Three growth-promoting bacteria combined with sinorhizobium fredii and application thereof

Abstract

The invention discloses three growth-promoting bacteria combined with sinorhizobium freudenreanum and application thereof, and belongs to the technical field of microorganisms. According to the invention, three growth-promoting bacteria of fish pseudomonas ZMC01, pseudomonas puticola ZMC129 and cellulomicrobe vinckii ZMC94 are separated from the root of an oxytropis glabra plant in a saline-alkali beach, and the three growth-promoting bacteria have the ability to grow in a saline-alkali environment. Tests prove that the growth-promoting bacteria ZMC01, ZMC129 and ZMC94 can promote the growth of soybeans in a saline-alkali environment or a non-saline-alkali environment by independently acting or jointly acting with the sinorhizobium freudenreanum, and indexes such as plant height, root length, stem and leaf fresh weight, root fresh weight, root nodule number and root nodule fresh weight of plants are remarkably improved or increased. The growth-promoting bacteria can also improve the nodulation capacity of the sinorhizobium freudenreanum in a saline-alkali environment, the nodulation and nitrogen fixation capacity of the soybeans under saline-alkali stress is expected to be fundamentally improved, and the growth-promoting bacteria have important significance for guaranteeing safe production of the soybeans.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to three strains of growth-promoting bacteria combined with Bradyrhizobium diazoefficiens and application thereof. BACKGROUND

[0002] Soybean is an important food and oil crop, and its production is often restricted by salt-alkali stress. Salt-alkali soil not only directly inhibits the growth and development of soybean, but also more seriously damages the symbiotic nitrogen fixation process with rhizobia, resulting in a sharp decrease in nodule number and a decrease in nitrogen fixation efficiency, thereby causing a substantial reduction in yield. Bradyrhizobium diazoefficiens is an efficient symbiotic nitrogen-fixing bacterium for soybean, but its survival, infection and nodulation ability are severely inhibited in salt-alkali environment.

[0003] In the prior art, single strain inoculation or conventional agricultural measures are often used to cope with salt-alkali problems, but the effect is often limited and unstable. Single inoculation of Bradyrhizobium diazoefficiens cannot guarantee its colonization and competition ability under stress; and the method of physically and chemically improving soil is costly and may cause environmental burden.

[0004] Therefore, there is an urgent need in the art for a biological technology solution that can effectively enhance the salt-alkali tolerance of the soybean-rhizobium symbiotic system, thereby stably achieving efficient nodulation and nitrogen fixation in salt-alkali soil. SUMMARY

[0005] The present application provides three strains of growth-promoting bacteria combined with Bradyrhizobium diazoefficiens and application thereof to solve the problems existing in the prior art. The growth-promoting bacteria ZMC01, ZMC129 and ZMC94 can promote the growth of soybean in salt-alkali environment or non-salt-alkali environment alone or in combination with Bradyrhizobium diazoefficiens. The growth-promoting bacteria can also improve the nodulation ability of Bradyrhizobium diazoefficiens in salt-alkali environment, which is expected to fundamentally improve the nodulation and nitrogen fixation ability of soybean under salt-alkali stress, and has important significance for ensuring the safe production of soybean.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0007] The present application provides three strains of growth-promoting bacteria, which are Pseudomonas piscicola ZMC01, Pseudomonas peli ZMC129 or Cellulosimicrobium funkei ZMC94.

[0008] The Pseudomonas piscicola ZMC01 has been preserved in the China General Microbiological Culture Collection Center on October 15, 2025, with the preservation number of CGMCC No. 36187 and the preservation address of No. 3, Institute of Microbiology, Chinese Academy of Sciences, 1, Beichen West Road, Chaoyang District, Beijing;

[0009] The Pseudomonas mudii ZMC129 has been preserved in the China General Microbiological Culture Collection Center on October 15, 2025, with a preservation number of CGMCC No. 36189 and a preservation address of No. 3, Yikuangli, Beichen West Road, Beijing, China Institute of Microbiology, Chinese Academy of Sciences;

[0010] The Caloimonas finkii ZMC94 has been preserved in the China General Microbiological Culture Collection Center on October 15, 2025, with a preservation number of CGMCC No. 36188 and a preservation address of No. 3, Yikuangli, Beichen West Road, Beijing, China Institute of Microbiology, Chinese Academy of Sciences.

