Microbial seed coating agent and pseudomonas bacteria used therein

Abstract

This invention discloses a microbial seed coating agent and the *Pseudomonas* strain used therein. This invention relates to the field of biotechnology and provides a strain of *Pseudomonas* found in saline-alkali soils. Pseudomonas alkalisoli D44, its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 36294. This strain is... Pseudomonas This is a novel strain capable of producing IAA (inorganic acid), which promotes plant growth under saline-alkali stress. Seed coating agents formulated with this strain can improve the germination rate of forage seeds and increase forage yield. The strain provided by this invention is of great significance for alleviating saline-alkali stress in plants and improving their adaptability to saline-alkali environments.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to microbial seed coating agents and the Pseudomonas bacteria used therein. Background Technology

[0002] Traditional methods of applying microbial inoculants are time-consuming, labor-intensive, and cumbersome, and are easily limited by environmental conditions and application timing, making efficient promotion difficult. In recent years, seed coating technology, which is easy to operate, has gradually become a research hotspot and application trend in the agricultural field.

[0003] Seed pelleting technology is a high-tech development based on seed coating. By mixing and processing seeds with specific additives, irregularly shaped and unevenly sized small seeds are transformed into standardized pellets of uniform size and shape. This significantly improves the rolling and flowability of seeds, facilitating machine sowing and aerial sowing operations. It is an effective way to achieve seed quality standardization, mechanized processing, cost reduction and efficiency improvement.

[0004] Alfalfa, known as the "king of forage," possesses extremely high economic and ecological value and is a key species for grassland degradation control and ecological restoration. However, alfalfa seeds are characterized by irregular shape, small size, and low thousand-seed weight (1.5-2.5g), making precise control difficult with conventional sowing methods. Uneven sowing often leads to time-consuming and labor-intensive thinning. Furthermore, in degraded grasslands severely stressed by wind erosion, salinity, and drought, seed germination and seedling survival rates are further limited, posing a severe challenge to large-scale mechanized and aerial seeding operations.

[0005] Currently, domestic alfalfa seed coating pelleting still mainly relies on traditional chemical coating pans, which easily cause seed damage during processing. Existing formulas mostly focus on physical shaping or conventional chemical agents, lacking targeted stress resistance and growth promotion functions. The pelleting effect is poor, and there are generally problems such as exposure, fragility, and poor transportability. Furthermore, there is a severe lack of specialized pelleting formulas for harsh environments such as saline-alkali and drought.

[0006] Meanwhile, existing growth-promoting microbial agents for alfalfa mostly use publicly available common strains. Their salt and alkali tolerance and growth-promoting effects are unstable in high-salt and alkali environments, and these strains have not been reported for alfalfa seed pelleting. Therefore, they cannot simultaneously solve the problems of precise alfalfa seed sowing, survival under adverse conditions, and efficient growth promotion.

[0007] Therefore, there is an urgent need in this field for a novel formulation adapted for alfalfa seed pelleting, especially one that combines previously unreported salt-tolerant and alkali-promoting microbial strains. This would enable standardized and mechanized seed sowing while improving seed germination rate and seedling survival rate under salt-alkali stress, thus providing technical support for the ecological restoration of degraded grasslands. Summary of the Invention

[0008] The purpose of this invention is to provide a microbial seed coating agent and the Pseudomonas bacteria used therein.

[0009] In a first aspect, the present invention claims protection for *Pseudomonas saline-alkali* or its progeny.

[0010] The *Pseudomonas saline-alkali* strain claimed in this invention is *Pseudomonas saline-alkali* ( Pseudomonas alkalisoli D44, its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 36294.

[0011] Pseudomonas aeruginosa ( Pseudomonas alkalisoli D44 is a Gram-negative bacterium. After 3 days of growth on TSA solid medium, the colonies are whitish-brown, round, raised, and easy to pick up, with a colony 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.

[0012] The term "offspring" refers to those produced by *Pseudomonas saline-alkali soil* (…). Pseudomonas alkalisoli The progeny strains of D44 obtained through artificial subculturing and natural propagation, as well as Pseudomonas aeruginosa (…), Pseudomonas alkalisoli Derivative strains obtained by mutagenesis, domestication, and genetic modification of D44 or its progeny all retain Pseudomonas saline-alkali soil bacteria ( Pseudomonas alkalisoli D44 original functional characteristics.

[0013] Secondly, the present invention claims protection for bacterial agents containing *Pseudomonas saline-alkali* or its progeny as described in the first aspect above.

[0014] The active ingredient of the bacterial agent is *Pseudomonas saline-alkali* or its progeny.

[0015] The bacterial agent may also contain cultures and / or metabolites of the aforementioned Pseudomonas saline-alkali or its progeny.

[0016] The culture is the substance obtained by culturing *Pseudomonas saline-alkali* or its progeny in a bacterial culture medium (all substances within the culture container, i.e., fermentation products; such as fermentation broth containing *Pseudomonas saline-alkali* or its progeny and substances secreted into a liquid culture medium, or solid fermentation product containing *Pseudomonas saline-alkali* or its progeny and substances secreted into a solid culture medium).

[0017] 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.

[0018] In the above-mentioned cultures, the bacterial culture medium can be a solid culture medium or a liquid culture medium.

[0019] 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.

[0020] In the aforementioned microbial agent, the active ingredient may be *Pseudomonas saline-alkali* or its progeny, metabolites of *Pseudomonas saline-alkali* or its progeny, and / or cultures of *Pseudomonas saline-alkali* or its progeny, 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 agent can be determined by those skilled in the art based on the desired effect.

[0021] 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.

[0022] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0023] 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.

[0024] In the bacterial agent, the *Pseudomonas saline-alkali* or its progeny and / or the metabolites of the *Pseudomonas saline-alkali* or its progeny may be present in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate.

