Flavobacterium capable of producing siderophores, promoting growth and relieving saline-alkali stress of plants and application of flavobacterium

Abstract

The invention discloses flavobacterium capable of producing siderophores, promoting growth and relieving saline-alkali stress of plants and application of flavobacterium. The invention provides Flavobacterium xylanum, the strain number of the Flavobacterium xylanum is 3 to 210, and the registration number of the Flavobacterium xylanum in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms is CGMCC No.30572. The Flavobacterium zanigense 3-210 can be used as a microbial organic fertilizer, and is used for improving the soil fertility, relieving the saline-alkali stress of crops and improving the saline-alkali adaptability of the crops.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a Flavobacterium that produces iron carriers, promotes growth, and alleviates salt and alkali 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 rhizosphere growth-promoting bacteria (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 plants' 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. Flavobacterium ( Flavobacterium Gram-negative bacteria are a class of bacteria that are widely distributed in the environment and have been reported to promote plant growth, resist stress (drought, cold, etc.) and treat wastewater. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a Flavobacterium genus ( Flavobacterium A new strain of bacteria was discovered, which has the functions of producing iron carriers, promoting growth, and alleviating salt and alkali stress in plants.

[0005] Firstly, the present invention claims protection for Flavobacterium tibetans or its descendants.

[0006] The *Flavobacterium tibeticum* claimed in this invention is *Flavobacterium tibeticum* (… Flavobacterium xizangense The strain number is 3-210, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 30572.

[0007] Flavobacterium tibetans ( Flavobacterium xizangense 3-210 is a Gram-negative bacterium. After 3 days of growth on R2A solid medium, the colonies are yellow, round, raised, and slimy, easily picked up, with a diameter of 1-2 mm. This strain has excellent plant growth-promoting properties and can significantly alleviate the stress of salt and alkali on plants.

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

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

[0010] The substances in the above-mentioned cultures include Flavobacterium tibetans or its progeny (the bacterial cells themselves) and its metabolites as described in the first aspect above.

[0011] 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.

[0012] In the above-mentioned cultures, the bacterial culture medium can be a solid culture medium or a liquid culture medium.

[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 bio-fertilizers, the active ingredient may be *Flavobacterium tibetense*, its metabolites, and / or its culture, as described in the first aspect above. The active ingredient may also contain other biological and / or non-biological components. Other active ingredients in the microbial agents, microecological preparations, or bio-fertilizers can be determined by those skilled in the art based on the desired effect.

[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 bacterial agent, microecological preparation, or bio-fertilizer, the Tibetan Flavobacterium and / or its metabolites 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 *Flavobacterium tibetense* (or its progeny) can be obtained from the fermentation broth of *Flavobacterium tibetense* (or its progeny). The metabolites of *Flavobacterium tibetense* (or its progeny) can be either sterile metabolites of *Flavobacterium tibetense* or bacterial metabolites of *Flavobacterium tibetense*. Specifically, the sterile metabolites of *Flavobacterium tibetense* (or its progeny) (sterile fermentation filtrate) can be prepared as follows: *Flavobacterium tibetense* (or its progeny) is cultured in a liquid culture medium, and the *Flavobacterium tibetense* is removed from the liquid culture (fermentation broth) by filtration, thus obtaining the sterile metabolites of *Flavobacterium tibetense*. Specifically, the bacterial metabolites of *Flavobacterium tibetense* (or its progeny) can be prepared as follows: *Flavobacterium tibetense* (or its progeny) is cultured in a liquid fermentation medium, and the fermentation broth—containing *Flavobacterium tibetense* (or its progeny) and substances secreted into the liquid culture medium—is collected; this fermentation broth is the bacterial metabolites of *Flavobacterium tibetense* (or its progeny).

[0021] Furthermore, the composition may have at least one of the following properties: A1) Iron-producing carrier; A2) Relieve salt and alkali stress in plants; A3) Promotes seed germination; A4) Improve the germination potential of plants; A5) Improve the germination rate of plants; A6) Promotes plant growth; A7) Promotes increased stem diameter in plants; A8) Enhances CAT activity in plants; A9) Increases the PRO content in plants.

