Azotobacter chroococcum with the functions of producing protease, releasing potassium, degrading cellulose, promoting growth and relieving NaCl stress on plants and application thereof

Abstract

The present application discloses a strain of azotobacter chroococcum with the abilities of producing protease, decomposing potassium and cellulose, promoting growth and relieving plant NaCl stress, and application thereof. Azospirillum dongyingense The present application provides the azotobacter chroococcum or its offspring, the azotobacter chroococcum is azotobacter chroococcum (Azotobacter chroococcum) of Dongying, the strain number is RS252, and the registration number of the Center of General Microbiological Culture Collection of China Microbial Culture Collection Committee is CGMCC No. 31494. The strain is a new strain of azotobacter chroococcum genus, has the abilities of producing protease, decomposing potassium and cellulose. Potted test shows that compared with the negative control group without inoculation, the strain can improve the root length and underground fresh weight of rice under NaCl stress, and the strain can be used as microbial organic fertilizer to improve soil fertility, relieve crop NaCl stress and improve the adaptability of crops to NaCl stress.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a nitrogen-fixing spirochete that produces proteases, solubilizes potassium, degrades cellulose, promotes growth, and alleviates NaCl stress in plants, and its applications. Background Technology

[0002] The rhizosphere microbiome plays a crucial role in plant growth and health, improving nutrient utilization, and protecting plants from biotic and abiotic stresses. Plant growth-promoting rhizobacteria (PGPRs) have been increasingly used as bio-agents and are playing an increasingly important role in agricultural production. Recent studies have shown that PGPRs can not only promote plant growth, control diseases, and increase crop yields, but also enhance plant resistance to various abiotic stresses such as drought, salt, and heavy metals, thereby improving plant adaptability to various environmental stresses.

[0003] Numerous PGPR groups have been discovered both domestically and internationally, possessing functions such as secreting plant hormones, solubilizing phosphorus, solubilizing iron, and solubilizing potassium. Among them, the genus *Azospirillum* (…) Azospirillum Members of the genus *Azospirillum* are widely distributed in nature and have been found in the rhizosphere of various crops, including corn, wheat, and rice. Strains of *Azospirillum* possess multiple functions, such as producing indole-3-acetic acid (IAA) and symbiotic nitrogen fixation. Furthermore, they have the ability to enhance plant stress resistance (such as drought and disease resistance). Some strains can also enhance the absorption of micronutrients such as iron, improve plant nutrition, and promote plant growth. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a nitrogen-fixing spirochete that produces protease, solubilizes potassium, degrades cellulose, promotes growth and alleviates NaCl stress in plants, and its applications.

[0005] In a first aspect, the present invention claims protection for a nitrogen-fixing spirochete and its progeny.

[0006] The *Azotrophus* species for which this application seeks protection is *Azotrophus dongyingense*. Azospirillum dongyingense Its strain number is RS252, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 31494.

[0007] Dongying Azotospirobacter ( Azospirillum dongyingense RS252 is a Gram-positive bacterium. After 3 days of growth on TSA solid medium, the colonies are white, round, raised, with smooth edges, viscous, and glossy, with a colony diameter of 1-2 mm. This strain has excellent plant growth-promoting properties and can significantly alleviate the stress of NaCl on plants.

[0008] Secondly, the present invention claims protection for compositions containing the azospirobacter or its progeny described in the first aspect above.

[0009] Further, the composition may be a culture, which is a substance obtained by culturing the azospira or its progeny described in the first aspect above in a microbial culture medium (all substances in the culture container, i.e., fermentation products, such as fermentation broth containing the azospira or its progeny and substances secreted into the liquid culture medium, or solid fermentation product containing the azospira or its progeny and substances secreted into the solid culture medium).

[0010] The microbial culture medium may be a bacterial culture medium. The bacterial culture medium may be a solid culture medium or a liquid culture medium.

[0011] In the above-mentioned culture, the substances include the azospirobacter or its progeny (the bacterial cells themselves) and / or its metabolites described in the first aspect above.

[0012] The term "metabolite" refers to the primary and / or secondary metabolites produced during microbial metabolism. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the external environment and, through catabolism and anabolism, generate substances and energy to sustain life activities. The products of primary metabolism are called primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolism refers to the process by which microorganisms, at a certain growth stage, use primary metabolites as precursors to synthesize substances that have no clearly defined function for their life activities. The products of secondary metabolism are called secondary metabolites, and are mostly compounds with relatively complex molecular structures. Based on their functions, they can be classified into types such as antibiotics, hormones, alkaloids, and toxins.

[0013] The term "culture" refers to any liquid or solid culture medium that has grown a microbial community after artificial inoculation and cultivation. It is the product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0014] Furthermore, the composition may be a microbial agent, a microecological preparation, or a bio-fertilizer.

