Cold-resistant bacillus subtilis and application thereof in improving stress resistance of plants

Abstract

The invention discloses an enterobacter strain and application thereof in improving stress resistance of plants. The invention relates to the field of biological agriculture, and provides bacillus fruticola YJD5-23, and the registration number of the bacillus fruticola YJD5-23 in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms is CGMCC No.36069. The bacillus fruticola YJD5-23 has the advantages that the bacillus fruticola YJD5-23 can be used for preparing the bacillus fruticola YJD5-23; the enterobacter sphaeroides YJD5-23 provided by the invention can improve the resistance of arabidopsis thaliana, alfalfa, corn and soybean to saline-alkali stress, specifically, the dry weight, the fresh weight, the plant length, the leaf area and the chlorophyll content are increased, the oxidation resistance is improved, the wilt is reduced, and the physiological and biochemical indexes are improved. The YJD5-23 microbial inoculum improves the property and activity of saline alkali soil and improves the abundance and composition of microbial communities. Detection shows that the YJD5-23 strain has the capabilities of secreting IAA, ACC deaminase and siderophore, dissolving phosphorus, fixing nitrogen and dissolving potassium. The indexes show that the YJD5-23 microbial inoculum has the potential of growth-promoting bacteria and has important application value in the field of agriculture.

Description

Technical Field

[0001] This invention relates to the field of bio-agriculture, specifically to a Bacillus strain and its application in improving plant stress resistance. Background Technology

[0002] Plants face both biotic and abiotic stresses in their natural environment, with salinity stress becoming one of the major abiotic stresses threatening sustainable crop production globally. Human activities combined with climate change have exacerbated soil salinization. Excessive salt concentrations, on the one hand, cause clay swelling and aggregate disintegration through the accumulation of exchangeable Na⁺ and high pH, ​​leading to decreased soil permeability, water holding capacity, and infiltration rate; on the other hand, they trigger ion imbalances, osmotic stress, reactive oxygen species (ROS) bursts, hindered nutrient mobilization, and hormonal imbalances within plants, ultimately inhibiting crop growth and significantly reducing yield. Furthermore, salinity also disrupts soil microbial diversity and weakens soil health.

[0003] Bacillus sp., widely distributed in the rhizosphere and roots, is an important member of the plant microbiome. It possesses characteristics such as salt tolerance, drought tolerance, acid and alkali tolerance, and the ability to form heat-resistant spores, enabling it to survive long-term and recover rapidly under adverse conditions. Numerous studies have shown that Bacillus sp. can alleviate the toxicity of salt stress on plants and improve crop salt tolerance and yield through multiple mechanisms, including phosphorus and potassium solubilization, production of plant hormones (such as IAA and cytokinins), induction of systemic resistance (ISR), and synthesis of antibacterial active substances.

[0004] *Peribacillus frigoritolerans*, a representative strain of the genus *Peribacillus* in the family Bacillusaceae, possesses the dual advantages of both low-temperature tolerance and salt-alkali tolerance. This bacterium maintains high survival rates and metabolic activity under conditions of 0–10℃, pH 8.5–10.0, and NaCl concentrations ≥8%. Its cold-adaptive extracellular polysaccharides, osmotically compatible solutes (such as proline and betaine), and antioxidant enzyme systems (SOD, CAT) significantly reduce salt stress-induced ROS accumulation, maintaining osmotic balance and membrane integrity in plant cells. Furthermore, *Peribacillus frigoritolerans* can colonize the root surface and endodermis of plants, enhancing plant adaptability to saline-alkali environments and nutrient acquisition by inducing the expression of host salt-tolerance genes (such as SOS1 and NHX1), promoting root auxin synthesis and root hair proliferation. Therefore, exploring and utilizing cold-resistant Bacillus subtilis resources is of great theoretical and practical significance for achieving green and sustainable agricultural production in areas under the dual stress of seasonal low temperatures and soil salinization. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to improve the normal growth of plants in saline-alkali land, increase plant yield and biomass, and enhance plant resistance.

[0006] To solve the above-mentioned technical problems, the present invention first screened a cold-resistant strain of *Bacillus subtilis*.

[0007] The cold-resistant *Ligusticum striatum* provided in this invention is *Ligusticum striatum* CGMCC No. 36069, which is registered with the China General Microbiological Culture Collection Center under the China General Microbiological Culture Collection Center as CGMCC No. 36069.

[0008] The present invention also provides a microbial agent.

[0009] The bacterial agent provided by the present invention contains the above-mentioned cold-resistant Bacillus subtilis and / or the metabolites of the cold-resistant Bacillus subtilis.

[0010] In the aforementioned microbial agent, in addition to the active ingredient, a carrier is also included. The carrier can be a commonly used and biologically inert carrier in the pesticide field. The carrier can be a solid or liquid carrier; the solid carrier can be a mineral material, plant material, or polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0011] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0012] 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 bacterial agent.

[0013] In a specific embodiment of the present invention, the bacterial agent is 2×10 8 The fermentation broth of the cold-resistant Peribacillus frigoritolerans YJD5-23 at CFU / mL was further prepared by a method including the following steps: inoculating the cold-resistant Peribacillus frigoritolerans YJD5-23 into a bacterial culture medium and culturing it to obtain OD. 600 The bacterial solution with a value of 0.5-1.0 (e.g., 1.0) (2×10⁸ CFU / mL) is the bacterial agent.

[0014] Furthermore, the bacterial culture medium is LB liquid medium.

[0015] In the preparation method of the bacterial agent, the culture conditions may be: 25-35℃ (e.g., 25-30℃, 30-35℃, 25℃, 30℃ or 35℃), 100-150 r / min (e.g., 100-130 r / min, 130-150 r / min, 100 r / min, 130 r / min or 150 r / min) for 30-60 h (e.g., 30-48 h, 48-60 h, 30 h, 48 h or 60 h).

[0016] 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, antioxidants, hormones, alkaloids, and toxins.

[0017] In the above text, the metabolites can be obtained from the fermentation broth of the *Bacillus frostbite*. The metabolites can be sterile metabolites of the *Bacillus frostbite* or bacterial metabolites of the *Bacillus frostbite*. Specifically, the sterile metabolites of the *Bacillus frostbite* (sterile fermentation filtrate) can be prepared by culturing the *Bacillus frostbite* in a liquid culture medium and filtering to remove the *Bacillus frostbite* from the liquid culture (fermentation broth). Specifically, the bacterial metabolites of the *Bacillus frostbite* can be prepared by culturing the *Bacillus frostbite* in a liquid fermentation medium and collecting the fermentation broth, which is the bacterial metabolite of the *Bacillus frostbite*.

[0018] The present invention also provides a culture of the above-mentioned cold-resistant Bacillus subtilis, which is a substance obtained by culturing the above-mentioned cold-resistant Bacillus subtilis in a bacterial culture medium.

[0019] The term "culture" refers to a liquid or solid product (all substances within the culture container) that has grown a microbial community after artificial inoculation and cultivation. It is a 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.

[0020] The use of the above-mentioned cold-resistant Bacillus subtilis, cold-resistant Bacillus subtilis inoculum, or cold-resistant Bacillus subtilis culture in at least one of the following is also within the scope of protection of this invention.

