Rhizobium LX with high exopolysaccharide yield and application of rhizobium LX

Abstract

The invention provides a rhizobium LX for high yield of exopolysaccharides and application thereof, and belongs to the technical field of biology, the rhizobium LX is preserved in 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 the exopolysaccharide in a conventional fermentation medium, the fermentation process has the characteristics of low cost, simplicity and convenience in operation, short period and high yield (30 g / L), and the obvious large-scale production advantage is shown; the fermentation liquor or exopolysaccharide of the rhizobium LX can obviously enhance the salt and alkali resistance and drought stress resistance of plants, and the required dosage is small.

Description

Technical Field

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

[0002] Salinized soils in China are characterized by significant distribution breadth and ecological diversity, with a total area approaching 100 million hectares. In agricultural production, salt accumulation and water scarcity have become key environmental stressors restricting crop growth. High concentrations of salt disrupt normal metabolic activities of plant cells by altering the cellular osmotic balance, leading to retarded development and plant senescence. Soil salinization subjects plants to salt stress damage, resulting in a dual decline in crop yield and quality, and even crop failure, severely restricting the sustainable development of the protected horticulture industry. Similarly, drought stress has multiple negative impacts on plants and crops, including inhibiting seed germination, slowing the growth of roots and above-ground parts, reducing photosynthesis efficiency, disrupting cell membrane stability, and causing water and nutrient uptake disorders. At the same time, it induces the accumulation of reactive oxygen species, leading to oxidative damage, ultimately significantly reducing crop yield and quality, and even causing plant death in severe cases. The regulatory strategies for the above stresses mainly focus on three dimensions: cultivating stress-resistant crop varieties, soil structure improvement projects, and adding exogenous biostimulants. Among them, biostimulants have attracted much attention because they can effectively activate the plant metabolic system, optimize nutrient utilization efficiency, and enhance the plant's stress resistance mechanism. Typical representatives such as chitosan oligosaccharides and betaine have been proven to significantly improve the salt and drought tolerance of crops through simple application.

[0003] Rhizobia are typical representatives of plant rhizosphere microorganisms. In the field of microbial inoculant applications, rhizobia, as typical plant symbiotic bacteria, are often used in farmland improvement in the form of compound biofertilizers. However, traditional live bacterial preparations have significant technical bottlenecks, including the susceptibility of cell viability to environmental fluctuations, low soil colonization efficiency, and niche competition with native microbial communities, all of which restrict their actual application effects. Rhizobial extracellular polysaccharide (EPS), as an important medium for the interaction between plant growth-promoting rhizobacteria (PGPR) and plants, exhibits multiple mechanisms in stress regulation: by chelating salt ions, scavenging reactive oxygen species, and activating plant endogenous hormone pathways, it synergistically enhances the plant's resistance to salinity and drought and promotes root development. As a functional metabolite, it can exert its function without relying on live bacteria colonization, avoiding the competition risk between exogenous microbial inoculants and native flora; and EPS can be produced on a large scale through low-cost fermentation, is resistant to storage, and is suitable for industrial coating or foliar spraying, having both ecological safety and economic feasibility in saline soil improvement and water-saving agriculture, providing 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 extracellular polysaccharide production. This rhizobium LX is isolated from a vegetable field, can efficiently produce extracellular polysaccharides, and has the functions of promoting plant growth and improving plant salt tolerance or drought tolerance.

[0005] To solve the above technical problems: The first object of the present invention is to provide a rhizobium with high extracellular polysaccharide production, classified and named Rhizobium sp. LX. The rhizobium LX is preserved in the China Center for Type Culture Collection (Wuhan University) on February 28, 2025, with the preservation number CCTCC M 2025321; The structural components of the extracellular polysaccharide include glucose, galactose, xylose, fucose, mannose, etc., among which glucose and galactose are the main structural components.

[0006] Furthermore, the mass of the main structural components accounts for more than 95% of the total mass.

[0007] Furthermore, the molar ratio of glucose to galactose in the main structural components of the extracellular polysaccharide is 7 - 8:1.

[0008] Furthermore, the preparation method of the extracellular polysaccharide includes the following steps: Inoculate the seed liquid of rhizobium LX into a fermentation medium to obtain a polysaccharide fermentation broth. Subject the polysaccharide fermentation broth to alcohol precipitation and drying to constant weight to obtain a crude extracellular polysaccharide. Dissolve the crude extract in water, add an organic solvent to remove proteins, and then perform dialysis and drying to obtain a pure extracellular polysaccharide.

