City, Shandong Province, China. The specific separation and screening methods are as follows: 10.0g of soil was weighed and placed in an Erlenmeyer flask containing sterile water, then placed on a constant temperature shaker at 37℃ and 180r / min for 30min to obtain a soil suspension. The soil suspension was then serially diluted using a gradient dilution method, and soil samples were collected at a dilution of 10... -4 10 -5 10 -6Soil suspensions were spread onto LB solid medium and incubated at 37°C. After single colonies grew, they were picked out with toothpicks and purified on the medium using the three-zone streak method until single colonies were obtained. The colonies were then screened for functions such as organic phosphorus solubilization, inorganic phosphorus solubilization, potassium solubilization, nitrogen fixation, lipid degradation, cellulose degradation, extracellular polysaccharide production, and glycogen production.

[0058] Through the above separation and screening work, a strain with the characteristics of degrading fat, solubilizing potassium, fixing nitrogen, solubilizing organic phosphorus, dissolving inorganic phosphorus, producing iron carriers, producing extracellular polysaccharides, producing auxin, and improving sandy soil was finally obtained and named "SK1".

[0059] The colony morphology of the above-screened strain SK1 on LB solid medium and its microscopic observation after Gram staining are shown in the following figures. Figure 1 As shown; Figure 1 As shown in Figure A, the colonies of strain SK1 are off-white, irregularly shaped, smooth, and viscous; Figure 1 As shown in Figure B, the strain is a typical rod-shaped Gram-negative bacterium.

[0060] In addition, the physiological and biochemical characteristics of the selected strain SK1 were identified, and the results are as follows: strain SK1 can utilize trehalose, sucrose, xylose, and maltose.

[0061] The 16S rRNA gene sequence of strain SK1 was sequenced by Sangon Biotech (Shanghai) 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. Strains with similar homology were selected, and a phylogenetic tree was constructed using MEGA 5.0 software with the Neighborjoining method. The results are as follows. Figure 2 As shown, the results showed that the strain SK1 obtained by the above screening had 100% similarity to Sinorhizobium melilotistrain NBRC 14782 and was relatively close in evolutionary distance. Based on the physiological and biochemical characteristics of the strain, it was identified as Sinorhizobium meliloti.

[0062] Sinorhizobium meliloti SK1 was deposited on October 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.32282.

[0063] Example 2

[0064] The specific method for culturing the SK1 bacterial culture of *Osmanthus fragrans* is as follows:

[0065] (1) The SK1 strain of Rhizobium sinense was transferred into a test tube containing 5 mL of LB liquid medium and placed in a constant temperature shaker at 180 rpm and 37 ℃ for 12 h to obtain activated bacterial solution.

[0066] (2) The activated bacterial solution obtained in step (1) was inoculated into 50 mL of sterilized LB liquid medium at an inoculation rate of 1% (0.5 mL) and cultured for 24 h to obtain the *Osmanthus fragrans* SK1 bacterial solution. The viable count in the bacterial solution was determined to be 2.32 × 10⁻⁶. 8 CFU / mL.

[0067] The above LB liquid culture medium formula is as follows: 5g yeast extract, 10g peptone, 10g sodium chloride, and distilled water to a final volume of 1L, pH 7.0.

[0068] Experimental Example 1

[0069] Determination of the abilities of *Osmanthus f. sinensis* rhizobium SK1 in degrading fats, solubilizing potassium, fixing nitrogen, solubilizing organic phosphorus, dissolving inorganic phosphorus, producing siderophores, producing extracellular polysaccharides, improving sandy soil, and producing auxin:

[0070] (1) The fat-degrading ability test of strain SK1 is as follows:

[0071] Strains SK1 were inoculated onto glyceryl tribose medium with sterile toothpicks and incubated in a constant temperature incubator at 28℃ for 3 days. Colony growth was then observed.

[0072] The above-mentioned tributylate culture medium formula is as follows: 10g peptone, 5g yeast extract, 10g NaCl, 2mL tributylate, 15-20g agar, and deionized water to a final volume of 1L.

[0073] Colony growth status as follows Figure 3 As shown, strain SK1 exhibits a transparent degradation zone in tribocylate medium, indicating that strain SK1 has the ability to degrade lipids.

