Synthetic flora capable of improving salt tolerance of alfalfa and application of synthetic flora

Abstract

The invention discloses a synthetic flora capable of improving salt tolerance of alfalfa. The synthetic flora consists of a pseudomonas strain EC5, an agrobacterium strain EC17, a streptomyces strain EC15, a paracoccus strain RH39, a pseudomonas strain RH6, a pseudomonas strain RH37, a sporosarcina strain RH34 and a bacillus strain RH32. The synthetic flora provided by the invention has the functions of dissolving organic phosphorus, dissolving inorganic phosphorus, dissolving potassium, producing iron carriers, producing biological membranes, producing IAA and producing ACC deaminase. Seed soaking and germination under the stress of 200 mmol / L and 250 mmol / L NaCl can obviously improve the germination potential, germination rate, germination index, radicle length, germ length and colony seedling embryo fresh weight of alfalfa seeds. Under the stress of 200 mmol / L NaCl, the plant height, stem diameter, leaf area and overground part fresh and dry weight of alfalfa plants can be obviously improved after the seeds are soaked by the synthetic flora.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a synthetic microbial community that can improve the salt tolerance of alfalfa and its application. Background Technology

[0002] Soil salinization is a global problem, with at least 20% of the world's arable land suffering from severe salinization erosion, making it one of the core causes of soil degradation. In my country, the total area of ​​saline-alkali land reaches hundreds of millions of hectares, and soil salinization is posing a serious challenge to my country's agricultural development. However, saline-alkali land is not entirely unusable; rather, it is a type of land resource with great development potential. Improving and efficiently utilizing it can effectively alleviate the current shortage of arable land resources in my country and solidify the foundation of land resources for sustainable agricultural development.

[0003] Synthetic microbial communities, composed of multiple strains with synergistic functional effects, exhibit superior environmental adaptability and plant growth-promoting efficiency compared to single-strain communities. While research on single-strain enhancement of plant salt tolerance is extensive, studies on the improvement of plant growth under salt stress by synthetic microbial communities remain relatively scarce. Existing research has confirmed that mixed microbial communities can enhance seed tolerance under adverse conditions. Current research on the growth-promoting effects of synthetic microbial communities primarily focuses on model plants such as maize, Arabidopsis thaliana, and duckweed, with very limited research on alfalfa.

[0004] Enhancing plant salt tolerance by leveraging soil microbial communities, creating a synergistic effect with the host's own salt tolerance, and thus enabling the cultivation of target plants in highly saline-alkali land, is an efficient way to revitalize idle saline-alkali land resources. Current technology urgently needs a synthetic microbial community that can improve alfalfa salt tolerance and its application. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a synthetic microbial community that can improve the salt tolerance of alfalfa and its application.

[0006] The objective of this invention is achieved through the following technical solution: a synthetic microbial community that can improve the salt tolerance of alfalfa, wherein the synthetic microbial community is composed of Pseudomonas (… Pseudomonas sp. ) strain EC5, Agrobacterium ( Agrobacterium sp. ) strain EC17, Streptomyces ( Streptomyces sp. ) strain EC15, Paracoccus ( Paracoccus sp. ) strain RH39, Pseudomonas ( Pseudomonas sp. ) strain RH6, Pseudomonas ( Pseudomonas sp. ) strain RH37, Bacillus occulta ( Sporosarcina sp. ) strain RH34 and Bacillus ( Bacillus sp. Composition of strain RH32; The strains EC5, EC17, RH39, RH6, RH37, RH34, and RH32 are all deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 25, 2024. The accession number for strain EC5 is CGMCC NO. 33193; for strain EC17, it is CGMCC NO. 33194; for strain EC15, it is CGMCC NO. 33195; for strain RH39, it is CGMCC NO. 33196; for strain RH6, it is CGMCC NO. 33197; for strain RH37, it is CGMCC NO. 33198; and for strain RH34, it is CGMCC NO. 33198. NO.33199; The preservation number of the strain RH32 is: CGMCC NO.33200; In the synthetic microbial community, the viable cell ratio of strain EC5, strain EC17, strain EC15, strain RH39, strain RH6, strain RH37, strain RH34 and strain RH32 is 1:1:1:1:1:1:1:1:1.

[0007] The present invention also provides the application of the synthetic microbial community described above in improving the germination index of alfalfa seeds under salt stress.