[0022] Thirdly, the present invention claims protection for any of the following uses of Flavobacterium tibetans or its descendants as described in the first aspect above, or of the compositions described in the second aspect above: B1) Application in the production of ferrogenes or in the preparation of products for the production of ferrogenes; B2) Applications in alleviating salt and alkali stress in plants or in the preparation of products for alleviating salt and alkali stress in plants; B3) Application in promoting plant seed germination or in the preparation of products for promoting plant seed germination; B4) Applications in improving the germination potential of plants or in the preparation of products for improving the germination potential of plants; B5) Applications in improving the germination rate of plants or in the preparation of products for improving the germination rate of plants; B6) Use in promoting plant growth or in the preparation of products for promoting plant growth; B7) Application in promoting the increase of plant stem diameter or in the preparation of products for promoting the increase of plant stem diameter; B8) Applications in enhancing plant CAT activity or in the preparation of products for enhancing plant CAT activity; B9) Application in increasing the PRO content of plants or in the preparation of products for increasing the PRO content of plants.

[0023] In the second and third aspects mentioned above, promoting plant seed germination can be done by promoting plant seed germination under alkaline stress conditions; increasing plant germination potential can be done by increasing plant germination potential under alkaline stress conditions; increasing plant germination rate can be done by increasing plant germination rate under alkaline stress conditions. Promoting plant growth can be done by promoting plant growth under alkaline stress conditions; promoting increased plant stem diameter can be done by promoting increased plant stem diameter under alkaline stress conditions. Increasing plant CAT activity can be done by increasing plant CAT activity under alkaline stress conditions; increasing plant PRO content can be done by increasing plant PRO content under alkaline stress conditions.

[0024] Fourthly, the present invention claims a method for alleviating salt and alkali stress in plants.

[0025] The method for alleviating salt and alkali stress in plants claimed by the present invention may include the following steps: treating the plant to be treated or its growth substrate with the Bacillus tibetica or its progeny as described in the first aspect above or the composition described in the second aspect above, thereby alleviating the salt and alkali stress in the plant.

[0026] Fifthly, the present invention claims a method for promoting plant growth.

[0027] 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 Flavobacterium tibetans or its progeny as described in the first aspect above or the composition as described in the second aspect above, thereby promoting the growth of the plant.

[0028] In one embodiment of the present invention, the promotion of plant growth refers to promoting plant growth under alkaline stress conditions. Specifically, the promotion of plant growth is manifested in promoting an increase in plant stem diameter.

[0029] The present invention also provides a method for preparing the microbial agent described in the third aspect above.

[0030] The method for preparing the bacterial agent provided by the present invention includes using Flavobacterium tibetans ( Flavobacterium xizangense )3-210 (or its progeny) or / and Flavobacterium tibetans ( Flavobacterium xizangense The step of obtaining the bacterial agent by using metabolites of 3-210 (or its descendants) and / or the above-mentioned culture as components of the bacterial agent.

[0031] This invention also provides a method for promoting plant seed germination under alkaline stress conditions, comprising the following steps: using *Flavobacterium tibetans* as described in the first aspect above (… Flavobacterium xizangense (or its descendants) or the aforementioned Flavobacterium tibetans ( Flavobacterium xizangense The plant seeds are treated with metabolites of (or their progeny) or the cultures described in the second aspect above or the fungal agents described in the third aspect above (e.g., soaking or dressing) to promote seed germination under alkaline stress.

[0032] In some embodiments of the present invention, the salt-alkali stress is simulated using NaCl, Na₂SO₄, and NaHCO₃ in a molar ratio of 1:1:1. + A mixed solution with a final concentration of 80 mmol / L; mix 50 mL of the mixed solution with 100 g of plant culture medium (such as vermiculite or soil) in proportion.

[0033] In the aforementioned relevant aspects, the plant may be any of the following: C1) Angiosperms; C2) Monocotyledons; C3) Plants of the order Poales; C4) Gramineae plants; C5) Plants of the subfamily Sorghum; C6) Plants of the Zea tribe; C7) Maize (commonly known as corn).

[0034] This invention, through a secretory siderophore test, showed that *Flavobacterium tibetense* (… Flavobacterium xizangense 3-210 has the ability to secrete siderophores. Pot experiments showed that, compared with the uninoculated negative control group, inoculation with *Flavobacterium tsevieriae* ( ) Flavobacterium xizangense After 3-210, it can increase the stem diameter, chlorophyll content, fresh weight, and dry weight of maize; it also significantly increases the activity of antioxidant enzymes in some maize parts. (Flavobacterium tibetans) Flavobacterium xizangense 3-210 can be used as a microbial organic fertilizer to improve soil fertility, alleviate crop salt and alkali stress, and enhance crop adaptability to salt and alkali environments.