[0021] The above applications can specifically be any of the following:

[0022] (a1) Enhances the salt and alkali resistance of plants;

[0023] (a2) To prepare products that enhance the salt and alkali resistance of plants;

[0024] (a3) Promotes plant growth;

[0025] (a4) Prepare products that promote plant growth;

[0026] (a5) Produces IAA;

[0027] (a6) Prepare products that produce IAA;

[0028] (a7) Produces ACC deaminase;

[0029] (a8) Prepare products that produce ACC deaminase;

[0030] (a9) produces heparin;

[0031] (a10) Preparation of products producing ferrophiles

[0032] (a11) Phosphorus dissolution;

[0033] (a12) Preparation of phosphorus-soluble products;

[0034] (a13) Nitrogen fixation;

[0035] (a14) Preparation of nitrogen-fixing products;

[0036] (a15) Formation of biofilms;

[0037] (a16) Products for preparing biofilms.

[0038] In the above applications, the promotion of plant growth is manifested in all or part of the following:

[0039] (b1) Promotes root elongation in plants under saline-alkali stress or non-saline-alkali stress conditions;

[0040] (b2) Promotes the increase of plant dry weight under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0041] (b3) Promotes the increase of plant fresh weight under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0042] (b4) Promote plant growth under saline-alkali stress or non-saline-alkali stress conditions;

[0043] (b5) Promotes increased leaf length in plants under saline-alkali stress or non-saline-alkali stress conditions;

[0044] (b6) Promotes an increase in plant leaf width under saline-alkali stress or non-saline-alkali stress conditions;

[0045] (b7) Reduce leaf wilting in plants under saline-alkali stress or non-saline-alkali stress conditions;

[0046] (b8) Promotes an increase in chlorophyll content in plants under saline-alkali stress or non-saline-alkali stress conditions;

[0047] (b9) Promotes a decrease in proline content in plants under saline-alkali stress or non-saline-alkali stress conditions;

[0048] (b10) Promotes the reduction of malondialdehyde content in plants under saline-alkali stress or non-saline-alkali stress conditions;

[0049] (b11) Promotes the increase of plant antioxidant enzyme activity under saline-alkali stress or non-saline-alkali stress conditions;

[0050] The present invention also provides a method for preparing the above-mentioned bacterial agent, comprising the following steps: using the above-mentioned cold-resistant Bacillus subtilis as the active ingredient to obtain the bacterial agent.

[0051] The present invention also provides a bio-organic fertilizer containing the above-mentioned cold-resistant Bacillus subtilis or bacterial agent or the above-mentioned culture.

[0052] The present invention also provides a method for promoting plant growth.

[0053] The present invention also provides a method for promoting plant growth, which may include the following steps: applying the above-mentioned cold-resistant Bacillus subtilis or the above-mentioned inoculum or the above-mentioned culture to the test plant under saline-alkali stress conditions or non-saline-alkali stress conditions, thereby promoting plant growth.

[0054] The application can be done by dripping.

[0055] The non-saline-alkali stress conditions can be, for example, under normal growth conditions (without any stress). In a specific embodiment of the present invention, water treatment is used as the non-saline-alkali stress condition.

[0056] In the above application or method, the plant can be a monocotyledonous plant or a dicotyledonous plant.

[0057] In the above application or method, the dicotyledonous plant can be a leguminous plant or a cruciferous plant.

[0058] Furthermore, the gramineous plant can be maize, the leguminous plant can be alfalfa or soybean, and the cruciferous plant can be Arabidopsis thaliana.

[0059] In a specific embodiment of the present invention, the plant is specifically the wild-type Arabidopsis thaliana Columbia-0 subtype. In another specific embodiment of the present invention, the plant is specifically the commercially available soybean Qianjindou, the plant is specifically the maize inbred line B73. In yet another specific embodiment of the present invention, the plant is specifically the alfalfa variety Longmu 806.

[0060] Experimental results show that the cold-tolerant Peribacillus frigoritolerans YJD5-23 provided by the present invention can improve the resistance of Arabidopsis thaliana, alfalfa, maize and soybean to saline-alkali stress, specifically manifested as significant increases in dry weight, fresh weight and root length, increase in the height of the above-ground part (i.e., plant height increase), increase in leaf length and width, and changes in physiological and biochemical properties. Through biochemical experiments detecting YJD5-23 as a growth-promoting bacterium, it is proved that YJD5-23 has strong saline-alkali stress tolerance and has the ability to dissolve phosphorus, fix nitrogen, secrete H + , siderophores, IAA and ACC deaminase, as well as the ability to form biofilms. These indicators are important evidences that YJD5-23 has the potential to be a saline-alkali-tolerant growth-promoting bacterium. The above experiments show that the cold-tolerant Peribacillus frigoritolerans YJD5-23 has important application value in the field of saline-alkali soil remediation.

[0061] Preservation Instructions

[0062] Name of the strain: Cold-tolerant Peribacillus frigoritolerans

[0063] Latin name: Peribacillus frigoritolerans <0所000161>Strain number: YJD5-23

[0065] Depository: China General Microbiological Culture Collection Center

[0066] Abbreviation of the depository: CGMCC

[0067] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0068] Deposit date: September 25, 2025

[0069] CGMCC Registration Number: CGMCC No. 36069 Attached Figure Description

[0070] Figure 1 The results show the morphological and genus identification of the cold-resistant Bacillus repens strain YJD5-23. A shows the Gram staining results and cell morphology of YJD5-23; B shows the phylogenetic tree analysis.

[0071] Figure 2 The effect of YJD5-23 inoculant on the growth of Arabidopsis thaliana seedlings under carbonate treatment. The left side shows the alkaline solid culture medium; the right side shows the alkaline + inoculant solid culture medium.

[0072] Figure 3 The effect of YJD5-23 inoculant on the overall growth of alfalfa seedlings under alkaline stress was investigated. Group 1 was the water control group; group 2 was the water + inoculant group; group 3 was the alkaline solution control group; and group 4 was the alkaline solution + inoculant group.

[0073] Figure 4 The effects of YJD5-23 inoculant on the physiological and biochemical parameters of alfalfa seedlings under alkaline stress were investigated. A represents plant height (aboveground part); B represents root length; C represents average fresh weight; and D represents leaf area.

[0074] Figure 5 The effect of YJD5-23 inoculant on the antioxidant enzyme activity of alfalfa seedlings under alkaline stress. A represents MDA content; B represents total chlorophyll content; C represents proline content; D represents SOD activity; E represents POD activity; F represents CAT activity; and G represents APX activity.

[0075] Figure 6 The study investigated the effects of YJD5-23 microbial agent on soybean seedling growth under alkaline stress. Group 1 represented a water control group; group 2 represented water plus microbial agent; group 3 represented an alkaline solution control group; and group 4 represented an alkaline solution plus microbial agent.

[0076] Figure 7 The effects of YJD5-23 inoculant on the physiological and biochemical parameters of soybean seedlings under alkaline stress. A represents plant height; B represents root length; C represents fresh weight.

[0077] Figure 8 The effect of YJD5-23 microbial agent on maize seedling growth under alkaline stress was investigated. A shows the phenotype of maize seedlings under various treatments; B shows the overall growth of maize seedlings under various treatments. 1 represents the water control group; 2 represents the water + microbial agent group; 3 represents the alkaline solution control group; and 4 represents the alkaline solution + microbial agent group.

[0078] Figure 9 The effects of YJD5-23 inoculant on physiological and biochemical parameters of maize seedlings under alkaline stress. A represents plant height (aboveground part); B represents root length; C represents fresh weight.

[0079] Figure 10 The results show the pH range of YJD5-23 and its tolerance to NaHCO3 and NaCl. A shows the proliferation ability of YJD5-23 under different pH conditions; B shows the proliferation ability of YJD5-23 under different NaHCO3 concentrations; C shows the proliferation ability of YJD5-23 under different NaCl concentrations.