[0008] Furthermore, the salt stress is 200 mmol / L NaCl stress, and the germination indicators include germination potential, germination rate, germination index, radicle length, plumule length, and fresh weight of the seedling population.

[0009] Furthermore, the salt stress is 250 mmol / L NaCl stress, and the germination indicators include germination potential, germination rate, germination index, radicle length, plumule length, and fresh weight of the seedling population.

[0010] The present invention also provides the application of the synthetic microbial community described above in improving the growth of alfalfa seedlings under salt stress.

[0011] Furthermore, the salt stress is 200 mmol / L NaCl stress, and the seedling growth status includes plant height, stem diameter, leaf area, fresh weight of above-ground parts, and dry weight of above-ground parts.

[0012] The beneficial effects of this invention are as follows: The synthetic microbial community of this invention has the functions of dissolving organic phosphorus, dissolving inorganic phosphorus, solubilizing potassium, producing iron carriers, producing biofilms, producing IAA and ACC deaminases. Soaking and germinating alfalfa seeds under 200 mmol / L and 250 mmol / L NaCl stress significantly improves seed germination potential, germination rate, germination index, radicle length, plumule length, and fresh weight of seedling embryos. Under 200 mmol / L NaCl stress, soaking alfalfa seeds with the synthetic microbial community significantly increases plant height, stem diameter, leaf area, and aboveground fresh and dry weight. Attached Figure Description

[0013] Figure 1 These are the colony morphologies of each strain in the synthetic microbial community; where A is EC5; B is EC17; C is EC15; D is RH39; E is RH6; F is RH37; G is RH34; and H is RH32. Figure 2 The results are the culture results of the synthetic bacteria in solid culture media containing inorganic phosphorus and solid culture media containing organic phosphorus; where A is the culture result of the synthetic bacteria in solid culture media containing inorganic phosphorus, and B is the culture result of the synthetic bacteria in solid culture media containing organic phosphorus. Figure 3 It is the result of culturing the synthetic microbial community in a potassium-containing solid culture medium; Figure 4 This shows the staining results of crystal violet dye on a 96-well plate of synthetic bacteria. Figure 5 This is the result of culturing synthetic microorganisms in a siderophore-selective solid medium; Figure 6 This shows the staining results of the synthetic bacterial community with Sachs reagent in a 96-well plate; where CK is the control group and S1 is the experimental group. Figure 7 This is the germination phenotype of alfalfa seeds soaked under 200 mmol / L NaCl stress; Figure 8 This is a bar chart of various germination indicators of alfalfa seeds under 200 mmol / L NaCl stress; where A is the germination potential bar chart, B is the germination rate bar chart, C is the germination index bar chart, D is the radicle length bar chart, E is the plumule length bar chart, and F is the fresh weight of the seedling population bar chart. Figure 9 This is the germination phenotype of alfalfa seeds soaked under 250 mmol / L NaCl stress; Figure 10 This is a bar chart of various germination indicators of alfalfa seeds under 250 mmol / L NaCl stress; where A is the germination potential bar chart, B is the germination rate bar chart, C is the germination index bar chart, D is the radicle length bar chart, E is the plumule length bar chart, and F is the fresh weight of the seedling population bar chart. Figure 11 It is 200 mmol / L Bar charts showing the growth indicators of alfalfa seedlings under NaCl stress; where A is the plant height bar chart; B is the stem diameter bar chart; C is the leaf area bar chart; D is the fresh weight of aboveground parts bar chart; and E is the dry weight of aboveground parts bar chart. Detailed Implementation

[0014] The present invention will now be described in detail with reference to the accompanying drawings.

[0015] Unless otherwise specified, the chemical reagents used in the examples are all commercially available conventional reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0016] The TSA solid culture medium used in the examples included: 15 g / L tryptone, 5 g / L soybean peptone, 5 g / L NaCl and 15 g / L agar.

[0017] The TSB liquid culture medium used in the examples included: 8.5 g / L tryptone, 1.5 g / L plant peptone, 2.5 g / L NaCl, 1.25 g / L K2HPO4, 1.25 g / L glucose, and distilled water.

[0018] The inorganic phosphorus-containing solid culture medium used in the examples included: KCl 0.2 g / L, glucose 10 g / L, Ca3(PO4) 5 g / L, MgCl2·6H2O 0.5 g / L, MgSO4·7H2O 0.25 g / L, (NH4)2SO4 0.1 g / L and agar 20 g / L.