[0009] The second object of the present invention is to provide an application of the above-mentioned rhizobium LX with high polysaccharide production in improving plant salt or drought stress resistance.

[0010] Furthermore, add the polysaccharide fermentation broth or extracellular polysaccharide of rhizobium LX during plant cultivation.

[0011] Furthermore, the concentration of the polysaccharide fermentation broth or extracellular polysaccharide is 20 - 300 mg / L.

[0012] The beneficial technical effects achieved by the present invention: (1) The rhizobium LX strain can efficiently synthesize extracellular polysaccharides in a conventional fermentation medium. Its fermentation process has the characteristics of low cost, simple operation, short cycle, and high yield (up to 30 g / L), showing significant advantages in large-scale production; (2) The fermentation broth 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 less. Description of the Drawings

[0013] Figure 1 : The present invention constructs a phylogenetic tree of strain Rhizobium sp. LX based on the 16S rDNA gene sequence; Figure 2 : The rhizobium strain of the present invention Rhizobium sp. LX colony morphology; Figure 3 : The morphology of the extracellular polysaccharide fermentation broth of rhizobium LX in the examples of the present invention; Figure 4 : The fermentation curve of rhizobium LX in a 5L stirred reactor in the examples of the present invention; Figure 5 : The high performance liquid chromatography chart of the extracellular polysaccharide produced by rhizobium LX in the examples of the present invention; Figure 6 : The gel permeation chromatography chart of the extracellular polysaccharide produced by rhizobium LX in the examples of the present invention; Figure 7 : The picture of the growth status of cucumber seedlings under salt stress in the examples of the present invention; Figure 8 : The picture of the growth status of tomato seedlings under salt stress in the examples of the present invention; Figure 9 : The picture of the growth status of wheat seedlings on the 14th day under extreme drought stress in the examples of the present invention; Figure 10 : The picture of the growth status of wheat seedlings under salt stress in the examples of the present invention.

[0014] The preservation information of the rhizobium in this application is as follows: Rhizobium: The classification name is Rhizobium sp. LX, preserved in the China Center for Type Culture Collection (Wuhan University), the preservation date is February 28, 2025, and the preservation number is CCTCC M 2025321; Detailed implementation manners

[0015] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and cannot be used to limit the protection scope of the present invention.

[0016] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0017] Example 1 Isolation, extraction and identification of rhizobium LX 1. Isolation and extraction of Rhizobium LX: Take the soil cultivated in the vegetable field in Nanjing, remove plant debris, weigh about 1 g of soil, add it to a 250 mL Erlenmeyer flask containing 100 mL of sterile water and glass beads, shake at 100 rpm for 60 min to fully swell the soil particles and release microorganisms. Let the Erlenmeyer flask stand for 60 min to allow solid substances such as sand grains to fully settle. Take the supernatant and dilute it stepwise with sterile water to 10 -5 . Take 100 μL of the diluted solution onto the screening medium plate, evenly spread it using a triangular spreader, spread a total of 10 plates, and incubate at 30 °C for 24 - 72 h. Observe the colony morphology on the plate, select single colonies with smooth, convex, moist, and viscous surfaces, number them, and use them for subsequent identification. Screening medium formula: glucose 20 g / L, yeast powder 2 g / L, peptone 3 g / L, NaCl 10 g / L, KH 2 PO 4 2 g / L, agar powder 16 g / L, pH 7.5.

[0018] 2. Identification of Rhizobium LX: 2.1 Extraction of total DNA: Take 200 μL of the bacterial solution obtained in step 1 into LB medium, culture it in a shaker at 30 °C and 200 rpm for 24 h, and extract total DNA according to the operation instructions of the genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., DP - 302).

[0019] 2.2 PCR amplification of the 16S rDNA of bacteria: Use the universal bacterial primers 27F and 1492R, detect the amplification products by 1% agarose gel electrophoresis, and send them to a sequencing company (General Biology Co., Ltd.) for sequencing identification. The gene sequence is shown as SEQ ID NO.1:

[0020] Among them, the PCR amplification system includes: ddH 2 O 20 μL, high-fidelity DNA polymerase and mixed substrates 25 μL, primer 1 2 μL, primer 2 2 μL, template 1 μL; 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.