[0035] Depositing Instructions Classification and nomenclature: Flavobacterium tibetans ( Flavobacterium xizangense ); Biological materials from ginseng: 3-210; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: May 9, 2024; Registration number at the Preservation Center: CGMCC No. 30572. Attached Figure Description

[0036] Figure 1 For Tibetan Flavobacterium ( Flavobacterium xizangense Colony morphology of 3-210 after 3 days of incubation on R2A plates.

[0037] Figure 2 To construct *Flavobacterium tibetense* (Tibetan strain) using the neighbor-joining method based on the 16S rRNA gene sequence. Flavobacterium xizangense Phylogenetic tree of strain 3-210 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. Escherichia coli ATCC 11775 T (X80725) is an outgroup.

[0038] Figure 3This refers to the construction of a gene pool containing *Flavobacterium tibetans* based on 836 orthologous genes using the maximum likelihood method. Flavobacterium xizangense )3-210 and Flavobacterium Phylogenetic tree of protein sequences of closely related species within the genus.

[0039] Figure 4 For Tibetan Flavobacterium ( Flavobacterium xizangense 3-210 Results of test for the ability to secrete siderophores. CK is the control without bacterial inoculation.

[0040] Figure 5 The growth status of maize seedlings inoculated with strain 3-210 and uninoculated with strain 30 days under salt-alkali stress.

[0041] Figure 6 The values ​​represent the fresh weight, dry weight, plant height, and root length of maize seedlings under 30 days of salt-alkali stress. CK is the control group without bacterial inoculation.

[0042] Figure 7 The figures show the stem diameter and chlorophyll content of maize seedlings under 30 days of salt-alkali stress. CK is the uninoculated control. Note: * in the figure indicates a significant difference compared to the uninoculated control CK. P <0.05). Detailed Implementation

[0043] 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.

[0044] 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.

[0045] R2A liquid culture medium: 0.25g tryptone, 0.5g acid-hydrolyzed casein, 0.5g yeast extract, 0.5g soluble starch, 0.3g dipotassium hydrogen phosphate, 0.1g magnesium sulfate, 0.3g sodium pyruvate, 0.25g peptone, 0.5g glucose, diluted to 1000mL with water, pH 7.2±0.2, sterilized at 121℃ for 15min.

[0046] R2A solid medium: Add 20.0g agar to R2A liquid medium and sterilize at 121℃ for 15min.

[0047] 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.

[0048] TSA solid medium: Add 20.0g agar to TSB liquid medium and sterilize at 121℃ for 15min.

[0049] Starch medium: NA medium, 0.2% soluble starch (% means g / 100mL), pH 7.4, sterilized at 121℃ for 20min.

[0050] Casein culture medium: Solution a: 5g skim milk powder, 50mL distilled water; Solution b: 50mL NB, 1.5g agar. Sterilize solutions a and b separately at 121℃ for 15min, and after cooling to about 60℃, mix well and dispense into plates.

[0051] CAS Qualitative Culture Medium: Solution ①: Dissolve 0.012 g CAS in 10 mL of deionized water and mix with 2 mL of 5 mM ferric chloride; Solution ②: 0.015 g cetyltrimethylammonium bromide dissolved in 8 mL of deionized water; Dye solution ③: Slowly pour solution ① into solution ② to obtain dye solution ③, and sterilize at 115℃ for 20 minutes; Culture medium ④: Add 6.04g piperazine diethanol sulfonic acid and 10mL 0.1M phosphate solution to an Erlenmeyer flask containing 150mL distilled water and mix well. Adjust the pH to 6.8 with 50% NaOH, add 4.0g agar powder, and sterilize at 115℃ for 20min. Phosphate solution: 2.427g disodium hydrogen phosphate, 0.5905g sodium dihydrogen phosphate, 0.075g potassium dihydrogen phosphate, 0.125g sodium chloride, 0.25g ammonium chloride, 100mL deionized water. Mix well and dilute 10 times before use. Nutrient solution: 0.2 mL 1 mM calcium chloride solution, 4 mL 1 mM magnesium sulfate tetrahydrate solution, 6 mL 10% (w / v) casein amino acid solution, sterilized at 115℃ for 20 min (prepare 10 mL before use, and store in a 4℃ refrigerator away from light after use). CAS qualitative culture medium plates: When the staining solution, culture medium ④, and nutrient solution have cooled to about 65°C, add the nutrient solution to culture medium ④, then slowly add the staining solution ③, mix thoroughly, and then pour into plates.