[0019] The solid culture medium containing organophosphorus compounds used in the examples included: 10 g / L glucose, 0.5 g / L (NH4)2SO4, 0.3 g / L NaCl, 0.3 g / L KCl, 0.03 g / L FeSO4·7H2O, 0.03 g / L SO4Mn4·H2O, 0.2 g / L lecithin, 0.4 g / L yeast extract (Beijing Cooler Master Technology Co., Ltd., model: CY12011-500g), and 20 g / L agar, with a pH of 7.0-7.5.

[0020] The potassium-containing solid culture medium used in the examples included: sucrose 5 g / L, Na2HPO4 2 g / L, MgSO4·7H2O 0.005 g / L, FeCl3 0.005 g / L, CaCO3 0.1 g / L, potassium feldspar powder 0.1 g / L, and agar 20 g / L.

[0021] The siderophore-selective solid culture medium used in the examples included: CAS (Crazinium S) 60.5 mg / L, distilled water 50 ml / L, HCl 8 μL / L, 2 mol / L FeCl3·6H2O 5 μL / L, HDTMA (hexadecyltrimethylammonium bromide) 72.9 mg / L, proteose peptone 20 g / L, MgSO4·7H2O 1.5 g / L, KH2PO4 1.2 g / L, glycerol 10 g / L, agar 13 g / L, pipes (piperazine-1,4-diethanesulfonic acid) 30 g / L, and 50% NaOH 12 ml / L.

[0022] Example 1: Isolation, screening, and identification of strains (a) Separation and screening: The eight strains in the synthetic microbial community of this invention were all derived from the saline soil surrounding the roots of alfalfa planted at the Hailiutu Experimental Base of Inner Mongolia Agricultural University. Alfalfa seedlings were planted in saline soil from the Hailiutu Experimental Base and then subjected to salt stress treatment (the seedling pots were immersed in a 200 mmol / L NaCl solution for 30 min). After four weeks of continuous culture, samples were collected for subsequent analysis.

[0023] Preparation of plant root samples: Using sterile forceps, remove alfalfa roots and place them in a test tube containing 25 ml of phosphate buffer. Vortex for 15 seconds. Then, using sterile forceps, adhere the roots to the inner wall of the test tube, slowly pour out the phosphate buffer, and add 25 ml of fresh phosphate buffer. Repeat this step until there is no rhizosphere soil in the phosphate buffer. Place the alfalfa roots in a 15 ml test tube and add 12 ml of phosphate buffer. Place the test tube in an ultrasonic sample homogenizer and sonicate for 5 minutes, followed by vortexing for 5 seconds. Rinse the roots with phosphate buffer. Dry the roots on filter paper, flash-freeze in liquid nitrogen, and store at -80°C.

[0024] Plant root samples were collected and cultured in TSA solid medium at 25°C. Colonies were selected based on their color (orange, pale yellow, milky white) and morphology (smooth or irregular edges) and cloned. The colonies produced from the plant root samples were streaked onto fresh TSA solid medium for purification. The purified colonies were then transferred to TSB liquid medium with 40% (v / v) glycerol added and stored at -80°C for later use.

[0025] (II) Classification and Identification: 1. Activated strains: Multiple bacterial strains preserved using the above method were removed from the -80℃ ultra-low temperature freezer, thawed at room temperature, and then activated and cultured on TSA solid medium. Colony morphology was observed (see...). Figure 1 ).

[0026] 2. Morphological characteristics of the strain: Based on their cytological characteristics, these strains are designated as strains 1-8. Their cytological characteristics are as follows: 1. The cytological characteristics of strain 1 are: colonies are milky white, moist, and have irregular edges; 2. The cytological characteristics of strain 2 are: the colonies are pale yellow to translucent, moist on the surface, and smooth at the edges; 3. The cytological characteristics of strain 3 are: yellow colonies that tend to be transparent, moist surface, and irregular edges; 4. The cytological characteristics of strain 4 are: orange-yellow colonies with moist surfaces and smooth edges; 5. The cytological characteristics of strain 5 are: pale yellow in the center of the colony with transparent edges, moist surface, and irregular edges; 6. The cytological characteristics of strain 6 are: the colonies are light yellow and tend to be transparent, with a moist surface and irregular edges; 7. The cytological characteristics of strain 7 are: the colonies are light yellow to white, the surface is moist, and the edges are irregular; 8. The cytological characteristics of strain 8 are: the colonies are milky white, moist, and have smooth edges.