[0021] The above sequencing results were aligned using the NCBI BLAST analysis software. The results showed that the homology between the strain sequence obtained in step 1 and the 16S rDNA gene sequence of the Rhizobium strain was over 95%. Therefore, the strain obtained in this application is Rhizobium Rhizobium sp., named Rhizobium sp. LX, and its phylogenetic tree is as Figure 1 shown.

[0022] Example 2 Preparation of extracellular polysaccharide by fermentation of Rhizobium LX 1) Identification of the ability of Rhizobium LX to produce extracellular polysaccharide: Rhizobium LX was transferred to a medium, and the medium included 20 g / L glucose, 3 g / L peptone, 2 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar. It was cultured at 30 °C for 72 h, and the colony morphology was as Figure 2 shown: the colonies were light yellow, smooth and convex, and moist and mucous, indicating that Rhizobium LX has the ability to produce capsular polysaccharide.

[0023] 2) Fermentation of Rhizobium LX to produce extracellular polysaccharide: Preparation of seed liquid: Add 50 mL of seed medium to a 250 mL Erlenmeyer flask, add 100 μL of Rhizobium LX bacterial liquid, and place the Erlenmeyer flask on a shaker at 30 °C and 200 rpm for 24 h to obtain the primary seed liquid. The primary seed liquid was inoculated into a 1000 mL Erlenmeyer flask containing 300 mL of fermentation medium at an inoculation amount of 6% (v / v), and cultured on a shaker at 30 °C and 200 rpm for 12 h to obtain the secondary seed liquid.

[0024] Fermentation culture: The secondary seed liquid was inoculated into a 5 L stirred fermenter at an inoculation amount of 10% (v / v). The fermenter was filled with 3 L of fermentation medium. Fermentation culture was carried out at a temperature of 30 °C, pH 7.5 - 8.0, aeration rate of 0.5 - 1.0 vvm, and stirring speed of 200 - 1300 rpm for 52 h. The fermentation broth was a viscous liquid, as Figure 3 shown. The polysaccharide production fermentation curve is as Figure 4As shown, the viscosity of the fermentation broth reached 5500 mPa·s, and the yield of LX polysaccharide measured by alcohol precipitation was as high as 30 g / L. The polysaccharide fermentation broth was heat-treated at 70 °C for 2 h to inactivate microorganisms, and 0.2 g / L bronopol as a preservative was added to obtain the rhizobium LX polysaccharide fermentation broth for subsequent plant experiments.

[0025] Among them, the seed medium includes 20 g / L sucrose, 2.3 g / L (NH 4 ) 2 HPO 4 , 1 g / L yeast extract powder, 0.1 g / L MgSO 4 , 0.05 g / L FeSO 4 ·7H 2 O, 0.02 g / L MnSO 4 , 0.01 g / L CoCl 2 , 0.01 g / L ZnCl 2 ; The fermentation medium includes 40 g / L glucose, 2.3 g / L (NH 4 ) 2 HPO 4 , 1 g / L yeast extract powder, 0.1 g / L MgSO 4 , 0.05 g / L FeSO 4 ·7H 2 O, 0.02 g / L MnSO 4 , 0.01 g / L CoCl 2 , 0.01 g / L ZnCl 2 ; 1 g / L antifoaming agent, pH 7.0 - 7.5, sterilized at 121 °C for 20 min.

[0026] 3) Identification of the components of the polysaccharide fermentation broth: Extraction of extracellular polysaccharide: A Alcohol precipitation: Equal volume of distilled water was added to the polysaccharide fermentation broth, placed in a 70 °C water bath for 30 min, centrifuged at 8000 r / min for 30 min, the supernatant was collected, 3 times the volume of 95% ethanol was added and mixed evenly, and left to stand overnight at room temperature; centrifuged at 8000 r / min for 10 min, the precipitate was collected and dried in an oven at 60 °C to constant weight to obtain the crude extracellular polysaccharide; B Deproteinization: The crude polysaccharide extract was taken into 100 mL of distilled water, heated and stirred until dissolved, then 20 mL of a mixed solution of chloroform and n-butanol with a volume ratio of 4:1 was added, shaken for 30 min and then centrifuged at 8000 r / min for 30 min, the supernatant was collected, and repeated multiple times until there was no oily substance in the organic phase. C Freeze-drying: The deproteinized polysaccharide sample was placed in a dialysis bag with a molecular weight of 10000 Da, concentrated and dialyzed with PEG10000 for 3 days, and the dialyzed solution was freeze-dried to obtain a white and porous pure extracellular polysaccharide.