[0074] (2) Potassium solubilization ability test of strain SK1, the specific method is as follows:

[0075] Strains SK1 were inoculated onto silicate bacteria culture medium with sterile toothpicks and incubated in a 30°C incubator for 3 days. Colony growth was then observed.

[0076] The above-mentioned silicate bacteria culture medium formula is as follows: sucrose 5g, MgSO4 0.5g, CaCO3 0.1g, Na2HPO4 2g, FeCl3 0.005g, glass powder 1g, agar 15g, deionized water to a final volume of 1L, pH 7.0.

[0077] Colony growth status as follows Figure 4 As shown, SK1 forms smooth, transparent, oil-drop-shaped colonies on silicate bacteria culture medium, indicating that strain SK1 has potassium-solubilizing ability.

[0078] (3) The nitrogen fixation capacity test of strain SK1 was conducted using the following method:

[0079] Strains SK1 were inoculated onto Assumption medium with sterile toothpicks and incubated in a constant temperature incubator at 28°C for 5 days. Colony growth was then observed.

[0080] The above Assumption medium formula is as follows: mannitol 10g, CaCO3 5g, KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaSO4·2H2O 0.1g, agar 18g, and deionized water to a final volume of 1L.

[0081] Colony growth status as follows Figure 5 As shown, SK1 bulges slightly on Assumption medium, appearing as a slimy substance with a smooth surface and a transparent ring, indicating that strain SK1 has nitrogen-fixing ability.

[0082] (4) Test of the organophosphate solubility of strain SK1, the specific method is as follows:

[0083] Strains SK1 were inoculated onto Monkina organophosphate medium with sterile toothpicks and incubated in a 28°C electric thermostatic incubator for 7 days. The colony growth was then observed.

[0084] The above-mentioned Monkina organic phosphorus culture medium formula is as follows: glucose 10g, (NH4)2SO4 0.5g, MgSO4·7H2O 0.3g, MnSO4·4H2O 0.03g, KCl 0.3g, FeSO4·7H2O 0.03g, NaCl 0.3g, CaCO3 5.0g, lecithin 0.2g, agar 15g, and deionized water to a final volume of 1L, pH 7.0~7.5.

[0085] Colony growth status as follows Figure 6 As shown, strain SK1 exhibited a transparent degradation zone in the Monkina organic phosphorus medium, indicating that strain SK1 has the ability to degrade organic phosphorus.

[0086] (5) The inorganic phosphorus solubility test of strain SK1 was conducted using the following method:

[0087] Strains SK1 were inoculated onto the Mongkina inorganic phosphorus medium with sterile toothpicks and incubated in a 28°C electric thermostatic incubator for 7 days. The colony growth was then observed.

[0088] The above-mentioned Monkina inorganic phosphorus culture medium formula is as follows: glucose 10g, (NH4)2SO4 0.5g, MgSO4·7H2O 0.3g, MnSO4·4H2O 0.03g, KCl 0.3g, FeSO4·7H2O 0.03g, NaCl 0.3g, Ca3(PO4)2 10.0g, deionized water to a final volume of 1L, pH 7.0~7.5.

[0089] Colony growth status as follows Figure 7 As shown, strain SK1 exhibited a transparent degradation zone in the Monkina inorganic phosphorus medium, indicating that strain SK1 has the ability to dissolve inorganic phosphorus.

[0090] (6) The siderophore production capacity test of strain SK1 was conducted using the following method:

[0091] Activate the strain on LB solid plates, pick a single colony with a toothpick and place it on a prepared CAS solid plate, incubate upside down at 37°C for 3 days, and observe the size of the discoloration zone around the bacterial growth.

[0092] The above-mentioned CAS culture medium formula is as follows: 0.06g of Chromium Azurite S (CAS), 0.073g of Hexadecyltrimethylammonium bromide (HDTMA), 0.0026g of FeCl3·6H2O, 0.295g of NaH2PO4·2H2O, 1.213g of Na2HPO4·12H2O, 0.125g of NH4Cl, 0.037g of KH2PO4, 0.0625g of NaCl, 9g of agar, and deionized water to a final volume of 1L, pH 6.7–6.9.

[0093] Colony growth status as follows Figure 8 As shown, strain SK1 exhibited a transparent degradation zone in CAS medium, indicating that strain SK1 has the ability to produce siderophores.

