Paenibacillus riograndensis RA10 and application thereof in improving salt and alkali tolerance of plants

Abstract

The application belongs to the technical field of microorganisms, and provides a paraburkholderia kururiensis RA10 and application thereof in improving salt-tolerant and alkali-tolerant ability of plants. The preservation number of the paraburkholderia kururiensis RA10 is CGMCC No. 38835. The strain has wide pH adaptability, can stably colonize in an alkaline salt stress environment with pH>9, and has nitrogen fixation and potassium solubilization functions, and can provide plants with necessary nutrients such as nitrogen and potassium. Under salt-alkali combined stress, application of the strain can significantly promote root elongation of plants, and improve plant height, stem diameter, aboveground and underground biomass, so as to realize synergistic effect of nutrient supply and growth promotion. The application provides a multifunctional strain resource integrating alkali tolerance, nitrogen fixation, potassium solubilization and growth promotion and stress resistance, for new-type stress-resistant microbial fertilizer development, and has important application value for promoting sustainable development of green agriculture.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Parasporium tumefaciens RA10 and its application in improving the salt and alkali tolerance of plants. Background Technology

[0002] Currently, crop cultivation and production are constrained by abiotic stresses such as salinity and alkalinity. High concentrations of salt bring osmotic stress and ion toxicity, while alkaline salts cause an increase in rhizosphere pH and a decrease in nutrient availability. This continuous compound stress will interfere with water absorption, photosynthesis, and key metabolic processes throughout the crop's growth period, ultimately seriously affecting crop yield and quality.

[0003] The main approaches to enhancing plant tolerance to salt and alkali stress include breeding salt-tolerant varieties, exogenous chemical regulation, and supporting agronomic measures. While salt-tolerant breeding can genetically enhance a plant's adaptability to saline-alkali environments, it is limited by limited genetic resources, long breeding cycles, and the difficulty in converging superior traits, making it difficult to meet the urgent needs of diversified production in the short term. Exogenous application of hormones, osmotic regulators, or signaling molecules can alleviate stress symptoms to some extent, but is often limited by short-lasting effects, high costs, and potential environmental risks. Agronomic measures such as seed initiation and rational water and fertilizer management can improve seedling tolerance, but their effectiveness is greatly affected by cultivation conditions and lacks controllability. Therefore, existing strategies for enhancing plant tolerance to salt and alkali stress still have limitations such as low efficiency, narrow window of action, and difficulty in systematically addressing complex stress throughout the entire growth cycle. There is an urgent need to develop more efficient, stable, and sustainable technologies to enhance plant salt and alkali tolerance.

[0004] Microbial inoculants, by introducing specific beneficial microorganisms, can enhance plant tolerance to salt-alkali stress through multiple pathways, effectively alleviating osmotic stress, ion toxicity, and high pH damage. They are green, safe, and sustainable, providing a new technological pathway for improving crop productivity under salt-alkali stress. However, existing salt-tolerant microbial strains have relatively limited functions; nitrogen fixation, potassium solubilization, and other growth-promoting traits are difficult to integrate efficiently with salt-alkali tolerance. These strains are unstable in the rhizosphere of saline-alkali environments, and their functional expression is easily affected by complex environments, leading to fluctuating effects and limiting their large-scale application under salt-alkali stress conditions. Therefore, there is an urgent need to develop novel microbial resources that combine stable nitrogen fixation and potassium solubilization functions with highly efficient salt-alkali tolerance and growth promotion. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a strain of *Parabolic Bacillus subtilis* RA10 and its application in improving the salt and alkali tolerance of plants.

[0006] In a first aspect, the present invention provides a strain of *Parabolic Bacillus subtilis* (… Metabacillus sediminitorisRA10, with accession number CGMCC No. 38835, was deposited on May 22, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China (Postcode: 100101). Its taxonomic name is *Parasitic Parasporium spp.* Metabacillus sediminitoris The collection name is RA10.