[0027] 3.16S rRNA identification: The strains were taken from the -80℃ ultra-low temperature freezer and inoculated into TSB liquid medium. They were cultured at 25℃ and 220r / min for 3 days to obtain bacterial suspension. 1 mL of bacterial suspension was taken, centrifuged to collect the bacterial cells, and the gene DNA was extracted using the MoBio Power Soil DNAIsolation Kit (MoBio Corporation, USA). The DNA was stored at -20℃ for later use.

[0028] The 16S rRNA in the extracted genomic DNA was amplified using primers 16S-63F and 16S-1389R: Forward primer 16S-63F: AGTTTGATCMTGGCTCAG (SEQ ID NO:9); Reverse primer 16S-1389R: GGTTACCTTGTTACGACTT (SEQ ID NO:10).

[0029] Amplification system (total 50 μL): 10×Buffer 2.5 μL, 2 mmol / L dNTP 2.5 μL, forward primer 16S-63F 1 μL, reverse primer 16S-1389R 1 μL, dNTP (2 mmol / L) 2.5 μL, DNA 2 μL, Taq polymerase 0.1 μL and ddH2O 15.9 μL.

[0030] The PCR reaction program was as follows: denaturation at 94℃ for 5 min; 30 cycles of 94℃ for 1 min, 55℃ for 1 min, and 72℃ for 1 min; extension at 72℃ for 10 min. PCR products were detected by 1.5% agarose gel electrophoresis. After the extracted DNA passed purity and integrity tests, it was sent to a sequencing company (Beijing Novogene Technology Co., Ltd.) for sequencing.

[0031] After comparing the bacterial morphology, 16S gene sequence, and the NCBI (National Center for Biotechnology Information) database, strains 1-8 were identified as belonging to the genus *Pseudomonas*. Pseudomonas sp The strain, with its nucleotide sequence shown in SEQ ID NO:1, is named EC5; Agrobacterium genus ( Agrobacterium sp The strain, with its nucleotide sequence shown in SEQ ID NO:2, is named EC17; Streptomyces genus ( Streptomyces sp The strain, with its nucleotide sequence shown in SEQ ID NO:3, is named EC15; *Paracoccus* genus ( Paracoccus sp The strain, with its nucleotide sequence shown in SEQ ID NO:4, is named RH39; *Pseudomonas* genus ( Pseudomonas sp The strain, with its nucleotide sequence shown in SEQ ID NO:5, is named RH6; *Pseudomonas* genus ( Pseudomonas sp ) strain, with nucleotide sequence as shown in SEQ ID NO:6, named RH37; Bacillus spp. ( Sporosarcina sp The strain, with its nucleotide sequence shown in SEQ ID NO:7, is named RH34; Bacillus spp. ( Bacillus sp The strain, with its nucleotide sequence shown in SEQ ID NO:8, is named RH32.

[0032] Example 2: Preparation of synthetic bacterial community S1 Eight single bacterial strains were cultured on TSA solid medium for 3 days, followed by elution with sterilized 0.9% NaCl solution. The bacterial concentration was adjusted to OD using a UV spectrophotometer. 600 =1, mix the adjusted single-strain bacterial solutions in equal volumes to prepare a synthetic bacterial culture solution, named SynCom 1 (abbreviated as S1).

[0033] Example 3: Biological Function Test of Synthetic Microbial Community S1 1. Pretreatment of synthetic microbial community S1: Take the synthetic bacterial community S1 prepared in Example 2, remove the NaCl solution using a centrifuge, leaving the bacterial precipitate, add 20 ml of TSB liquid medium to the centrifuge tube, and incubate for 3 days on a shaker at 25°C and 220 r / min. Then, centrifuge at 4°C and 8000 r / min for 10 min and collect the bacterial precipitate. Subsequently, adjust the bacterial concentration to OD using a UV spectrophotometer. 600 =1, used for subsequent experiments.