[0094] (7) Test of the extracellular polysaccharide production capacity of strain SK1, the specific method is as follows:

[0095] Strain SK1 was inoculated into LB liquid medium and cultured with shaking at 180 rpm until the logarithmic growth phase. It was then transferred to PDA liquid medium and cultured for another 36 h at 30°C and 180 rpm. The culture was then centrifuged at 12,000 rpm for 20 min, and the supernatant was mixed with frozen acetone at a volume ratio of 1:2. After standing for 24 h, as follows... Figure 9 As shown, a precipitate was produced in the conical flask, which is an extracellular polysaccharide, proving that strain SK1 has the ability to produce extracellular polysaccharides.

[0096] The above PDA liquid culture medium formula is as follows: 200g of potato (peel the potato, cut it into small pieces, boil it in boiling water for 30 minutes, and filter it with four layers of gauze to remove the potato), add 20g of sucrose or glucose, and make up to 1L with deionized water.

[0097] (8) The test of the ability of strain SK1 to improve sandy soil was conducted using the following method:

[0098] Sandy soils were collected from a sandy soil experimental field and divided into three groups: Group A (CK1), Group B (CK2), and Group C (SK1). Group A sandy soils were not treated in any way, Group B sandy soils were irrigated with sterile water, and Group C sandy soils were irrigated with the same volume of SK1 bacterial solution (prepared in Example 2) as Group B. After irrigation, the three groups of sandy soils were dried at 105°C for 3 hours, 6 hours, and 12 hours, respectively. The changes in the sandy soils in the different treatment groups were photographed and observed.

[0099] The effect of strain SK1 on improving sandy soil is as follows: Figure 10 As shown, before drying, the sandy soil in group A was loose and dry. After being irrigated with the same volume of sterile water and bacterial solution SK1, the soil in groups B and C had a higher moisture content. After 3 hours of drying, the soil in group B had severe water loss, becoming compacted and cracked. The sandy soil in group C treated with strain SK1 remained moist compared to group B, with a high moisture content. After 6 hours of drying, the sandy soil in group B had become severely compacted, hard, and cracked. In contrast, the sandy soil in group C, irrigated with bacterial solution SK1, only showed signs of water loss at the edges and maintained high moisture content. After 12 hours of drying, both groups B and C showed signs of compaction, with the sandy soil in group B cracking and becoming severely compacted. Therefore, it can be concluded that the sandy soil treated with strain SK1 was more moist and experienced slower water loss than the sterile water treatment group under drying conditions at 105℃. Thus, it is determined that strain SK1 has the ability to improve sandy soil.

[0100] (9) The ability of strain SK1 to produce lignin (IAA) was tested, and the specific method is as follows:

[0101] Strain SK1 was inoculated into R2A liquid medium containing L-tryptophan and cultured at 28°C with shaking at 180 rpm for 4 days. 50 μL of the bacterial suspension was dropped onto a white ceramic plate, and 50 μL of Salkowski colorimetric solution was added. Simultaneously, 50 mg / L of IAA was added to the Salkowski colorimetric solution as a positive control. The white ceramic plate was stored at room temperature in the dark for 30 minutes, and the color change was observed. If the color turned red, it indicated that the strain had the function of producing IAA.

[0102] The above R2A liquid culture medium formula is as follows: 0.5g yeast extract, 0.5g glucose, 0.5g peptone, 0.5g casein amino acids (acid-hydrolyzed casein), 0.5g soluble starch, 0.3g K2HPO4, 0.05g MgSO4·7H2O, 0.3g sodium pyruvate, 200mg L-tryptophan, and deionized water to a final volume of 1L. The pH is adjusted to 7.2 with K2HPO4 and KH2PO4, and the medium is autoclaved at 121℃ for 20 minutes.

[0103] The preparation method of Salkowski colorimetric solution is as follows: prepare 50 mL of HClO4 with a volume fraction of 35% and 1 mL of FeCl3 with a concentration of 0.5 mol / L, mix well, and store in the dark.

[0104] like Figure 11 As shown, the liquid in the wells of strain SK1 and the liquid in the wells of the IAA control group both turned red, proving that strain SK1 has the ability to produce auxin.