[0015] In the aforementioned microbial agents, microecological preparations, or biofertilizers, the active ingredients may be *Azotrophus* or its progeny, metabolites of *Azotrophus* or its progeny, and / or cultures of *Azotrophus* or its progeny, as described in the first aspect above. The active ingredients may also contain other biological and / or non-biological components. Those skilled in the art can determine the other active ingredients of the microbial agents, microecological preparations, or biofertilizers based on the desired effects.

[0016] In addition to the active ingredients, the aforementioned microbial agents, microecological preparations, or biofertilizers may also contain a carrier. The carrier may be a biologically inert carrier commonly used in the pesticide field. The carrier may be a solid carrier or a liquid carrier; the solid carrier may be a mineral material, plant material, or polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of wheat flour, soybean flour, and starch; the polymer compound may be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier may be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane and / or dodecane.

[0017] In the above-mentioned microbial agents, microecological preparations, or bio-fertilizers, the dosage form of the microbial agents, microecological preparations, or bio-fertilizers can be various, such as liquid, emulsion, suspension, powder, granules, wettable powder, or water-dispersible granules.

[0018] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the microbial agents, microecological preparations, or bio-fertilizers.

[0019] In the microbial agent, microecological preparation, or biofertilizer, the azospira or its progeny and / or the metabolites of the azospira or its progeny may be present in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate.

[0020] In this document, the metabolites of *Azospirillum* or its progeny can be obtained from the fermentation broth of *Azospirillum* or its progeny. The metabolites of *Azospirillum* or its progeny can be sterile metabolites of *Azospirillum* or its progeny or bacterial metabolites of *Azospirillum* or its progeny. Specifically, the sterile metabolites of *Azospirillum* or its progeny (sterile fermentation filtrate) can be prepared as follows: *Azospirillum* or its progeny are cultured in a liquid culture medium, and the *Azospirillum* or its progeny are removed from the liquid culture (fermentation broth) by filtration, thus obtaining the sterile metabolites of *Azospirillum* or its progeny. Specifically, the bacterial metabolites of *Azospirillum* or its progeny can be prepared as follows: *Azospirillum* or its progeny are cultured in a liquid fermentation medium, and the fermentation broth—containing *Azospirillum* or its progeny and substances secreted into the liquid culture medium—is collected; this fermentation broth is the bacterial metabolites of *Azospirillum* or its progeny.

[0021] Furthermore, the composition may have at least one of the following properties:

[0022] A1) Protease production;

[0023] A2) Potassium dissolution;

[0024] A3) Degradation of cellulose;

[0025] A4) Relieves NaCl stress in plants;

[0026] A5) Enhances POD activity in plants;

[0027] A6) Increase the SOD activity of plants;

[0028] A7) Reduce the MDA content in plants;

[0029] A8) Promotes plant growth;

[0030] A9) Promotes an increase in the underground fresh weight of plants;

[0031] A10) promotes increased root growth in plants.

[0032] Thirdly, the present invention claims the use of the azospirobacter or its progeny described in the first aspect above, or the composition described in the second aspect above, wherein the use is any of the following:

[0033] B1) Application in the production of proteases or in the preparation of products for the production of proteases;

[0034] B2) Applications in potassium solubilization or in the preparation of products for potassium solubilization;

[0035] B3) Applications in the degradation of cellulose or in the preparation of products for the degradation of cellulose;

[0036] B4) Applications in alleviating NaCl stress in plants or in the preparation of products for alleviating NaCl stress in plants;

[0037] B5) Applications in enhancing the POD activity of plants or in the preparation of products for enhancing the POD activity of plants;

[0038] B6) Application in enhancing SOD activity in plants or in the preparation of products for enhancing SOD activity in plants;

[0039] B7) Application in reducing the MDA content of plants or in the preparation of products for reducing the MDA content of plants;

[0040] B8) Use in promoting plant growth or in the preparation of products for promoting plant growth;

[0041] B9) Application in promoting the increase of underground fresh weight of plants or in the preparation of products for promoting the increase of underground fresh weight of plants;

[0042] B10) Application in promoting plant root growth or in the preparation of products for promoting plant root growth.

[0043] Furthermore, the improvement of plant POD activity can be achieved by enhancing plant POD activity under NaCl stress conditions.

[0044] Furthermore, the improvement of SOD activity in plants can be achieved by increasing SOD activity in plants under NaCl stress conditions.

[0045] Furthermore, the reduction of plant MDA activity can be achieved by reducing plant MDA activity under NaCl stress conditions.

[0046] Furthermore, the promotion of plant growth can be achieved by promoting plant growth under NaCl stress conditions.