[0007] Secondly, the present invention also provides a microbial agent, the microbial agent comprising the *Parabacterium tumefaciens* (…). Metabacillus sediminitoris )RA10.

[0008] According to the present invention, a microbial agent is preferably provided, wherein the microbial agent comprises *Parabacterium tumefaciens* (…). Metabacillus sediminitoris RA10 bacterial culture, or the aforementioned *Parasitic bacillus* ( Metabacillus sediminitoris ) RA10 freeze-dried bacterial cells, or the aforementioned *Parabenia sericata* ( Metabacillus sediminitoris Inactivated bacterial cells of RA10, or the aforementioned *Parabolic Bacillus subtilis* ( Metabacillus sediminitoris ) RA10 fermentation supernatant.

[0009] According to the present invention, the microbial agent is preferably a solid agent or a liquid agent.

[0010] More preferably, the liquid bacterial agent contains *Parasitic bacillus* ( Metabacillus semi-military RA10 has a viable bacteria content ≥10 8 cfu / ml.

[0011] Thirdly, the present invention also provides a method for preparing the aforementioned microbial agent, comprising the following steps: culturing the *Parasitic bacillus* (… Metabacillus sediminitoris RA10, collect the culture product; culture conditions include: temperature 29℃~31℃, pH 6.8~11; the culture medium used contains 3.0g / L~5.0g / L soybean peptone and 15g / L~17g / L tryptone.

[0012] According to the preparation method of the microbial agent provided by the present invention, preferably, the culture conditions include: a temperature of 30°C and a pH of 7.1 to 7.5.

[0013] According to the preparation method of the microbial agent provided by the present invention, preferably, the culture conditions further include: shaking culture at 150 r / min to 250 r / min.

[0014] More preferably, the culture conditions also include: shaking culture at 200 r / min.

[0015] According to the method for preparing the microbial agent provided by the present invention, preferably, the culture medium used includes TSA solid culture medium and / or TSB liquid culture medium.

[0016] According to the preparation method of the microbial agent provided by the present invention, preferably, the preparation method includes the following steps: preparing the *Parasitic bacillus* (… Metabacillus sediminitoris RA10 cultured to the logarithmic growth phase, and the culture product was collected.

[0017] According to the preparation method of the microbial agent provided by the present invention, preferably, the preparation method further includes the following step: centrifuging the culture product at 7000r / min to 9000r / min for 5min to 15min.

[0018] More preferably, the preparation method further includes the following step: centrifuging the culture product at 8000 r / min for 10 min.

[0019] The present invention also provides a composition comprising the aforementioned microbial agent.

[0020] Preferably, the composition further includes other active ingredients with nitrogen-fixing and / or potassium-solubilizing functions.

[0021] The application of the composition in nitrogen fixation and / or potassium solubilization, or in improving plant salt and alkali tolerance, or in promoting plant growth under salt and / or alkali stress conditions, should also be within the scope of protection of this invention.

[0022] The present invention provides a plant growth promoter comprising the microbial agent or the composition thereof.

[0023] According to a plant growth promoter provided by the present invention, preferably, the plant growth promoter further includes excipients.

[0024] The application of the plant growth promoter in nitrogen fixation and / or potassium solubilization, or in improving plant salt and alkali tolerance, or in promoting plant growth under salt and / or alkali stress conditions, should also be within the scope of protection of this invention.

[0025] The present invention provides a fertilizer comprising the plant growth promoter.

[0026] According to the present invention, a fertilizer preferably further comprises a fertilizer-grade acceptable carrier.

[0027] The application of the fertilizer in nitrogen fixation and / or potassium solubilization, or in improving plant salt and alkali tolerance, or in promoting plant growth under salt and / or alkali stress conditions, should also be within the scope of protection of this invention.

[0028] The *Parabolic Bacillus subtilis* described in this invention (… Metabacillus sediminitorisRA10 has both nitrogen fixation and potassium solubilization functions.