[0025] In this article, the metabolites of *Pseudomonas saline-alkali* or its progeny can be obtained from the fermentation broth of *Pseudomonas saline-alkali* or its progeny. The metabolites of *Pseudomonas saline-alkali* or its progeny can be sterile metabolites of *Pseudomonas saline-alkali* or its progeny, or bacterial metabolites of *Pseudomonas saline-alkali* or its progeny. Specifically, the sterile metabolites of *Pseudomonas saline-alkali* or its progeny (sterile fermentation filtrate) can be prepared by the following method: culturing *Pseudomonas saline-alkali* or its progeny in a liquid culture medium, and filtering to remove the *Pseudomonas saline-alkali* or its progeny from the liquid culture (fermentation broth), thus obtaining the sterile metabolites of *Pseudomonas saline-alkali* or its progeny. The microbial metabolites of *Pseudomonas saline-alkali* or its progeny can be prepared by the following method: culturing *Pseudomonas saline-alkali* or its progeny in a liquid fermentation medium, and collecting the fermentation broth—containing *Pseudomonas saline-alkali* or its progeny and substances secreted into the liquid culture medium. This fermentation broth is the microbial metabolites of *Pseudomonas saline-alkali* or its progeny.

[0026] Furthermore, the microbial agent is a microbial agent for alleviating salt and alkali stress in plants.

[0027] Furthermore, the microbial agent is a microbial agent that promotes plant growth under salt-alkali stress conditions.

[0028] Furthermore, the microbial agent can be used as a microecological preparation, a bio-fertilizer, or a soil remediation agent.

[0029] Thirdly, the present invention claims protection for seed coating agents containing *Pseudomonas saline-alkali* or its progeny as described in the first aspect above, or the fungal agent as described in the second aspect above.

[0030] Furthermore, the seed coating agent contains *Pseudomonas saline-alkali* or its progeny and sodium alginate.

[0031] Furthermore, the seed coating agent may also contain one or more of biochar, rice husk powder, and superabsorbent resin.

[0032] In some embodiments of the present invention, the superabsorbent resin is sodium polyacrylate. The sodium polyacrylate has the following CAS number: 9003-04-7; molecular formula: (C3H3NaO2)n; EC number: 692-137-3; PubChem number: 4068533; purity ≥95%; and powder particle size 100-300 mesh.

[0033] In some embodiments of the present invention, the viscosity of the sodium alginate is 200±20 mPa·s.

[0034] In some embodiments of the present invention, the functional filler system of the seed coating agent is composed of biochar and rice husk powder, wherein the mass ratio of biochar to rice husk powder is 1:1. A superabsorbent resin is added at 10% of the total mass of the biochar-rice husk powder mixture, and the mixture is stirred again to obtain the composite filler. Further, using a microbial agent containing *Pseudomonas saline-alkali* or its progeny as a base, sodium alginate is added at 0.5% of the total mass of the microbial agent for thickening treatment, and then uniformly mixed with the above composite filler to obtain the seed coating agent.

[0035] Fourthly, this invention claims protection for any of the following applications: (A1) The application of *Pseudomonas saline-alkali* or its progeny described in the first aspect above, or the inoculant described in the second aspect above, or the seed coating agent described in the third aspect above, in alleviating plant salt-alkali stress; (A2) The application of *Pseudomonas saline-alkali* or its progeny described in the first aspect above, or the microbial agent described in the second aspect above, in the preparation of products for alleviating plant salt stress; (A3) The application of *Pseudomonas saline-alkali soils* or its progeny described in the first aspect above, or the inoculant described in the second aspect above, or the seed coating agent described in the third aspect above, in promoting plant growth; (A4) The application of *Pseudomonas saline-alkali* or its progeny described in the first aspect above, or the microbial agent described in the second aspect above, in the preparation of products for promoting plant growth; (A5) The application of *Pseudomonas saline-alkali* or its progeny described in the first aspect above, or the inoculant described in the second aspect above, or the seed coating agent described in the third aspect above, in improving the germination rate of plant seeds; (A6) The application of *Pseudomonas saline-alkali* or its progeny described in the first aspect above, or the inoculant described in the second aspect above, in the preparation of products for improving the germination rate of plant seeds; (A7) The application of *Pseudomonas saline-alkali soils* or its progeny described in the first aspect above, or the inoculant described in the second aspect above, or the seed coating agent described in the third aspect above, in improving plant yield; (A8) The application of *Pseudomonas saline-alkali* or its progeny described in the first aspect above, or the inoculant described in the second aspect above, in the preparation of products for increasing plant yield; (A9) The application of *Pseudomonas saline-alkali* or its progeny described in the first aspect above, or the inoculant described in the second aspect above, or the seed coating agent described in the third aspect above, in promoting nodulation in leguminous plants; (A10) The use of *Pseudomonas saline-alkali* or its progeny as described in the first aspect above, or the microbial agent as described in the second aspect above, in the preparation of products for promoting nodulation in leguminous plants.

[0036] Fifthly, the present invention claims a method for alleviating salt and alkali stress in plants.

[0037] The method for alleviating salt-alkali stress in plants claimed by this invention may include: applying *Pseudomonas salinis* or its progeny, as described in the first aspect above, or the microbial agent described in the second aspect above, to the plant to be treated or its cultivation substrate, so as to alleviate the salt-alkali stress in the plant.

[0038] The application methods may include seed soaking, seed coating treatment, root irrigation during the seedling stage, and soil inoculation with fungi.

[0039] In some embodiments of the present invention, specifically, before sowing, the plant seeds are soaked in a suspension of *Pseudomonas saline-alkali* (soaking time can be 0.5 h). Further, during the plant growth period, watering is performed every 2-3 days, and on the 15th day, the roots are treated with a root drench of the *Pseudomonas saline-alkali* suspension, 0.5 mL OD per plant. 600 =0.5 bacterial suspension.

[0040] In other embodiments of the present invention, the seeds of the plant to be treated are specifically coated using the seed coating agent described in the third aspect above.

[0041] Sixthly, the present invention also claims protection for any of the following methods: Method I: A method for promoting plant growth, comprising: applying *Pseudomonas saline-alkali* or its progeny, or the microbial agent described in the first aspect above, to the plant to be treated or its cultivation substrate, to achieve the promotion of plant growth.