[0011] The application also provides a microbial inoculant comprising the growth-promoting bacteria or a bacterial suspension thereof.

[0012] The application also provides the use of the growth-promoting bacteria or the microbial inoculant in any of the following aspects:

[0013] (1) promoting plant growth;

[0014] (2) preparing a product for promoting plant growth;

[0015] (3) promoting plant growth in combination with Sinorhizobium fredii;

[0016] (4) preparing a product for promoting plant growth in combination with Sinorhizobium fredii;

[0017] (5) improving the nodulation capacity of Sinorhizobium fredii in a saline-alkali environment.

[0018] Optionally, the plant growth promotion includes promoting plant growth in a saline-alkali environment and promoting plant growth in a non-saline-alkali environment.

[0019] Optionally, the growth-promoting bacteria and the Sinorhizobium fredii are mixed in the form of a bacterial suspension.

[0020] Optionally, the volume ratio of the growth-promoting bacterial suspension to the Sinorhizobium fredii bacterial suspension is 3:1, and the concentration of each is 1.5x10 8 CFU / mL.

[0021] Optionally, the plant includes soybean.

[0022] The application also provides a method for promoting plant growth, comprising any of the following steps:

[0023] (1) treating the plant with the growth-promoting bacteria or a bacterial suspension thereof;

[0024] (2) The plant is treated by the growth promoting bacteria and the Bradyrhizobium fengii.

[0025] The growth promoting bacteria and the Bradyrhizobium fengii are mixed in the form of bacterial suspension.

[0026] Optionally, the volume ratio of the growth promoting bacteria suspension and the Bradyrhizobium fengii suspension is 3:1, and the concentration of each is 1.5x10 8 CFU / mL.

[0027] Optionally, the plant growth promotion includes promoting the growth of the plant in a saline-alkali environment and promoting the growth of the plant in a non-saline-alkali environment.

[0028] The plant includes soybean.

[0029] The present application discloses the following technical effects:

[0030] The present application discloses the following technical effects:

[0031] Based on the characteristics of IAA production and salt and alkali tolerance of the three strains of growth-promoting bacteria ZMC01, ZMC129 and ZMC94 and the characteristics of co-growth promotion with rhizobium S.fredii, the three strains of bacteria provided by the application provide strain resources and technical support for the preparation of microbial fertilizers or microbial agents, alleviate the environmental hazards caused by excessive use of chemical fertilizers; on the other hand, safe and effective microbial resources are provided for microbial fertilizers or microbial agents suitable for saline-alkali areas. The three growth-promoting bacteria have broad application prospects in the fields of saline-alkali agricultural, ecological restoration and adversity crop breeding, and are expected to become an important tool for improving crop yield and improving soil quality. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0033] Figure 1 ZMC01, ZMC129 and ZMC94 colony growth on LB medium;

[0034] Figure 2 ZMC01, ZMC129 and ZMC94 phylogenetic tree;

[0035] Figure 3 ZMC01, ZMC129 and ZMC94 qualitative determination test of IAA secretion ability;

[0036] Figure 4 IAA standard curve;

[0037] Figure 5 Effect of ZMC01, ZMC129 and ZMC94 on soybean growth under salt and alkali stress;

[0038] Figure 6 Figure 5 Column chart of plant height (A), root length (B), stem and leaf fresh weight (C), and root fresh weight (D) of soybean plants in each group in Example 1;

[0039] Figure 7 Figure 5 Column chart of plant height (A), root length (B), stem and leaf fresh weight (C), and root fresh weight (D) of soybean plants under salt and alkali stress in each group in Example 2;

[0040] Figure 8 ZMC01, ZMC129 and ZMC94 compatibility experiment with S. fredii strain;

[0041] Figure 9 ​​Effects of ZMC01, ZMC129 and ZMC94 on S. fredii strain growth;

[0042] Figure 10 Effects of ZMC01, ZMC129 and ZMC94 on S. fredii strain growth;

[0043] Figure 11 Effects of ZMC01, ZMC129 and ZMC94 on S. fredii strain growth;

[0044] Figure 12 Effects of ZMC01, ZMC129 and ZMC94 on S. fredii strain growth; Figure 10 Column charts of plant height (A), root length (B), stem and leaf fresh weight (C), and root fresh weight (D) of soybean plants in each group in the normal condition;