[0052] CAS test solution: 10mM cetyltrimethylammonium bromide, 1.5mL 1mM ferric chloride, 7.5mL 2mM CAS, 4.307g anhydrous dimethylamine, pH adjusted with 12mM HCl to completely dissolve the anhydrous dimethylamine, and 100mL deionized water.

[0053] Example 1: Flavobacterium tibetans ( Flavobacterium xizangense Separation and identification of 3-210 I. Flavobacterium tibetans ( Flavobacterium xizangense )3-210 separation Rhizosphere soil samples of *Erigeron breviscapus* were collected along the Duilongqu River in Lhasa, Tibet (29°48′8″N, 93°54′32″E), and brought back to the laboratory for storage in a 4°C refrigerator using a 4°C ice box.

[0054] Shake off the soil adhering to the plant roots, retaining only the rhizosphere soil tightly adhering to the root surface. Immerse the *Erigeron canadensis* root system with rhizosphere soil in an Erlenmeyer flask containing 100 mL of sterile water and shake at 150 rpm for 30 min at room temperature. Centrifuge the collected suspension at 3000 rpm for 10 min at 4°C, discarding the supernatant; the remaining residue is the rhizosphere soil. Weigh 1 g of rhizosphere soil and resuspend it in 10 mL of sterile water for serial dilution. Spread 100 μL of each dilution onto R2A plates and incubate upside down at 30°C for 1 week. Based on physiological morphological characteristics, pick single colonies with a bamboo stick and inoculate them onto plates for purification. After confirming pure bacteria, transfer them to slant culture for short-term storage at 4°C, then transfer them to 20% glycerol tubes for long-term storage at -80°C. Name one of the isolated and purified strains 3-210.

[0055] II. Flavobacterium tibetans ( Flavobacterium xizangense ) 3-210 identification 1. Morphological identification of strains The strain 3-210, 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, Gram staining of strain 3-210 was performed using a Solarbio Gram staining kit, and the morphology of the bacteria was observed using an optical microscope.

[0056] Colonies of strain 3-210 on R2A plates are yellow, round, raised, and slimy, easily picked up, with a colony diameter of 1-2 mm. Figure 1 The cells are Gram-negative, rod-shaped, and do not form spores.

[0057] 2. Molecular identification Following the instructions, genomic DNA was extracted using the TIANamp bacterial genomic DNA extraction kit from Beijing Tiangen Biotech Co., Ltd. The genomic DNA was then sent to Annoroad Gene Technology (Beijing) Co., Ltd., where a draft genome sequence of strain 3-210 was performed using an Illumina NovaSeq 6000 sequencing system. Assembly was performed using SPAdes software, yielding 424 contigs with a coverage of approximately 150×N. 50 The length is 353504 bp. The genome size is 5.13 Mb, and the G+C content is 33.95%. The ANIm method in the pyANI software was used to analyze strain 3-210 and... Flavobacterium Genome-wide average nucleotide identity (ANI) analysis was performed on closely related strains. The digital DNA-DNA hybridization (dDDH) value between strain 3-210 and the reference strain was calculated using the Genome-to-Genome Distance Calculator (GGDC) 3.0 server (https: / / ggdc.dsmz.de / ggdc.php#). The sequencing length of the 16S rRNA gene of strain 3-210 was 1511 bp (SEQ ID No. 1). Alignment with the EzBioCloud database showed that strain 3-210 belongs to the genus *Flavobacterium* (…). Flavobacterium (members, with) Flavobacterium resistens BD-b365 T (97.91%) Flavobacterium psychrolimnae LMG 22018 T (97.08%) Flavobacterium ginsengisoli DCY54 T (97.08%) and Flavobacterium tructae CCUG 60100 T (97.00%) of the sequences showed high similarity, and were similar to... Flavobacterium The sequence similarity of the model bacteria from other species in the genus was <98.8%. Closely related 16S rRNA gene sequences were retrieved from the EzBioCloud server and aligned using the MUSCLE program. A phylogenetic tree was constructed using the Neighbour-Joining method with MEGA X software. The evolutionary distance using the NJ method was calculated using the Kimura two-parameter model, with a bootstrap value of 1000. The phylogenetic tree constructed using the Neighbour-Joining method is shown below. Figure 2 As shown, strain 3-210 and strain Flavobacterium resistens BD-b365 T , Flavobacterium procerum T3 T 5 plantsFlavobacterium They categorize together, yet each forms its own branch.