[0080] Figure 11 For YJD5-23 strain to secrete H + Ability test results.

[0081] Figure 12 The results show the ability of strain YJD5-23 to secrete IAA.

[0082] Figure 13 The results show the ACC deaminase secretion capacity of strain YJD5-23.

[0083] Figure 14 The results show the siderophore secretion ability of strain YJD5-23.

[0084] Figure 15 The results show the phosphorus solubility of strain YJD5-23.

[0085] Figure 16 The results show the nitrogen fixation capacity of strain YJD5-23.

[0086] Figure 17 Results of the detection of the biofilm formation ability of strain YJD5-23.

[0087] Figure 18 Results of potassium solubilization ability test of strain YJD5-23 Detailed Implementation

[0088] 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.

[0089] 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.

[0090] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0091] LB liquid medium: 10 g tryptone, 5 g yeast extract, 30 g sodium chloride, dissolved in distilled water, pH adjusted to 8.0, brought to a final volume of 1 L, sterilized at 121℃ for 15 min, and cooled for later use.

[0092] LB solid medium: Add agar to LB liquid medium to a final concentration of 15 g / L, sterilize at 121°C for 15 min. Cool to approximately 55°C, pour into petri dishes, and cool until solidified before use.

[0093] The wild-type Arabidopsis thaliana Columbia-0 subtype is described in the following literature: Kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim JA. Genetic link between cold responses and flowering time through FVE in Arabidopsis thaliana. Nature Genetics. 2004, 36:167-171. The wild-type Arabidopsis thaliana Columbia-0 subtype will be referred to as Arabidopsis thaliana in the following text.

[0094] Example 1: Isolation, identification, and preservation of cold-resistant Peribacillus frigoritolerans YJD5-23

[0095] I. Separation of YJD5-23

[0096] 1. Add 5 g of plant rhizosphere soil sample to 45 mL of sterile distilled water, stir for 15 min, let stand for 10 min, then take 1 mL of the supernatant and add it to a sterile test tube containing 9 mL of sterile water and mix thoroughly (the dilution at this point is recorded as 10). -1 Then, take 1 mL from this test tube and add it to another sterile test tube containing 9 mL of sterile water, mix well, and so on to make 10 mL. -2 10 -3 10 -4 10 -5 10 -6 10 -7 Different dilutions of bacterial suspension. Take 0.1 mL of each dilution and spread it evenly on LB solid medium. Incubate at 30°C for 2-3 days.

[0097] 2. After completing step 1, pick a single colony from the LB solid medium and purify it repeatedly at least 3 times to obtain a single strain. Name one of the strains YJD5-23.

[0098] II. Identification of YJD5-23

[0099] 1. Morphological identification

[0100] Gram staining:

[0101] ① Slide preparation: Take the bacterial culture, make a smear, dry and fix it according to routine procedures.

[0102] Gram staining should be performed using a culture of the actively growing strain YJD5-23; the smear should not be too thick to avoid incomplete decolorization and false positives; flame fixation should not be too hot (the slide should be warm to the touch).

[0103] ② Initial staining: Add crystal violet (just enough to cover the bacterial film) and stain for 1-2 minutes, then wash with water.

[0104] ③ Mordant: Rinse off the residual water with iodine solution, cover with iodine solution for about 1 minute, and then wash with water.

[0105] ④ Decolorization: Use filter paper to absorb the residual water on the slide, tilt the slide, and against a white background, use a dropper to add 95% ethanol to decolorize until the ethanol that flows out is no longer purple. Then immediately wash with water.

[0106] Ethanol destaining is a crucial step in Gram staining: insufficient destaining will cause Gram-negative bacteria to be mistakenly stained as Gram-positive bacteria; excessive destaining will cause Gram-positive bacteria to be mistakenly stained as Gram-negative bacteria. Therefore, the destaining time is generally 20-30 seconds.

[0107] ⑤ Counterstain: Counterstain with safranin solution for about 2 minutes, then rinse with water.

[0108] ⑥ Microscopic examination: After drying, observe under an oil immersion microscope. Gram-positive bacteria will be stained blue-purple, while Gram-negative bacteria will be stained red.

[0109] Gram staining of strain YJD5-23 yielded the following results: Figure 1 A is identified as a Gram-negative bacterium. The bacteria are 5-20 μm in length and are rod-shaped.

[0110] 2. Homology analysis of 16S rDNA sequences

[0111] The specific analysis steps are as follows:

[0112] (1) Bacterial genome extraction

[0113] (2) Amplification of 16S rDNA sequence using specific primers

[0114] (3) Purification of PCR products

[0115] (4) DNA sequencing to obtain 16S rDNA sequence

[0116] (5) Obtain sample species information by comparing with known bacteria in the NCBI database.

[0117] (6) Select similar bacterial species sequences and construct a phylogenetic tree.

[0118] The 16S rDNA of strain YJD5-23 is shown in SEQ ID No. 1.

[0119] The double-stranded DNA molecule shown in SEQ ID No. 1 was compared with sequences in GenBank using Clustal X software, and a phylogenetic tree was constructed by combining this with 16S rRNA sequences from similar species. The results are as follows: Figure 1 B indicates that strain YJD5-23 has the highest homology (99.45%) with the cold-resistant Peribacillus frigoritolerans strain DSM 8801.

[0120] III. Preservation of the cold-resistant Bacillus subtilis strain YJD5-23

[0121] Based on the above morphological, physiological and biochemical characteristics and 16S rDNA sequence homology analysis results, the bacterium YJD5-23 isolated and purified in step one was identified as *Peribacillus frigoritolerans*. It was deposited on September 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 36069. Hereinafter referred to as *Peribacillus frigoritolerans* CGMCC No. 36069 or *Peribacillus frigoritolerans* YJD5-23.

[0122] Strain preservation: Single colonies of the cold-resistant Peribacillus frigoritolerans (YJD5-23) were inoculated into LB liquid medium and cultured at 30°C for 16 h to obtain a culture solution. One volume of the culture solution was mixed with one volume of 80% (v / v) glycerol aqueous solution and stored at -80°C.

[0123] Example 2: Preparation of cold-resistant Bacillus subtilis YJD5-23 inoculum

[0124] The cold-resistant Peribacillus frigoritolerans YJD5-23 strain, preserved at -80℃ in Example 1, was revived on LB solid medium. A single colony was picked and inoculated into a 500 mL Erlenmeyer flask containing 100 mL of LB liquid medium. The flask was incubated at 30℃ and 160 r / min for 24-36 h to obtain the OD. 600 A bacterial culture with a value of approximately 1.0 (2 × 10⁻⁶) 8 (CFU / mL). This bacterial solution is the prepared YJD5-23 bacterial agent.

[0125] Example 3: Application of cold-resistant Bacillus subtilis YJD5-23 in improving the salt-alkali stress resistance of Arabidopsis thaliana.

[0126] The petri dish measures 10 cm × 10 cm.

[0127] I. Preparation of Culture Medium

[0128] Alkaline solid culture medium: Adjust the pH of 1 / 2MS solid culture medium to 8.0, sterilize at 121℃ for 60 min, add NaHCO3 to the culture medium cooled to about 55℃ to a final concentration of 1.5 mM, then pour the culture medium into petri dishes (20-25 mL per petri dish), and allow it to cool naturally to obtain alkaline solid culture medium.