[0047] Furthermore, the promotion of increased underground fresh weight of plants can be achieved by promoting increased underground fresh weight of plants under NaCl stress conditions.

[0048] Furthermore, the promotion of increased plant root length can be achieved by promoting increased plant root length under NaCl stress conditions.

[0049] Fourthly, the present invention claims a method for alleviating NaCl stress in plants.

[0050] The method for alleviating NaCl stress in plants claimed by this invention may include the following steps: treating the plant to be treated or its growth substrate with the nitrogen-fixing spirochete or its progeny as described in the first aspect above or the composition as described in the second aspect above, thereby alleviating the NaCl stress in the plant.

[0051] In some embodiments of the present invention, the treatment involves drenching the plant with a bacterial suspension containing the nitrogen-fixing spirochete or its progeny.

[0052] Fifthly, the present invention claims a method for promoting plant growth.

[0053] The method for promoting plant growth claimed in this invention may include the following steps: treating the plant to be treated or its growth substrate with the azotoxin bacteria or its progeny described in the first aspect above or the composition described in the second aspect above, thereby promoting the growth of the plant.

[0054] Furthermore, the promotion of plant growth can be achieved by promoting plant growth under NaCl stress conditions.

[0055] In some embodiments of the present invention, the treatment involves drenching the plant with a bacterial suspension containing the nitrogen-fixing spirochete or its progeny.

[0056] Sixthly, the present invention claims a method for culturing the azospirobacter or its progeny described in the first aspect above.

[0057] The method for culturing the azospirobacter or its progeny described in the first aspect of the present invention includes the step of culturing the azospirobacter or its progeny in a culture medium for culturing microorganisms.

[0058] In a seventh aspect, the present invention claims a method for preparing a composition.

[0059] The method for preparing the composition claimed in this invention includes the step of using the *Azotrophus* or its progeny described in the first aspect above as a component of the composition; the composition is the composition described in the second aspect above.

[0060] In some embodiments of the present invention, the NaCl stress is simulated using a NaCl solution with a final concentration of 120 mmol / L.

[0061] In all the aforementioned relevant aspects, the plant may be any of the following:

[0062] C1) Angiosperms;

[0063] C2) Monocotyledons;

[0064] C3) Plants of the order Poales;

[0065] C4) Gramineae plants;

[0066] C5) Plants of the genus *Oryza*;

[0067] C6) Rice.

[0068] Experiments have shown that the *Dinospirobacter dongyingensis* provided in this invention (…) Azospirillum dongyingense RS252 is a new species of the genus *Azotrophus*. *Azotrophus dongyingense* ( Azospirillum dongyingense RS252 possesses the ability to produce proteases, solubilize potassium, and degrade cellulose. Pot experiments showed that under NaCl stress, compared to the uninoculated negative control group, inoculation with *Dinospirillum dongyingense* (a strain of *Dinospirillum dongyingense*) significantly increased the mortality rate. Azospirillum dongyingense RS252 can increase root length and underground fresh weight in rice. (Dongying nitrogen-fixing spirochete) Azospirillum dongyingense RS252 can be used as a microbial organic fertilizer to improve soil fertility, alleviate NaCl stress in crops, and enhance crop adaptability to NaCl stress.

[0069] Depositing Instructions

[0070] Classification and nomenclature: Dongying azotoxinus ( Azospirillum dongyingense );

[0071] Biological material of ginseng: RS252;

[0072] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0073] The abbreviation for the depository institution is CGMCC.

[0074] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0075] Deposit date: July 30, 2024;

[0076] Registered with the China National Collection Center (CGMCC) No. 31494. Attached Figure Description

[0077] Figure 1 For Dongying Azotospirobacter ( Azospirillum dongyingense The colony morphology of RS252 after 3 days of incubation on TSA plates.

[0078] Figure 2 To construct *Dinospirobacter dongyingensis* using the neighbor-joining method based on the 16S rRNA gene sequence ( Azospirillum dongyingense Phylogenetic tree of RS252 and related model bacteria. Note: The numbers in parentheses are the GenBank sequence numbers of the strain's 16S rRNA gene sequence; the reference strains in the figure are all model strains of their respective species. Pseudomonas abietaniphila ATCC700689 T (FNCO01000040) is an outgroup.

[0079] Figure 3 A genome of *Dinospirobacter dongyingensis* was constructed using the maximum likelihood method by tandemly linking 92 bacterial core genes.Azospirillum dongyingense RS252 and Azospirillum Phylogenetic tree of closely related species within the genus.

[0080] Figure 4 For Dongying Azotospirobacter ( Azospirillum dongyingense Results of RS252's protease production, potassium solubilization, and cellulose degradation capabilities. The left side shows the morphology of strain RS252 after 2 days of growth on protease detection medium; the middle side shows the morphology of strain RS252 after 2 days of growth on modified potassium solubilization medium; and the right side shows the morphology of strain RS252 after 3 days of growth on cellulose Congo red medium.