[0045] Figure 13 Effects of ZMC01, ZMC129 and ZMC94 on S. fredii strain growth; Figure 10 Column charts of plant height (A), root length (B), stem and leaf fresh weight (C), and root fresh weight (D) of soybean plants under salt-alkali stress in each group;

[0046] Figure 14 Effects of ZMC01, ZMC129 and ZMC94 on S. fredii strain growth; Figure 11 Column charts of normal group nodule number (A), normal group nodule fresh weight (B), stress group nodule number (C), and stress group nodule fresh weight (D) of soybean plants in each group. DETAILED DESCRIPTION

[0047] The various illustrative embodiments of the present application will now be described in detail in connection with the accompanying drawings. This description is made for the purpose of illustrating the present application and is not intended to limit the application in its broader aspects to those details. Furthermore, where considered appropriate, steps can be practiced in an order different from that described.

[0048] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application. Additionally, where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of the ranges is also specifically disclosed. Each smaller range between any stated value or intervening value in the stated ranges and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0049] Unless otherwise indicated, all technical and scientific terms have the same meaning as those one of ordinary skill in the art of the present application would understand. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described herein. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0050] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application. The specification and examples are illustrative only.

[0051] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to mean including, but not limited to.

[0052] The culture media used in the present application are as follows:

[0053] LB solid medium: tryptone 10 g, yeast extract 5 g, NaCl 10 g, agar 15 g, H2O 1000 mL, pH 7.0~7.2, sterilized at 121°C for 30 min.

[0054] LB liquid medium: tryptone 10 g, yeast extract 5 g, NaCl 10 g, H2O 1000 mL, pH 7.0~7.2, sterilized at 121°C for 30 min.

[0055] YMA solid medium: mannitol 10 g, yeast powder 0.4 g, K2HPO4 0.25 g, KH2PO4 0.25 g, MgSO4·7H2O 0.2 g, NaCl 1 g, CaCl2·6H2O 0.1 g, Rh 4 mL (ammonium molybdate 5 g, boric acid 5 g, H2O 1000 mL), agar 16 g, H2O 1000 mL, pH 7.0~7.2, sterilized at 121°C for 30 min.

[0056] YMA liquid medium: mannitol 10 g, yeast powder 0.4 g, K2HPO4 0.25 g, KH2PO4 0.25 g, MgSO4·7H2O 0.2 g, NaCl 1 g, CaCl2·6H2O 0.1 g, Rh 4 mL (ammonium molybdate 5 g, boric acid 5 g, H2O 1000 mL), H2O 1000 mL, pH 7.0~7.2, sterilized at 121°C for 30 min.

[0057] IAA fermentation medium: 10g tryptone, 5g yeast extract, 10g NaCl, 1g L-tryptophan, 15g agar, 1000mL H2O, pH=7.0~7.2, sterilized at 121℃ for 30min.

[0058] The species *Sinorhizobium fredii* (S. fredii) has been published in the literature “Hu Fengxiao, Shen Hongling, Zheng Bo, et al. Diversity of indigenous rhizobia in soybean in Xinjiang and analysis of their matching relationships with different soybean varieties [J / OL]. Xinjiang Agricultural Sciences, 1-23 [2025-11-22]. https: / / link.cnki.net / urlid / 65.1097.S.20250224.1248.002.”

[0059] Example 1: Isolation and Identification of ZMC01, ZMC129 and ZMC94

[0060] 1. Source

[0061] ZMC01, ZMC129, and ZMC94 were all isolated from the roots of *Oxytropis florida* in the saline-alkali land of Alar City, Xinjiang Uygur Autonomous Region.

[0062] 2. Separation and purification

[0063] Collected *Oxytropis pulveratus* plants, rinse the roots with tap water to remove surface soil, cut the roots into several sections, and soak them successively in 2% sodium hypochlorite and 70% ethanol for 7 minutes each. Then rinse four times with sterile water. Next, grind the roots in a sterile mortar and pestle, and prepare a series of diluted grinding solutions with sterile water. A dilution gradient of 10 is selected. -3 and 10 -4 The grinding solution was plated onto solid LB medium containing 3% NaCl and incubated in a constant temperature incubator at 28℃. Single colonies of different shapes and colors were picked and streaked multiple times. After 2-3 generations of subculture, ZMC01, ZMC129 and ZMC94 with purified and consistent growth morphology were obtained.