[0029] Fourthly, the present invention provides the aforementioned *Parabolic Bacillus subtilis* (… Metabacillus sediminitoris The application of RA10 or the aforementioned microbial agent in nitrogen fixation and / or potassium solubilization.

[0030] Fifthly, the present invention provides the aforementioned *Parabolic Bacillus subtilis* (… Metabacillus sediminitoris The application of RA10 or the aforementioned microbial agent in improving the salt and alkali tolerance of plants.

[0031] According to the present invention, the *Parabolic Bacillus subtilis* ( Metabacillus sediminitoris The application of RA10 or the aforementioned microbial agent in improving the salt and alkali tolerance of plants, preferably, the plant is maize or Arabidopsis thaliana.

[0032] More preferably, the plant is corn.

[0033] According to the present invention, the *Parabolic Bacillus subtilis* ( Metabacillus sediminitoris In the application of RA10 or the aforementioned microbial agent to improve the salt and alkali tolerance of plants, preferably, the aforementioned *Parasitica parasitica* ( Metabacillus sediminitoris RA10 or the microbial agent described herein shall be applied to the roots and / or rhizosphere soil of the target plant.

[0034] More preferably, the method of application includes irrigation.

[0035] The shore-sinking parasitic bacillus ( Metabacillus sediminitoris The application of RA10 or the aforementioned microbial agents in promoting plant growth should also be within the scope of protection of this invention.

[0036] According to the present invention, the *Parabolic Bacillus subtilis* ( Metabacillus sediminitoris The application of RA10 or the aforementioned microbial agent in promoting plant growth, preferably, the plant is maize or Arabidopsis thaliana.

[0037] More preferably, the plant is corn.

[0038] According to the present invention, the *Parabolic Bacillus subtilis* ( Metabacillus sediminitoris The application of RA10 or the aforementioned microbial inoculant in promoting plant growth, preferably, involves using the aforementioned *Pseudomonas aeruginosa* ( Metabacillus sediminitoris RA10 or the microbial agent described herein shall be applied to the roots and / or rhizosphere soil of the target plant.

[0039] More preferably, the method of application includes irrigation.

[0040] Sixthly, the present invention also provides the aforementioned *Parabolic Bacillus subtilis* (… Metabacillus sediminitorisApplication of RA10 or the aforementioned microbial inoculants in promoting plant growth under salt and / or alkali stress conditions.

[0041] According to the present invention, the *Parabolic Bacillus subtilis* ( Metabacillus sediminitoris The application of RA10 or the microbial agent in promoting plant growth under salt stress and / or alkaline stress conditions, preferably, the salinity of the salt stress is 1% to 8%.

[0042] According to the present invention, the *Parabolic Bacillus subtilis* ( Metabacillus sediminitoris The application of RA10 or the microbial agent in promoting plant growth under salt and / or alkali stress conditions, preferably, the pH of the alkali stress is 9-11.

[0043] According to the present invention, the *Parabolic Bacillus subtilis* ( Metabacillus sediminitoris The application of RA10 or the microbial agent in promoting plant growth under salt and / or alkali stress conditions, preferably, the plant is maize or Arabidopsis thaliana.

[0044] More preferably, the plant is corn.

[0045] According to the present invention, the *Parabolic Bacillus subtilis* ( Metabacillus sediminitoris The application of RA10 or the aforementioned microbial agent in promoting plant growth under salt and / or alkali stress conditions, preferably, involves using the aforementioned *Pseudomonas aeruginosa* ( Metabacillus sediminitoris RA10 or the aforementioned microbial agent may be used on the roots and / or rhizosphere soil of the target plant.

[0046] More preferably, the method of application includes irrigation.

[0047] The shore-sinking parasitic bacillus ( Metabacillus sediminitoris The application of RA10, the microbial agent, the composition, the plant growth promoter, or the fertilizer in alleviating salt stress and / or alkali stress in plants should also be within the scope of protection of this invention.