[0129] Alkali + bacterial agent (cold-resistant Bacillus repens YJD5-23) solid culture medium: Take a petri dish containing alkaline solid culture medium, stand it upright, and use a toothpick to apply a small amount of the cold-resistant Bacillus repens YJD5-23 bacterial agent prepared in Example 2 above to the surface of the culture medium in the lower quarter of the dish. Let it air dry naturally.

[0130] II. Effects of cold-resistant Bacillus subtilis YJD5-23 inoculum on Arabidopsis thaliana's resistance to salt-alkali stress

[0131] The cultivation conditions were: 22℃, 10 h light / 14 h dark, and a light intensity of 5000 Lx.

[0132] 1) Take Arabidopsis thaliana (wild type Arabidopsis thaliana Columbia-0 subtype) seeds, sterilize with 2.6% (v / v) sodium hypochlorite aqueous solution for 10 min, and then wash with sterile water 3-4 times.

[0133] 2) After completing step 1), sow Arabidopsis seeds on a solid culture medium (alkaline solid culture medium or alkali + inoculant solid culture medium) and vernalize at 4°C for three days. It is important to note that the seeds should be sown on approximately one-quarter of the alkali + inoculant solid culture medium, and the inoculant should be applied to approximately one-quarter of the opposite side, ensuring that the roots of the seedlings do not come into direct contact with the inoculant for 7-14 days (this experiment mainly studies the effect of inoculant secretions on Arabidopsis growth).

[0134] 3) The solid culture medium from step 2) was cultured vertically, and the growth and development phenotype of Arabidopsis thaliana was observed starting on day 7.

[0135] The phenotype of Arabidopsis thaliana after 12 days of growth is shown in the figure. Figure 2 (Left side: Alkaline solid culture medium; Right side: Alkaline + Bacterial Agent solid culture medium). Results showed that under salt-alkali stress, the average taproot length of Arabidopsis seedlings on solid culture medium coated with the cold-resistant Bacillus repens YJD5-23 was significantly higher than that on the control alkaline solid culture medium (CK). Furthermore, the number and length of lateral roots in seedlings treated with YJD5-23 were also significantly higher than those in the control group. Figure 2 The leaves of seedlings treated with the microbial agent were larger and greener than those of the control group.

[0136] 4) Use ImageJ software to measure and count the taproot length of Arabidopsis seedlings from step 3 (n=30, repeated 3 times).

[0137] The statistical results are shown in Table 1. Significance was evaluated by one-way ANOVA, with different letters indicating significant differences (the same applies to the following statistics).

[0138] Table 1. Statistics on taproot length of Arabidopsis thaliana

[0139] Types of solid culture media Average taproot length (cm) Alkaline solid culture medium 0.4505 Alkali + Bacterial Agent Solid Culture Medium 1.6941 ***

[0140] The results showed that under salt-alkali stress, the average taproot length of Arabidopsis seedlings on solid culture medium coated with the cold-resistant Bacillus repens YJD5-23 was significantly higher than that on the control solid culture medium (CK). This indicates that the cold-resistant Bacillus repens YJD5-23 can significantly promote Arabidopsis growth and improve its salt-alkali tolerance.

[0141] Example 4: Application of cold-resistant Bacillus subtilis YJD5-23 inoculum in improving alfalfa's resistance to salt and alkali stress.

[0142] Culture conditions: 28℃; 10 h light / 14 h dark; light intensity 5000 Lx.

[0143] Alkaline solution (30 mM, Na2CO3: NaHCO3=1:9): Dissolve 0.636 g Na2CO3 and 4.536 g NaHCO3 in 2000 mL of distilled water, adjust the pH to 8.0, and set aside.

[0144] 1) Sow alfalfa seeds in 8 large pots containing the same volume of nutrient soil (40 seeds per pot). After 6 days of cultivation, a total of about 252 alfalfa seedlings were obtained, with an average of about 32 seedlings per pot and a germination rate of about 78.8%.

[0145] 2) Divide the seedlings into two groups of 4 pots each. One group is thoroughly irrigated with 800 mL of 30 mM alkaline solution (thorough irrigation means that a large amount of liquid flows out from the bottom of the pot, and the treatment substance is penetrated as much as possible to remove the original impurities). The other group is thoroughly irrigated with 800 mL of distilled water. After that, the treatment is repeated every 7 days, with 500 mL each time, for a total of 3 times.

[0146] 3) Four pots of alfalfa seedlings watered with alkaline solution were randomly divided into two groups: an alkaline solution control group and an alkaline solution + microbial agent group, with two pots in each group. Four pots of alfalfa seedlings watered with distilled water were randomly divided into two groups: a water control group and a water + microbial agent group, with two pots in each group. The experiment is as follows:

[0147] After the first thorough irrigation of the alkali solution + bacterial agent and water + bacterial agent groups, 5 mL of YJD5-23 bacterial agent prepared in Example 2 was evenly dripped onto the roots of the alfalfa seedlings and cultured for 30 days; the water and alkali solution control groups were cultured under the same conditions for 30 days.

[0148] 4) After 7 days of cultivation, the survival rate was calculated. The survival rate of alfalfa seedlings in the water control group was about 70%, the survival rate of seedlings in the water + inoculant group was about 86.7%, the survival rate of seedlings in the alkali control group was about 53.5%, and the survival rate of seedlings in the alkali + inoculant group was about 80%.

[0149] 5) Observation and analysis of the growth and development phenotypes of alfalfa in each group.

[0150] Phenotypic results of alfalfa plants treated for 30 days are shown below. Figure 3 (1 is the water control group; 2 is the water + microbial agent group; 3 is the alkali solution control group; 4 is the alkali solution + microbial agent group). Significance was evaluated using one-way ANOVA. Statistical results showed that under both water and alkali treatment conditions, the 30-day-old alfalfa seedlings in the YJD5-23 microbial agent group were taller and had a higher survival rate than the control group. Figure 3 This indicates that applying YJD5-23 inoculant under normal and alkaline treatment conditions can significantly promote plant growth and development.

[0151] 6) Effects of YJD5-23 inoculant treatment on alfalfa seedling biomass under normal and carbonate stress conditions.

[0152] Under both water and alkali treatment conditions, the average plant height, root length, fresh weight, and leaf area of ​​alfalfa seedlings in the alkali + inoculant group treated with YJD5-23 inoculant were significantly higher than those in the alkali control group without YJD5-23 inoculant treatment after 30 days. Figure 4 AD).

[0153] 7) Effects of YJD5-23 inoculant treatment on physiological indicators of alfalfa seedlings under normal and carbonate stress conditions.

[0154] The contents of malondialdehyde (MDA), total chlorophyll, and proline, as well as the activities of peroxidase (POD), superoxide dismutase (SOD), and ascorbate peroxidase (APX) in alfalfa leaves treated in 4) after 30 days were detected.

[0155] Principle: When plants are subjected to abiotic stress (such as alkali stress), the level of reactive oxygen species (ROS) rises sharply. ROS have strong oxidizing power and can damage the structure of macromolecules in cells. Therefore, excessive accumulation of ROS inevitably damages cells. For example, it causes peroxidation of cell membrane lipids, producing malondialdehyde (MDA). MDA content is an important parameter reflecting the body's antioxidant potential, indicating the rate and intensity of lipid peroxidation and indirectly reflecting the degree of tissue oxidative damage. Proline plays an important role in regulating cellular osmotic pressure and redox potential; its content reflects the degree of cellular stress. Chlorophyll content is an important indicator of plant photosynthetic capacity and growth status, commonly used to characterize plant growth. Antioxidant enzymes can convert excess ROS in plants into less toxic or harmless substances, balancing the ROS level in the body. Oxidative stress is a state in which the production of free radicals exceeds the body's antioxidant capacity, causing oxidative damage to cells and tissues. Defense against oxidative stress mainly relies on the body's antioxidant system. Based on different scavenging mechanisms, antioxidant systems can be broadly divided into two systems: enzymatic antioxidant systems and non-enzymatic antioxidant systems. Each system contains a variety of substances, including large and small antioxidant molecules and enzymes, reflecting the total antioxidant capacity within that system.