[0064] 3. Classification and identification

[0065] 3.1 Morphological identification

[0066] ZMC01, ZMC129, and ZMC94 were cultured on LB agar plates at 30°C for 2 days, and colony growth was observed as follows: Figure 1 As shown, ZMC01 colonies are milky white, opaque, smooth, moist, and non-fluorescent round colonies; ZMC129 colonies are small, round, smooth, moist, yellow, and convex in the center; ZMC94 colonies are bright yellow, smooth, moist, round, and opaque.

[0067] 3.2 Systematic identification

[0068] The ZMC01, ZMC129 and ZMC94 on the LB solid medium plate were picked up by a loop, and the bacterial mass was diluted with 15 μL of sterile water, centrifuged at 12000 r / min for 5 min, the supernatant was discarded, and the bacterial mass was suspended with 15 μL of sterile water, centrifuged again, and then placed in an ice bath for 15-20 min. The bacterial suspension after treatment was used as a PCR identification template, and the target gene fragment was amplified by using bacterial universal primers 27F and 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 1; 1492R: 5'-GGCTACCTTGTTACGACTT-3', SEQ ID NO. 2). The PCR reaction system was as follows: 27F and 1492R primers (10 mmol) 1 μL each, bacterial suspension 3 μL, 2x PCR mix 12.5 μL, sterile water 7.5 μL, and a total of 25 μL; and a control system without bacterial suspension. The amplification program was as follows: 95 °C for 3 min; 95 °C for 1 min, 56 °C for 1 min, 72 °C for 1 min, 25 cycles; 72 °C for 10 min. The PCR product was submitted for sequencing (Shengong Sequencing (Xi'an) Co., Ltd.), and the 16S rDNA sequences of the three strains of ZMC01, ZMC129 and ZMC94 were measured.

[0069] The 16S rDNA sequences of the three strains were subjected to homology comparison with the nucleic acid data in Ezbiocloud (https: / / www.ezbiocloud.net / identify), and the results showed that the homology of ZMC01 with Pseudomonas piscicola P50 strain was 99.43%, the homology of ZMC129 with Pseudomonas peli R-20805 strain was 99.37%, and the homology of ZMC94 with Cellulosimicrobium funkei ATCC BAA-886 strain was 99.29%.

[0070] The phylogenetic tree of the three strains was constructed by using software MOLECULAR EVOLUTIONARY GENETIC ANALYSIS software (MEGA 7.0), as shown in Figure 2As shown, the sequences of ZMC01 and Pseudomonas piscicola P50, ZMC129 and Pseudomonas peli R-20805, and ZMC94 and Cellulosimicrobium funkei ATCC BAA-886 can constitute a stable evolutionary branch.

[0071] Therefore, ZMC01 was identified as Pseudomonas piscicola, named ZMC01, and strain ZMC01 has been preserved in the China General Microbiological Culture Collection Center on October 15, 2025, with the preservation number of CGMCC No. 36187 and the preservation address of No. 3, Yuanmingyuan West Road, Beijing City, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences; ZMC129 was identified as Pseudomonas peli, named ZMC129, and strain ZMC129 has been preserved in the China General Microbiological Culture Collection Center on October 15, 2025, with the preservation number of CGMCC No. 36189 and the preservation address of No. 3, Yuanmingyuan West Road, Beijing City, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences; ZMC94 was identified as Cellulosimicrobium funkei, named ZMC94, and strain ZMC94 has been preserved in the China General Microbiological Culture Collection Center on October 15, 2025, with the preservation number of CGMCC No. 36188 and the preservation address of No. 3, Yuanmingyuan West Road, Beijing City, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences.

[0072] Example 2 Detection of salt (NaCl) and alkali (NaOH) tolerance of ZMC01, ZMC129 and ZMC94

[0073] 1. Evaluation of salt tolerance

[0074] ZMC01, ZMC129 and ZMC94 were inoculated in LB liquid medium containing 1%, 3%, 5%, 7%, 9% and 11% (w / v) NaCl, respectively, at an inoculation amount of 1%, and cultured at 30°C for 48 h with shaking, and the absorbance value (OD 600 ) at 600 nm was measured. The seed bacteria solution was not inoculated as a control group. The results showed that ZMC01, ZMC129 and ZMC94 could grow in LB medium with NaCl concentration of 1-5% (w / v), 1-9% (w / v) and 1-7% (w / v), respectively.

