Rhizobium with high yield of exocellular polysaccharide LX and application thereof

Abstract

The application provides a high-yield exocellular polysaccharide rhizobium LX and application thereof, and belongs to the technical field of biology, wherein the rhizobium LX is preserved in the China Center for Type Culture Collection, the preservation time is February 28, 2025, and the preservation number is CCTCC M 2025321; the rhizobium LX strain can efficiently synthesize exocellular polysaccharide in a conventional fermentation medium, the fermentation process has the characteristics of low cost, simple operation, short cycle and high yield (up to 30 g / L), and exhibits significant scale production advantages; the fermentation liquor or exocellular polysaccharide of the rhizobium LX can significantly enhance the ability of plants to resist saline-alkali and drought stress, and the required amount is less.

Description

Technical Field

[0001] The invention relates to a rhizobium LX with high extracellular polysaccharide production and application thereof, belonging to the field of biotechnology. Background Art

[0002] China's salinized soils are remarkably widespread and ecologically diverse, covering nearly 100 million hectares. In agricultural production, salt accumulation and water scarcity have become key environmental stressors that restrict crop growth. High salt concentrations disrupt normal plant metabolic activity by altering osmotic balance, leading to stunted growth and plant death. Soil salinization causes salt stress in plants, resulting in reduced crop yield and quality, even to the point of total crop failure, severely hindering the sustainable development of protected horticulture. Similarly, drought stress has multiple negative effects on plants and crops, including inhibiting seed germination, slowing root and aboveground growth, reducing photosynthetic efficiency, disrupting cell membrane stability, and impairing water and nutrient absorption. It also induces the accumulation of reactive oxygen species, resulting in oxidative damage, ultimately significantly reducing crop yield and quality, and in severe cases, even leading to plant death. Strategies to regulate these stresses focus on three key areas: cultivating stress-resistant crop varieties, improving soil structure, and adding exogenous biostimulants. Among them, biostimulants have attracted much attention because they can effectively activate plant metabolic systems, optimize nutrient utilization efficiency and enhance plant stress resistance mechanisms. Typical representatives such as oligochitosan and betaine have been proven to significantly improve crop salt and drought tolerance through simple application.

[0003] Rhizobia are typical representatives of plant rhizosphere microorganisms. In the field of microbial inoculants, rhizobia, as typical plant symbiotic bacteria, are often used in the form of compound fertilizers for farmland improvement. However, traditional live bacterial formulations face significant technical bottlenecks, including vulnerability of bacterial activity to environmental fluctuations, low soil colonization efficiency, and niche competition with native microbial communities. These factors restrict their practical application. Rhizobium exopolysaccharides (EPS), a key mediator of plant growth-promoting rhizobium (PGPR), exhibit multiple mechanisms in stress regulation: by chelating salt ions, scavenging reactive oxygen species, and activating endogenous plant hormone pathways, they synergistically enhance plant resistance to salinity and drought, and promote root development. As functional metabolites, EPS do not require live bacterial colonization to exert their functions, thus avoiding the risk of competition between exogenous microbial agents and native microbial communities. Furthermore, EPS can be produced on a large scale through low-cost fermentation, is durable in storage, and is suitable for industrial coating or foliar spraying. It combines ecological safety with economic feasibility in saline soil improvement and water-saving agriculture, offering a new path for the development of green agricultural technologies. Summary of the Invention

[0004] In order to solve the above-mentioned defects and deficiencies in the prior art, the present invention provides a rhizobium LX with high exopolysaccharide production. The rhizobium LX is isolated from a vegetable field and can efficiently produce exopolysaccharides, thereby promoting plant growth and improving plant salt tolerance or drought tolerance.

[0005] To solve the above technical problems:

[0006] The first object of the present invention is to provide a rhizobium with high production of extracellular polysaccharides, which is classified as Rhizobium sp. LX, the rhizobium LX was deposited in the China Center for Type Culture Collection (Wuhan University) on February 28, 2025, with the deposit number CCTCC M 2025321;

[0007] The extracellular polysaccharide structural components include glucose, galactose, xylose, fucose and mannose, among which glucose and galactose are the main structural components.