[0027] Analysis of extracellular polysaccharide components: Take 5 mg of the pure extracellular polysaccharide extract in an ampoule, add 1 mL of sulfuric acid with a mass fraction of 72%, keep it in a water bath at 30 °C for 1 h, then make up the volume to 10 mL with water. Hydrolyze it in an oven at 110 °C for 2 h under nitrogen atmosphere, and cool it to room temperature after taking it out. Take 0.5 mL and put it into a 4 mL centrifuge tube, adjust the pH to neutral, and make up 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, keep it in a water bath at 70 °C for 60 min under nitrogen atmosphere, cool it to room temperature after taking it out, add 0.2 mL of 0.3 M HCl, and make up the volume to 2 mL with water. Then add 1.5 mL of chloroform, shake it well and let it stand for layering, discard the lower chloroform layer, filter the water layer through a 0.45 μm filter membrane, and determine it by high performance liquid chromatography. The results are as Figure 5 shown. Among them, A is the high performance liquid chromatography diagram of 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 (30.166 min); 13 is Xyl (31.103 min); 14 is Ara (31.531 min); 15 is Fuc (33.631 min); B is the high performance liquid chromatography diagram after hydrolysis of extracellular polysaccharide. It can be seen from the figure that the monosaccharide component peaks in the pure extracellular polysaccharide of Rhizobium LX are: galactose (30.166 min), glucose (28.487 min), mannose (19.478 min), xylose (31.103 min) and fucose (33.631 min). According to the peak area, galactose and glucose are the main structural components of monosaccharides, with a proportion > 95%, and the molar ratio of glucose to galactose is 7 - 8:1.

[0028] Determination of the molecular weight of extracellular polysaccharide: Analysis was carried out using an Agilent 1260 Infinity Ⅱ MDS gel permeation chromatograph (equipped with a differential refractive index detector and a dual-angle laser scattering detector). The chromatographic column used was PL aquagel-OH Mixed-H, 8 μm (7.5 × 300 mm) (Agilent, USA). The detection conditions were 45 °C, 1.0 ml / min; the injection volume was 50 μl; the mobile phase was 0.1 M sodium nitrate (0.01% sodium azide). The gel permeation chromatogram of the extracellular polysaccharide of Rhizobium is as Figure 6 shown: The weight-average molecular weight (Mw) of the extracellular polysaccharide of Rhizobium LX is 6.8×106 Da, the number-average molecular weight (Mn) is 5.7×10 6 Da, and the polydispersity index (PDI) (Mw / Mn) = 1.19. Therefore, the extracellular polysaccharide of Rhizobium LX prepared in this application has a high molecular weight polymerization degree and better homogeneity. The molecular weight of extracellular polysaccharide depends on the type of strain. With a high molecular weight polymerization degree, it can form a uniform protective layer with consistent film-forming property and moisture retention, helping plants retain water and reducing salt stress.

[0029] To better illustrate the effects of the Rhizobium LX polysaccharide fermentation broth or extracellular polysaccharide of this application on plant drought tolerance or salt tolerance, the following examples select extracellular polysaccharide for specific illustration, and the plants selected are cucumber seedlings, tomato seedlings, wheat seeds, and wheat seedlings.

[0030] Example 3 Effects of Rhizobium LX polysaccharide fermentation broth or extracellular polysaccharide on the salt tolerance of cucumber seedlings Place 60 g of nutrient soil in a small pot, add 60 ml of deionized water to reach 50% of the maximum field water holding capacity. Select "Jinchun No. 4" cucumber seeds, soak them in clear water for 12 h, wrap them with a moist gauze and germinate in the dark. After 24 h, rinse the gauze with clear water for 1 min, and continue to germinate in the dark for 24 h. After germination, sow them in the nutrient soil holes of the small pot, cover them with 1 cm thick of nutrient soil, and supplement water every day to keep the total mass at about 120 g. Cultivate at an air humidity of 55%±5%, 25°C, and in a ventilated place for about 10 d. When the cucumber seedlings grow to "two leaves and one heart", select seedlings of the same size for grouping treatment, and set 10 parallel samples for each experimental group. According to the grouping, add 60 ml of each group of solutions to the nutrient cups to make the NaCl concentration under salt stress conditions be 100 mM. Continue to cultivate, supplement water every day to keep the total mass at about 180 g. The grouping is as follows: CK: Deionized water; S: 17.55 g / L NaCl solution; S+E 1 : 17.55 g / L NaCl + 60 mg / L Rhizobium LX extracellular polysaccharide; S+E 2 : 17.55 g / L NaCl + 120 mg / L Rhizobium LX extracellular polysaccharide; S+E 3 : 17.55 g / L NaCl + 180 mg / L Rhizobium LX extracellular polysaccharide; S+E 4 : 17.55 g / L NaCl + 240 mg / L Rhizobium LX extracellular polysaccharide; S+E 5 : 17.55 g / L NaCl + 300 mg / L Rhizobium LX extracellular polysaccharide.