[0058] Table 1. Flavobacterium tibetans ( Flavobacterium xizangense )3-210 and Flavobacterium Values ​​of ANI and dDDH for closely related species within the genus

[0059] Strain 3-210 and other strains with publicly available genome sequences Flavobacterium Compared to the type strains of the same genus, the ANI values ​​were 83.6-92.8%, lower than the previously proposed critical value of 95-96% for species delimitation; the dDDH values ​​of strain 3-210 and its type strain were between 20.2-47.1%, far below the 70% species delimitation threshold. See Table 1 for details. Both the ANI and dDDH results indicate that strain 3-210 is a... Flavobacterium A new species of the genus.

[0060] Simultaneously, the Bacterial Pan Genome Analysis tool (BPGA) software was used to analyze strain 3-210 and... Flavobacterium Analysis was performed on 31 type strains of the genus, and 836 orthologous genes were tandemly analyzed. A protein sequence phylogenetic tree was constructed using the maximum likelihood method. Figure 3 The Bootstrap value was 1000. The results showed that strains 3-210 were similar to strain... Flavobacterium resistens DSM 19382 T The clustering of several strains of the genus Flavobacterium, and their separate branching, indicates that strain 3-210 is a potential new species of the genus Flavobacterium.

[0061] 4. Physiological and chemical classification and identification Add several drops of 5% H2O2 to a glass petri dish, pick strain 3-210 and react with it. If bubbles are produced, it proves that the strain can produce catalase. Spot strain 3-210 on 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 3-210 on five points on starch medium, setting up three replicates, and incubate at 30℃ 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 substrate, 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 3-210 were inoculated onto casein medium, with three replicates. The plates were incubated at 30°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.

[0062] The results showed that strain 3-210 produced bubbles after contact with 5% H2O2, indicating that its catalase result was positive; after contact with filter paper moistened with 1% p-aminoxylamine hydrochloride, strain 3-210 produced a light rose-red color, indicating that its oxidase result was weakly positive; the strain produced clear zones on both casein medium and starch medium with added iodine solution, indicating that strain 3-210 has the ability to hydrolyze casein and starch.

[0063] The enzyme activity and carbohydrate utilization of strain 3-210 and related model strains were determined using API 20NE, ZYM and 50 CH test strips from bioMérieux, France.

[0064] The 20NE test results showed that strain 3-210 was positive for heptathion hydrolysis, and could assimilate glucose, arabinose, and mannose. It also underwent nitrate reduction, indole reaction, glucose acidification, arginine hydrolysis, urease hydrolysis, gelatin hydrolysis, and p-nitro- β -d-galactoside hydrolysis reaction is negative, and it cannot assimilate mannitol. N - Acetyl-glucosamine, gluconate, decanoic acid, adipic acid, apple acid, citric acid, phenylacetic acid, and tetramethyl-1,1-phenylenediamine.

[0065] In the ZYM enzyme activity identification assay, alkaline phosphatase, leucine aromatic aminoaminase, valine aromatic aminoaminase, acid phosphatase, α -glucosidase and N The result for acetyl-glucosaminease was positive; lipase (C14), cystine aromatic aminoaminase, trypsin, chymotrypsin, naphthol-as-bi-phosphohydrolase,α -Galactosidase, β -Galactosidase, β - Glucuronidase, β -glucosidase, α -Mannosidase and α - Fucosidase results were negative; esterase (C4) and lipoesterase (C8) results were weakly positive.