[0008] Furthermore, the mass of the main structural components accounts for more than 95% of the total mass.

[0009] Furthermore, the molar ratio of glucose to galactose in the main structural components of the extracellular polysaccharide is 7-8:1.

[0010] Furthermore, the preparation method of the exopolysaccharide comprises the following steps:

[0011] The seed liquid of Rhizobium LX is inoculated into a fermentation medium to obtain a polysaccharide fermentation liquid. The polysaccharide fermentation liquid is precipitated with alcohol and dried to a constant weight to obtain a crude exopolysaccharide. The crude extract is dissolved in water and then an organic solvent is added to remove protein. The extract is then dialyzed and dried to obtain a pure exopolysaccharide.

[0012] The second object of the present invention is to provide a use of the above-mentioned high-polysaccharide-producing Rhizobium LX in improving plant resistance to salt or drought stress.

[0013] Furthermore, the polysaccharide fermentation liquid or exopolysaccharide of the Rhizobium LX is added during the plant cultivation process.

[0014] Furthermore, the concentration of the polysaccharide fermentation broth or extracellular polysaccharide is 20-300 mg / L.

[0015] The beneficial technical effects achieved by the present invention are:

[0016] (1) Rhizobium LX strain can efficiently synthesize exopolysaccharides in conventional fermentation medium. Its fermentation process is characterized by low cost, simple operation, short cycle, and high yield (up to 30g / L), showing significant advantages in large-scale production;

[0017] (2) The fermentation liquid or extracellular polysaccharide of Rhizobium LX can significantly enhance the ability of plants to resist saline-alkali and drought stress, and the required dosage is relatively small. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :The present invention constructs strains based on 16S rDNA gene sequence Rhizobium sp.LX phylogenetic tree;

[0019] Figure 2 :Rhizobium strains of the present invention Rhizobium sp.LX colony morphology;

[0020] Figure 3 : The morphology of the Rhizobium LX polysaccharide fermentation broth of the embodiment of the present invention;

[0021] Figure 4 : Fermentation curve of Rhizobium LX in a 5L stirred reactor according to an embodiment of the present invention;

[0022] Figure 5 : HPLC chromatogram of exopolysaccharide produced by Rhizobium LX in the embodiment of the present invention;

[0023] Figure 6 : Gel permeation chromatogram of exopolysaccharide produced by Rhizobium LX in the embodiment of the present invention;

[0024] Figure 7 : Pictures of the growth status of cucumber seedlings under salt stress in the embodiment of the present invention;

[0025] Figure 8 : Pictures of the growth status of tomato seedlings under salt stress in the embodiment of the present invention;

[0026] Figure 9 : A picture of the growth status of wheat seedlings under extreme drought stress on the 14th day in an embodiment of the present invention;

[0027] Figure 10 : Pictures of the growth status of wheat seedlings under salt stress in the examples of the present invention.

[0028] The deposit information of the rhizobium of this application is as follows:

[0029] Rhizobium: Classification and naming Rhizobium sp. LX, deposited in China Center for Type Culture Collection (Wuhan University) on February 28, 2025, with the accession number CCTCC M 2025321; DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0031] The present invention is further described below with reference to the accompanying drawings and embodiments.

[0032] Example 1 Isolation, extraction and identification of Rhizobium LX

[0033] 1. Isolation and extraction of Rhizobium LX: Take the soil from the vegetable field in Nanjing, remove the plant debris, weigh about 1g of soil, add 100mL of sterile water and glass beads to a 250mL triangular flask, and shake at 100rpm for 60min to allow the soil particles to swell fully and release the microorganisms. Let the triangular flask stand for 60min to allow the sand and other solids to fully settle. Take the supernatant and dilute it with sterile water to 10 -5 . Take 100μL of the dilution to the screening medium plate, spread it evenly with a triangular spreading stick, spread it on 10 plates in total, and incubate at 30℃ for 24-72h. Observe the colony morphology on the plate, select single colonies with smooth, raised, moist and sticky surfaces, and number them for subsequent identification. Screening medium formula: glucose 20g / L, yeast powder 2g / L, peptone 3g / L, NaCl 10g / L, KH2PO4 2g / L, agar powder 16g / L, pH 7.5.