[0081] Figure 5 The growth status of rice seedlings inoculated with strain RS252 and uninoculated with strain RS252 under NaCl stress at 27 days. The left side is the experimental group (RS252), and the right side is the control group (CK).

[0082] Figure 6 The figures show the fresh weight of the underground parts and root length of rice seedlings under NaCl stress at 27 days. The CK group was the control group without inoculation. ** in the figure indicate... P <0.01.

[0083] Figure 7 This figure shows the determination of the environmental stress response and antioxidant capacity of rice seedlings under NaCl stress at 27 days. In the figure, ** and *** indicate... P <0.01. Detailed Implementation

[0084] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0085] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0086] The formulations of the various culture media and solutions involved in the following examples are as follows:

[0087] (1) TSB liquid culture medium: 15.0g tryptone, 5.0g soybean peptone, 5.0g sodium chloride, 1000mL distilled water, adjust pH to 7.3±0.2, sterilize at 121℃ for 15min.

[0088] (2) TSA solid medium (TSA plate): Add 20.0g agar to TSB liquid medium and sterilize at 121℃ for 15min.

[0089] (3) Starch medium: NA medium, 2 g / L soluble starch, pH 7.4, 121℃, sterilized for 20 min.

[0090] (4) Casein culture medium: Solution a: 5g skim milk powder, 50mL distilled water; Solution b: 50mL NB, 1.5g agar. Sterilize solutions a and b at 121℃ for 15min, and after cooling to about 60℃, mix well and dispense into plates.

[0091] (5) Protease detection medium:

[0092] 2g peptone, 1g NaCl, 0.02g CaCl2, 2ml skim milk (add milk before pouring into plates, when the culture medium is not hot to the touch), 2% agar, sterilize at 121℃ for 15 minutes.

[0093] (6) Modified potassium-solubilizing medium:

[0094] Alexander Buff medium: sucrose 5.0g, disodium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.5g, ferric chloride 5.0mg, calcium carbonate 0.1g, potassium feldspar powder 1.0g, distilled water 1000mL, agar 20.0g, pH 7.2, sterilized at 121℃ for 15 minutes.

[0095] Modified potassium-solubilizing medium: Add 100 mg / L bromothymol blue to Alexander Bough medium.

[0096] (7) Cellulose Congo Red Medium:

[0097] Sodium nitrate 1g, dimethyl hydrogen phosphate 1.2g, potassium dihydrogen phosphate 0.9g, magnesium sulfate 0.5g, potassium chloride 0.5g, yeast extract powder 0.5g, acid-hydrolyzed casein 0.5g, Congo red 0.2g, cellulose powder 5.0g, agar 20g, distilled water 1000ml. Sterilize at 121℃ for 15 minutes.

[0098] Example 1: Dongying Azotospirobacter ( Azospirillum dongyingense Separation and identification of RS252

[0099] I. Dongying Azotospirobacter ( Azospirillum dongyingense RS252 separation

[0100] Rice rhizosphere soil samples were collected in Kenli District, Dongying City, Shandong Province (118.58°E, 37.58°N). The samples were transported back to the laboratory and stored at 4°C using an ice box. Intact rice roots were taken, and excess soil was removed using sterile forceps. The roots were placed in Erlenmeyer flasks containing sterile water and shaken at 150 rpm for 30 minutes at room temperature. Root tissue was then picked out with forceps, centrifuged, and the supernatant was discarded; the precipitate was the rhizosphere soil. 1 g of rhizosphere soil was weighed and resuspended in 10 mL of sterile water for serial dilution. 100 μL of each dilution was spread onto TSA plates and incubated upside down at 30°C for one week. Based on physiological morphological characteristics, single colonies were picked with a bamboo stick and inoculated onto plates for purification. After confirming pure cultures, the cultures were transferred to slant culture for short-term storage at 4°C, and then transferred to 20% glycerol tubes for long-term storage at -80°C. One of the isolated and purified strains was named RS252.

[0101] II. Dongying Azotobacter ( Azospirillum dongyingense RS252 identification

[0102] 1. Morphological identification of strains

[0103] The RS252 strain, isolated and purified in step one above and in the logarithmic growth phase with stable colony size, was described as a single colony, including colony size, color, transparency, colony surface condition, and colony edge condition. Following the manufacturer's instructions, a Gram staining kit from Solarbio Technology Co., Ltd. (Beijing) was used to Gram stain the RS252 smears, and the morphology of the bacteria was observed using an optical microscope.

[0104] The colonies of strain RS252 on TSA plates are white, round, raised, with smooth edges, viscous, and glossy, with a colony diameter of 1-2 mm. Figure 1 The cells are Gram-positive, rod-shaped, and do not form spores.