[0105] Experimental Example 2

[0106] The specific steps for determining the wheat growth-promoting effect of *Osmanthus fragrans* rhizobium SK1 in saline-alkali and sandy soil environments are as follows:

[0107] (1) Soak Jinchun No. 6 wheat seeds in a 1% sodium hypochlorite solution for 10 minutes. Rinse the soaked wheat seeds with sterile water. Then soak the rinsed wheat seeds in 75% ethanol for 5 minutes. Rinse them again with sterile water. Wrap the rinsed wheat seeds in a damp gauze for 12 hours for later use.

[0108] (2) Prepare 18 cultivation pots for wheat cultivation. Divide the 18 cultivation pots into 6 groups: saline-alkali soil CK-LB group, saline-alkali soil CK-sterile water group and saline-alkali soil SK1 experimental group, sandy soil CK-LB group, sandy soil CK-sterile water group and sandy soil SK1 experimental group. Each group has 3 replicates.

[0109] The saline-alkali soil added to the cultivation pots was taken from the saline-alkali soil experimental field in Liaocheng City, Shandong Province, with a salt content of 3.45 g / kg and a soil pH of 8.5; the sandy soil added to the cultivation pots was taken from the sandy soil experimental field in Liaocheng City, Shandong Province, with a salt content of 1.1 g / kg and a soil pH of 8.27.

[0110] Among them, the bacteria added to the cultivation pot of SK1 experimental group was Rhizobium sinense SK1. The application method was as follows: the bacterial solution prepared in Example 2 was diluted with water by 200 times and the application amount was 70mL / Kg. The CK-LB group and the CK-sterile water group were given the same volume of LB liquid culture medium and sterile water as the bacterial solution of SK1 experimental group in the cultivation pot.

[0111] (3) Select uniform wheat seeds from step (1) and plant them in cultivation pots. After the seedlings emerge, thin them out so that there are 3 seedlings in each cultivation pot. After thinning, treat with bacterial solution. After planting in cultivation pots for 10 days, take pictures of the growth morphology of the wheat. After 14 days, harvest the seedlings and measure the agronomic traits of the wheat, such as physiological plant height (the maximum distance after straightening), above-ground fresh weight, above-ground dry weight, underground fresh weight, and underground dry weight.

[0112] Photos of wheat morphology in different treatment groups after 10 days of cultivation in saline-alkali soil are shown below. Figure 12 As shown, wheat morphology photographs from different treatment groups 14 days after cultivation are as follows. Figure 13 As shown, by Figure 12 and Figure 13 It can be seen that the wheat plants in the SK1 experimental group grew more vigorously than those in the CK-LB group and the CK-sterile water group.

[0113] The results of the determination of the growth-promoting effect of strain SK1 on wheat in different treatment groups under saline-alkali soil environment after 14 days of cultivation are as follows: Figure 14 As shown; by Figure 14 As shown in Figure A, the wheat plant height in the SK1 experimental group was significantly higher than that in the CK-LB group. On day 10, the wheat plant height in the SK1 experimental group increased by 33.46% compared with that in the CK-LB group (p<0.01); after 14 days of wheat cultivation, the wheat plant height in the SK1 experimental group increased by 19.49% compared with that in the CK-LB group (p<0.01).

[0114] The weight of wheat measured 14 days after cultivation is as follows: Figure 14 As shown in Figures B-C, it can be seen that, under saline-alkali conditions, the above-ground fresh weight of wheat in the SK1 experimental group increased by 66.67% compared to the CK-LB group on day 14 (p<0.01); the above-ground dry weight increased by 78.23% compared to the CK-LB group on day 14 (p<0.01); the underground fresh weight increased by 63.69% compared to the CK-LB group on day 14 (p<0.01); and the underground dry weight increased by 48.61% compared to the CK-LB group on day 14.

[0115] Photographs of wheat morphology in different treatment groups after 10 days of cultivation in sandy soil environment are shown below. Figure 15 As shown, wheat morphology photographs from different treatment groups 14 days after cultivation are as follows. Figure 16 As shown, by Figure 15 and Figure 16 It can be seen that the wheat plants in the SK1 experimental group grew more vigorously than those in the CK-LB group and the CK-sterile water group.