[0034] 2. Phosphorus solubility test: Take 2 μL of bacterial culture and inoculate it onto the surface of solid culture medium containing organic phosphorus and solid culture medium containing inorganic phosphorus, respectively. Incubate at 25℃, with 3 biological replicates for each treatment. After 3-5 days, observe whether a phosphate-solubilizing zone appears in the culture medium. The presence or absence of a phosphate-solubilizing zone indicates whether the treatment has the function of dissolving insoluble organic and inorganic phosphorus.

[0035] The results are as follows Figure 2 A, Figure 2 As shown in Figure B, transparent phosphorus-solubilizing zones appeared in all culture media, proving that the synthetic bacterial group S1 has a strong ability to dissolve both organic and inorganic phosphorus.

[0036] 3. Determination of potassium solubilization capacity: Take 2 μL of bacterial culture and inoculate it into potassium-containing solid medium. Incubate at 25℃ with three biological replicates. After 3-5 days, observe the growth of colonies on the medium. The appearance of transparent, oil-drop-like colonies indicates potassium-solubilizing ability.

[0037] like Figure 3 As shown, oil droplet-shaped colonies appeared on the culture medium, proving that the synthetic bacterial group S1 has potassium-solubilizing ability.

[0038] 4. Determination of biofilm production capacity: Take 20 μL of bacterial culture and add it to a 96-well plate pre-filled with 180 μL of TSB liquid medium. Incubate at 25℃ and 220 r / min for 2-3 days. After standing, add 0.1 μL of crystal violet dye (containing 0.1% Crytorl) to each well and incubate for 15 min. Wash three times with sterile water. Add 200 μL of 96% ethanol to dissolve the dye remaining on the tube wall for 2 min. Observe whether there is purple staining in the 96-well plate. Determine whether the plate has the ability to produce a bacterial film and the strength of the bacterial film production ability based on the presence and degree of purple staining.

[0039] like Figure 4As shown, a purple-colored bacterial film appeared at the bottom of the 96-well plate, proving that the synthetic bacterial group S1 has a very strong ability to produce biofilms.

[0040] 5. Determination of iron production capacity: Take 2 μL of bacterial culture and inoculate it onto the surface of siderophore-selective solid medium. Incubate at 25°C with three biological replicates. After 3-5 days, observe whether there is an orange-yellow secretion zone in the siderophore-selective solid medium. The presence of an orange-yellow secretion zone indicates the presence of siderophore production capacity.

[0041] like Figure 5 As shown, the colonies in the culture medium have a large orange-yellow secretion zone, proving that the synthetic bacterial group S1 has a very strong ability to produce siderophores.

[0042] 6. Assay for ACC deaminase production capacity: Take 10 ml of bacterial culture into a 15 ml centrifuge tube, centrifuge at 4℃ and 8000 r / min for 10 min, discard the supernatant, leaving the bacterial precipitate, add 7.5 ml of nitrogen-free DF medium (Beijing Cooler Technology Co., Ltd., model: MM6150-2) to the centrifuge tube, and add 45 μL of filtered and sterilized 0.5 mol / L ACC (1-aminocyclopropane-1-carboxylic acid) solution to make the ACC solution concentration 3 mmol / L. Then, culture at 25℃ and 220 r / min with shaking for 24 h (to induce the ACC deaminase activity of bacteria) to obtain the bacterial suspension. Centrifuge the above bacterial suspension at 4°C and 8000 rpm for 10 min, remove the supernatant, collect the bacterial pellet, add 5 ml of Tris-HCl buffer (0.1 mol / L, pH 7.6) to the pellet to resuspend the bacteria, centrifuge at 4°C and 8000 rpm for 10 min, and collect the pellet. Repeat this step 3 times to completely remove the nitrogen-free DF medium. Add 1 ml of Tris-HCl buffer (0.1 mol / L, pH 7.6) to the pellet to resuspend the bacteria, then transfer to a 1.5 ml centrifuge tube, centrifuge at 15000 rpm for 5 min, remove the supernatant, and collect the bacterial pellet. Then add 600 μL of Tris-HCl buffer (0.1 mol / L, pH 8.5) to the pellet again, add 30 μL of toluene, and vortex for 30 s to disrupt the bacteria. Take 200 μL of the lysed bacterial suspension, add 20 μL of ACC solution (0.5 mol / L), mix well, and incubate at 30°C for 15 min. Then add 1 mL of HCl solution (0.56 mol / L), mix well, and centrifuge at 15000 rpm for 5 min at room temperature to obtain the supernatant. Take 1 mL of the supernatant, add 800 μL of HCl solution (0.56 mol / L), mix well, and then add 300 μL of 2,4-dinitrophenylhydrazine (2 g / L, dissolved in 2 mol / L HCl), and incubate at 30°C for 30 min. Subsequently, measure the absorbance at 540 nm using a UV-Vis spectrophotometer, with distilled water as a control. Determine whether the synthetic bacterial group S1 can produce ACC deaminase based on the OD value.