[0075] 2. Evaluation of alkali tolerance

[0076] The following pH gradient was set: 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, and the pH value was adjusted with 1M NaOH or 1M HCl. Seed bacterial liquid of ZMC01, ZMC129 and ZMC94 was inoculated in LB liquid medium at different pH values, and the inoculation amount was 1%. The culture was incubated at 30°C for 48h, and the OD was measured 600 . The control group was not inoculated with seed bacterial liquid. The results showed that ZMC01, ZMC129 and ZMC94 could grow in the pH range of 7.0~10.0, 7.0~10.0 and 7.0~11.0, respectively.

[0077] Example 3: Detection of the ability of ZMC01, ZMC129 and ZMC94 to secrete IAA

[0078] Salkowski colorimetric reagent: 12g FeCl3 was dissolved in 300mL deionized water, and 429.7mL concentrated H2SO4 was slowly added. After cooling, it was diluted to 1L.

[0079] Preparation of IAA standard curve: 50mL of standard IAA solution with a concentration of 100µg / mL was prepared, and was diluted to 6.25, 12.5, 25, 50µg / mL, respectively. The IAA solution was mixed with Salkowski colorimetric solution at a volume ratio of 1:1, and reacted at room temperature for 30min in the dark. Distilled water was mixed with an equal volume of Salkowski colorimetric solution as a control. Then the OD value at 530nm of each concentration was measured by UV spectrophotometry (OD 530 ). The IAA concentration was taken as the abscissa, and the OD 530 was taken as the ordinate, and the IAA standard curve was obtained. Figure 4

[0080] Quantitative determination of IAA concentration of strains: ZMC01, ZMC129 and ZMC94 were respectively taken in LB liquid medium, and the seed culture liquid was prepared by incubating at 30°C, 150r / min for 12h. 100µL of seed culture liquid of ZMC01, ZMC129 and ZMC94 was inoculated in IAA fermentation medium, and was incubated at 30°C, 180r / min for 3d. Then it was centrifuged at 12000r / min for 2min, and 500μL of supernatant was mixed with an equal volume of Salkowski colorimetric solution, and was shaken well. Then it was placed at room temperature for 30min in the dark. The OD 530 was measured. The control group was the mixture of fermentation medium and an equal volume of Salkowski colorimetric solution. Finally, the corresponding IAA content was calculated according to the standard curve.

[0081] From the above experiments, it can be seen that ZMC01, ZMC129 and ZMC94 can grow in the pH range of 7.0~10.0, 7.0~10.0 and 7.0~11.0, respectively, and can secrete IAA. The ability of ZMC01 to secrete IAA is stronger than that of ZMC129 and ZMC94. Figure 3 ​It can be seen that ZMC01, ZMC129 and ZMC94 all have the ability to produce IAA. The secretion of IAA under the induction of L-tryptophan can reach 55.37 mg / L, 71.34 mg / L and 114.32 mg / L, respectively.

[0082] Example 4: Effect of ZMC01, ZMC129 and ZMC94 on the growth of soybeans under salt stress

[0083] Preparation of sterile soybean seedlings: Soybean seeds were soaked in 2% sodium hypochlorite for 3 min, rinsed with sterile water for 3 times, then soaked in 70% ethanol for 2 min, rinsed with sterile water for 5 times, and germinated in water at 25°C for 3 days, then transferred to a flowerpot containing nutrient soil:vermiculite with a volume ratio of 2:1 (total volume 180 cm 3 ).

[0084] Preparation of bacterial suspension: ZMC01, ZMC129 and ZMC94 bacterial bodies were picked and inoculated into LB liquid medium, cultured at 37°C, 150 r / min, when the OD 600 value was greater than 1.5, centrifuged at 5000 rmp / min for 5 min to discard the supernatant, washed the bacterial bodies with 30 mL sterile water twice, removed the culture medium components, and prepared ZMC01, ZMC129 and ZMC94 bacterial suspensions with a concentration of 1.5 x 10 8 CFU / mL, respectively.