[0116] The results of the determination of the growth-promoting effect of strain SK1 on wheat in different treatment groups under sandy soil environment after 14 days of cultivation are as follows: Figure 17 As shown; by Figure 17 As shown in Figure A, the wheat plant height in the SK1 experimental group was significantly higher than that in the CK-LB group. On day 10, the wheat plant height in the SK1 experimental group increased by 51.31% compared with that in the CK-LB group (p<0.01); after 14 days of wheat cultivation, the wheat plant height in the SK1 experimental group increased by 32.42% compared with that in the CK-LB group (p<0.01).

[0117] The weight of wheat measured 14 days after cultivation is as follows: Figure 17 As shown in Figures B-C, it can be seen that: for wheat grown in sandy soil, on day 14, the SK1 experimental group showed a 95.62% increase in above-ground fresh weight compared to the CK-LB group (p<0.01); the SK1 experimental group showed a 105.66% increase in above-ground dry weight compared to the CK-LB group (p<0.01); the SK1 experimental group showed a 33.44% increase in underground fresh weight compared to the CK-LB group; and the SK1 experimental group showed a 19.54% increase in underground dry weight compared to the CK-LB group.

[0118] Experimental Example 3

[0119] The specific steps for determining the growth-promoting effect of *Osmanthus fragrans* SK1 on soybeans in saline-alkali and sandy soil environments are as follows:

[0120] (1) Select plump and uniform soybean seeds, treat them with 75% ethanol for 5 minutes, then sterilize them with 1% sodium hypochlorite solution for 3 minutes, and then wash them with sterile water 8-10 times; spread them evenly on a damp gauze and germinate them at 28℃ for 2-3 days.

[0121] (2) Prepare 18 cultivation pots for soybean cultivation. The group settings, soil sources, and application methods are the same as in Experiment 2.

[0122] (3) Select soybean seeds of uniform size from step (1) and plant them in cultivation pots. After the seedlings emerge, thin them out so that there are 3 seedlings in each cultivation pot. After thinning, treat with bacterial solution. After planting in cultivation pots for 12 days, take pictures of the growth morphology of soybeans in saline-alkali environment and measure the physiological plant height and stem diameter. After 16 days, harvest the seedlings and measure the physiological plant height, stem diameter, above-ground fresh weight, underground fresh weight, above-ground dry weight, and underground dry weight of soybeans in saline-alkali environment.

[0123] Fourteen days after being planted in cultivation pots, the growth morphology of soybeans in the sandy soil environment was photographed and the physiological plant height and stem diameter were measured. Twenty-one days later, the seedlings were harvested and the physiological plant height, stem diameter, above-ground fresh weight, underground fresh weight, above-ground dry weight, and underground dry weight of soybeans in the sandy soil environment were measured.

[0124] Photos of soybean morphology in different treatment groups after 12 days of cultivation in saline-alkali soil environment are shown below. Figure 18 As shown in the images, soybean morphology photos from different treatment groups were taken 16 days after cultivation. Figure 19 As shown, by Figure 18 and Figure 19 It can be seen that the soybean plants in the SK1 experimental group grew more vigorously than those in the CK-LB group and the CK-sterile water group.

[0125] The results of the determination of the growth-promoting effect of strain SK1 on soybeans in different treatment groups under saline-alkali soil environment after 16 days of cultivation are as follows: Figure 20 As shown; by Figure 20 As shown in Figure A, compared with the CK-LB group, the soybean plant height in the SK1 experimental group was significantly increased. On day 12, the soybean plant height in the SK1 experimental group was 24.59% higher than that in the CK-LB group (p<0.01); after 16 days of soybean cultivation, the wheat plant height in the SK1 experimental group was 20.78% higher than that in the CK-LB group (p<0.01). Figure 20 As shown in Figure B, the soybean stem diameter in the SK1 experimental group was significantly increased compared with that in the CK-LB group. On the 12th day, the soybean stem diameter in the SK1 experimental group increased by 21.21% compared with that in the CK-LB group (p<0.01); after 16 days of soybean cultivation, the soybean stem diameter in the SK1 experimental group increased by 14.21% compared with that in the CK-LB group (p<0.01).