[0042] Method II: A method for improving the germination rate of plant seeds, comprising: coating the seeds of the plant to be treated with the seed coating agent described in the third aspect above, so as to improve the germination rate of plant seeds.

[0043] Method III: A method for increasing plant yield, comprising: coating the seeds of the plant to be treated with the seed coating agent described in the third aspect above, so as to increase plant yield.

[0044] Method IV: A method for promoting nodulation in legumes, comprising: coating the seeds of the legume to be treated with the seed coating agent described in the third aspect above to promote nodulation in legumes.

[0045] Regarding the aforementioned aspects, in some embodiments of the present invention, the seed coating treatment of the plant to be treated is specifically carried out as follows: Biochar and rice husk powder are mixed at a mass ratio of 1:1, and then 10% of the total mass of the mixed powder with superabsorbent resin is added and mixed thoroughly to obtain a composite powder; sodium alginate is added to the microbial agent containing *Pseudomonas saline-alkali* or its progeny and stirred to obtain a liquid binder solution; wherein, the amount of sodium alginate added is 2 × 10⁻⁶ per 10⁻⁶ spores. 11 1g of sodium alginate was added to the *Pseudomonas saline-alkali* strain (bacterial count in 200mL bacterial suspension) in CFU. The seeds were then treated using a pelleting process: first, the liquid binder solution was sprayed onto the seed surface, then the composite powder was added in several batches, and this process was repeated to complete the seed coating and pelleting.

[0046] Among the aforementioned aspects, promoting plant growth can refer to promoting plant growth under salt-alkali stress conditions.

[0047] In the aforementioned aspects, improving the germination rate of plant seeds can refer to increasing the germination rate of plant seeds under saline-alkali stress conditions. In some embodiments of the present invention, improving the germination rate of plant seeds means that seeds treated with the seed coating agent described in the third aspect above have a higher germination rate compared to seeds treated with the control seed coating agent. The control seed coating agent does not contain the *Pseudomonas saline-alkali* bacteria, and its remaining composition is identical to that of the seed coating agent described in the third aspect above.

[0048] Among the aforementioned related aspects, increasing plant yield can refer to increasing plant yield under salt-alkali stress conditions.

[0049] Among the aforementioned related aspects, the promotion of nodulation in legumes can be described as promoting nodulation in legumes under saline-alkali stress conditions.

[0050] In some embodiments of the present invention, promoting plant growth under salt-alkali stress conditions means promoting the growth of the taproot length and / or promoting the increase of the dry weight of the underground parts of the plant under salt-alkali stress conditions.

[0051] In some embodiments of the present invention, increasing plant yield under salt-alkali stress conditions means promoting the growth of the taproot, increasing the aboveground biomass, and / or increasing the underground biomass of plants under salt-alkali stress conditions.

[0052] In all of the above-mentioned relevant aspects, the plant is any one of the following: (B1) Dicotyledonous plants; (B2) Legumes or Malvaceae; (B3) Plants of the genus *Alfalfa* or *Gossypium*; (B4) Alfalfa or cotton.

[0053] In some embodiments of the present invention, the plant is cotton. In other embodiments of the present invention, the plant is alfalfa.

[0054] In the aforementioned relevant aspects, the salt-alkali stress may be salt stress and / or alkali stress.

[0055] In some embodiments of this invention, a salt-alkali stress solution is specifically used to simulate salt-alkali stress. The salt-alkali stress solution is a mixture of NaCl, Na₂SO₄, and NaHCO₃ in a molar ratio of 1:1:1. + A mixed solution with a final concentration of 120-200 mmol / L, the natural pH of this salt-alkali stress solution is 8.6.

[0056] Experiments have shown that the *Pseudomonas aeruginosa* provided in this invention (…) Pseudomonas alkalisoli D44 is Pseudomonas This is a new species, possessing the ability to secrete IAA (intracellular acid). Pot experiments showed that under salt-alkali stress, compared with the uninoculated negative control group, the inoculation with *Pseudomonas saline-alkali* (…) significantly improved the bacterial count. Pseudomonas alkalisoli The D44 strain can promote cotton growth. Furthermore, coating alfalfa seeds with a seed coating agent containing strain D44 significantly improves seed germination rate, promotes alfalfa nodulation, increases forage yield, and enhances alfalfa's tolerance to salt and alkali stress. In summary, the strain D44 provided by this invention is of great significance for alleviating plant salt and alkali stress and improving plant adaptability to salt and alkali environments.

[0057] Deposit Description Classification and nomenclature: Pseudomonas saline-alkali soil ( Pseudomonas alkalisoli ); Reference biological material (strain): D44; 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: October 22, 2025; Registration number at the Collection Center: CGMCC No. 36294. Attached Figure Description

[0058] Figure 1 The *Pseudomonas saline-alkali soil* in Example 1 of this invention (… Pseudomonas alkalisoliColony morphology of D44 after 3 days of incubation on TSA plates.

[0059] Figure 2 The *Pseudomonas saline-alkali soils* strain constructed using the neighbor-joining method based on the 16S rRNA gene sequence in Example 1 of this invention (…). Pseudomonas alkalisoli Phylogenetic tree of D44 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. Acinetobacter baumannii ATCC 19606 T (ACQB01000091) is an outgroup.

[0060] Figure 3 This refers to the *Pseudomonas saline-alkali* strain constructed using the maximum likelihood method based on 1714 orthologous genes in Example 1 of this invention. Pseudomonas alkalisoli D44 and Pseudomonas Phylogenetic tree of protein sequences of closely related species within the genus.

[0061] Figure 4 The figure shows the statistical analysis of the main root length of cotton potted plants in the water treatment group, the salt-alkali stress treatment group, and the D44 strain salt-alkali stress treatment group in Example 3 of this invention. In the figure, different lowercase letters indicate significant differences (P < 0.05).

[0062] Figure 5 The aboveground fresh weight of cotton potted plants in Example 3 of this invention is statistically analyzed in the water treatment group, the salt-alkali stress treatment group, and the D44 strain salt-alkali stress treatment group. In the figure, different lowercase letters indicate significant differences (P < 0.05).