[0031] After 14 days of culturing in each group, the growth of cucumber seedlings was as follows Figure 7 As shown, from the appearance of the plants, after applying LX polysaccharide, the growth of cucumber seedlings under salt stress conditions was significantly improved. Data was collected and then sorted using Excel 2021. Finally, the data was processed using the LSD method, and the results are shown in Table 1.

[0032] Table 1 Effects of extracellular polysaccharide of Rhizobium LX on the growth traits of cucumber seedlings under salt stress

[0033] Note: The table only records the data of surviving samples. Different survival rates result in different numbers of data in each group.

[0034] As can be seen from Table 1 and Figure 7 : Compared with the salt stress group S, the survival rate of the experimental group after adding extracellular polysaccharide of Rhizobium LX increased significantly; when 60, 120, 180, 240, and 300 mg of extracellular polysaccharide were added respectively, the plant height increased by 23.35%, 25.90%, 32.95%, 30.98%, and 24.74% respectively, the root length increased by 25.38%, 52.26%, 17.02%, 15.28%, and 19.62% respectively, the number of leaves increased by 21.07%, 37.55%, 34.09%, 12.26%, and 16.48% respectively, the fresh weight increased by 20.16%, 30.65%, 38.71%, 6.45%, and 10.48% respectively, and the dry weight increased by 20.55%, 29.63%, 37.3%, 20.62%, and 12.31% respectively. Therefore, when the extracellular polysaccharide addition amount is 180 mg, the growth promotion effect on cucumber seedlings under salt stress is the best, and it has good economy in actual use.

[0035] Example 4 Effects of fermentation broth or extracellular polysaccharide of Rhizobium LX on the salt tolerance of tomato seedlings Select "Cooperation 903" tomato seeds, soak them in warm water at 55°C for 15 minutes, then soak them in normal temperature water for 6 hours to make them fully imbibed, rinse them 3 times with water, and sow them in 100-hole white seedling-raising sponges. The sponges were always kept moist. After sowing, they were covered with transparent plastic wrap and germinated in the dark. The germinated tomato seedlings were irradiated with a pure white LED light source, the photoperiod was set to 12 hours, and they were cultured in a ventilated place with an air humidity of 55% ± 5% and a temperature of 25°C. Healthy seedlings with basically the same growth conditions were selected, and Hoagland nutrient solution was added to increase the growth rate of tomatoes. Its concentration increased sequentially from 1 / 8, 1 / 4, to 1 / 2. After about 18 days, the tomato seedlings grew to one leaf and one heart, and the seedlings were randomly grouped, with ectoine (ectoine) as the control group. The grouping is as follows: CK: 1.6 L of deionized water; S: 1.6 L of deionized water + 14.04 g of NaCl; S + E 1 : 1.6 L of deionized water + 14.04 g of NaCl + 32 mg of extracellular polysaccharide of Rhizobium LX; S + E 2 : 1.6 L of deionized water + 14.04 g of NaCl + 64 mg of extracellular polysaccharide of Rhizobium LX; S + E 3 : 1.6 L of deionized water + 14.04 g of NaCl + 96 mg of extracellular polysaccharide of Rhizobium LX; S + E 4 : 1.6 L of deionized water + 14.04 g of NaCl + 128 mg of extracellular polysaccharide of Rhizobium LX; S + E 5 : 1.6 L of deionized water + 14.04 g of NaCl + 160 mg of extracellular polysaccharide of Rhizobium LX; S + Ect: 1.6 L of deionized water + 14.04 g of NaCl + 32 mg of ectoine; Each group was cultured for 21 days, and the growth of tomato seedlings was as Figure 8 shown. From the perspective of plant growth appearance, after applying extracellular polysaccharide of Rhizobium LX and ectoine, the growth of tomato seedlings under salt stress was significantly improved. Data were collected and then the experimental data were sorted out using Excel 2021 software; the LSD method was used for multiple comparisons of differences (P < 0.05); the results are shown in Table 2.