[0085] Inoculation: The prepared ZMC01, ZMC129 and ZMC94 bacterial suspensions were inoculated into the roots of sterile cultured dicotyledonous soybean seedlings, 2 mL per plant, and continued to grow at 25°C for 30 days, during which the compound saline solution was added to each pot to reach a concentration of 150 mmol / L (compound saline solution: NaCl:Na2SO4:NaHCO3:Na2CO3 molar ratio = 1:9:9:1; 150 mmol / L compound salt formula: NaCl:Na2SO4:NaHCO3:Na2CO3 (g / L) = 0.4383:9.5877:5.6705:0.7949), pH 8.0-8.5, and the growth status of soybeans was observed. The plant height, root length, stem and leaf fresh weight and fresh weight of soybeans were measured after 30 days of growth. The bacterial suspension alone was used as a negative control; the compound saline solution alone was used as a positive control (CK-150); and sterile water was used as a blank control (CK).

[0086] It can be seen from Figure 5 and Figure 6 that ZMC01, ZMC129 and ZMC94 have a significant promoting effect on the plant height, stem and leaf fresh weight and root fresh weight of soybeans, and Figure 5 and Figure 7It can be seen that ZMC01, ZMC129 and ZMC94 also have a significant promoting effect on the plant height, stem and leaf fresh weight and root fresh weight of soybeans under saline-alkali stress.

[0087] Example 5 Detection of ZMC01, ZMC129 and ZMC94 and S. fredii microorganisms.

[0088] The ZMC01, ZMC129 and ZMC94 bacterial bodies were respectively picked and inoculated into LB liquid medium, and cultured at 37°C and 150r / min. When the OD value was greater than 1.5, 10 mL of sterile water was added to wash the bacterial bodies twice, and the medium components were removed to prepare the ZMC01, ZMC129 and ZMC94 bacterial suspension with OD value of 0.5. 600 600 The S. fredii bacterial bodies were picked and inoculated into YMA liquid medium, and cultured at 30°C and 150r / min. When the OD value was greater than 1.5, 10 mL of sterile water was added to wash the bacterial bodies twice, and the medium components were removed to prepare the S. fredii bacterial suspension with OD value of 0.5. 600 600 The S. fredii bacterial bodies were picked and inoculated into YMA liquid medium, and cultured at 30°C and 150r / min. When the OD value was greater than 1.5, 10 mL of sterile water was added to wash the bacterial bodies twice, and the medium components were removed to prepare the S. fredii bacterial suspension with OD value of 0.5.

[0089] 100 μL of the ZMC01, ZMC129 and ZMC94 bacterial suspensions were respectively mixed with 100 μL of the S. fredii bacterial suspension, and 5 μL of the mixed solution was spotted in the center of the YMA medium plate. The single-strain bacterial suspensions contained in the mixed solution were respectively spotted at a distance of 2 cm from the inoculation site to detect whether the strains were repelled. Then, the two kinds of single-strain bacterial suspensions were respectively spotted on the YMA plates at a distance of 2 cm (ZMC01-ZMC01, ZMC01-S. fredii, S. fredii-S. fredii, ZMC129-S. fredii, ZMC129-ZMC129, ZMC94-S. fredii and ZMC94-ZMC94), and the influence of the strains on the growth of S. fredii was observed.

[0090] It can be seen from Figure 8 and Figure 9 that ZMC01, ZMC129 and ZMC94 all have no antagonism to the S. fredii strain.

[0091] Example 6 Influence of ZMC01, ZMC129 and ZMC94 and S. fredii on the growth of soybeans under salt stress

[0092] Preparation of sterile soybean seedlings: same as in Example 4.

[0093] ​​Preparation of bacterial suspensions: The preparation of bacterial suspensions for ZMC01, ZMC129, and ZMC94 was the same as in Example 4. Preparation of S. fredii bacterial suspension: S. fredii cells were picked and inoculated into YMA liquid medium and cultured at 30°C and 150 r / min until OD... 600 When the value is greater than 1.5, centrifuge at 5000 rpm for 5 min, discard the supernatant, add 30 mL of sterile water to wash the bacterial cells twice, remove the culture medium components, and prepare 1.5 × 10⁻⁶ cells / mL. 8 CFU / mL bacterial suspension.

[0094] Inoculations of ZMC01, ZMC129, and ZMC94 with a bacterial suspension of *S. fredii* at a 3:1 volume ratio were applied to the roots of aseptically cultured soybean seedlings with fully opened dicotyledons. Each seedling was inoculated with 4 mL of the solution. The seedlings were allowed to grow at 25°C for 30 days, during which time a compound saline-alkali solution was added to each pot until the concentration reached 150 mmol / L, pH 8.0–8.5. Soybean growth was observed, and plant height, root length, stem and leaf fresh weight, and root fresh weight were measured after 30 days. Inoculation with the bacterial suspension alone served as a negative control; inoculation with the compound saline-alkali solution alone served as a positive control (*S. fredii-150*); and inoculation with *S. fredii* alone served as a control (*S. fredii*).