[0063] Figure 6 The figure shows the statistical analysis of the fresh weight of the underground parts of cotton potted plants in the water treatment group, the salt-alkali stress treatment group, and the D44 strain salt-alkali stress treatment group in Example 3 of this invention. In the figure, different lowercase letters indicate significant differences (P < 0.05).

[0064] Figure 7 This image shows the morphology of coated and pelleted alfalfa seeds prepared in Example 4 of this invention. The left side shows naked alfalfa seeds; the right side shows pelleted alfalfa seeds.

[0065] Figure 8 This is a statistical analysis of the germination rates of potted plants subjected to salt and alkali stress using naked seeds, sterile pellets, and inoculant pellets in Example 5 of this invention.

[0066] Figure 9 This is a statistical analysis of the plant height of potted plants subjected to salt and alkali stress in Example 5 of the present invention, including naked seeds, sterile agent pellets, and inoculant pellet seeds.

[0067] Figure 10This figure shows the statistical analysis of the taproot length of potted plants subjected to salt and alkali stress in Examples 5 of this invention, including naked seeds, sterile seed pellets, and seed pellets treated with fungicide. Different lowercase letters in the figure indicate significant differences (P < 0.05).

[0068] Figure 11 This figure shows the statistical analysis of the number of nodules formed in potted plants subjected to salt and alkali stress using naked seeds, sterile seed pellets, and seed pellets treated with fungicide in Example 5 of this invention. In the figure, different lowercase letters indicate significant differences (P < 0.05).

[0069] Figure 12 This figure shows the statistical analysis of the aboveground fresh weight of potted plants subjected to salt and alkali stress, including naked seeds, sterile pellets, and inoculant pellets, in Example 5 of this invention. In the figure, different lowercase letters indicate significant differences (P < 0.05).

[0070] Figure 13 This figure shows the statistical analysis of the underground fresh weight of potted plants subjected to salt and alkali stress, including naked seeds, sterile pellets, and inoculant pellets, in Example 5 of this invention. In the figure, different lowercase letters indicate significant differences (P < 0.05).

[0071] Figure 14 These are actual photos of potted plants subjected to salt and alkali stress, including naked seeds, sterile agent pellets, and inoculant pellet seeds, as described in Example 5 of this invention. Detailed Implementation

[0072] 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.

[0073] 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.

[0074] The various culture media involved in the following examples: (1) TSB liquid culture medium: 15.0g tryptone, 5.0g soybean peptone, 5.0g sodium chloride, 1000mL distilled water, adjust pH to 7.3±0.2, sterilize at 121℃ for 15min.

[0075] (2) TSA solid medium: Add 20.0g agar to TSB liquid medium and sterilize at 121℃ for 15min.

[0076] (3) Anthrone solution: Slowly add 760 ml of concentrated sulfuric acid to ultrapure water and bring the volume to 1000 mL. Weigh 1 g of purified anthrone and dissolve it in 1000 mL of sulfuric acid to obtain anthrone reagent.

[0077] The superabsorbent polymer used in the following examples is sodium polyacrylate, a product of Aladdin Reagent (Shanghai) Co., Ltd., with product number S301686; CAS number: 9003-04-7; molecular formula: (C3H3NaO2)n; EC number: 692-137-3; PubChem number: 4068533; purity ≥95%, and powder particle size 100-300 mesh.

[0078] The sodium alginate used in the following examples is a product of Sinopharm Chemical Reagent Co., Ltd. (Shanghai), with Sinopharm code: 30164426, CAS number: 9005-38-3, and viscosity of 200±20 mPa.s.

[0079] Example 1: Pseudomonas aeruginosa ( Pseudomonas alkalisoli Isolation and Identification of D44 I. Pseudomonas aeruginosa ( Pseudomonas alkalisoli Separation of D44 Soybean rhizosphere soil samples from the Yellow River Delta, a severely saline-alkali land in the agricultural highland area of ​​Guangrao County, Dongying City, Shandong Province (118°17′04″-118°57′11″E, 36°56′09″-37°21′23″N), were brought back to the laboratory and stored in a 4°C refrigerator using a 4°C ice box.

[0080] Shake off the soil adhering to the plant roots, retaining only the rhizosphere soil tightly adhering to the root surface. Immerse soybean roots 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 TSA 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 D44.

[0081] II. Pseudomonas aeruginosa ( Pseudomonas alkalisoli Identification of D44 1. Morphological identification of strains The D44 strain, isolated and purified in step one above and in the logarithmic growth phase with stable colony size, was described as a single colony, including colony size, color, transparency, colony surface condition, and colony edge condition. Following the manufacturer's instructions, Gram staining of strain D44 was performed using a Solarbio Gram staining kit, and the morphology of the bacteria was observed using an optical microscope.

[0082] The colonies of strain D44 on TSA plates are whitish-brown, round, raised, and easy to pick up, with a colony diameter of 1-2 mm. Figure 1 The cells are Gram-negative, rod-shaped, and do not form spores.

[0083] 2. Molecular identification Following the instructions, genomic DNA was extracted using the TIANamp bacterial genomic DNA extraction kit from Beijing Tiangen Biotech Co., Ltd. 16S rRNA gene amplification was performed using universal bacterial primers 27F (SEQ ID NO:1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO:2: 5'-GGTTACCTTGTTACGACTT-3'). The 50 μL PCR amplification system consisted of: 25 μL 2×Taq PCR Mix, 2 μL 27F (10 μmol / L), 2 μL 1492R (10 μmol / L), 19 μL ddH2O, and 2 μL DNA template. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 1 min, 72℃ extension for 90 s, 30 cycles; and a final extension at 72℃ for 10 min. PCR amplification products were detected by 1% agarose gel electrophoresis. The amplified fragment was approximately 1500 bp. After electrophoresis verification, positive PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequenced sequences were uploaded to Ezbiocloud (www.ezbiocloud.net / eztaxon) for sequence alignment.