[0105] 2. Molecular identification

[0106] Following the instructions, genomic DNA was extracted using the TIANamp bacterial genomic DNA extraction kit from Beijing Tiangen Biotech Co., Ltd. 16S rRNA gene amplification was performed using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The 50 μL PCR amplification system consisted of: 25 μL 2×Taq PCRMix, 2 μL 27F (10 μmol / L), 2 μL 1492R (10 μmol / L), 19 μL ddH2O, and 2 μL DNA template. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 1 min, 72℃ extension for 90 s, 30 cycles; and a final extension at 72℃ for 10 min. After verification by 1% agarose gel electrophoresis, positive PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequences obtained from sequencing are uploaded to Ezbiocloud (www.ezbiocloud.net / eztaxon) for sequence alignment.

[0107] The sequencing length of the 16S rRNA gene of strain RS252 is 1406 bp (SEQ ID No. 1). Comparison results from the EzBioCloud database show that strain RS252 is similar to... Azospirillum palustre B2 T (98.44%) Azospirillum [[ID=二十九]]oryzae COC8 T (98.32%) Azospirillum humicireducens SgZ-5 T (98.30%) and Azospirillum ramasamyi M2T2B2 T (98.11%) of the sequences showed high similarity and were all below the classification threshold (98.8%) for describing the species. 16S rRNA gene sequences highly similar to strain RS252 were retrieved from the EzBioCloud server and aligned using MUSCLE. A phylogenetic tree was constructed using the Neighbour-Joining (NJ) method with MEGA X software. The evolutionary distance using the Kimura two-parameter model was calculated, with a bootstrap value of 1000. The phylogenetic tree constructed using the NJ method is shown below. Figure 2 As shown, strain RS252 forms a separate branch, indicating that strain RS252 is... Azospirillum A potential new species of the genus.

[0108] 3. Genome analysis

[0109] Genomic DNA was sent to Annoroad Gene Technology (Beijing) Co., Ltd., where a draft genome sequence of strain RS252 was performed using an Illumina NovaSeq 6000 sequencing system. Assembly was performed using SPAdes software, yielding 124 contigs, N 50 The length is 215272 bp. The genome size is 6.69 Mb, and the G+C content is 67.51%. The ANIm method in the pyANI software was used to analyze strain RS252 and... Azospirillum Genome-wide average nucleotide identity (ANI) analysis of closely related strains. The digital DNA-DNA hybridization (dDDH) values ​​between strain RS252 and the reference strain were compared using the Genome-to-Genome Distance Calculator (GGDC) 3.0 server (https: / / ggdc.dsmz.de / ggdc.php#).

[0110] Strain RS252 and others with publicly available genome sequences Azospirillum Compared to the type strains of the same genus, the ANI values ​​ranged from 76.43% to 93.16%, lower than the previously proposed critical value of 95% to 96% for species delimitation; the dDDH values ​​of strain RS252 and its type strain ranged from 21.10% to 50.80%, far below the 70% species delimitation threshold. See Table 1 for details. Both the ANI and dDDH results indicate that strain RS252 is a... Azospirillum A new species of the genus.

[0111] Table 1. *Diazospira* species from Dongying ( Azospirillum dongyingense RS252 and Azospirillum Values ​​of ANI and dDDH for closely related species within the genus

[0112]

[0113] To further clarify the taxonomic position of strain RS252, this invention performed a genomic phylogenetic analysis, using UBCG software to classify strain RS252 and... Azospirillum Analysis was performed on 23 type strains of the genus, and 92 orthologous genes were tandemly analyzed. A gene sequence phylogenetic tree was constructed using the maximum likelihood method. Figure 3 The Bootstrap value was 1000. The results showed that strain RS252 was similar to strain... Azospirillum oryzae COC8 T (GCA_013347285.1) and Azospirillum Several strains of the genus clustered together, indicating that strain RS252 is... Azospirillum A potential new species of the genus.

[0114] Furthermore, KEGG annotation results show that RS252 contains a complete nitrogen fixation biosynthetic pathway (K02588, K02586, and K02591), indicating potential nitrogen fixation capacity. (Azospirobacter) Azospirillum As a common nitrogen-fixing bacterium in the rhizosphere of grasses, it can regulate root flavonoid synthesis and hormone signal transduction during the colonization of rice roots, thereby promoting rice growth.