[0034] 2. Identification of Rhizobium LX:

[0035] 2.1 Extraction of total DNA: 200 µL of the bacterial solution obtained in step 1 was added to LB medium and cultured in a shaking incubator at 30°C and 200 rpm for 24 h. Total DNA was extracted according to the operating instructions of the genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., DP-302).

[0036] 2.2 PCR amplification of bacterial 16S rDNA: Using bacterial universal primers 27F and 1492R, the amplified product was detected by 1% agarose gel electrophoresis and sent to a sequencing company (General Biotech Co., Ltd.) for sequencing and identification. The gene sequence is shown in SEQ ID NO. 1:

[0037]

[0038] The PCR amplification system includes: ddH2O 20µL, high-fidelity DNA polymerase and mixed substrate 25µL, primer 1 2µL, primer 2 2µL, template 1µL;

[0039] Reaction conditions: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 30 s / 1 kb, and extension at 72°C for 5 min after 30 cycles.

[0040] The above sequencing results were compared using NCBI BLAST analysis software. The results showed that the strain sequence obtained in step 1 had a homology of more than 95% with the 16S rDNA gene sequence of the Rhizobium strain. Therefore, the strain obtained in this application is Rhizobium Rhizobium sp., named Rhizobium sp. LX, whose phylogenetic tree is as follows Figure 1 shown.

[0041] Example 2 Preparation of exopolysaccharides by fermentation of Rhizobium LX

[0042] 1) Identification of the ability of Rhizobium LX to produce extracellular polysaccharides: Rhizobium LX was transferred to a culture medium containing 20 g / L glucose, 3 g / L peptone, 2 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar. Cultured at 30°C for 72 hours, the colony morphology was as follows: Figure 2 As shown: The colonies are light yellow, smooth and raised, and moist and mucous, indicating that Rhizobium LX has the ability to produce capsular polysaccharides.

[0043] 2) Rhizobium LX fermentation to produce exopolysaccharides:

[0044] Seed solution preparation: Add 50 mL of seed culture medium to a 250 mL Erlenmeyer flask, add 100 µL of Rhizobium LX culture, and incubate the flask in a shaker at 30°C, 200 rpm for 24 hours to obtain the primary seed solution. Inoculate the primary seed solution at a 6% (v / v) concentration into a 1000 mL Erlenmeyer flask containing 300 mL of fermentation medium and incubate in a shaker at 30°C, 200 rpm for 12 hours to obtain the secondary seed solution.

[0045] Fermentation: Inoculate the secondary seed solution at 10% (v / v) into a 5L stirred fermenter containing 3L of fermentation medium. Fermentation was carried out at 30°C, pH 7.5-8.0, aeration 0.5-1.0 vvm, and stirring speed 200-1300 rpm for 52 hours. The fermentation liquid was a viscous liquid, such as Figure 3 The polysaccharide fermentation curve is shown in Figure 4As shown, the fermentation broth had a viscosity of 5500 mPa·s, and the LX polysaccharide yield was as high as 30 g / L, as measured by the alcohol precipitation method. The polysaccharide fermentation broth was heat-treated at 70°C for 2 h to inactivate the microorganisms, and a preservative, 0.2 g / L Bropol, was added to obtain the Rhizobium LX polysaccharide fermentation broth for subsequent plant experiments.

[0046] The seed culture medium includes 20 g / L sucrose, 2.3 g / L (NH4)2HPO4, 1 g / L yeast extract powder, 0.1 g / LMgSO4, 0.05 g / L FeSO4·7H2O, 0.02 g / L MnSO4, 0.01 g / L CoCl2, and 0.01 g / L ZnCl2;

[0047] The fermentation medium includes 40 g / L glucose, 2.3 g / L (NH4)2HPO4, 1 g / L yeast extract, 0.1 g / L MgSO4, 0.05 g / L FeSO4·7H2O, 0.02 g / L MnSO4, 0.01 g / L CoCl2, 0.01 g / L ZnCl2; 1 g / L defoamer, pH 7.0-7.5, and sterilization at 121°C for 20 min.