[0084] The sequencing length of the 16S rRNA gene of strain D44 is 1530 bp (SEQ ID NO:3). Comparison with the EzBioCloud database shows that strain D44 belongs to the genus *Pseudomonas*. Pseudomonas (members, with) Pseudomonas inefficax JV551A3 T (99.93%) Pseudomonas monteilii NBRC 103158 T (99.86%) Pseudomonas plecoglossicida NBRC 103162 T(99.86%) of the sequences showed high similarity, and were similar to... Pseudomonas The sequence similarity of the model bacteria from other species in the genus was <99.8%. 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 D44 and strain Pseudomonas plecoglossicida NBRC 103162 T (BBIV01000080) and Pseudomonas The strains of the genus cluster together, and each forms a separate branch.

[0085] 3. Genome analysis Genomic DNA was sent to Annoroad Gene Technology (Beijing) Co., Ltd., where a draft genome sequence of strain D44 was performed using an Illumina NovaSeq 6000 sequencing system. Assembly was performed using SPAdes software, yielding 81 contigs with a coverage of approximately 150×N. 50 The length is 289229 bp. The genome size is 5.47 Mb, and the G+C content is 63.1%. Strain D44 was compared with... Pseudomonas Genome-wide average nucleotide identity (ANI) analysis was performed on closely related strains. The digital DNA-DNA hybridization (dDDH) values ​​between strain D44 and the reference strain were compared using the Genome-to-Genome Distance Calculator (GGDC) 3.0 server (https: / / ggdc.dsmz.de / ggdc.php#). See Table 1 for details.

[0086] Table 1. The present invention's *Pseudomonas aeruginosa* D44 and... Pseudomonas Values ​​of ANI and dDDH for closely related species within the genus

[0087] As shown in Table 1, strain D44 and other strains with publicly available genome sequences... PseudomonasCompared to the type strains of the same genus, the ANI values ​​were 79.3-94.8%, lower than the previously proposed critical value of 95-96% for species delimitation; the dDDH values ​​of strain D44 and its type strain were between 24.4-61.6%, far below the 70% species delimitation threshold. The results of ANI and dDDH indicate that strain D44 is a... Pseudomonas A new species of the genus.

[0088] Simultaneously, the Bacterial Pan Genome Analysis tool (BPGA) software was used to analyze strain D44 and... Pseudomonas Analysis was performed on 33 type strains of the genus, and 1714 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 strain D44 was similar to strain... Pseudomonas asiatica RYU5 T (GCA_009932335.1) and Pseudomonas Several strains of the genus clustered together and formed a separate branch, indicating that strain D44 is... Pseudomonas A potential new species of the genus.

[0089] 4. Physiological and chemical classification and identification Add several drops of 5% H2O2 to a glass petri dish, pick strain D44 and react with it. If bubbles are produced, it proves that the strain can produce catalase. Spot strain D44 onto filter paper soaked in 1% p-aminoxylamine hydrochloride, using Pseudomonas aeruginosa and Escherichia coli as positive and negative controls, respectively. If a rose-red ring appears around the colony, it indicates that it can produce catalase.

[0090] The results showed that strain D44 produced bubbles after contact with 5% H2O2, indicating that its catalase result was positive; after contact with filter paper soaked in 1% p-aminoxylamine hydrochloride, strain D44 produced a light rose-red color, indicating that its oxidase result was weakly positive.

[0091] Strain D44 was inoculated into liquid culture medium and cultured at 37°C with shaking (200 rpm) for 48-72 h to obtain fermentation broth. 3 mL of fermentation broth was taken, centrifuged at 10000 rpm for 15 min, and 1 mL of the supernatant was collected. Three times the volume (3 mL) of anhydrous ethanol was added, and the mixture was placed at 4°C for 24 h to precipitate. After centrifugation at 10000 rpm for 15 min, the supernatant was discarded, yielding a polysaccharide precipitate. The precipitate was washed with 1 mL of anhydrous ethanol, and the precipitate was re-dissolved in deionized water (generally 1 mL, until completely dissolved). 0.5 mL of Sevag solution (chloroform:n-butanol = 4:1, freshly prepared) was added, and the mixture was shaken to mix. The mixture was centrifuged at 10000 rpm for 15 min, and the upper aqueous phase was collected (to remove the white viscous precipitate between the two phases and the lower organic reagent), yielding a polysaccharide solution. 5 mL of anthrone sulfate solution was then added to each solution, and the mixture was cooled in ice water. Place it in a boiling water bath for 10 minutes, then cool it down and measure the OD using a spectrophotometer. 620 Value. Calculate the total sugar content according to the glucose standard curve.

[0092] After being cultured at 37℃ and 200 rpm for 48-72 h with shaking, strain D44 was centrifuged, precipitated with ethanol, and deproteinized using the Sevag method. The extracellular polysaccharide content was determined at 620 nm using the anthrone-sulfuric acid colorimetric method. A standard curve was plotted using glucose as the standard, and the resulting regression equation was y = 2.8102x - 0.0024, with a coefficient of determination R0. 2 =0.9981, indicating a good linear relationship between glucose concentration and absorbance value within the experimental concentration range. OD values ​​from five parallel measurements... 620 The values ​​were 0.067, 0.070, 0.060, 0.070 and 0.062, respectively. After conversion using the standard curve, the average content of extracellular polysaccharides in the fermentation broth of strain D44 was 0.0243 mg / mL.

[0093] The enzyme activity and carbohydrate utilization of strain D44 and related model strains were determined using API 20NE, ZYM and 50 CH test strips from bioMérieux, France.

[0094] The 20NE test results showed that strain D44 was weakly positive (W) for nitrate reduction and indole reaction, positive (+) for urease hydrolysis reaction, and weakly positive (W) for aesculin hydrolysis reaction. It could assimilate glucose, arabinose, mannose, maltose, and potassium gluconate, but could not assimilate mannitol, N-acetylglucosamine, decanoic acid, adipic acid, malic acid, citric acid, and phenylacetic acid. It was negative (-) for glucose acidification, arginine double hydrolysis, gelatin hydrolysis, and p-nitro-β-D-galactoside hydrolysis reactions.