[0095] Depend on Figure 10- Figure 14 It can be seen that double inoculation of S. fredii with ZMC01, ZMC129 or ZMC94 significantly increased the plant height, stem and leaf fresh weight and root fresh weight of soybean plants compared with single inoculation of S. fredii. In salt stress environment, it can significantly increase the number of root nodules and root nodule fresh weight, that is, ZMC01, ZMC129 and ZMC94 can promote the nodulation ability of S. fredii in saline-alkali environment.

[0096] In summary, this invention isolated three growth-promoting bacteria (ZMC01, ZMC129, and ZMC94) from the roots of *Oxytropis rubrum* plants in saline-alkali areas. All three strains possess the ability to grow in saline-alkali environments and can secrete a certain amount of IAA (intracellular acid). Verification showed that ZMC01, ZMC129, and ZMC94 can all promote soybean growth under salt stress, helping the plants resist this condition. Furthermore, co-inoculation with *S. fredii* enhanced the soybean's tolerance to salt-alkali nodulation. ZMC01, ZMC129, and ZMC94 show promising applications in the preparation of microbial fertilizers and inoculants suitable for saline-alkali areas.

[0097] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. 3 Strains of growth promoting bacteria, characterized in that, the Pseudomonas piscicola ZMC01, the Pseudomonas peli ZMC129 or the Cellulosimicrobium funkei ZMC94; the Pseudomonas piscicola ZMC01 has been deposited with the China General Microbiological Culture Collection Center on October 15, 2025, and has the accession number of CGMCC No. 36187, and the deposit address is No. 3, Yuanmingyuan West Road, Beijing City, China; the Pseudomonas peli ZMC129 has been deposited with the China General Microbiological Culture Collection Center on October 15, 2025, and has the accession number of CGMCC No. 36189, and the deposit address is No. 3, Yuanmingyuan West Road, Beijing City, China; the Cellulosimicrobium funkei ZMC94 has been deposited with the China General Microbiological Culture Collection Center on October 15, 2025, and has the accession number of CGMCC No. 36188, and the deposit address is No. 3, Yuanmingyuan West Road, Beijing City, China.

2. A microbial inoculant, characterized in that, a microbial inoculant comprising the growth-promoting bacteria of claim 1 or a bacterial suspension thereof.

3. Use of the growth-promoting bacteria of claim 1 or the microbial inoculant of claim 2 in any one of the following: (1) promoting plant growth; (2) preparing a product for promoting plant growth; (3) promoting plant growth in combination with the Sinorhizobium fredii; (4) preparing a product for promoting plant growth in combination with the Sinorhizobium fredii; (5) improving the nodulation ability of the Sinorhizobium fredii in a saline-alkali environment.

4. The use according to claim 3, wherein the compound is ###0002### The plant growth promotion includes promoting plant growth in a saline-alkali environment and promoting plant growth in a non-saline-alkali environment.

5. The use according to claim 3, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The growth-promoting bacteria and the Sinorhizobium fredii are mixed in the form of a bacterial suspension.

6. The use according to claim 5, wherein the compound is ###0002### The volume ratio of the growth-promoting bacteria suspension and the Mesorhizobium huanghuaiensis suspension is 3:1, and the concentration of each is 1.5×10 8 CFU / mL.

7. The use according to claim 3, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The plant includes soybean.

8. A method of promoting plant growth, characterized by, comprising any one of the following steps: (1) treating a plant with the growth-promoting bacteria of claim 1 or a bacterial suspension thereof; (2) treating a plant with the growth-promoting bacteria of claim 1 in combination with the Sinorhizobium fredii; The growth-promoting bacteria and the Sinorhizobium fredii are mixed in the form of a bacterial suspension.

9. The method of claim 8, wherein, The volume ratio of the growth-promoting bacteria suspension and the Mesorhizobium huanghuaiensis suspension is 3:1, and the concentration of each is 1.5×10 8 CFU / mL.

10. The method of claim 8, wherein, The plant growth promotion includes promoting plant growth in a saline-alkali environment and promoting plant growth in a non-saline-alkali environment. The plant includes soybean.

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