[0048] 3) Identification of components of polysaccharide fermentation broth:

[0049] Extraction of exopolysaccharides: A. Alcohol precipitation: Add an equal volume of distilled water to the polysaccharide fermentation broth, place in a 70°C water bath for 30 minutes, centrifuge at 8000 rpm for 30 minutes, collect the supernatant, add 3 volumes of 95% ethanol, mix well, and let stand at room temperature overnight. Centrifuge at 8000 rpm for 10 minutes, collect the precipitate, and oven-dry at 60°C to constant weight to obtain crude exopolysaccharide. B. Deproteinization: Add the crude polysaccharide extract to 100 mL of distilled water, heat and stir until dissolved, then add 20 mL of a 4:1 mixture of chloroform and n-butanol, shake for 30 minutes, centrifuge at 8000 rpm for 30 minutes, collect the supernatant, and repeat this process multiple times until no oil appears in the organic phase. C. Freeze drying: Place the deproteinized polysaccharide sample in a 10,000 Da dialysis bag, concentrate against PEG 10000, and dialyze for 3 days. The dialyzed solution is freeze-dried to obtain pure white, fluffy exopolysaccharide.

[0050] Analysis of extracellular polysaccharide components: Take 5 mg of pure extracellular polysaccharide extract and put it in an ampoule, add 1 mL of sulfuric acid with a mass fraction of 72%, and place it in a 30°C water bath for 1 hour. Then, dilute the volume to 10 mL with water and hydrolyze it in a nitrogen-filled, 110°C oven for 2 hours. Take it out and cool it to room temperature. Take 0.5 mL in a 4 mL centrifuge tube, adjust the pH to neutral, dilute the volume to 1 mL with water; then add 0.2 mL of 0.3 M NaOH and 0.4 mL of PMP methanol solution, fill it with nitrogen and place it in a 70°C water bath for 60 minutes. Take it out and cool it to room temperature, add 0.3 M HCL 0.2 mL, and dilute the volume to 2 mL with water. Then add 1.5 mL of chloroform, shake it evenly, and let it stand to separate the layers. Discard the lower layer of chloroform, filter the water layer through a 0.45 μm filter membrane, and measure it on a high performance liquid chromatograph. The results are as follows Figure 5 As shown in FIG, wherein A is the HPLC chromatogram of the standard monosaccharide components, 1 is GulA (17.096 min); 2 is ManA (18.112 min); 3 is Man (19.478 min); 4 is GlcN (21.661 min); 5 is Rib (22.579 min); 6 is Rha (23.534 min); 7 is GlcA (24.696 min); 8 is GalA (26.243 min); 9 is GalN (27.746 min); 10 is Glc (28.487 min); 11 is GlcNAc (29.612 min); 12 is Gal (30. 166min); 13 is Xyl (31.103min); 14 is Ara (31.531min); 15 is Fuc (33.631min); B is the HPLC chromatogram after hydrolysis of extracellular polysaccharide. From the figure, we can see the monosaccharide component peaks in the pure extracellular polysaccharide of Rhizobium LX: galactose (30.166min), glucose (28.487min), mannose (19.478min), xylose (31.103min) and fucose (33.631min). According to the peak area calculation, galactose and glucose are the main structural components of monosaccharides, accounting for >95%, and the molar ratio of glucose to galactose is 7~8:1.