[0126] The weight of soybeans measured 16 days after cultivation is as follows: Figure 20 As shown in Figures C and D, under saline-alkali conditions, the above-ground fresh weight of soybeans increased by 41.64% compared to the CK-LB group on day 16 (p<0.01); the above-ground dry weight increased by 53.68% compared to the CK-LB group on day 16 (p<0.01); the underground fresh weight increased by 45.44% compared to the CK-LB group on day 16 (p<0.01); and the underground dry weight increased by 89.32% compared to the CK-LB group on day 16 (p<0.01).

[0127] Photos of soybean morphology in different treatment groups after 14 days of cultivation in sandy soil environment are shown below. Figure 21 As shown in the images, soybean morphology photos from different treatment groups were taken 21 days after cultivation. Figure 22 As shown, by Figure 21 and Figure 22 It can be seen that the soybean plants in the SK1 experimental group grew more vigorously than those in the CK-LB group and the CK-sterile water group.

[0128] The results of the determination of the growth-promoting effect of strain SK1 on soybeans in different treatment groups under sandy soil environment after 21 days of cultivation are as follows: Figure 23 As shown; by Figure 23 As shown in Figure A, the soybean plant height in the SK1 experimental group was significantly higher than that in the CK-LB group. On day 14, the soybean plant height in the SK1 experimental group was 37.09% higher than that in the CK-LB group (p<0.01); after 21 days of soybean cultivation, the soybean plant height in the SK1 experimental group was 31.44% higher than that in the CK-LB group (p<0.01). Figure 23 As shown in Figure B, the soybean stem diameter in the SK1 experimental group was significantly increased compared with that in the CK-LB group. On the 14th day, the soybean stem diameter in the SK1 experimental group increased by 11.64% compared with that in the CK-LB group; after 21 days of soybean cultivation, the soybean stem diameter in the SK1 experimental group increased by 13.90% compared with that in the CK-LB group (p<0.05).

[0129] The weight of soybeans measured 21 days after cultivation is as follows: Figure 23 As shown in Figures C and D, under sandy soil conditions, the aboveground fresh weight of soybeans on day 21 was 25.80% higher than that of the CK-LB group (p<0.01); the aboveground dry weight was 40.20% higher than that of the CK-LB group (p<0.01); the underground fresh weight was 30.67% higher than that of the CK-LB group on day 21; and the underground dry weight was 36.68% higher than that of the CK-LB group on day 21.

[0130] Experiment Example 4

[0131] The specific steps for determining the growth-promoting effect of *Osmanthus fragrans* SK1 on maize in saline-alkali and sandy soil environments are as follows:

[0132] (1) Select plump and uniform corn seeds, treat them with 75% ethanol for 5 minutes, then wash them with 2% antifomin (prepared fresh) for 10 minutes, and finally wash them with sterile water for 3 minutes. Then place the seeds on a damp gauze and germinate at 30°C for 24 hours in complete darkness until the seeds sprout.

[0133] (2) Prepare 18 cultivation pots for growing corn. The group settings, soil sources, and application methods are the same as in Experiment 2.

[0134] (3) Select uniformly sized corn seeds from step (1) and plant them in cultivation pots. After emergence, thin the seedlings to ensure that there are 3 seedlings in each cultivation pot. After thinning, irrigate with bacterial solution. Nine days after planting in cultivation pots, measure the corn plant height in the saline-alkali soil environment. Twelve days after planting, photograph the growth morphology of corn in the saline-alkali soil environment and measure agronomic traits such as physiological plant height, above-ground fresh weight, above-ground dry weight, underground fresh weight, and underground dry weight. Fourteen days after planting in cultivation pots, photograph the growth morphology of corn in the sandy soil environment and measure agronomic traits such as physiological plant height, above-ground fresh weight, above-ground dry weight, underground fresh weight, and underground dry weight.

[0135] Photos of maize morphology in different treatment groups after 12 days of cultivation in saline-alkali soil are shown below. Figure 24 As shown, by Figure 24 It can be seen that the corn plants in the SK1 experimental group grew more vigorously than those in the CK-LB group and the CK-sterile water group.