[0156] Under alkaline treatment conditions, the malondialdehyde (MDA) content in alfalfa leaves increased significantly compared to the water control group. Figure 5 A) indicates increased oxidative damage to alfalfa; total chlorophyll content decreased significantly ( Figure 5 B indicates that photosynthesis in alfalfa is inhibited; proline content increased significantly ( Figure 5 C) indicates that alkaline stress causes osmotic stress in alfalfa, activating the synthesis of osmotic regulatory substances; antioxidant enzyme activity is significantly increased ( Figure 5 The results (DG) indicate that increased oxidative stress activated the antioxidant system in alfalfa. These results demonstrate that alkaline stress severely affects plant physiological processes and growth, activating the plant stress response.

[0157] Under normal and alkaline treatment conditions, the malondialdehyde (MDA) content in alfalfa leaves significantly decreased after application of YJD5-23 inoculant, and was lower than that in their respective control groups. Figure 5 A) indicates that YJD5-23 inoculant significantly reduced oxidative stress; total chlorophyll content significantly increased ( Figure 5 B) indicates that the YJD5-23 bacterial agent has a protective effect on the photosynthetic system and can improve photosynthetic efficiency; the proline content increased significantly ( Figure 5 C), indicating that YJD5-23 bacterial agent can activate the biosynthesis of osmotic regulators and improve environmental adaptability; the activities of antioxidant enzymes were significantly increased ( Figure 5 The presence of DG indicates that the YJD5-23 microbial agent activates the antioxidant system, enhances the plant's antioxidant capacity, and thus alleviates oxidative damage caused by reactive oxygen species.

[0158] The above results demonstrate that under normal and alkaline stress conditions, the application of cold-resistant Bacillus subtilis YJD5-23 inoculant can significantly promote plant growth and enhance the plant's resistance to alkaline stress.

[0159] Example 5: Application of cold-resistant Bacillus subtilis YJD5-23 in improving the salt-alkali stress resistance of soybeans

[0160] Alkaline solution (80 mM, Na2CO3: NaHCO3=1:9): Dissolve 2.544 g Na2CO3 and 18.144 g NaHCO3 in 3000 mL of distilled water for later use.

[0161] Soybean seedling cultivation conditions: 22℃; 10 h light / 14 h dark; light intensity of 5000 Lx.

[0162] 1) Sow soybean seeds in 8 large pots containing the same volume of nutrient soil (30 seeds per pot), and divide them into two groups. One group is thoroughly irrigated with 800 mL of 80 mM alkaline solution per pot, and the other group is thoroughly irrigated with 800 mL of distilled water per pot. Then treat once every 7 days, with 500 mL each time, for a total of 3 times.

[0163] 2) After the first thorough irrigation of the water + bacterial agent and alkali solution + bacterial agent groups, the YJD5-23 bacterial agent (OD) prepared in Example 2 was evenly dripped onto each pot of soybean seeds. 600 =1), totaling 3 mL, cultured for 21 days. The water control group and the alkaline control group were cultured under the same conditions for 21 days.

[0164] 3) Germination rate was calculated after 7 days of cultivation. The germination rate of soybean seedlings in the water control group was approximately 58%, the germination rate of seedlings in the water + bacterial agent group was approximately 62%, the germination rate of seedlings in the alkali solution control group was approximately 36%, and the germination rate of seedlings in the alkali solution + bacterial agent group was 48%.

[0165] 4) Observation and analysis of the growth and development phenotypes of soybean seedlings in each group.

[0166] Phenotypic characteristics of soybean seedlings at 21 days of age are shown below. Figure 6(1 is the water control group, 2 is the water + microbial agent group, 3 is the alkali control group, and 4 is the alkali + microbial agent group). Under normal conditions, application of YJD5-23 microbial agent promoted the growth of soybean seedlings. Under alkali treatment conditions, the growth of soybean seedlings was inhibited, while the soybean seedlings in the alkali + microbial agent group treated with YJD5-23 were taller, had larger and greener leaves, and had longer roots.

[0167] 5) Effects of YJD5-23 inoculant treatment on soybean seedling biomass under normal and alkaline stress conditions.

[0168] Statistical results showed that under normal conditions, the average plant height, root length, and fresh weight of soybean seedlings treated with YJD5-23 inoculant were significantly higher than those in the water control group. Under alkaline treatment, the average plant height, root length, and fresh weight of soybean seedlings were significantly lower than those in the water control group, while these indicators of seedlings in the alkaline + inoculant group treated with YJD5-23 inoculant were significantly higher. Figure 7 AC).

[0169] The above results indicate that under both normal and alkaline conditions, YJD5-23 inoculant can significantly promote the growth and development of soybean seedlings.

[0170] Example 6: Application of cold-resistant Bacillus subtilis YJD5-23 in improving maize's resistance to salt and alkali stress

[0171] Alkaline solution (80 mM, Na2CO3: NaHCO3=1:9): Dissolve 2.544 g Na2CO3 and 18.144 g NaHCO3 in 3000 mL of distilled water for later use.

[0172] Maize seedling cultivation conditions: 22℃; 10 h light / 14 h dark; light intensity of 5000 Lx.

[0173] 1) Sow corn seeds in 8 large pots containing the same volume of nutrient soil (30 seeds per pot), and divide them into two groups. One group is thoroughly irrigated with 800 mL of 80 mM alkaline solution (the solution is considered effective when a large amount of liquid flows out from the bottom of the pot), and the other group is thoroughly irrigated with 800 mL of distilled water. Then, treat once every 7 days, with 500 mL each time, for a total of 3 times.

[0174] 2) After the first thorough irrigation of the water + bacterial agent and alkali solution + bacterial agent groups, the YJD5-23 bacterial agent (OD) prepared in Example 2 was evenly dripped onto each pot of corn seeds. 600 =1), a total of 3 mL, and cultured for 21 days; the water control group and the alkaline control group were cultured under the same conditions for 21 days.

[0175] 3) Germination rate was calculated after 7 days of cultivation. The germination rate of corn seedlings in the water control group was about 73.3%, the germination rate of seedlings in the water + bacterial agent group was 83.3%, the germination rate of seedlings in the alkali solution control group was about 68.3%, and the germination rate of seedlings in the alkali solution + bacterial agent group was about 75%.

[0176] 4) Observation and analysis of the growth and development phenotypes of corn seedlings in each group.

[0177] Phenotypic characteristics of maize seedlings at 21 days of growth are shown below. Figure 8 (1 is the water control group, 2 is the water + bacterial agent group, 3 is the alkali control group, and 4 is the alkali + bacterial agent group). It can be observed that under normal conditions, application of YJD5-23 bacterial agent significantly promoted the growth of maize seedlings. Under alkali treatment conditions, the growth of maize seedlings was severely inhibited, while the growth of maize seedlings in the alkali + bacterial agent group treated with YJD5-23 was significantly promoted.