[0036] Table 2 Effects of extracellular polysaccharide of Rhizobium LX on growth traits of tomato seedlings under salt stress

[0037] Note: Only survival data are recorded in the table, and one tomato seedling died in the S + Ect group.

[0038] It can be seen from Table 2 that: compared with the salt stress group S, the salt stress on tomato seedlings was significantly alleviated after adding extracellular polysaccharide of Rhizobium LX. When the added concentrations were 20, 40, 60, 80, and 100 mg / L respectively, the plant height increased by 16.54%, 6.27%, 11.41%, 11.22%, and 10.84% respectively, the root length increased by 25.38%, 52.26%, 17.02%, 15.28%, and 19.62% respectively, the stem diameter increased by 13.07%, 6.54%, 13.73%, 20.26%, and 12.42% respectively, and the root fresh weight increased by 127.79%, 101.88%, 145.68%, 100.00%, and 118.84% respectively. Adding 20 mg / L of polysaccharide can have a significant growth-promoting effect on tomato seedlings under salt stress.

[0039] Effect of Rhizobium LX Polysaccharide Fermentation Broth or Exopolysaccharide on Stress Resistance of Wheat Seeds under Extreme Drought Conditions Select "Jimai 22" wheat seeds with plump grains and uniform size. Wash them with water and knead them thoroughly wet for 2 min, soak them in 70% alcohol and stir for 2 min for disinfection, then wash them with distilled water for 2 min and 1 min respectively. The total time for disinfection and cleaning is 7 min. Finally, blot the water dry with absorbent paper. Put 350 g of quartz sand in a small pot, evenly sprinkle 20 wheat seeds, cover the upper layer with 1 cm of quartz sand (50 g), add 55 ml of each group of solution in batches to make it reach 55% of the maximum water holding capacity, and germinate in the dark for 3 - 4 days. Then water every day to keep the weight of each potted plant consistent, maintaining at 455 g. Cultivate in a ventilated place with an air humidity of 55% ± 5% and a temperature of 25 °C. After 10 d, select seedlings with the same size and conduct the following experimental treatments: CK 2 The CK group is the normal water-added culture control group, adding water every day to maintain a total weight of 455 g; the remaining 4 groups do not add water and are subjected to drought stress treatment. Each group sets 10 parallel samples. The grouping is as follows: CK 1 : 55 ml of deionized water + no water added later; CK 2 : 55 ml of deionized water + continuous water added later; E: 55 ml of deionized water + 9.1 mg of Rhizobium LX exopolysaccharide + no water added later; F: 55 ml of deionized water + 0.30 ml of Rhizobium LX polysaccharide fermentation broth + no water added later; Ect: 55 ml of deionized water + 9.1 mg of ectoine + no water added later; Ectoine (ectoine) is a product that is currently widely considered to have the performance of improving the salt and drought tolerance of plants. Ectoine is selected as the control group.

[0040] Wheat seedlings have a certain ability to tolerate drought stress. However, when using quartz sand as the growth medium, due to the relatively large gap between the pores of quartz sand itself, the water loss situation will be relatively serious. Therefore, according to the speed of its water loss and the survival time of the plants under extreme drought conditions, the use effects of LX polysaccharide fermentation broth and LX polysaccharide pure product under drought conditions are judged. Among them, "*" represents the number of days when reaching the extreme drought level, that is, when the maximum water holding capacity is 25%; "#" represents the number of days when half of the plants in the same group die. Collect the data, and then use Excel 2021 software to organize the experimental data. The results are shown in Table 3.