[0051] Exopolysaccharide molecular weight determination: Analyses were performed using a 1260 Infinity II MDS gel permeation chromatograph (equipped with a differential refractive index detector and a dual-angle laser light scattering detector) using a PL aquagel-OH Mixed-H column, 8 μm (7.5 × 300 mm) (Agilent, USA). Detection conditions were 45°C, 1.0 ml / min; injection volume 50 μl; mobile phase 0.1 M sodium nitrate (0.01% sodium azide). The gel permeation chromatogram of rhizobium exopolysaccharides is shown in Figure 2. Figure 6 As shown: The weight average molecular weight (Mw) of Rhizobium LX exopolysaccharide is 6.8×10 6Da, number average molecular weight (Mn) is 5.7×10 6 Da, polydispersity index (PDI) (Mw / Mn) = 1.19. Therefore, the higher the degree of polymerization, the better the uniformity of the exopolysaccharide molecular weight of the Rhizobium LX prepared in this application. The molecular weight of the exopolysaccharide depends on the strain type. A high degree of polymerization provides consistent film-forming and moisture-retaining properties, forming a uniform protective layer that helps plants retain water and reduce salt stress.

[0052] In order to better illustrate the effects of the Rhizobium LX polysaccharide fermentation liquid or exopolysaccharide of the present application on plant drought resistance or salt tolerance, the following examples use exopolysaccharides for specific illustration, and the plants used are cucumber seedlings, tomato seedlings, wheat seeds, and wheat seedlings.

[0053] Example 3 Effects of Rhizobium LX Polysaccharide Fermentation Broth or Exopolysaccharide on Salt Tolerance of Cucumber Seedlings

[0054] Place 60g of nutrient soil in a small pot, and add 60ml of deionized water to reach 50% of the maximum field water holding capacity. Select "Jinchun No. 4" cucumber seeds and soak them in clean water for 12h, wrap them with moist gauze in the dark for germination, rinse the gauze with clean water for 1min after 24h, and continue to germinate in the dark for 24h. After germination, sow them in the holes of the nutrient soil in the small pot, cover them with 1cm thick nutrient soil, and replenish water every day to maintain the total mass at about 120g. Cultivate them in a ventilated place at an air humidity of 55%±5% and 25℃ for about 10 days. When the cucumber seedlings grow to "two leaves and one heart", pick seedlings of the same size and group them. Each experimental group has 10 parallel samples. According to the grouping, add 60ml of each group's solution to the nutrient cup so that the NaCl concentration under salt stress conditions is 100mM. Continue to cultivate and replenish water every day to maintain the total mass at about 180g. The groups are as follows:

[0055] CK: deionized water;

[0056] S: 17.55 g / L NaCl solution;

[0057] S+E1: 17.55 g / L NaCl + 60 mg / L Rhizobium LX exopolysaccharide;

[0058] S+E2: 17.55 g / L NaCl + 120 mg / L Rhizobium LX exopolysaccharide;

[0059] S+E3: 17.55 g / L NaCl + 180 mg / L Rhizobium LX exopolysaccharide;

[0060] S+E4: 17.55 g / L NaCl + 240 mg / L Rhizobium LX exopolysaccharide;

[0061] S+E5: 17.55 g / L NaCl + 300 mg / L Rhizobium LX exopolysaccharide.

[0062] After 14 days of cultivation, the growth of cucumber seedlings in each group was as follows: Figure 7 As shown in the figure, from the appearance of the plants, after applying LX polysaccharide, the growth of cucumber seedlings under salt stress conditions was significantly improved. The data was collected and then sorted using Excel 2021. Finally, the data was processed using the LSD method. The results are shown in Table 1.

[0063] Table 1 Effects of Rhizobium LX exopolysaccharide on growth characteristics of cucumber seedlings under salt stress

[0064]

[0065] Note: The table only records the data of surviving samples. Different survival rates result in different numbers of data in each group.

[0066] From Table 1 and Figure 7 It can be seen that compared with the salt stress group S, the survival rate of the experimental group after adding Rhizobium LX exopolysaccharide was greatly improved; when the exopolysaccharide was added at 60, 120, 180, 240, and 300 mg, respectively, the plant height increased by 23.35%, 25.90%, 32.95%, 30.98%, and 24.74%, the root length increased by 25.38%, 52.26%, 17.02%, 15.28%, and 19.62%, the number of leaves increased by 21.07%, 37.55%, 34.09%, 12.26%, and 16.48%, the fresh weight increased by 20.16%, 30.65%, 38.71%, 6.45%, and 10.48%, and the dry weight increased by 20.55%, 29.63%, 37.3%, 20.62%, and 12.31%. Therefore, when the amount of extracellular polysaccharide added is 180 mg, it has the best effect on promoting the growth of cucumber seedlings under salt stress and has better economic efficiency in actual use.