[0178] 5) Effects of YJD5-23 inoculant treatment on the biomass of maize seedlings under normal and alkaline stress conditions.

[0179] Statistical results showed that under normal conditions, the average plant height, root length, and fresh weight of maize seedlings treated with YJD5-23 inoculant were significantly higher than those of the water control group without YJD5-23 inoculant. Under alkaline treatment, the average plant height, root length, and fresh weight of maize seedlings were significantly lower than those of the water control group; however, these indicators of seedlings in the alkaline + inoculant group treated with YJD5-23 inoculant were significantly higher. Figure 9 (AC) indicates that applying YJD5-23 inoculant under normal and alkaline treatment conditions can significantly promote the growth of maize seedlings.

[0180] In summary, under normal and alkaline stress conditions, the application of YJD5-23 inoculant can significantly promote plant growth, and its growth-promoting effect is broad-spectrum.

[0181] Example 7: Determination of the pH tolerance range of cold-resistant Bacillus repens strain YJD5-23

[0182] Adjust the pH of the LB liquid medium to the range of 3-10 using hydrochloric acid or sodium hydroxide, and then add 1 mL of YJD5-23 bacterial suspension (OD500) to each 100 mL of LB liquid medium. 600 =1) Incubate the cells, and measure the OD using a spectrophotometer every 2 hours. 600 The culture was carried out for 18 hours. The results showed that the cold-resistant Bacillus subtilis YJD5-23 strain had the lowest proliferation ability at pH 3, the strongest proliferation ability at pH 6-8, slightly lower proliferation ability at pH 5 and pH 9 than at pH 6-8, and lower proliferation ability at pH 4 and pH 10 than at pH 5 and pH 9. Figure 10A). These results indicate that strain YJD5-23 has a wide pH adaptation range.

[0183] Example 8: Analysis of the tolerance of cold-resistant Bacillus repens strain YJD5-23 to alkaline and neutral salts.

[0184] Add NaHCO3 to 100 mL of LB liquid medium to final concentrations of 0 mM, 30 mM, 60 mM, 90 mM, and 120 mM, then add 1 mL of YJD5-23 bacterial suspension (OD) to each concentration. 600 =1) Incubate the cells, and measure the OD using a spectrophotometer every 2 hours. 600 The culture was carried out for 18 hours. The results showed that the cold-resistant *Bacillus subtilis* YJD5-23 exhibited the best proliferation ability in the control solution (0 mM NaHCO3). As the NaHCO3 concentration increased, the proliferation ability of strain YJD5 gradually decreased, but it still maintained a certain growth rate and survived well even at 120 mM. Figure 10 B) indicates that strain YJD5-23 has a high ability to resist alkaline salt stress.

[0185] Add NaCl to 100 mL of LB liquid medium to final concentrations of 0 M, 0.8 M, 1.2 M, 1.6 M, and 2.0 M. Then add 1 mL of YJD5-23 bacterial suspension (OD) to each. 600 =1) Incubate the cells, and measure the OD using a spectrophotometer every 12 h. 600 The values ​​were determined by incubation for 12 or 24 hours. The results showed that the cold-resistant Bacillus subtilis YJD5-23 strain could still survive in a high NaCl solution of 1.6 M, but could not survive at a NaCl concentration of 2.0 M. Figure 10 C). This indicates that strain YJD5-23 has a high resistance to neutral salt stress.

[0186] Example 9: Cold-resistant Bacillus repens YJD5-23 secretes H + Capability testing

[0187] It has been reported that most plant growth-promoting bacteria have the ability to secrete organic acids. The secretion of H by the cold-resistant Bacillus subtilis strain YJD5-23 is also noted. + The capabilities were measured.

[0188] Adjust the pH of the LB liquid medium to 8, then add 1 mL of YJD5-23 bacterial suspension (OD) to 50 mL of pH 8 LB liquid medium. 600 =1) Incubate the cells and measure the OD using a spectrophotometer every 1.5 h. 600The culture medium was incubated for 4.5 hours. The results showed that after 4.5 hours of incubation, the pH of the culture medium rapidly decreased to below 6.5. Figure 11 This indicates that strain YJD5-23 has a strong ability to secrete hydrogen ions.

[0189] Example 10: Detection of the ability of cold-resistant *Bacillus repens* YJD5-23 to secrete indoleacetic acid (IAA)

[0190] It has been reported that plant rhizosphere growth-promoting bacteria can generally secrete auxin (IAA) to promote plant growth. The IAA secretion capacity of the cold-resistant Bacillus subtilis strain YJD5-23 was determined.

[0191] Salkowski colorimetric solution (250 mL): 150 mL concentrated H₂SO₄, 37.5 mL 0.5 M FeCl₃, diluted to volume with distilled water.

[0192] The cold-resistant Bacillus subtilis strain YJD5-23 was inoculated into 1 mL of LB liquid medium containing 200 mg / L L-tryptophan (L-Try), with a strain that could not produce IAA used as a negative control. After incubation at 30℃ and 180 r / min for 36–48 h, 200 μL of bacterial suspension (OD) was collected. 600 =1) Transfer the solution to a 48-well plate, add 800 μL of Salkowski chromogenic solution, and incubate at room temperature in the dark for 20 min. Observe the plate; a red color indicates a positive result, signifying that strain YJD5-23 has the ability to secrete IAA. Using IAA as a standard sample, the IAA standard curve equation was obtained as: y = 0.0142x + 0.0325 (R² = 0.9925). Quantitative detection results showed that when the tryptophan concentration was 500 ug / ml, the IAA synthesis reached 13.047 mg / ml·OD. 600 ( Figure 12 This indicates that the cold-resistant Bacillus repens YJD5-23 has a strong IAA secretion capacity.

[0193] Example 11. Analysis of the growth-promoting characteristics of cold-resistant *Bacillus subtilis* YJD5-23—its ability to secrete 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase.

[0194] Plants produce large amounts of ethylene when under stress. High concentrations of ethylene can inhibit plant growth. Some microorganisms contain ACC deaminase, which can inhibit the production of α-butanol, a precursor to ethylene, thereby reducing ethylene production and its inhibitory effect on plant growth. The secretion capacity of ACC deaminase in the cold-resistant Bacillus subtilis strain YJD5-23 was determined.

[0195] DF medium (1 L): Component a 0.1 mL, Component b 0.1 mL, KH2PO4 4.0 g, Na2HPO4 6.0 g, MgSO4·7H2O 0.2 g, glucose 2.0 g, 50% D-gluconic acid solution 4 mL, citric acid 2.0 g, (NH4)2SO4 2.0 g, deionized water to volume, pH 7.0-7.2.

[0196] Component a (100 mL): H3BO3 10 mg, MnSO4·H2O 11.19 mg, ZnSO4·7H2O 124.6 mg, CuSO4·5H2O 78.22 mg, MoO3 10 mg, and deionized water to a final volume.

[0197] Component b (10 mL): 100 mg FeSO4·7H2O, diluted to volume with deionized water.

[0198] ADF medium (1 L): Replace (NH4)2SO4 in DF medium with 0.5 M 1-aminocyclopropane-1-carboxylic acid (ACC).

[0199] A small amount of the cold-resistant Bacillus subtilis strain YJD5-23 was suspended in 10 μL of sterile water. The bacterial suspension was then inoculated onto DF solid medium (DF liquid medium with 10% added agar). After culturing for 3-4 days, the strain was transferred to ADF solid medium (ADF liquid medium with 1% added agar) with ACC as the sole nitrogen source. The plates were then placed in an incubator at 28°C and inverted for incubation. The growth of the strain was observed.