[0048] The present invention has the following beneficial effects: The present invention provides *Parasporinus parasiticus* ( Metabacillus sediminitoris RA10 exhibits broad pH adaptability and can stably colonize under alkaline saline stress conditions with pH > 9. This strain possesses both nitrogen-fixing and potassium-solubilizing functions, providing plants with essential nutrients such as nitrogen and potassium. Under combined saline-alkali stress, application of this strain significantly promotes root elongation, increases plant height, stem diameter, and aboveground and belowground biomass, achieving a synergistic effect of nutrient supply and growth promotion. This invention provides a multifunctional strain resource integrating alkali tolerance, nitrogen fixation, potassium solubilization, and growth promotion for the development of novel stress-resistant microbial fertilizers, possessing significant application value for promoting the sustainable development of green agriculture. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0050] Figure 1 This is a colony morphology diagram of strain RA10 provided by the present invention inoculated on R2A solid culture medium.

[0051] Figure 2 This is a Gram staining image of strain RA10 provided by the present invention.

[0052] Figure 3 This is a transmission electron microscope image of strain RA10 provided by the present invention.

[0053] Figure 4 This is a phylogenetic tree diagram constructed based on the 16S rRNA sequence of strain RA10, provided by the present invention.

[0054] Figure 5 This is the growth of strain RA10 provided by the present invention on Assumption solid medium.

[0055] Figure 6 This shows the dissolution zone of strain RA10 provided by this invention on potassium-solubilizing medium.

[0056] Figure 7 This shows the growth of strain RA10 provided by this invention on TSA solid medium with different NaCl concentrations.

[0057] Figure 8 This shows the growth of strain RA10 provided by this invention on TSA solid medium at different pH values.

[0058] Figure 9 This invention describes the growth promotion of Arabidopsis thaliana under NaHCO3 stress by strain RA10; A shows the growth of Arabidopsis thaliana seedlings in the control group under 3mM NaHCO3 stress; B shows the growth of Arabidopsis thaliana seedlings in the experimental group inoculated with strain RA10 under 3mM NaHCO3 stress; C shows the root length statistics of the two groups of Arabidopsis thaliana seedlings; different lowercase letters indicate significant differences (…). p <0.05).

[0059] Figure 10This invention describes the growth promotion of maize seedlings under salt-alkali combined stress by strain RA10. A represents the growth of maize seedlings in each group; B represents the plant height of maize seedlings in each group; C represents the stem diameter of maize seedlings in each group; D represents the aboveground fresh weight of maize seedlings in each group; E represents the underground fresh weight of maize seedlings in each group; F represents the aboveground dry weight of maize seedlings in each group; G represents the underground dry weight of maize seedlings in each group; CK represents the blank control group without salt-alkali stress; SS represents the control group under salt-alkali combined stress; SS+RA10 represents the treatment group treated with RA10 bacterial solution for root irrigation under salt-alkali combined stress; different lowercase letters indicate significant differences (…). p <0.05). Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0061] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0062] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0063] The culture media involved in the following examples are as follows: (1) TSA solid culture medium: 15.0g tryptone, 5.0g soybean peptone, 5.0g sodium chloride and 15.0g agar powder were dissolved in 1000mL distilled water, the pH was adjusted to 7.3±0.2, and sterilized at 121℃ for 15min; (2) Assumption solid culture medium: 0.2g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 0.2g sodium chloride, 5.0g calcium carbonate, 10.0g mannitol, 0.1g calcium sulfate and 15.0g agar powder were dissolved in 1000mL distilled water, the pH was adjusted to 7.0±0.1, and sterilized at 121℃ for 15min; (3) Potassium-solubilized solid culture medium Culture medium (Alexandrov solid medium): 5.0g sucrose, 0.5g magnesium sulfate, 2.0g disodium hydrogen phosphate, 0.005g ferric chloride, 0.1g calcium carbonate, 1.0g soil minerals and 18.0g agar powder were dissolved in 1000mL distilled water, the pH was adjusted to 7.2±0.2, and sterilized at 121℃ for 15min; (4) TSB liquid medium: 17.0g tryptone, 3.0g soybean peptone, 5.0g sodium chloride, 2.5g dipotassium hydrogen phosphate and 2.5g glucose were dissolved in 1000mL distilled water, the pH was adjusted to 7.3±0.2, and sterilized at 121℃ for 15min; (5) 1 / 2 MS solid culture medium: Potassium nitrate 950mg, ammonium nitrate 825mg, calcium chloride 165mg, potassium iodide 0.83mg, zinc sulfate 8.6mg, cobalt chloride 0.025mg, ferrous sulfate 15.2mg, glycine 2.0mg, nicotinic acid 0.5mg, potassium dihydrogen phosphate 85mg, magnesium sulfate 90mg, boric acid 6.2mg, manganese sulfate 16.9mg, sodium molybdate 0.25mg, copper sulfate 0.025mg, inositol 100mg, thiamine hydrochloride 0.1mg, pyridoxine hydrochloride 0.5mg, disodium EDTA 37.26mg, and agar powder 15.0g were dissolved in 1000mL of distilled water. The pH was adjusted to 6.0±0.2, and the medium was sterilized at 115℃ for 30min.