[0043] As shown in Table 1, the OD value of the synthetic bacterial group S1 is 0.030, which proves that the synthetic bacterial group S1 has a very strong ability to produce ACC deaminase.

[0044] Table 1. ACC deaminase and IAA OD values ​​of synthetic bacterial community S1

[0045] 7. IAA production capacity determination: In a 96-well plate, pre-add 180 μL of TSB liquid medium and 1.5 μL of tryptophan solution (0.11 g / L, sterilized by filtration). Add 20 μL of bacterial culture to the 96-well plate and incubate at 25℃ and 220 r / min for 2-3 days. Then add Sachs' reagent (3.51 mL of 35% HClO4 + 2 μL of 0.5 mol / L FeCl3) and incubate in the dark for 30 min. Observe whether the 96-well plate becomes turbid. The OD value is then measured. 600 The absorbance was measured under the specified conditions, with distilled water as the control (CK), to determine whether the synthetic bacterial group S1 had the function of producing IAA.

[0046] like Figure 6 As shown in Table 1, the color of the 96-well plate of the synthetic bacterial community S1 was significantly darker than that of CK, and the OD value was 0.872, proving that the synthetic bacterial community S1 has a very strong ability to produce IAA.

[0047] Example 4: Germination of alfalfa seeds after soaking under 200 mmol / L NaCl stress In this embodiment, the preparation method of the single-strain bacterial solution is the same as the preparation method of the synthetic bacterial group S1 in Example 2. The difference is that in this embodiment, the OD values ​​of the single-strain bacterial solution and the synthetic bacterial group S1 solution are... 600 Adjusted to 0.1.

[0048] Plump and intact alfalfa seeds (“Prairie No. 3” hybrid alfalfa) were selected and soaked for 8 hours in single-strain bacterial solution and synthetic bacterial group S1 bacterial solution, with sterile water as a control. Germination tests were conducted on a paper bed containing 200 mmol / L NaCl solution. 50 seeds were placed in each petri dish, with 3 biological replicates. The petri dishes were pre-sealed with 1-2 turns of sealing film to prevent moisture evaporation, and water was replenished daily by weighing to maintain a stable salt concentration. The number of germinated seeds in each dish was recorded daily until day 7. Germination potential, germination rate, and germination index were calculated. Then, 10 seedlings were randomly selected from each petri dish to measure the radicle length and plumule length, and the fresh weight of all seedlings in the dish was determined.

[0049] Seed germination phenotypes such as Figure 7 As shown, seedlings soaked in the bacterial solution exhibited greater cotyledon opening, significantly thicker and longer radicles, and a markedly faster seed germination rate. Figure 7 It can be seen that after soaking in single-strain bacterial solution and synthetic bacterial group S1, all germination indicators of the seeds were higher than those of CK, especially after soaking in synthetic bacterial group S1. Seeds soaked in single synthetic bacterial group S1 showed significantly higher germination potential and germination index than CK, reaching 52.28% and 69.13% of CK, respectively. P< 0.05) (see) Figure 8A, 8C); germination rate, radicle length, plumule length, and fresh weight of seedlings were all significantly higher than CK, at 113.04%, 56.67%, 24.01%, and 157.64% of CK, respectively. P< 0.01)(see Figure 8 B, 8D, 8E, and 8F).

[0050] Example 5 250 mmol / L Alfalfa seeds germinated after soaking under NaCl stress The experimental steps in this embodiment are the same as in Example 3, except that the 200 mmol / L NaCl solution is replaced with a 250 mmol / L NaCl solution.