[0066] The results of 50 CH showed that strain 3-210 could hydrolyze l-arabinose, d-galactose, d-glucose, d-fructose, d-mannose, N-acetylglucosamine, amygdalin, arecoline, ferric citrate of aesculin, d-cellobiose, d-maltose, d-sucrose, d-raffinose, starch, glycogen, and d-gentiobiose; it weakly hydrolyzed d-lactose and inulin; and it could not hydrolyze mannitol, erythritol, d-arabinose, d-ribose, l-xylose, d-calendulol, or methyl- β d-xylanoside, L-sorbose, L-rhamnose, eurythritol, inositol, mannitol, sorbitol, methyl- α d-Mannopyranoside, methyl- α d-glucopyranoside, salicin, d-merbiose, d-trehalose, d-melinotriose, xylitol, d-thulose, d-lythose, d-tagatose, d-fucose, l-fucose, d-arabinol, l-arabinol, potassium gluconate, potassium 2-ketogluconate, and potassium 5-ketogluconate. The physiological and biochemical characteristics of strain 3-210 differ from those of related model strains, as shown in Table 2.

[0067] Table 2. Differences in physiological and biochemical characteristics between strain 3-210 and related model strains

[0068] Note: + indicates positive or usable; - indicates negative or unusable; w indicates weak positive.

[0069] Based on homology comparison, phylogenetic tree construction, and combined with morphological, physiological and biochemical identification, and genomic analysis results, strain 3-210 can be identified as... Flavobacterium A new species of the genus, the proposed classification name is... Flavobacterium xizangense Its Chinese name is *Flavobacterium tibetum*. Flavobacterium xizangense )3-210 was deposited at the China General Microbiological Culture Collection Center on May 9, 2024, with the registration number CGMCC No.30572.

[0070] Example 2, Flavobacterium tibetans ( Flavobacterium xizangense Detection of the secretory siderophore capacity of 3-210 Tibetan Flavobacterium (Flavobacterium xizangense 3-210 points were inoculated onto CAS qualitative medium and incubated at 30℃ for 4 days. The presence of orange-yellow rings was observed; if present, it preliminarily indicates that the strain possesses the ability to secrete siderophores. (The text then abruptly shifts to a different topic: "Tibetan Flavobacterium (...") Flavobacterium xizangense 3-210 was inoculated into R2A liquid medium and incubated in the dark at 30℃ for 2 days at 150 rpm. 1 mL of the supernatant was collected by centrifugation at 12000 rpm and mixed with an equal volume of CAS detection solution. The mixture was then allowed to stand at room temperature in the dark for 1 hour. A represents the absorbance of the mixed solution at 630 nm, and Ar represents the absorbance of the CAS detection solution and R2A liquid medium mixture (1:1) at 630 nm. The absorbance was zeroed using double-distilled water. The ability of the strain to produce siderophores was evaluated by the ratio of A to Ar, with 0-0.6 indicating strong, 0.6-0.8 indicating moderate, and 0.8-1 indicating weak (Yang Hongru, Yuan Bo, Zhao Xia, et al. Three culturable nitrogen-fixing bacteria groups in the rhizosphere of desert shrubs and their nitrogen fixation and siderophore production abilities [J]. Bulletin of Microbiology, 2016, 43(11): 2366-2373. DOI:10.13344 / j.microbiol.china.150967).

[0071] The results showed that after 4 days of growth on CAS qualitative medium, *Flavobacterium tibetense* (… Flavobacterium xizangense )3-210 can produce an orange-yellow transparent ring, and its preliminary quantitative result for secreting siderophores is 0.65±0.04 ( Figure 4 ).

[0072] Example 3, Flavobacterium tibetans ( Flavobacterium xizangense 3-210 Relieves Salt and Alkali Stress in Plants 1. Promotes corn seed germination under salt and alkali stress Selecting Tibetan Flavobacterium ( Flavobacterium xizangense 3-210 single colonies were inoculated into 500 mL Erlenmeyer flasks containing 200 mL of R2A 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 R2A liquid medium to obtain OD values. 600 =1.0 bacterial suspension (with uninoculated R2A liquid medium as the control group for seed soaking). Select large and plump corn (Zhengdan 958) seeds and place them in the bacterial suspension, soaking at 30℃ for 4 hours.

[0073] After soaking, spread the seeds evenly on a saline-alkali solution (composed of NaCl, Na₂SO₄, and NaHCO₃ in a molar ratio of 1:1:1). +Filter paper (with a final concentration of 80 mmol / L) was moistened, and then placed on seedling trays. The trays were then placed in a 30℃ constant temperature incubator, with 100 seeds per treatment. The germination of maize seeds was monitored and observed, the number of germinated seeds was recorded, and the effect of the experimental strain on promoting maize seed germination was analyzed.