[0064] Example 1: Isolation, Identification and Preservation of Strain RA10 1. Isolation of strain RA10 In a saline-alkali corn-growing area in Quzhou, Handan City, Hebei Province, China (latitude: 36°45'58.85"N, longitude: 114°57'27.18"E), intact corn roots and attached soil were dug up and brought back to the laboratory. The outer layer of root periphery soil was shaken off, and the rhizosphere soil attached to the root surface of 1mm to 2mm was gently brushed off to obtain soil samples.

[0065] Weigh 10g of soil sample and place it in a conical flask containing 50mL of sterile water. Shake at 150rpm for 30min on a shaker at 30℃, then let stand for 30min. Perform serial dilutions, taking 100μL of each sample as a 10⁻⁶ dilution. -410 -5 and 10 -6 The bacterial suspension was spread onto TSA solid medium with pH ≥ 9.0, with three replicates for each concentration. The plates were inverted and incubated at 30°C for 2–3 days. Single colonies were then picked and streaked for purification. The purified strain was designated as strain RA10.

[0066] 2. Identification of strain RA10 (1) Colony morphology identification Strain RA10 was cultured on R2A solid medium, as follows Figure 1 As shown, its colonies are milky white, round and raised, with neat edges and a smooth surface.

[0067] (2) Gram staining identification Gram staining was performed on bacteria RA10 for identification, such as... Figure 2 As shown, it is a Gram-positive rod-shaped bacterium.

[0068] (3) Microscopic morphological identification The microstructure of strain RA10 was observed under a transmission electron microscope, such as... Figure 3 As shown, each bacterial cell is approximately 1 μm to 2 μm long and 0.5 μm to 0.75 μm wide.

[0069] (4) Molecular biological identification Genomic DNA was extracted from the bacterial culture of strain RA10 and used as a template for PCR amplification using primers 16S-27F (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1)) and 16S-1492R (5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO.2)).

[0070] The PCR amplification system consisted of: 25 μL of 2×PCR Mix, 2 μL of 16S-27F (SEQ ID NO.1), 2 μL of 16S-1492R (SEQ ID NO.2), 1 μL of DNA template, and 20 μL of sterile enzyme-free water.

[0071] The PCR amplification program was as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 55℃ for 15 s, extension at 72℃ for 1 min, for 30 cycles; final extension for 10 min.

[0072] The amplified products were sequenced, and a phylogenetic tree was constructed based on the obtained 16S rRNA sequences. Comparative analysis was performed, and so on... Figure 4 As shown, strain RA10 and Metabacillus sediminitorisstrain DSL-17 showed the highest similarity (99%), therefore strain RA10 belongs to the Parasporal Bacillus subtilis family. Metabacillus semi-military ).

[0073] 3. Preservation of strain RA10 Strain RA10 was deposited on May 22, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 38835, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China (Postcode: 100101). Its taxonomic name is *Parasporium parasporum*. Metabacillus semi-military The collection name is RA10.