[0115] 4. Physiological and chemical classification and identification

[0116] Add several drops of 5% H2O2 to a glass petri dish, pick strain RS252 and react with it. If bubbles are produced, it proves that the strain can produce catalase. Spot strain RS252 onto filter paper soaked in 1% p-aminoxylamine hydrochloride, using Pseudomonas aeruginosa and Escherichia coli as positive and negative controls, respectively. If a rose-red ring appears around the colony, it indicates that it can produce catalase. Spot strain RS252 on five points on a starch medium, setting up three replicates, and incubate at 25°C for 2-5 days. After removing the plates, add iodine solution around the colonies and observe the color change around the colonies. If a colorless transparent ring appears around the colony, it indicates that the bacteria have produced amylase and diffused into the matrix, hydrolyzing the starch in the medium into a substance that does not react with iodine; if the area around the colony is blue, it indicates that the bacteria do not produce amylase. Five spots of strain RS252 were inoculated onto casein medium, with three replicates. The plates were incubated at 25°C for 7 days. After removing the plates, the casein around and below the colonies was observed to see if it was decomposed into clear zones. If it was clear, it indicated that the strain had the ability to hydrolyze casein.

[0117] The results showed that strain RS252 did not produce bubbles after contact with 5% H2O2, indicating that its catalase result was negative; after contact with filter paper soaked in 1% p-aminoxylamine hydrochloride, strain RS252 produced a color change, indicating that its oxidase result was positive; the strain did not produce a clear zone on casein medium and starch medium with added iodine solution, indicating that strain RS252 does not have the ability to hydrolyze casein and starch.

[0118] The enzyme activity and carbohydrate utilization of strain RS252 and related model strains were determined using API 20NE, ZYM and 50 CH test strips from bioMérieux, France.

[0119] The 20NE test results showed that strain RS252 was positive for nitrate reduction, urease hydrolysis, and aesculin hydrolysis, and positive for nitro- β -d-galactoside hydrolysis reaction showed a weak positive result, while indole reaction, arginine hydrolysis reaction, acidification reaction, and gelatin hydrolysis reaction were negative. It can assimilate mannitol. NAcetyl-glucosamine, gluconate, adipic acid, malic acid, citric acid, and phenylacetic acid cannot assimilate glucose, arabinose, mannose, maltose, and decanoic acid.

[0120] In the ZYM enzyme activity assay, esterase (C4), leucine aromatic aminoaminase, and naphthol-AS-BI-phosphohydrolase were positive; alkaline phosphatase, lipase (C14), valine aromatic aminoaminase, cystine aromatic aminoaminase, trypsin, chymotrypsin, and acid phosphatase were also positive. α -Galactosidase, β -Galactosidase, β - Glucuronidase, α -glucosidase, β -glucosidase, N -acetyl-glucosaminease, α -Mannosidase, α - Fucosidase result was negative; lipoesterase (C8) result was weakly positive.

[0121] Results from 50 CH showed that strain RS252 could hydrolyze L-arabinose, D-glucose, and D-fructose. N - Acetylglucosamine, aesculin, d-raffinose, d-fucose, and potassium gluconate; cannot be hydrolyzed. Hydrolyzes erythritol, d-arabinose, l-xylose, d-calendol I, and methyl- β -d-xylanoside, d-mannose, l-rhamnose, l-sorbose, euonymol, inositol, methyl- α -d-glucopyranoside, methyl- α -d-Mannopyranoside, amygdalin, d-lactose, d-cellobiose, d-maltose, d-merbiose, d-sucrose, d-trehalose, d-minotriose, inulin, glycogen, starch, d-gentiobiose, d-thulose, d-lysose, l-fucose, d-tagatose and l-arabinol; weakly utilized mannitol, d-ribose, d-xylose, d-galactose, mannitol, sorbitol, salicin, xylitol, d-arabinol, 2-keto-gluconate and 5-keto-gluconate.

[0122] The physiological and biochemical characteristics of strain RS252 differ from those of related model strains, as shown in Table 2.

[0123] Table 2. Phenotypic differences between strain RS252 and reference strains of *Azospirillum*.

[0124]

[0125] Note: "+" indicates a positive test result, "-" indicates a negative test result, and "w" indicates a weak positive test result.

[0126] Based on the above identification, it can be confirmed that the strain RS252 obtained in this invention is a nitrogen-fixing spirochete ( Azospirillum A new bacterial species has been named *Dinospirobacter dongyingensis*. Azospirillum dongyingense The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 30, 2024, with the registration number CGMCC No. 31494. Hereinafter referred to as *Dinospirobacter dongyingensis*. Azospirillum dongyingense RS252.

[0127] Example 2, Dongying nitrogen-fixing spirochete ( Azospirillum dongyingense Detection of RS252's ability to produce protease, solubilize potassium, and degrade cellulose.

[0128] Dongying Azotoxin ( Azospirillum dongyingense RS252 was inoculated onto the protease detection medium and incubated at 30°C for 2 days. The presence or absence of a clear zone was observed. If a clear zone was observed, it would preliminarily indicate that the strain has the ability to produce protease.