[0200] Using α-butanone (Shanghai Maclean Biochemical Technology Co., Ltd., product number: K835540-1g) as the standard sample, the standard curve equation was obtained as: y = 0.114x + 0.0584 (R² = 0.9737). Quantitative detection results showed that the ACC deaminase activity of strain YJD5-23 was 0.208 U / mg. Figure 13 This indicates that the cold-resistant Peribacillus frigoritolerans YJD5-23 has a strong ACC deaminase secretion capacity.

[0201] Example 12: Analysis of the growth-promoting characteristics of cold-resistant Bacillus repens YJD5-23—its ability to secrete siderophores.

[0202] CAS medium (250 mL): Add 0.2 mL of pre-sterilized 1 mM CaCl2, 4 mL of 1 mM MgSO4, 2 mL of 20% glucose, and 6 mL of 10% acid-hydrolyzed casein to 200 mL of sterilized medium d, mix well, and then add 37.8 mL of pre-sterilized staining solution c, mix well. Solution a: 0.024 g resazurite (CAS) dissolved in 20 mL of distilled water, then mixed with 4 mL of 1 mM FeCl3 solution. Solution b: 0.03 g hexadecyltrimethylammonium bromide dissolved in 16 mL of distilled water. Slowly add 24 mL of solution a to 16 mL of solution b, mix thoroughly to prepare staining solution c. Medium d (200 mL): 20 mL of 10 × MM9 salt solution, dissolve 6.04 g piperazine diethanolsulfonic acid in 150 mL of distilled water, mix well, adjust the pH to 6.8 with 50% NaOH solution, and add 4 g of agar.

[0203] MKB medium (100 mL): 0.5 g tyrosine, 1.5 mL glycerol, 0.25 g KH2PO4, 0.25 g MgSO4·7H2O, mix well, and adjust the pH to 7.2 with 50% NaOH solution.

[0204] A trace amount of the cold-resistant Bacillus subtilis strain YJD5-23 was suspended in 10 μL of sterile water. The bacterial suspension was inoculated into 5 mL of MKB liquid medium and cultured at 150 r / min and 30℃ for 48 h. The culture was centrifuged at 5000 rpm for 10 min, and 1 mL of the supernatant (reference value, Ar) was taken and mixed with 1 mL of MKB liquid medium. The mixture was then mixed with the CAS detection solution at a 1:1 ratio. After reacting at room temperature for 1 h, the OD value (A) was measured at 630 nm, using distilled water as a control. The relative content of ferroptamine was expressed as the A / Ar ratio; the smaller the ratio, the stronger the ferroptamine production capacity of the strain. Quantitative detection results showed that the ferroptamine secretion capacity of YJD5-23 was 1.333 A / Ar ( Figure 14 This indicates that the cold-resistant Peribacillus frigoritolerans YJD5-23 has a strong ability to secrete iron-phagocytic substances.

[0205] Example 13: Analysis of the phosphorus-solubilizing ability of cold-resistant Bacillus subtilis YJD5-23

[0206] Organic and inorganic phosphorus in soil play a crucial role in plant growth. In alkaline soils, inorganic phosphorus is mainly insoluble Ca3(PO4)2, which cannot be directly absorbed and utilized by plants, thus limiting their growth and development. Phosphorus-solubilizing bacteria can secrete organic acids to dissolve insoluble phosphorus-containing substances in the soil, converting them into phosphorus-containing substances that plants can utilize. If bacteria possess phosphorus-solubilizing ability, a transparent ring will appear around the colony after a period of cultivation on PKO medium (containing insoluble calcium phosphate).

[0207] PKO medium (1L): glucose 10.0 g, tricalcium phosphate 5.0 g, magnesium chloride 5 g, ammonium sulfate 0.1 g, potassium chloride 0.2 g, magnesium sulfate 0.25 g, agar 17 g, the remainder being water, pH 7.0.

[0208] The phosphorus solubilizing ability of strain YJD5-23 was quantitatively determined using the molybdenum-antimony colorimetric method. A standard curve was obtained using a phosphorus standard solution as the standard sample, calculated as: y = 0.183x + 0.2651 (R² = 0.9907). The results showed that the phosphorus solubilizing ability of strain YJD5-23 was 130.404 mg / L. Figure 15 This indicates that the cold-resistant Bacillus subtilis YJD5-23 has a strong phosphorus-solubilizing ability.

[0209] Example 14: Analysis of the growth-promoting characteristics—nitrogen fixation ability—of the cold-resistant Bacillus subtilis YJD5-23

[0210] When microorganisms grow in nitrogen-free culture media, if they possess nitrogen-fixing capabilities, they can convert atmospheric nitrogen (N2) into bioavailable nitrogen compounds (such as ammonia and amino acids). The nitrogen-fixing capacity of the strain can be indirectly reflected by measuring changes in the total nitrogen content of the fermentation broth. Alkaline potassium persulfate (K2S2O8) was used as a strong oxidant to oxidize organic nitrogen to nitrate nitrogen (NO3⁻-N) under high temperature and pressure. Nitrate ions absorb in the ultraviolet region (220 nm), and 275 nm was used to correct for background absorbance. The nitrogen concentration was finally calculated using a standard curve.

[0211] Ashby medium (1 L): KH2PO4 0.2 g, NaCl 0.2 g, MgSO4·7H2O 0.2 g, K2SO4·2H2O 0.2 g, CaCO3 5 g, glucose 5 g, mannitol 5 g, agar 10 g, pH 7.0, diluted to volume with deionized water.

[0212] A beaker containing 1000 mL of ammonia-free water was heated in a 50°C water bath. K₂S₂O₈ was then gradually added until it could not be dissolved. The completely dissolved saturated solution was then allowed to cool naturally at room temperature, followed by recrystallization in a 4°C refrigerator overnight. The next day, the supernatant was discarded, and the crystals were washed several times with ice-cold ammonia-free water. The crystals were then dried in a 50°C oven. This process was repeated four times, and the absorbance of a blank sample was measured after each recrystallization.

[0213] To prepare the standard curve, 0, 1, 3, 5, and 7 mL of KNO3 standard solution were placed in 25 mL containers, diluted to 10 mL with ammonia-free water, and then 5 mL of alkaline K2S2O8 solution was added. The containers were then capped, the mouths of the containers were tightly wrapped with aluminum foil, and digested at 121–124 °C for 45 min. After cooling, 1 mL of HCl solution was added to each container, and the solution was brought to volume with ammonia-free water. The results showed that within the mass range of 0–70 μg, the absorbance value had a good linear relationship with the mass of nitrogen, with the linear equation Y = 0.0104X – 0.0023 and R² = 0.9997.

[0214] Nitrogen fixation capacity determination: The fermentation broth of nitrogen-fixing bacteria fermented for 7 days was centrifuged at 10000 r / min for 15 min. 1 mL of the supernatant was transferred to a 25 mL volumetric flask, diluted to 10 mL with ammonia-free water, and 5 mL of alkaline K₂S₂O₈ (obtained by recrystallization) solution was added. The flask was tightly sealed, wrapped in aluminum foil, and digested at 121–124℃ for 45 min. After cooling, 1 mL of HCl solution was added, and the volume was adjusted to 10 mL with ammonia-free water. The absorbance at A220 nm and A275 nm was measured as a blank control. The results showed that the nitrogen fixation capacity of strain YJD5-23 was 19.022 mg / L. Figure 16 This indicates that the cold-resistant Peribacillus frigoritolerans YJD5-23 has a strong nitrogen-fixing ability.