[0095] In the ZYM enzyme activity assay: acid phosphatase and naphthol-AS-BI-phosphohydrolase were positive (+), and α-galactosidase was weakly positive (W); alkaline phosphatase, esterase (C4), lipoesterase (C8), lipoesterase (C14), leucine aromatic aminoaminase, valine aromatic aminoaminase, cystine aromatic aminoaminase, trypsin, chymotrypsin, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-glucosidase, α-mannosidase, and α-fucosidase were all negative (-).

[0096] The 50CH results showed that strain D44 could hydrolyze glycerol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, D-merbiose, starch, glycogen, and D-gentiobiose; but could not hydrolyze erythritol, D-ribose, L-rhamnose, D-agonol, D-mannitol, L-sorbose, inositol, and D-mannitol. D-sorbitol, D-melatoninose, methyl-β-D-xylopyranoside, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, sucrose, trehalose, inulin, raffinose, D-melinobiose, D-lythose, D-tagatose, D-fucose, L-fucose, xylitol, D-arabinol, L-arabinol, potassium gluconate, potassium 2-ketogluconate, and potassium 5-ketogluconate; weakly utilized D-mannose, D-mannitol, D-merinobiose, and D-gentibiose. The physiological and biochemical characteristics of strain D44 differ from those of related model strains in Table 2.

[0097] Table 2. Differences in physiological and biochemical characteristics between strain D44 and related model strains

[0098] Note: + indicates a positive result or that the condition may be available; , negative or unusable; w, weakly positive.

[0099] Based on homology comparison, phylogenetic tree construction, and combined with morphological, physiological and biochemical identification, and genomic analysis results, strain D44 can be identified as... Pseudomonas A new species of the genus, the proposed classification name is... Pseudomonas erigeronis Its Chinese name is *Pseudomonas saline-alkali*. Pseudomonas alkalisoli D44 was deposited on October 22, 2025, at the China General Microbiological Culture Collection Center (GMCC), with the registration number GMCC No. 36294. Hereafter referred to as *Pseudomonas saline-alkali soils* (…). Pseudomonas alkalisoli D44, or simply strain D44.

[0100] Example 2, Pseudomonas aeruginosa ( Pseudomonas alkalisoli D44 IAA-induced fertility testing 1. Add 0.5 g / L L-tryptophan to LB medium. The LB medium is sterilized using an autoclave. Tryptophan is prepared separately as a high-concentration solution, sterilized by filtration, and added individually. Dispense the prepared L-tryptophan-containing LB medium into test tubes, 3 mL per tube. After inoculation, incubate at 28℃ and 180 rpm for 4 days. Prepare the Salkowski colorimetric solution (formula: 50 mL 35% HClO4 + 1 mL 0.5 mol / L FeCl3).

[0101] 2. Culture 3 ml of D44 bacterial suspension in a test tube, centrifuge 1 ml at 10000 rpm for 10 min, and collect the supernatant; separately, measure the OD of the remaining bacterial suspension using a spectrophotometer. 600 To characterize cell concentration, 500 μL of the supernatant and Salkowski colorimetric solution were mixed in equal volumes (500 μL + 500 μL). The control was a mixture of LB medium and colorimetric solution alone. The mixtures were incubated at room temperature in the dark for 30 min, and the OD value was measured. 530 Value. Analytical grade IAA standards were used, dissolved in anhydrous ethanol. IAA concentrations of 0, 50, 100, 200, and 250 mg / L were used. After the same colorimetric treatment, the OD value was determined. 530 The values ​​are used to plot a standard curve, and then the IAA content in the sample is quantitatively calculated based on the standard curve.

[0102] The results are shown in Table 3. The results indicate that strain D44 has the ability to produce IAA, with an average yield of 0.036 mg / L, and has the potential to promote growth.

[0103] Table 3. IAA Production Capacity Detection of Strains D44

[0104] Example 3, Pseudomonas aeruginosa ( Pseudomonas alkalisoli The alleviating effect of D44 on salt-alkali stress in cotton Test plant: Cotton pot experiment variety "Zhongmian 113".

[0105] 1. Experimental Methods This experiment used a nutrient soil:vermiculite ratio of 1:1 (volume ratio) as the substrate, and applied 0.5 mL of saline-alkali solution per gram of substrate to induce stress. The sodium content in this saline-alkali solution was... + The concentration was 200 mmol / L, and it was prepared by mixing NaCl, Na₂SO₄, and NaHCO₃ in a 1:1:1 molar ratio. The natural pH of this salt-alkali stress solution was 8.6. Three treatment groups were set up in the experiment: Treatment 1: A combination of salt and alkali stress and soaking in the fermentation broth of strain D44 for 30 minutes. This treatment is referred to as D44. Treatment 2: The combination of no salt and alkali stress and fermentation broth of strain D44, this treatment is denoted as water; Treatment 3: The treatment group subjected to salt stress but not to fermentation broth of strain D44 was denoted as salt.

[0106] Thirty days after sowing, the taproot length and above-ground / below-ground fresh weight of the plants in each group were measured. At least 5 plants were measured in each group, and the average of the results was taken.

[0107] 2. Experimental Results Depend on Figure 4 It can be seen that the main root length of cotton in the D44 strain treatment group was significantly higher than that in the water and saline-alkali control groups. Figure 5 and Figure 6 It can be seen that the aboveground fresh weight of the D44 treatment group was slightly higher than that of the saline-alkali treatment group, but not significantly higher, while the underground fresh weight was significantly higher than that of the saline-alkali treatment group and the same as that of the water control group. The D44 strain treatment group effectively promoted the growth and development of plant roots and formed a stable root environment.

[0108] Example 4: Alfalfa Seed Pelletizing 1. Take 100g of naked seeds of alfalfa variety No. 2, remove impurities larger than alfalfa seeds by using a 12-mesh sieve, and remove impurities and small seeds by using a 20-mesh sieve, keeping large, plump, and regularly shaped pre-coated seeds for later use.