[0129] Dongying Azotoxin ( Azospirillum dongyingense RS252 was inoculated onto a modified potassium-solubilizing medium and cultured at 30°C for 2 days. The presence of a specific halo was observed. If present, it preliminarily indicates that the strain has potassium-solubilizing ability.

[0130] Dongying Azotoxin ( Azospirillum dongyingense RS252 was inoculated onto cellulose Congo red medium and incubated at 30°C for 3 days. Observe whether there is a light red transparent ring around the colony. If there is, it indicates that the colony can secrete cellulase.

[0131] The results showed that after 2 days of growth on the protease detection medium, *Dinospirillum dongyingense* (… Azospirillum dongyingense RS252 can produce a clear zone; after growing for 2 days on a modified potassium-solubilizing medium, *Dinospirillum dongyingense* ( Azospirillum dongyingense RS252 can produce a specific halo. After growing on cellulose Congo red medium for 3 days and then staining, *Dinospirillum dongyingense* ( Azospirillum dongyingense RS252 can produce a pale red transparent ring; this indicates that *Dinospirobacter dongyingense* (… Azospirillum dongyingense RS252 possesses the ability to produce protease, solubilize potassium, and degrade cellulose. Figure 4 ).

[0132] Example 3, Dongying nitrogen-fixing spirochete ( Azospirillum dongyingense RS252 alleviates NaCl stress in plants

[0133] Selecting Dongying Azotoxin ( Azospirillum dongyingenseA single colony of RS252 was inoculated into a 500 mL Erlenmeyer flask containing 200 mL of TSB liquid medium and incubated at 30 °C for 48 h. The cells were then collected by centrifugation at 8000 rpm for 10 min and resuspended in TSB liquid medium to obtain an OD value. 600 =1.0 bacterial suspension (with uninoculated TSB liquid medium as the control group for seed soaking). Uniformly sized rice varieties (SE362) were selected (described in "R. Quan, J. Wang, H. Qin, L. Chen, D. Xiao, Z. Zhao, Z. Zhang, X. Zhu, Z. Li, R. Huang, Improving grain yield and salttolerance by optimizing plant height with beneficial haplotypes in rice"). Oryza sativa Seeds (Table S1) from the supplementary materials of the paper "(2024) S2090123224005630)" were placed on sterile, moist filter paper and incubated at 30°C for 2-3 days, with regular water spraying to maintain humidity until the rice seeds sprouted. Seeds were sown in 32-cell seedling trays containing a total of 3713g of a mixture of vermiculite and nutrient soil (vermiculite: nutrient soil mass ratio = 1:1), with 6 seeds per cell. An experimental group (labeled RS252) and a control group (labeled CK) each had 8 cells. Seeds were sown after most of the rice seeds had germinated. After thinning, four rice seedlings of similar growth were left per hill. When the seedlings reached the one-leaf-one-heart stage (approximately 7 days), NaCl stress treatment was applied by watering each hill with 8 L of salt solution (final NaCl concentration 120 mmol / L). 24 hours after salt treatment, a bacterial suspension was applied to the roots of the seedlings in the treatment group, with 0.5 mL of the bacterial suspension per seedling. The control group was watered with uninoculated pure TSB liquid medium. Watering was done every 3 days during the plant growth period. The experiment was conducted in a greenhouse.

[0134] Figure 5 The growth status of rice seedlings at 27 days. Under NaCl stress, rice seedlings exhibited symptoms such as leaf drying, curling, and shedding; plant height was significantly reduced; and the overall plant showed a wilting trend. Different treatments resulted in varying degrees of salt damage. Agronomic traits of rice seedlings were also observed. Figure 6 As shown, the underground fresh weight and root length of rice seedlings in the experimental group (RS252 inoculated with potted seedling serial number YB 122) were 0.3392±0.1103g and 17.12±4.444cm, respectively, while those of the uninoculated control group (CK, potted seedling number y122) were 0.2154±0.0741g and 12.90±3.692cm, respectively. Both the underground fresh weight and root length of the experimental group were significantly higher than those of the control group.P <0.01). This indicates inoculation with *Dinospirobacter dongyingense* (…). Azospirillum dongyingense Treatment with RS252 can promote the growth of rice seedlings under NaCl stress.

[0135] To further investigate the ability of strain RS252 to alleviate NaCl stress in rice, the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), malondialdehyde (MDA), and reduced glutathione (GSH) in the leaves of 27-day-old rice seedlings were measured. Appropriate amounts of rice seedling leaves were cut into 2.0 mL centrifuge tubes, quickly placed in liquid nitrogen, and ground into powder. SOD, CAT, POD, MDA, and GSH in the leaves of the experimental and control groups were extracted and measured according to the instructions provided in the respective kits: Superoxide Dismutase (SOD) Activity Kit (Beijing Sangon Biotech, China), Catalase (CAT) Activity Kit (Beijing Sangon Biotech, China), Peroxidase (POD) Activity Kit (Beijing Sangon Biotech, China), Malondialdehyde (MDA) Content Kit (Beijing Sangon Biotech, China), and Reduced Glutathione (GSH) Content Kit (Beijing Sangon Biotech, China).