[0136] The results of the determination of the growth-promoting effect of strain SK1 on maize in different treatment groups under saline-alkali soil environment after 12 days of cultivation are as follows: Figure 25 As shown; by Figure 25 As shown in Figure A, compared with the CK-sterile water group, the maize plant height in the SK1 experimental group was significantly increased. After 9 days of maize cultivation, the wheat plant height in the SK1 experimental group increased by 5.3% compared with the CK-sterile water group; after 12 days of maize cultivation, the wheat plant height in the SK1 experimental group increased by 5.63% compared with the CK-sterile water group; the weight measurement results of maize after 12 days of cultivation are as follows. Figure 25 As shown in Figures B and C, it can be seen that under saline-alkali soil conditions, the above-ground fresh weight of maize on day 12 increased by 14.05% compared to the CK-sterile water group; the above-ground dry weight increased by 17.87% compared to the CK-sterile water group; the underground fresh weight increased by 27.51% compared to the CK-sterile water group; and the underground dry weight increased by 9.06% compared to the CK-sterile water group.

[0137] Photos of maize morphology in different treatment groups after 14 days of cultivation in sandy soil environment are shown below. Figure 26 As shown, by Figure 26 It can be seen that the corn plants in the SK1 experimental group grew more vigorously than those in the CK-LB group and the CK-sterile water group.

[0138] The results of the determination of the growth-promoting effect of strain SK1 on maize in different treatment groups under sandy soil conditions after 14 days of cultivation are as follows: Figure 27 As shown; by Figure 27As shown in Figure A, the maize plant height in the SK1 experimental group was significantly higher than that in the CK-LB group. After 14 days of maize cultivation, the wheat plant height in the SK1 experimental group was 1.58% higher than that in the CK-LB group.

[0139] The results of the weight measurement of corn 14 days after cultivation are as follows: Figure 27 As shown in Figures B and C, it can be seen from Figures B and C that, under sandy soil conditions, the aboveground fresh weight of maize on day 14 increased by 47.25% compared to the CK-LB group; the aboveground dry weight increased by 50% compared to the CK-LB group on day 14; the underground fresh weight increased by 53.76% compared to the CK-LB group on day 14; and the underground dry weight increased by 21.50% compared to the CK-LB group on day 14.

Claims

1. A strain of Rhizobium sinense ( Sinorhizobium meliloti SK1 was deposited on October 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.32282.

2. The culture method of Methylobacterium mespalii SK1 according to claim 1, wherein, comprising the following steps: The Sinorhizobium meliloti SK1 strain is inoculated into a liquid culture medium for activation culture to obtain an activated bacterial liquid; the activated bacterial liquid is inoculated into a liquid culture medium at a volume ratio of 1%-2% for expansion culture to obtain the Sinorhizobium meliloti SK1 bacterial liquid.

3. The culture method according to claim 2, wherein The liquid culture medium is an LB liquid culture medium.

4. The culture method according to claim 2, wherein The activation culture condition is 120-180 rpm, 36-38℃ culture for 12h.

5. The culturing method according to claim 2, wherein The expansion culture condition is 120-180 rpm, 36-38℃ culture for 12h.

6. A microbial inoculant, characterized in that, The Sinorhizobium meliloti SK1 of claim 1.

7. The Sinorhizobium meliloti SK1 of claim 1 or the microbial inoculant of claim 6 is applied in degrading fat, releasing potassium, fixing nitrogen, degrading organic phosphorus, dissolving inorganic phosphorus, producing siderophore, producing exopolysaccharide, producing auxin, and improving one or more than two of sandy soil.

8. The Sinorhizobium meliloti SK1 of claim 1 or the microbial inoculant of claim 6 is applied in promoting the growth of wheat, corn, and soybean.

9. Use according to claim 8, wherein the compound is ###0002### The application method is to prepare the Sinorhizobium meliloti SK1 into a viable bacteria preparation with a viable bacteria content of 2.0-3.0×10 8 CFU / mL, and to apply in the soil at an application amount of 2-5 L / mu after dilution by 100 times or more.

10. Use according to claim 9, wherein The soil is saline-alkali soil with a salt content of ≤10g / Kg and a pH of ≤9, or sandy soil with a salt content of ≤2g / Kg and a pH of ≤9. The soil is saline-alkali soil with a salt content of ≤10g / Kg and a pH of ≤9, or sandy soil with a salt content of ≤2g / Kg and a pH of ≤9.

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