[0109] 2. Weigh 200 g each of 200-mesh biochar and rice husk powder according to a mass ratio of 1:1 to obtain 400 g of mixed powder. Then add 10% (i.e., 40 g) of superabsorbent resin to the total mass of the mixed powder, and mix thoroughly again to obtain the composite powder for later use.

[0110] 3. Add 200 mL of microbial inoculum (i.e., fermentation broth of *Pseudomonas saline-alkali soil* D44 cultured for two days, with a bacterial count of approximately 10). 9 Add 1g of sodium alginate to (CFU / mL) and stir until homogeneous to obtain a liquid adhesive solution.

[0111] 4. Put the seeds into the coating pelleting machine, and use an air compressor to evenly spray a small amount of the liquid adhesive solution prepared in step 3 above onto the seed surface, so that the seed surface is moist but not sticky. Then, evenly add a small amount of the composite powder prepared in step 2 above, so that the composite powder fully adheres to the moist seed surface. Repeat this process several times until the seed particle size increases significantly.

[0112] 5. When the seed particle size reaches the target range, stop the machine and remove the material. Use a combination sieve (with apertures of 10 mesh, 12 mesh, and 20 mesh from top to bottom) for grading and screening. Particles that are trapped above the 10 mesh sieve are due to excessive coating and should be removed. Particles that pass through the 10 mesh sieve and are trapped on the 12 mesh sieve are qualified pellets. Particles that pass through the 12 mesh sieve and are trapped on the 20 mesh sieve are too small and have insufficient coating. These particles should be put back into the pelletizing machine to continue the coating process. Particles that pass through the 20 mesh sieve are coated powder.

[0113] 6. Place the qualified pellets obtained from screening back into the pelletizing machine and polish them for 10 minutes under no-load conditions to make their surface smooth and their structure dense.

[0114] 7. The obtained seed pellets are dried at low temperature to obtain the alfalfa seed coating product, such as... Figure 7 As shown, its shape is regular, its size is uniform, and its pellets are round.

[0115] 8. The quality inspection of the obtained alfalfa seed coating product is as follows: (1) Inspection of appearance integrity: The appearance inspection is carried out by visual inspection. Take 3 mixed samples, 200 seeds in each sample, and observe whether the coating layer is intact and uniform, whether the particle size is uniform, and whether there are any pellet seeds with exposed seed skin.

[0116] (2) Thousand-grain weight: 100 pelleted seeds were randomly selected from each treatment and weighed using an electronic balance (accurate to 0.01g). The process was repeated 3 times, and the average value was calculated and converted into the thousand-grain weight (g).

[0117] (3) Bulb diameter: Randomly select 10 pelleted seeds and measure their diameter (mm) with a vernier caliper. Repeat 10 times and calculate the average value.

[0118] (4) Compressive strength: Randomly select one seed, place it on the platform of the push-pull force gauge, apply pressure at a constant speed until the seed deforms or breaks, record the maximum force required (Newtons, N), repeat 10 times, and calculate the average value.

[0119] (5) Single seed rate: Randomly select 100 pelleted seeds, destroy the seed coating layer, and count the percentage of single seeds.

[0120] (6) Test for viable count of microorganisms in pelleted seeds: On day 0 and day 90 after pelleting, 100 seed pellets were randomly selected and placed in sterile 50 mL centrifuge tubes. 10 mL of sterile water was added, and the tubes were cultured at 30℃ and 200 r / min for 1-2 h with shaking to fully dissolve the pellet coating and obtain the initial bacterial suspension.

[0121] Take the initial bacterial suspension and perform a 10-fold serial dilution to 10⁻⁶. -6For each dilution, take 10 μL and spot it onto the surface of TY solid medium containing the corresponding resistance. Each dilution should be replicated three times. After the droplets are absorbed by the medium, invert the petri dishes and incubate at 28°C for 3 days. Count the number of colony-forming units (CFU) on each dilution plate. Calculate the colony count at the lowest dilution.

[0122] Select the dilution plate with the lowest colony count for counting, and calculate the viable count per seed using the following formula:

[0123] In the formula, 10 is the total volume of the initial bacterial suspension (mL), and 100 is the number of seed samples taken.

[0124] The results are shown in Table 4.

[0125] Table 4. Quality Inspection of Coated and Pelleted Seeds of Zhongmu No. 2

[0126] Note: " / " indicates that it is not involved or not detected.

[0127] The results shown in Table 4 indicate that the coated pelleted seeds of Zhongmu No. 2 prepared in this embodiment significantly increased in weight and volume compared with naked seeds, and the number of viable strains and seed compressive strength both met market standards.

[0128] Example 5: Test of alfalfa seed pellets under salt and alkali stress in pots Test plant: Alfalfa pot experiment variety "Zhongmu No. 2".

[0129] 1. Test Methods This experiment used a nutrient soil:vermiculite ratio of 1:1 (volume ratio) as the substrate. 0.5 mL of saline-alkali solution was applied per gram of substrate to induce stress. The sodium content in this saline-alkali solution was... + The concentration was 120 mmol / L, and it was prepared by mixing NaCl, Na₂SO₄, and NaHCO₃ in a 1:1:1 molar ratio. The natural pH of this salt-alkali stress solution was 8.6. Three treatment groups were set up in the experiment: (1) Untreated naked seedlings; this treatment is referred to as naked seedlings. (2) Based on Example 4, replace the microbial agent with clean water and sterile agent seed pellets. This treatment is called pellet coating. (3) The pellet seeds prepared in Example 4 of this invention are coated with Pseudomonas saline-alkali soil ( Pseudomonas alkalisoli Seed pellets of D44, this treatment is denoted as D44 pellet coating; Each treatment was set up with 6 replicates, and each replicate consisted of 5-6 plants. Relevant indicators (germination rate, plant height, taproot length, number of nodules, aboveground fresh weight, and underground fresh weight) were statistically analyzed 38 days after sowing.