[0074] Example 2: Identification of the physiological and biochemical capabilities of strain RA10 1. Nitrogen fixation capacity (1) Experimental methods Strawberry strain RA10 was streaked onto Assumption solid medium and incubated upside down at 30°C for 2–3 days. After incubation, the growth of the strain was observed. The strain was subcultured three times. If it still grew well on the third subculture, the strain was considered to have nitrogen-fixing function.

[0075] (2) Experimental results like Figure 5 As shown, strain RA10 can be passaged three times on Assumption solid medium, indicating that strain RA10 has nitrogen-fixing ability.

[0076] 2. Potassium solubility (1) Experimental methods Strain RA10 was inoculated onto potassium-solubilizing solid medium and incubated upside down at 30°C for 4–5 days. After incubation, the presence of a clear zone around the colonies was observed. If a clear zone was present, the strain was identified as having potassium-solubilizing ability. The potassium-solubilizing ability of the strain was determined by the ratio of the diameter D of the clear zone to the diameter d of the colony.

[0077] (2) Experimental results like Figure 6 As shown, a clear zone exists around strain RA10 on potassium-solubilizing solid medium, indicating that strain RA10 has potassium-solubilizing ability. The diameters of the clear zones and colonies of three single colonies of strain RA10 were measured and are shown in Table 1. Its potassium-solubilizing ability is 4.48 ± 0.43 (D / d).

[0078] Table 1. Evaluation results of potassium solubilization ability of strain RA10

[0079] Under saline-alkali stress, high concentrations of sodium ions in the soil not only cause ion toxicity to plants but also competitively inhibit root absorption of potassium ions. Potassium, as an activator of many key enzymes and an osmotic regulator in plants, is deficient in, exacerbating cell membrane damage and metabolic disorders. Simultaneously, the high pH of the saline-alkali environment reduces the availability of mineral potassium in the soil, further limiting plant potassium uptake. The superior potassium-solubilizing ability of strain RA10 in this invention ensures that it can convert insoluble potassium-containing minerals in the soil into available potassium that can be absorbed and utilized by plants, increasing the supply of available potassium to the rhizosphere and maintaining a high intracellular K level. + / Na + The ratio reduces sodium ion toxicity and stabilizes cell osmotic potential. Under conditions of sufficient potassium, the activity of plant antioxidant enzymes and photosynthetic capacity are maintained, and overall stress resistance is enhanced.

[0080] 3. Salt tolerance (1) Experimental methods To verify the salt tolerance of RA10, strain RA10 was inoculated onto TSA solid medium with pH 7.0±0.2 and NaCl contents of 1%, 2%, 4%, 6%, 8% and 10%, respectively. The growth of the strain was observed to determine the salt tolerance of RA10.

[0081] (2) Experimental results like Figure 7 As shown, RA10 can grow on TSA solid medium containing 1% to 8% NaCl, but cannot grow on TSA solid medium containing 10% NaCl, indicating that RA10 has a salt tolerance of up to 8%.

[0082] 4. Alkali resistance (1) Experimental methods Strains RA10 were inoculated onto TSA solid medium with pH values ​​of 9, 9.5, 10, 10.5, 11, 11.5 and 12, and the growth of the strains was observed to determine the alkali tolerance of RA10.

[0083] (2) Experimental results like Figure 8 As shown, RA10 can grow on TSA solid medium with pH 9–11, but cannot grow on TSA solid medium with pH 11.5 and 12, indicating that RA10 has a salt tolerance of up to 11.

[0084] Example 3: Effects of strain RA10 on Arabidopsis thaliana under alkaline salt stress 1. Preparation of bacterial culture Strain strain RA10 was activated using TSA solid medium. Single colonies were picked up using an inoculation loop and transferred to TSB liquid medium. The culture was then shaken at 30℃±1℃ and 200r / min±50r / min until the logarithmic growth phase.