[0215] Example 15: Analysis of the biofilm-forming ability of cold-resistant Bacillus repens YJD5-23

[0216] Add 100 μL of LB liquid medium to each well of a 96-well plate, and inoculate with 10 μL of overnight cultured YJD5-23 bacterial suspension. Incubate at 37°C for 36 h. Aspirate the bacterial suspension, add 200 μL of sterile PBS buffer to each well, and wash the wells three times. Add 100 μL of methanol to each well for fixation for 15 min, discard the methanol, and allow to air dry. Add 100 μL of 1% crystal violet solution to each well, incubate at room temperature for 5 min, discard unbound crystal violet staining solution, wash away excess staining solution with distilled water, and dry at 37°C or room temperature. Add 100 μL of 33% glacial acetic acid solution to each well, and incubate at 37°C for 30 min to dissolve the crystal violet. Measure the OD of the solution in the culture wells using a microplate reader. 590 Value. Using uninoculated culture medium as a negative control, twice the negative value was taken as the limit value. Figure 17 Quantitative experimental results showed that the cold-resistant Bacillus subtilis YJD5-23 has a strong ability to form biofilms.

[0217] Example 16: Analysis of the potassium-solubilizing ability of cold-resistant Bacillus subtilis YJD5-23

[0218] Sodium tetraphenylborate turbidimetric method: Under weakly alkaline conditions (pH 8–10), potassium ions (K⁺) in the sample react with sodium tetraphenylborate to form a sparingly soluble potassium tetraphenylborate precipitate: K + +NaB(C6H5)4→KB(C6H5)4↓+Na + After filtration, washing, drying, and weighing, the potassium content is calculated based on the mass of the precipitate. The linear equation is A = 0.0454ρ − 0.2291, where A is the absorbance (680 nm) and ρ is the mass concentration of the potassium standard solution (mg / L).

[0219] PDA medium (1 L): 6 g potato extract powder, 20 g glucose, pH 5.6±0.2.

[0220] Strain YJD5-23 was inoculated onto PDA solid medium, and the control group was inoculated with an equal volume of PDA solid medium. The cultures were incubated at 25℃ and 150 r / min with shaking for 10 days. 2 mL of fermentation broth was collected daily at the same time, centrifuged at 12,000 r / min for 5 min, and the supernatant was collected. The soluble potassium content in the supernatant was determined by the sodium tetraphenylborate turbidimetric method. The results showed that the maximum potassium solubilization capacity of YJD5-23 was 130.157 mg / 100 ml. Figure 18 ).

[0221] In summary, the cold-resistant *Bacillus cereus* YJD5-23 can survive over a wide pH range, exhibits strong salt and alkali tolerance, can produce organic acids, IAA, ACC deaminase, and iron phosphate, and possesses nitrogen-fixing and phosphorus-solubilizing capabilities, as well as the ability to form biofilms. These characteristics endow *Bacillus cereus* YJD5-23 with strong plant growth-promoting and stress-resistance abilities.

[0222] 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. Bacillus, characterized in that: The Bacillus species in question is Peribacillus frigoritolerans, strain number YJD5-23, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 36069.

2. The culture of Bacillus as described in claim 1 is a substance obtained by culturing the Bacillus as described in claim 1 in a microbial culture medium.

3. A microbial agent, characterized in that: The microbial agent contains the Bacillus of claim 1 and / or the metabolites of the Bacillus of claim 1 and / or the culture of claim 2.

4. An anti-salt and / or alkali stress agent, characterized in that, The salt and / or alkali stress resistant preparation contains the Bacillus of claim 1 or / and the metabolites of the Bacillus of claim 1 or / and the culture of claim 2 or / and the bacterial agent of claim 3.

5. Any one of the following applications of the Bacillus of claim 1, the metabolite of the Bacillus of claim 1, the culture of claim 2, the bacterial agent of claim 3, or the salt and / or alkali stress resistant agent of claim 4: The above applications can specifically be any of the following: (a1) Enhances the plant's resistance to salt and alkali; (a2) To prepare products that enhance the salt and alkali resistance of plants; (a3) Promotes plant growth; (a4) Prepare products that promote plant growth; (a5) Produces IAA; (a6) Prepare products that produce IAA; (a7) Produces ACC deaminase; (a8) Prepare products that produce ACC deaminase; (a9) produces heparin; (a10) Preparation of products producing ferrophiles (a11) Phosphorus dissolution; (a12) Preparation of phosphorus-soluble products; (a13) Nitrogen fixation; (a14) Preparation of nitrogen-fixing products; (a15) Biofilm; (a16) Products for preparing biofilms; In the above applications, the promotion of plant growth is manifested in all or part of the following: (b1) Promotes the increase of plant fresh weight under saline-alkali stress conditions or non-saline-alkali stress conditions; (b2) Promotes the increase of plant dry weight under saline-alkali stress conditions or non-saline-alkali stress conditions; (b3) Promotes root elongation in plants under saline-alkali stress or non-saline-alkali stress conditions; (b4) Promote plant growth under saline-alkali stress or non-saline-alkali stress conditions; (b5) Promotes increased leaf length in plants under saline-alkali stress or non-saline-alkali stress conditions; (b6) Promotes an increase in plant leaf width under saline-alkali stress or non-saline-alkali stress conditions; (b7) Reduce leaf wilting in plants under saline-alkali stress or non-saline-alkali stress conditions; (b8) Promotes an increase in chlorophyll content in plants under saline-alkali stress or non-saline-alkali stress conditions; (b9) Promotes a decrease in proline content in plants under saline-alkali stress or non-saline-alkali stress conditions; (b10) Promotes the reduction of malondialdehyde content in plants under saline-alkali stress or non-saline-alkali stress conditions; (b11) Promotes the increase of plant antioxidant enzyme activity under saline-alkali stress or non-saline-alkali stress conditions; (b12) Adjusting plant secondary metabolites under saline-alkali stress or non-saline-alkali stress conditions; (b13) Improve the vitality of saline-alkali soil and improve the physical and chemical properties of saline-alkali soil; (b14) Improve the abundance and composition of microbial communities in saline-alkali soils.

6. The application according to claim 5, characterized in that... The plant families mentioned are Brassicaceae, Poaceae, and Leguminosae. The plants mentioned are Brassicaceae, specifically a1), a2), or a3) below: a1) Brassicaceae; a2) Arabidopsis; a3) Arabidopsis; The grasses mentioned may be b1), b2), or b3): b1) Poaceae (grass family); b2) Oryza or Zea; b3) Rice or Maize; The legumes mentioned may be c1), c2), or c3): c1) Leguminosae; c2) Alfalfa or Glycine; c3) Alfalfa or Glycine.

7. The method for preparing the bacterial agent according to claim 3, comprising the following steps: using the Bacillus subtilis according to claim 1 as the active ingredient to obtain the bacterial agent.

8. A bio-organic fertilizer, characterized by: The bio-organic fertilizer contains the Bacillus spp. of claim 1, the culture of claim 2, or the microbial agent of claim 3.

9. The application according to any one of claims 5 or the method according to claim 8, characterized in that: The plant is a monocotyledonous plant or a dicotyledonous plant.

10. A method for improving plant stress resistance and / or promoting plant growth, characterized in that: The method includes treating plants or plant culture media with the Bacillus of claim 1, metabolites of the Bacillus of claim 1, the culture of claim 2, or the salt and / or alkali stress resistant agent of claim 5.

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