[0130] 2. Results and Analysis Depend on Figure 8 It can be seen that after sowing, the germination rate of the inoculant-coated pellet treatment group was higher than that of the sterile-coated pellet treatment group.

[0131] Depend on Figure 9 It can be seen that, compared with the naked seed, the plant height of the pellet-coated group and the D44 pellet-coated group was increased compared with the naked seed control group.

[0132] Depend on Figure 10 It can be seen that the main root length of the pellet-treated group and the D44 pellet-treated group was significantly longer than that of the bare seed.

[0133] Depend on Figure 11 It can be seen that, compared with the naked seed and pellet treatment groups, the D44 pellet treatment group significantly increased the number of alfalfa nodules.

[0134] Depend on Figure 12 , Figure 13 and Figure 14 It can be seen that, compared with naked seed, pelleting treatment significantly increased the biomass of both aboveground and belowground parts, and the biomass of the D44 pelleting treatment group was significantly higher than that of the pelleting treatment group. It can be visually seen that the forage yield of the D44 pelleting treatment group was significantly improved.

[0135] The above-mentioned efficacy experiments show that the alfalfa seed microbial pelleting scheme of the present invention can effectively encapsulate microbial agents, increase forage yield, and enhance alfalfa's tolerance to salt and alkali stress.

[0136] 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. *Pseudomonas saline-alkali* or its progeny, characterized by: The *Pseudomonas saline-alkali* species is *Pseudomonas saline-alkali* (… Pseudomonas alkalisoli D44, its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 36294.

2. An inoculum containing the *Pseudomonas saline-alkali* or its progeny as described in claim 1.

3. A seed coating agent containing *Pseudomonas saline-alkali soilii* or its progeny as described in claim 1, or the inoculum as described in claim 2; Furthermore, the seed coating agent contains *Pseudomonas saline-alkali* or its progeny and sodium alginate; Furthermore, the seed coating agent also contains one or more of biochar, rice husk powder, and superabsorbent resin.

4. Any of the following applications: (A1) The application of the *Pseudomonas saline-alkali* or its progeny as described in claim 1, or the inoculum as described in claim 2, or the seed coating agent as described in claim 3, in alleviating plant salt-alkali stress; (A2) The use of the *Pseudomonas saline-alkali* or its progeny as described in claim 1 or the fungal agent as described in claim 2 in the preparation of products for alleviating plant salt stress; (A3) The application of the *Pseudomonas saline-alkali soils* or its progeny as described in claim 1, or the inoculant as described in claim 2, or the seed coating agent as described in claim 3, in promoting plant growth; (A4) The use of the *Pseudomonas saline-alkali* or its progeny as described in claim 1 or the inoculum as described in claim 2 in the preparation of products for promoting plant growth; (A5) The application of the *Pseudomonas saline-alkali soil* or its progeny as described in claim 1, or the inoculum as described in claim 2, or the seed coating agent as described in claim 3, in improving the germination rate of plant seeds; (A6) The use of the *Pseudomonas saline-alkali* or its progeny as described in claim 1 or the inoculum as described in claim 2 in the preparation of products for improving the germination rate of plant seeds; (A7) The application of the *Pseudomonas saline-alkali soil* or its progeny as described in claim 1, or the inoculant as described in claim 2, or the seed coating agent as described in claim 3, in improving plant yield; (A8) The use of the *Pseudomonas saline-alkali* or its progeny as described in claim 1 or the inoculum as described in claim 2 in the preparation of products for increasing plant yield; (A9) The application of the *Pseudomonas saline-alkali* or its progeny as described in claim 1, or the inoculum as described in claim 2, or the seed coating agent as described in claim 3, in promoting nodulation in leguminous plants; (A10) The use of the *Pseudomonas saline-alkali* of claim 1 or its descendants or the fungal agent of claim 2 in the preparation of a product for promoting nodulation in leguminous plants.

5. A method for alleviating salt and alkali stress in plants, comprising: Applying the *Pseudomonas saline-alkali* or its progeny as described in claim 1 or the inoculant as described in claim 2 to the plants to be treated or their cultivation substrate can alleviate the salt stress of the plants.

6. The method according to claim 5, characterized in that: In the method, the seeds of the plant to be treated are coated with the seed coating agent of claim 3.

7. Any of the following methods: Method I: A method for promoting plant growth, comprising: Apply the *Pseudomonas saline-alkali soils* or its progeny as described in claim 1 or the inoculant as described in claim 2 to the plants to be treated or their cultivation substrate to promote plant growth; Method II: A method for improving the germination rate of plant seeds, comprising: coating the seeds of the plant to be treated with the seed coating agent described in claim 3 to improve the germination rate of plant seeds; Method III: A method for increasing plant yield, comprising: coating the seeds of the plant to be treated with the seed coating agent of claim 3 to increase plant yield; Method IV: A method for promoting nodulation in legumes, comprising: coating the seeds of the legume to be treated with the seed coating agent of claim 3 to promote nodulation in legumes.

8. The application or method according to any one of claims 4-7, characterized in that: The promotion of plant growth refers to promoting plant growth under salt-alkali stress conditions; and / or, The improvement of plant seed germination rate refers to improving the plant seed germination rate under salt-alkali stress conditions; and / or, The aforementioned increase in plant yield refers to increasing plant yield under salt-alkali stress conditions; and / or, The method of promoting nodulation in legumes refers to promoting nodulation in legumes under saline-alkali stress conditions.

9. The application or method according to claim 8, characterized in that: The promotion of plant growth under saline-alkali stress conditions refers to promoting the growth of the taproot length and / or promoting the increase of the dry weight of the underground parts of the plant under saline-alkali stress conditions; and / or, The method of increasing plant yield under saline-alkali stress includes promoting the growth of the taproot, increasing the aboveground biomass, and / or increasing the underground biomass of plants under saline-alkali stress.

10. The application or method according to any one of claims 4-9, characterized in that: The plant is any one of the following: (B1) Dicotyledonous plants; (B2) Legumes or Malvaceae; (B3) Plants of the genus *Alfalfa* or *Gossypium*; (B4) Alfalfa or cotton.

Images