[0074] Germination potential % = (Number of germinated seeds at peak germination period / Number of seeds tested) × 100% Germination rate % = (Number of germinated seeds in a specified number of days / Number of seeds tested) × 100% After 7 days of cultivation, the germination potential and germination rate of the control group seeds were 62.22% and 68.89%, respectively, while those of the experimental group seeds were 71.11% and 82.22%, respectively, indicating that *Flavobacterium tibetans* (…) Flavobacterium xizangense 3-210 can promote the germination of maize seeds under salt-alkali stress. The specific results are shown in Table 3.

[0075] Table 3. Statistical results of germination potential and germination rate

[0076] 2. Alleviating growth stress in maize seedlings under saline-alkali conditions Selecting Tibetan Flavobacterium ( Flavobacterium xizangense 3-210 single colonies were inoculated into 500 mL Erlenmeyer flasks containing 200 mL of R2A 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 R2A liquid medium to obtain OD values. 600 =1.0 bacterial suspension (with uninoculated R2A liquid medium as the control group for seed soaking). Select maize (Zhengdan 958) seedlings of similar growth, transplant 3 seedlings per pot into pots containing 100g vermiculite substrate. Set up 9 pots each for the experimental group and control group. Three days after transplanting and allowing the seedlings to recover, apply salt-alkali stress by watering each pot with a salt-alkali solution (NaCl, Na2SO4, and NaHCO3, in a molar ratio of 1:1:1). + The final concentration was 80 mmol / L (50 mL). On the second day of stress treatment, the bacterial suspension was applied as a root drenching treatment. 5 mL of the bacterial suspension was poured onto the roots of corn seedlings in the experimental group (strain 3-210), while the control group (CK) was drenched with uninoculated pure R2A liquid medium. Watering was done every 2-3 days during the plant growth period, and the bacterial agent was replenished on day 15. The experiment was conducted in a light incubator. The day and night temperatures were 25℃ / 20℃, and the day duration was 14 hours.

[0077] Fresh and dry weight, plant height (from the base of the stem to the tip of the leaf), root length, stem diameter, and chlorophyll content of maize seedlings were measured 30 days after salt-alkali stress treatment.

[0078] Figure 5The growth status of maize seedlings under salt and alkali stress for 30 days is shown. The final experimental data is the average value of each potted plant.

[0079] Results of agronomic traits of maize seedlings Figure 6 As shown, the fresh weight and dry weight of maize seedlings in the experimental group were 2.56±0.75g and 0.22±0.06g, respectively, while those in the control group (CK) were 2.40±0.19g and 0.21±0.06g, respectively. Both the fresh weight and dry weight of the experimental group were better than those of the control group, but the differences were not statistically significant.

[0080] Figure 7 As shown, the stem diameter and chlorophyll content of the experimental group of maize seedlings were 3.36±0.50 cm and 29.77±5.41 SPAD, respectively, while those of the control group (CK) were 3.00±0.58 cm and 27.16±4.28 SPAD, respectively. The experimental group showed better stem diameter and chlorophyll content than the control group, and the stem diameter was significantly higher than that of the control group. P <0.05), indicating that Flavobacterium tibetans ( Flavobacterium xizangense 3-210 can promote the growth of maize seedlings and the accumulation of chlorophyll under saline-alkali conditions, and significantly increase the stem diameter of maize seedlings.

[0081] To further investigate the ability of the strain to alleviate salt-alkali stress in maize seedlings, the activities of catalase (CAT), peroxidase (POD), malondialdehyde (MDA), and proline (PRO) in the leaves were measured. Appropriate amounts of maize seedling leaves were cut into 2.0 mL centrifuge tubes, quickly placed in liquid nitrogen, and ground into powder. CAT, POD, MDA, and PRO were extracted and measured from the leaves of maize seedlings in the experimental and control groups according to the instructions provided in the catalase (CAT) activity assay kit (Solarbio, China), peroxidase (POD) activity assay kit (Solarbio, China), malondialdehyde (MDA) content assay kit (Solarbio, China), and proline (PRO) content assay kit (Solarbio, China).