[0067] Example 4 Effects of Rhizobium LX Polysaccharide Fermentation Broth or Exopolysaccharide on Salt Tolerance of Tomato Seedlings

[0068] Select "Hezuo 903" tomato seeds, soak them in 55℃ warm water for 15min, then soak them in room temperature water for 6h to make them completely swell, rinse them with water 3 times, and sow them in a 100-hole white seedling sponge. The sponge is always kept in an infiltrated state. After sowing, cover with transparent plastic wrap and protect from light to accelerate germination. After germination, the tomato seedlings are irradiated with a positive white LED light source, the photoperiod is set to 12h, the air humidity is 55%±5%, and the temperature is 25℃. Healthy seedlings with basically the same growth conditions are selected, and Hoagland nutrient solution is added to increase the growth rate of tomatoes. The concentration increases from 1 / 8, 1 / 4, and 1 / 2 in sequence. After about 18 days, the tomato seedlings grow to one leaf and one heart. The seedlings are randomly grouped, and Etodoxa (tetrahydropyrimidine) is used as the control group. The groups are as follows:

[0069] CK: 1.6 L deionized water;

[0070] S: deionized water 1.6L + 14.04g NaCl;

[0071] S+E1: 1.6 L deionized water + 14.04 g NaCl + 32 mg Rhizobium LX exopolysaccharide;

[0072] S+E2: 1.6 L deionized water + 14.04 g NaCl + 64 mg Rhizobium LX exopolysaccharide;

[0073] S+E3: 1.6 L deionized water + 14.04 g NaCl + 96 mg Rhizobium LX exopolysaccharide;

[0074] S+E4: 1.6 L deionized water + 14.04 g NaCl + 128 mg Rhizobium LX exopolysaccharide;

[0075] S+E5: 1.6 L deionized water + 14.04 g NaCl + 160 mg Rhizobium LX exopolysaccharide;

[0076] S+Ect: 1.6 L deionized water + 14.04 g NaCl + 32 mg tetrahydropyrimidine;

[0077] Each group was cultured for 21 days, and the growth of tomato seedlings was as follows Figure 8 As shown in the results, the growth of tomato seedlings under salt stress was significantly improved after the application of Rhizobium LX exopolysaccharides and ectoine. Data were collected and organized using Excel 2021 software. Multiple comparisons were performed using the LSD method (P < 0.05). The results are shown in Table 2.

[0078] Table 2 Effects of Rhizobium LX exopolysaccharide on growth characteristics of tomato seedlings under salt stress

[0079]

[0080] Note: Only survival data are recorded in the table. One tomato seedling died in the S+Ect group.

[0081] As shown in Table 2, compared with the salt stress group S, the addition of Rhizobium LX exopolysaccharide significantly alleviated the salt stress of tomato seedlings. At concentrations of 20, 40, 60, 80, and 100 mg / L, plant height increased by 16.54%, 6.27%, 11.41%, 11.22%, and 10.84%, respectively; root length increased by 25.38%, 52.26%, 17.02%, 15.28%, and 19.62%, respectively; stem diameter increased by 13.07%, 6.54%, 13.73%, 20.26%, and 12.42%, and root length increased by 127.79%, 101.88%, 145.68%, 100.00%, and 118.84%. Adding 20 mg / L of polysaccharide significantly promoted the growth of tomato seedlings under salt stress.