[0085] The bacterial culture was collected and centrifuged at 4℃ and 8000 r / min for 10 min. The supernatant was discarded, and the bacterial cells were resuspended in 10 mM magnesium sulfate solution to obtain a bacterial concentration of 10. 8 Prepare a CFU / ml RA10 bacterial suspension for later use.

[0086] 2. Disinfection treatment of Arabidopsis thaliana seeds Disinfect Arabidopsis seeds by soaking them in 0.3% sodium hypochlorite disinfectant for 10 minutes, and then rinse them 5 to 10 times with sterile ultrapure water.

[0087] 3. Cultivation of Arabidopsis thaliana under alkali stress The sterilized Arabidopsis seeds were sown in square petri dishes (13cm×13cm) containing 1 / 2 MS solid medium (pH 6.0) and cultured for about 5 days. At this time, Arabidopsis entered the seedling stage, grew the first pair of true leaves, and the root length was about 1.5cm.

[0088] Twelve Arabidopsis thaliana seedlings with uniform root length were selected and transferred to 1 / 2 MS solid medium containing 3 mM NaHCO3 (pH 7.5 ± 0.2). These twelve seedlings constituted twelve biological replicates. Two μL of RA10 bacterial suspension was evenly applied to the roots of each Arabidopsis thaliana seedling, forming the experimental group (denoted as RA10).

[0089] Twelve Arabidopsis thaliana seedlings with uniform root length were transferred to 1 / 2 MS solid medium containing 3 mM NaHCO3 (pH 7.5 ± 0.2). 2 μL of 10 mM magnesium sulfate solution was evenly applied to the roots of each Arabidopsis thaliana seedling as a control group (CK).

[0090] Two groups of Arabidopsis seedlings were placed in an incubator and cultured for another 10 days. Then, their leaves and roots were photographed to observe their growth status, and their root length (i.e., the length from the base of the seedling stem to the longest part of the root system) was measured.

[0091] Arabidopsis seed germination and seedling culture were conducted in an artificial climate incubator under the following conditions: 10 hours of light and a light intensity of 60 μmol·m⁻². -2 s -1 ~70 μmol·m -2 s -1 Temperature 21℃; after 14 hours of darkness, temperature 19℃.

[0092] 4. Experimental Results like Figure 9 As shown in A to C, compared to the control group, the root length of Arabidopsis seedlings in the experimental group increased by 2.1 times ( ). p <0.05). This indicates that strain RA10 significantly promotes the growth of Arabidopsis seedlings under alkaline salt stress.

[0093] Example 4: Effects of strain RA10 on maize growth under saline-alkali stress. 1. Potted plant treatment A stress resistance and growth-promoting experiment on maize using strain RA10 was conducted in an artificial greenhouse at the Beijing Academy of Agricultural and Forestry Sciences.

[0094] The experiment included a blank control group (CK) without salt and alkali stress, a control group (SS) under combined salt and alkali stress, and a treatment group (SS+RA10) that received root irrigation with RA10 bacterial solution under combined salt and alkali stress. Each group had 6 maize seedlings as biological replicates.

[0095] 2. Preparation of bacterial culture Strain RA10 was activated using R2A solid medium, and single colonies were picked and cultured in R2A liquid medium at 30°C and 200 rpm for 48 h.

[0096] After cultivation, collect the bacterial suspension and centrifuge at 4℃ and 8000 rpm for 10 min. Discard the supernatant, resuspend the bacterial cells in phosphate buffer, and adjust the OD of the bacterial suspension. 600 The concentration was approximately 1.0, resulting in a bacterial concentration of 10. 8 Prepare RA10 bacterial culture at CFU / mL for later use.

[0097] 3. Preparation of salt-alkali composite salt solution NaCl, Na2SO4, NaHCO3, and Na2CO3 were dissolved in deionized water in a molar ratio of 1:9:9:1 to obtain a salt-alkali complex solution with a concentration of 250 mM and a pH of 8.5 ± 0.3.