[0041] Table 3 Statistics of the survival time of wheat seedlings under extreme drought conditions Group <![CDATA[CK 1 > 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 Extreme Drought Time (d) 4 5 5 4 Half Mortality Time (d) 8 9 12 11 After 14 days of cultivation, the growth of wheat seedlings is asFigure 9 as shown. From Table 3 and Figure 9 it can be seen that under drought conditions, applying the fermentation broth of Rhizobium LX or exopolysaccharide can significantly extend the survival time of wheat seedlings; under drought stress conditions, the decrease in water holding capacity in the system is significantly alleviated. During the process from when wheat seedlings enter the extreme drought condition to when half of them die in the same group, in the experimental groups adding the fermentation broth of Rhizobium LX and exopolysaccharide, the wheat survived for 7 days, while in the control group CK 1 and the ectoricin group, the wheat seedlings only survived for 4 days, indicating that the addition of the fermentation broth of Rhizobium LX and exopolysaccharide can significantly extend the survival time of wheat seedlings under extreme drought conditions.

[0042] Example 6 Effect of the polysaccharide fermentation broth of Rhizobium LX or exopolysaccharide on the salt tolerance of wheat seedlings Select "Jimai 22" wheat with plump grains and uniform size. Wash it with water and fully knead it wet for 2 min, soak it in 70% alcohol and stir for 2 min for disinfection, then wash it with distilled water for 2 min and 1 min respectively. The total time for disinfection and cleaning is 7 min. Finally, dry the water with absorbent paper. Place 60 g of nutrient soil in each small pot, evenly sow 20 wheat seeds into the cups, add 60 mL of deionized water, and supplement water every day to keep the total weight at about 120 g. Cultivate at 25 °C in a ventilated place with an air humidity of 55% ± 5% for 7 days. Select seedlings with the same size and randomly group them. Set 10 parallel samples for each experimental group, add 60 mL of each group of solutions to the nutrient cups, and continue to cultivate. During this period, supplement water every day to keep the total mass at 180 g. Each group is as follows: (1) CK: Deionized water (blank control group); (2) S: 17.55 g / L NaCl solution; (3) S+E 1 : 17.55 g / L NaCl + 1 mL / L fermentation broth of Rhizobium LX polysaccharide; (4) S+E 2 : 17.55 g / L NaCl + 2 mL / L fermentation broth of Rhizobium LX polysaccharide; (5) S+E 3 : 17.55 g / L NaCl + 60 mg / L exopolysaccharide; (6) S+Ect: 17.55 g / L NaCl + 60 mg / L ectoricin; After each experimental group was cultured for 7 days, the growth of wheat seedlings was as Figure 10 shown. From the appearance of the plants, after applying the fermentation broth of Rhizobium LX or exopolysaccharide, the growth of wheat seedlings under salt stress conditions was significantly improved. Collect data, then organize the data using Excel 2021, and finally process the data using the LSD method. The results are shown in Table 4.

[0043] Table 4 Growth traits of wheat seedlings with fermentation broth or exopolysaccharide of Rhizobium LX under salt stress

[0044] It can be seen from Table 4 that compared with the salt stress S group, the addition of the fermentation broth of Rhizobium LX polysaccharide and exopolysaccharide 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%; the dry weight increased by 43.40%, 35.22%, and 30.82%.

[0045] In summary, the fermentation broth or exopolysaccharide of Rhizobium LX provided by the present invention has obvious effects in improving plant salt tolerance or drought stress resistance, and has broad application prospects.

[0046] The present invention has been published with preferred embodiments, but it is not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation are within the protection scope of the present invention.

Claims

1. A rhizobium LX with high exopolysaccharide production, characterized in that: The rhizobium LX was deposited in China Center for Type Culture Collection on February 28, 2025, with a deposit number of CCTCC M 2025321; The main structural components of the extracellular polysaccharide include glucose, galactose, xylose, fucose and mannose.

2. The high polysaccharide-producing rhizobium LX according to claim 1, characterized in that: 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, characterized in that: The molar ratio of glucose to galactose in the main structural components of the extracellular polysaccharide is 7-8:

1.

4. The high polysaccharide-producing rhizobium LX according to claim 1, characterized in that: The preparation method of the extracellular polysaccharide comprises the following steps: The seed liquid of Rhizobium LX is inoculated into the fermentation medium to obtain polysaccharide fermentation liquid, the polysaccharide fermentation liquid is subjected to alcohol precipitation and drying to constant weight to obtain crude extracellular polysaccharide, the crude product is dissolved in water and then an organic solvent is added to remove protein, and then dialyzed and dried to obtain pure extracellular polysaccharide.

5. Use of the high-polysaccharide-producing rhizobium LX according to claim in improving plant resistance to salt or drought stress.

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

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

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