[0051] Seed germination phenotypes such as Figure 9 As shown, seedlings soaked in the synthetic bacterial culture S1 solution had significantly longer radicles and more robust seeds. Figure 10 It can be seen that the germination indicators of seeds soaked in the synthetic microbial community S1 solution are better than those of seeds soaked in the single-strain microbial solution. After soaking in the synthetic microbial community S1 solution, the germination potential, germination rate, germination index, radicle length, plumule length, and embryo fresh weight of the seeds were all significantly higher than those of the control (CK), at 76.67%, 405.41%, 217.06%, 47.87%, 19.13%, and 1390.89% of CK, respectively. P < 0.01)(see Figure 10 A-10F).

[0052] Example 6 200 mmol / L Alfalfa seedling growth under NaCl stress Select plump and intact alfalfa seeds (“Prairie No. 3” mixed alfalfa) and germinate them in sterile petri dishes. After 6-7 days, select seedlings with uniform growth and transplant them into pots (pot dimensions: 10cm×10cm×8cm; the substrate in the pot consists of sterilized vermiculite and volcanic rock, with the weight of the volcanic rock being twice the weight of the vermiculite). Three days after transplanting, inoculate with single-strain bacterial solution and synthetic bacterial group S1 bacterial solution (OD of single-strain bacterial solution and synthetic bacterial group S1 bacterial solution). 600 =1. Inoculation is completed by injecting 1 ml of single-strain bacterial solution or synthetic bacterial group S1 bacterial solution into the soil around the roots of alfalfa using a pipette (with no single-strain bacterial solution or synthetic bacterial group S1 bacterial solution as a control). Salt stress treatment is carried out 2 weeks later (the potted plants are soaked in 200 mM NaCl solution for 30 min). After 4 weeks, the seedlings are harvested and the plant height, stem diameter, leaf area, fresh weight of above-ground parts and dry weight of above-ground parts are measured.

[0053] Depend on Figure 11As shown in A-11E, after inoculation with single-strain bacterial solution and synthetic bacterial group S1 bacterial solution, the plant height, stem diameter, leaf area, aboveground fresh weight and aboveground dry weight of seedlings under salt stress were significantly higher than those of CK. Seedlings inoculated with synthetic bacterial group S1 bacterial solution had a more obvious growth-promoting effect and could significantly improve the growth indicators of seedlings under salt stress.

[0054] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A synthetic microbial community that can improve the salt tolerance of alfalfa, characterized in that: The synthetic microbial community consists of Pseudomonas (… Pseudomonas sp. ) strain EC5, Agrobacterium ( Agrobacterium sp. ) strain EC17, Streptomyces ( Streptomyces sp. ) strain EC15, Paracoccus ( Paracoccus sp. ) strain RH39, Pseudomonas ( Pseudomonas sp. ) strain RH6, Pseudomonas ( Pseudomonas sp. ) strain RH37, Bacillus occulta ( Sporosarcina sp. ) strain RH34 and Bacillus ( Bacillus sp. Composition of strain RH32; The strains EC5, EC17, RH39, RH6, RH37, RH34, and RH32 are all deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 25, 2024. The accession number for strain EC5 is CGMCC NO. 33193; for strain EC17, it is CGMCC NO. 33194; for strain EC15, it is CGMCC NO. 33195; for strain RH39, it is CGMCC NO. 33196; for strain RH6, it is CGMCC NO. 33197; for strain RH37, it is CGMCC NO. 33198; and for strain RH34, it is CGMCC NO. 33198. NO.33199; The preservation number of the strain RH32 is: CGMCC NO.33200; In the synthetic microbial community, the viable cell ratio of strain EC5, strain EC17, strain EC15, strain RH39, strain RH6, strain RH37, strain RH34 and strain RH32 is 1:1:1:1:1:1:1:1:

1.

2. The application of the synthetic microbial community described in claim 1 to improve alfalfa seed germination indicators under salt stress.

3. The application according to claim 2, characterized in that: The salt stress is 200 mmol / L NaCl stress, and the germination indicators include germination potential, germination rate, germination index, radicle length, plumule length, and fresh weight of seedling population.

4. The application according to claim 2, characterized in that: The salt stress is 250 mmol / L NaCl stress, and the germination indicators include germination potential, germination rate, germination index, radicle length, plumule length, and fresh weight of seedling population.

5. The application of the synthetic microbial community described in claim 1 to improve the growth of alfalfa seedlings under salt stress.

6. The application according to claim 5, characterized in that: The salt stress is 200 mmol / L NaCl stress, and the seedling growth status includes plant height, stem diameter, leaf area, fresh weight of above-ground parts, and dry weight of above-ground parts.

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