[0098] 4. Corn processing The treatment method for the CK group was as follows: watering with 200mL of sterile deionized water every 5 days; applying 2mL of phosphate buffer to the roots of corn seedlings by root irrigation every 7 days, for a total of 4 applications during the growing season.

[0099] The treatment method for the SS group was as follows: every 5 days, water with 200 mL of sterile saline-alkali compound salt solution; every 7 days, apply 2 mL of phosphate buffer to the roots of corn seedlings by root irrigation, for a total of 4 applications during the growing season.

[0100] The treatment method for the SS+RA10 group was as follows: every 5 days, 200 mL of sterile saline-alkali compound salt solution was applied; every 7 days, 2 mL of RA10 bacterial solution was applied to the roots of the corn seedlings by root irrigation, for a total of 4 applications during the growing season.

[0101] The growth period is one month.

[0102] Regularly photograph and record the growth of corn seedlings. After the growing season, measure the plant height, stem diameter, fresh and dry weight of the above-ground parts, and fresh and dry weight of the underground parts of each group of corn.

[0103] 5. Experimental Results Table 2. Effects of RA10 bacterial solution on maize plant height and stem diameter under combined salt and alkali stress.

[0104] Table 3. Effects of RA10 bacterial culture on maize biomass under combined saline-alkali stress.

[0105] Note: Different lowercase letters in Tables 2 and 3 indicate significant differences. p <0.05).

[0106] As shown in Tables 2 and 3, and Figure 10 As shown in Figures A through G, compared to the control group (CK), after applying salt-alkali combined stress, the plant height, stem diameter, aboveground fresh weight, aboveground dry weight, underground fresh weight, and underground dry weight of maize in the SS group decreased by 37.77%, 36.60%, 69.94%, 74.00%, 80.15%, and 95.40%, respectively. However, after applying RA10 bacterial solution, compared to the SS group, the plant height, stem diameter, aboveground fresh weight, aboveground dry weight, underground fresh weight, and underground dry weight of maize in the SS+RA10 group increased by 23.47%, 51.96%, 85.95%, 69.23%, 107.69%, and 525%, respectively.

[0107] This indicates that strain RA10 can effectively alleviate the stress of saline-alkali environment on maize, improve maize's resistance to adverse conditions, and promote its growth and development.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of *Parabolic Bacillus parasiticus* ( Metabacillus sediminilitoris RA10, characterized in that, The shore-sinking parasitic bacillus ( Metabacillus sediminilitoris The accession number for RA10 is CGMCC No. 38835.

2. A microbial inoculant, characterized in that, The microbial agent comprises the *Parabenchoides spp.* as described in claim 1. Metabacillus sediminilitoris )RA10.

3. The method for preparing the microbial inoculant according to claim 2, characterized in that, Includes the following steps: culturing the *Parasitic bacillus* as described in claim 1 (… Metabacillus sediminilitoris RA10, collect the culture product; The cultivation conditions include: temperature of 29℃~31℃, pH of 6.8~11; The culture medium used contained 3.0 g / L to 5.0 g / L soybean peptone and 15 g / L to 17 g / L trypsin.

4. The preparation method according to claim 3, characterized in that, The culture conditions also include: shaking culture at 150 r / min to 250 r / min.

5. The *Parabolic Bacillus subtilis* as described in claim 1 (… Metabacillus sediminilitoris RA10, or the application of the microbial agent of claim 2 in nitrogen fixation and / or potassium solubilization.

6. The *Parabolic Bacillus subtilis* as described in claim 1 (… Metabacillus sediminilitoris The application of RA10, or the microbial agent of claim 2, in improving the salt and alkali tolerance of plants.

7. The *Parabolic Bacillus subtilis* as described in claim 1 (… Metabacillus sediminilitoris RA10, or the application of the microbial agent of claim 2 in promoting plant growth under salt stress and / or alkali stress conditions.

8. The application according to claim 6 or 7, characterized in that, The plant in question is either maize or Arabidopsis thaliana.

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