[0082] Example 5 Effects of Rhizobium LX Polysaccharide Fermentation Broth or Exopolysaccharide on the Stress Resistance of Wheat Seeds under Extreme Drought Conditions

[0083] Select "Jimai 22" wheat seeds with full grains and uniform size, wash them with water and squeeze them thoroughly for 2 minutes, soak them in 70% alcohol and stir them for 2 minutes to disinfect, then wash them with distilled water for 2 minutes and 1 minute respectively. The total time for disinfection and washing is 7 minutes. Finally, use absorbent paper to absorb the water. Put 350g of quartz sand in a small pot, evenly sprinkle 20 wheat seeds, cover the top with 1cm of quartz sand (50g), add 55ml of each group of solutions in batches to make it reach 55% of the maximum water holding capacity, and germinate in the dark for 3-4 days. Then water it every day to keep the weight of each pot consistent and maintain it at 455g. Cultivate in a ventilated place with air humidity of 55%±5% and 25℃. After 10 days, take seedlings of uniform size and perform the following experimental treatments: CK2 group is the control group with normal water addition, and water is added every day to maintain the total weight of 455g; the other four groups are not watered and are subjected to drought stress treatment. Each group has 10 parallel samples. The groups are as follows:

[0084] CK1: 55ml deionized water + no water added later;

[0085] CK2: 55ml deionized water + continuous addition of water later;

[0086] E: 55 ml deionized water + 9.1 mg Rhizobium LX exopolysaccharide + no water added in the later stage;

[0087] F: 55ml deionized water + 0.30ml Rhizobium LX polysaccharide fermentation broth + no water added in the later stage;

[0088] Ect: 55ml deionized water + 9.1mg icodine + no water added in the later stage;

[0089] Icodone (tetrahydropyrimidine) is currently widely believed to have the ability to improve the salt, alkali and drought resistance of plants. Icodone was selected as the control group.

[0090] Wheat seedlings have a certain tolerance to drought stress. However, when growing in quartz sand, water loss is more severe due to the large interstices within the sand. Therefore, the effectiveness of LX polysaccharide fermentation broth and pure LX polysaccharide under drought conditions was determined based on the rate of water loss and the survival time of the plants under extreme drought conditions. "*" indicates the number of days until the maximum water holding capacity reached 25% of the extreme drought level; "#" indicates the number of days until half of the seedlings in the group died. Data were collected and organized using Excel 2021 software. The results are shown in Table 3.

[0091] Table 3 Statistics of wheat seedling survival time under extreme drought conditions

[0092] Group <![CDATA[CK1]]> Ectoin LX polysaccharide fermentation broth LX exopolysaccharide Day 1 0 0 0 0 Day 2 0 0 0 0 Day 3 0 0 0 0 Day 4 0* 0 0 0* Day 5 0 0* 0* 0 Day 6 1 0 0 0 Day 7 2 0 0 0 Day 8 <![CDATA[5 # ]]> 1 0 0 Day 9 8 <![CDATA[5 # ]]> 0 1 Day 10 10 7 1 4 Day 11 10 9 4 <![CDATA[6 # ]]> Day 12 10 10 <![CDATA[7 # ]]> 9 Day 13 10 10 9 10 Day 14 10 10 10 10 Entering the extreme drought period (d) 4 5 5 4 Half death time (d) 8 9 12 11

[0093] After 14 days of cultivation, the growth of wheat seedlings is as follows Figure 9 As shown in Table 3 and Figure 9 It can be seen that under drought conditions, the application of Rhizobium LX fermentation liquid or exopolysaccharides can significantly prolong the survival of wheat seedlings, and the decline in water holding capacity in the system under drought stress conditions is significantly alleviated. When the wheat seedlings entered extreme drought conditions and half of the seedlings in the same group died, the wheat seedlings in the experimental group supplemented with Rhizobium LX fermentation liquid and exopolysaccharides survived for 7 days, while the wheat seedlings in the control group CK1 and the icodoin group survived only 4 days. This shows that the addition of Rhizobium LX fermentation liquid and exopolysaccharides can significantly prolong the survival of wheat seedlings under extreme drought conditions.