[0082] The results are shown in Table 4. The MDA content in the experimental group was higher than that in the control group (CK), but the difference was not significant. P >0.05), indicating that both groups were subjected to a certain degree of abiotic stress. The experimental group had a higher content, which is speculated to be related to the plant's stress response when the strain, as an "invasive species," establishes an interaction relationship with the crop. The CAT activity in the experimental group was significantly higher than that in the control group, but the POD activity was significantly lower than that in the control group. P <0.05), indicating that Flavobacterium tibetans ( Flavobacterium xizangense)3-210 mainly alleviates salt-alkali stress in maize by stimulating plants to produce highly active CAT rather than POD; at the same time, the PRO content in the experimental group was significantly higher than that in the control group. P <0.05), as an important osmotic regulator in plants under abiotic stress, an increase in its content indicates enhanced plant resistance. In summary, it is speculated that *Flavobacterium tibetans* (… Flavobacterium xizangense )3-210 alleviates the growth stress of maize seedlings under saline-alkali conditions and improves the saline-alkali adaptability of maize by inducing plants to produce a large amount of PRO and highly active CAT.

[0083] Table 4. Enzyme activities and compound contents related to abiotic stress in maize seedling leaves at 30 days.

[0084] Note: Different lowercase numbers in the same column indicate significant differences. P <0.05).

[0085] 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. Flavobacterium tibetans or its progeny, characterized in that: The *Flavobacterium tibeticum* is *Flavobacterium tibeticum* (… Flavobacterium xizangense The strain number is 3-210, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 30572.

2. A composition containing the Tibetan Flavobacterium of claim 1 or its progeny.

3. The composition according to claim 2, characterized in that: The composition is a culture; the culture is a substance obtained by culturing the Tibetan Flavobacterium or its progeny in a microbial culture medium.

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

5. The composition according to claim 4, characterized in that: The composition has at least one of the following properties: A1) Iron-producing carrier; A2) Relieve salt and alkali stress in plants; A3) Promotes seed germination; A4) Improve the germination potential of plants; A5) Improve the germination rate of plants; A6) Promotes plant growth; A7) Promotes increased stem diameter in plants; A8) Enhances CAT activity in plants; A9) Increases the PRO content in plants.

6. The use of the Flavobacterium tibetans or its descendants as described in claim 1, or the composition as described in any one of claims 2-5, wherein the use is any one of the following: B1) Application in the production of ferrogenes or in the preparation of products for the production of ferrogenes; B2) Applications in alleviating salt and alkali stress in plants or in the preparation of products for alleviating salt and alkali stress in plants; B3) Application in promoting plant seed germination or in the preparation of products for promoting plant seed germination; B4) Application in improving the germination potential of plants or in the preparation of products for improving the germination potential of plants; B5) Applications in improving the germination rate of plants or in the preparation of products for improving the germination rate of plants; B6) Use in promoting plant growth or in the preparation of products for promoting plant growth; B7) Application in promoting the increase of plant stem diameter or in the preparation of products for promoting the increase of plant stem diameter; B8) Applications in enhancing plant CAT activity or in the preparation of products for enhancing plant CAT activity; B9) Application in increasing the PRO content of plants or in the preparation of products for increasing the PRO content of plants.

7. The composition according to claim 5 or the application according to claim 6, characterized in that: The promotion of plant seed germination is to promote plant seed germination under alkaline stress conditions; and / or, the improvement of plant germination potential is to improve plant germination potential under alkaline stress conditions; and / or, the improvement of plant germination rate is to improve plant germination rate under alkaline stress conditions. and / or The promotion of plant growth is to promote plant growth under alkaline stress conditions; and / or, the promotion of increased plant stem diameter is to promote increased plant stem diameter under alkaline stress conditions. and / or The improvement of plant CAT activity is to increase plant CAT activity under alkaline stress conditions; and / or, the improvement of plant PRO content is to increase plant PRO content under alkaline stress conditions.

8. A method for alleviating salt and alkali stress in plants, comprising the following steps: treating the plant to be treated or its growth substrate with the *Flavobacterium tibetans* or its progeny as described in claim 1 or any of the compositions described in claims 2-5, thereby alleviating the salt and alkali stress in the plant.

9. A method for promoting plant growth, comprising the steps of: treating a plant or its growth substrate with the *Flavobacterium tibetans* of claim 1 or its progeny, or the composition of any one of claims 2-5, thereby promoting the growth of the plant.

10. The composition, application, or method according to any one of claims 5-9, characterized in that: The plant is any one of the following: C1) Angiosperms; C2) Monocotyledons; C3) Plants of the order Poales; C4) Gramineae plants; C5) Plants of the subfamily Sorghum; C6) Plants of the Zea tribe; C7) Corn.

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