[0136] The results showed no significant differences in catalase (CAT) activity and reduced glutathione (GSH) content between the experimental group (RS252) and the control group (CK). The POD and SOD activities in the experimental group (RS252) were significantly higher than those in the control group (CK). Figure 7 , P <0.01), SOD catalyzes the disproportionation of superoxide anions to generate H2O2 and O2, while POD catalyzes the conversion of H2O2 to H2O and O2. SOD and POD play important roles in the reactive oxygen species scavenging system, and higher POD and SOD activities indicate that the plant possesses strong antioxidant capacity and resistance to stress. These results demonstrate that *Azotrophus dongyingensis* (… Azospirillum dongyingense RS252 can stimulate plants to produce highly active POD and SOD, enabling them to better cope with NaCl stress and promoting plant growth; at the same time, the MDA content of the experimental group (RS252) was significantly lower than that of the control group ( Figure 7 , P <0.01), reactive oxygen species (ROS) are metabolic products in plants. Stress exacerbates the production of large amounts of ROS. ROS attacks unsaturated fatty acids in the cell membrane, triggering lipid peroxidation and producing MDA. The accumulation of MDA further damages the structure and function of the cell membrane, leading to increased cell membrane permeability, leakage of intracellular substances, and affecting the normal physiological activities of the plant. In summary, it is speculated that *Dinospirobacter dongyingensis* (… Azospirillum dongyingenseRS252 alleviates NaCl stress in rice seedlings and improves rice's NaCl stress tolerance by inducing plants to produce large amounts of POD and SOD and reducing MDA accumulation.

[0137] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Azotospirobacter, characterized by: The nitrogen-fixing spirochete is *Deniospirobacter dongyingensis* (… Azospirillum dongyingense Its strain number is RS252, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 31494.

2. A composition containing the azospirobacter of claim 1.

3. The composition according to claim 2, characterized in that: The composition is a culture, which is a substance obtained by culturing the azospirobacter in a microbial culture medium.

4. The composition according to claim 2 or 3, characterized in that: The composition is a microbial agent, a microecological preparation, or a bio-fertilizer.

5. The use of the nitrogen-fixing spirochete according to claim 1 or the composition according to any one of claims 2-4, wherein the use is any one of the following: B1) Applications in the production of proteases or in the preparation of products for the production of proteases; B2) Applications in potassium solubilization or in the preparation of products for potassium solubilization; B3) Applications in the degradation of cellulose or in the preparation of products for the degradation of cellulose; B4) Applications in alleviating NaCl stress in plants or in the preparation of products for alleviating NaCl stress in plants; B5) Applications in enhancing the POD activity of plants or in the preparation of products for enhancing the POD activity of plants; B6) Application in enhancing SOD activity in plants or in the preparation of products for enhancing SOD activity in plants; B7) Application in reducing the MDA content of plants or in the preparation of products for reducing the MDA content of plants; B8) Use in promoting plant growth or in the preparation of products for promoting plant growth; B9) Application in promoting the increase of underground fresh weight of plants or in the preparation of products for promoting the increase of underground fresh weight of plants; B10) Application in promoting plant root growth or in the preparation of products for promoting plant root growth; The plant in question is rice.

6. The application according to claim 5, characterized in that: The enhancement of plant POD activity is achieved by increasing plant POD activity under NaCl stress; and / or, the enhancement of plant SOD activity is achieved by increasing plant SOD activity under NaCl stress; and / or, the reduction of plant MDA activity is achieved by reducing plant MDA activity under NaCl stress; and / or The promotion of plant growth is to promote plant growth under NaCl stress conditions; and / or, the promotion of increased underground fresh weight of plants is to promote increased underground fresh weight of plants under NaCl stress conditions; and / or, the promotion of increased root length of plants is to promote increased root length of plants under NaCl stress conditions.

7. A method for alleviating NaCl stress in plants, comprising the following steps: treating the plant or its growth substrate with the nitrogen-fixing spirochete according to claim 1 or the composition according to any one of claims 2-4, thereby alleviating the NaCl stress in the plant; The plant in question is rice.

8. A method for promoting plant growth, comprising the steps of: treating the plant to be treated or its growth substrate with the nitrogen-fixing spirochete of claim 1 or the composition of any one of claims 2-4, thereby promoting the growth of the plant; The plant in question is rice.

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