[0094] Example 6 Effects of Rhizobium LX Polysaccharide Fermentation Broth or Extracellular Polysaccharide on Salt Tolerance of Wheat Seedlings

[0095] Select "Jimai 22" wheat with full grains and uniform size, wash with water and fully squeeze for 2 minutes, soak and stir in 70% alcohol for 2 minutes for disinfection, wash with distilled water for 2 minutes and 1 minute respectively, and the disinfection and cleaning time is 7 minutes in total. Finally, use absorbent paper to absorb the water. Place 60g of nutrient soil in each small pot, sow 20 wheat seeds evenly in the cup, add 60mL of deionized water, and replenish water every day to maintain the total weight at about 120g. Cultivate in a ventilated place with an air humidity of 55%±5% and 25℃ for 7 days, select seedlings of the same size and randomly group them. Set up 10 parallel samples for each experimental group, add 60mL of each group's solution to the nutrient cup, and continue to cultivate. During this period, replenish water every day to keep the total mass at 180g. The groups are as follows:

[0096] (1) CK: deionized water (blank control group);

[0097] (2) S: 17.55 g / L NaCl solution;

[0098] (3) S+E1: 17.55 g / L NaCl + Rhizobium LX polysaccharide fermentation broth 1 ml / L;

[0099] (4) S+E2: 17.55 g / L NaCl + Rhizobium LX polysaccharide fermentation broth 2 ml / L;

[0100] (5) S+E3: 17.55 g / L NaCl + 60 mg / L exopolysaccharide;

[0101] (6) S+Ect: 17.55 g / L NaCl + 60 mg / L Ecodone;

[0102] After 7 days of cultivation, the growth of wheat seedlings in each experimental group was as follows: Figure 10 As shown in the figure, from the appearance of the plants, the growth of wheat seedlings under salt stress conditions was significantly improved after the application of rhizobium LX fermentation liquid or extracellular polysaccharides. The data were collected and then sorted using Excel 2021. Finally, the data were processed using the LSD method. The results are shown in Table 4.

[0103] Table 4 Effects of Rhizobium LX fermentation broth or exopolysaccharide on wheat seedling growth under salt stress

[0104]

[0105] It can be seen from Table 4 that compared with the salt stress S group, the addition of Rhizobium LX polysaccharide fermentation broth and extracellular polysaccharides increased the stem length of wheat seedlings by 39.00%, 47.93% and 57.95% respectively; the root length increased by 33.81%, 37.21% and 33.37%; the fresh weight increased by 69.47%, 86.30% and 86.87%; and the dry weight increased by 43.40%, 35.22% and 30.82%.

[0106] In summary, the Rhizobium LX fermentation liquid or exopolysaccharide provided by the present invention has a significant effect on improving plant salt tolerance or drought stress resistance and has broad application prospects.

[0107] The present invention has been disclosed above with preferred embodiments, which are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. A high-yield exopolysaccharide rhizobium LX ( Rhizobium sp. LX ), characterized by: The rhizobium LX was deposited in the China Center for Type Culture Collection on March 7, 2025, with the deposit number CCTCC M 2025321; The main structural components of the extracellular polysaccharide include glucose, galactose, xylose, fucose and mannose; the molar ratio of glucose to galactose in the main structural components of the extracellular polysaccharide is 7-8:

1.

2. The high-polysaccharide-producing rhizobium LX according to claim 1 ( Rhizobium sp. LX ), characterized by: The mass of the main structural components accounts for more than 95% of the total mass of the monosaccharide components.

3. The high-polysaccharide-producing rhizobium LX according to claim 1 ( Rhizobium sp. LX ), characterized in that, The preparation method of the exopolysaccharide comprises the following steps: The seed liquid of Rhizobium LX is inoculated into a fermentation medium to obtain a polysaccharide fermentation liquid. The polysaccharide fermentation liquid is precipitated with alcohol and dried to a constant weight to obtain a crude exopolysaccharide. The crude product is dissolved in water and then an organic solvent is added to remove protein. The product is then dialyzed and dried to obtain a pure exopolysaccharide.

4. The high-polysaccharide-producing rhizobium LX according to claim 1 ( Rhizobium sp. LX ) in improving plant resistance to salt or drought stress.

5. The use according to claim 4, characterized in that: The polysaccharide fermentation liquid or extracellular polysaccharide of the rhizobium LX is added during the plant cultivation process.

6. The use according to claim 5, characterized in that: The concentration of the polysaccharide fermentation broth or extracellular polysaccharide is 20-300 mg / L.

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