Preparation method and application of microcapsule seed coating agent containing pseudoxanthomonas hydrophila GM-4

Abstract

The invention belongs to the technical field of microbial seed coating agents, and particularly relates to a preparation method and application of a microcapsule seed coating agent containing pseudoxanthomonas aquaticum GM-4. The invention discloses a Pseudoxanthomonas sp. GM-4, which can generate indoleacetic acid (IAA), catalase and siderophore production capacity, and has the effects of promoting growth and improving the drought resistance and salt resistance of plants. The invention provides a preparation method of a probiotic microcapsule, which comprises the following steps: mixing a mixed solution of potassium alginate and pectin, pseudoxanthomonas hydrophila GM-4 and calcium carbonate to obtain a first mixture; mixing the first mixture with a first oil phase and a second oil phase, and standing to obtain a water-phase substance; and centrifuging and drying the water-phase substance to obtain the probiotic microcapsule. The invention provides a seed coating agent which can improve drought resistance and salt resistance of plants.

Description

Technical Field

[0001] This invention belongs to the technical field of microbial seed coating agents, specifically relating to a method for preparing and applying a microcapsule seed coating agent containing *Xanthomonas hydrateus* GM-4. Background Technology

[0002] In actual agricultural production, seed germination rate and seedling growth have a significant impact on crop yield. Especially under abiotic stress conditions (such as salinity and drought), seed germination rate and seedling growth are closely related to yield and quality.

[0003] Seed coating agents are preparations used for seed treatment. They form a protective film on the seed surface, providing functions such as controlling underground pests and soil-borne and seed-borne diseases, improving seed germination rate, promoting healthy crop growth, increasing crop yield, and improving crop quality. Seed coating agents prepared using biocontrol bacteria can control pests and diseases, improve germination rate, promote seedling growth, and reduce pesticide use, offering advantages such as environmental friendliness and high efficiency. However, existing technology CN105994379A discloses a seed coating agent based on actinomycetes, its preparation method, and coating method, wherein the actinomycetes have the effect of promoting root development and increasing yield.

[0004] Microencapsulation technology can effectively protect probiotics by embedding them in the cell wall material; however, the encapsulation rate of probiotics in microcapsules is currently low. For example, in the preparation method and application of microbial microcapsules disclosed in prior art CN117562080A, the encapsulation rate of *Vitis vinifera* in sea anemones only reaches a maximum of 61.5%–69.3%.

[0005] Furthermore, the types of microorganisms available for preparing seed coating agents are relatively scarce, and there is a lack of seed coating agents that simultaneously possess salt tolerance and growth-promoting effects. Summary of the Invention

[0006] This invention provides a method for preparing and applying a microcapsule seed coating agent containing *Xanthomonas suis* GM-4. The invention relates to a strain of *Xanthomonas suis* GM-4, which can produce indoleacetic acid (IAA), catalase, and siderophore production. The seed coating agent prepared using *Xanthomonas suis* GM-4 has the function of promoting growth and alleviating drought and salt tolerance in plants, and the prepared seed coating agent has a high encapsulation rate of *Xanthomonas suis* GM-4.

[0007] To address the aforementioned technical problems, the present invention proposes the following technical solution:

[0008] This invention provides a strain of Pseudoxanthomonas suwonensis GM-4, with accession number CGMCC No.34616.

[0009] This invention provides a method for preparing probiotic microcapsules, comprising:

[0010] A first mixture is obtained by mixing a mixture of potassium alginate and pectin, the *Xanthomonas suis* GM-4 strain described in the above technical solution, and calcium carbonate; the volume-to-mass ratio of the mixture of potassium alginate and pectin to calcium carbonate is 50-200 mL: 0.5-2 g.

[0011] The mass concentration of potassium alginate in the mixture of potassium alginate and pectin is 10-55 g / L, and the mass concentration of pectin is 5-55 g / L; and the mass ratio of potassium alginate to pectin in the mixture is (2.5-3.5):1.

[0012] The first mixture is emulsified with the first oil phase, then mixed with the second oil phase and allowed to stand to obtain an aqueous phase substance.

[0013] The aqueous phase material was centrifuged and dried to obtain probiotic microcapsules.

[0014] Preferably, the application form of *Xanthomonas suis* includes a bacterial suspension, wherein the viable count of *Xanthomonas suis* in the bacterial suspension is ≥10. 8 CFU / mL.

[0015] Preferably, the volume ratio of the bacterial suspension, the potassium alginate and pectin mixture is (10-30):(100-300).

[0016] Preferably, the first oil phase comprises soybean oil or paraffin oil; the second oil phase comprises soybean oil or paraffin oil.

[0017] The first oil phase contains Span80, and the concentration of Span80 in the first oil phase is 0.5-2 g / mL;

[0018] The second oil phase contains Span80 and glacial acetic acid; the concentration of Span80 in the second oil phase is 0.5-2 g / mL; the volume ratio of the second oil phase to glacial acetic acid is 50-100:0.2-0.6; the volume ratio of the first oil phase to the second oil phase is 100-400:50-100.

[0019] This invention provides the application of *Xanthomonas suis* GM-4 as described in the above-described technical solution or the probiotic microcapsules obtained by the preparation method described in the above-described technical solution in the preparation of seed coating agents.

[0020] This invention provides a seed coating agent, comprising, by volume of solvent: 1 g to 20 g / L probiotic microcapsules and 5 to 100 g / L film-forming agent; wherein the probiotic microcapsules are probiotic microcapsules prepared by the preparation method described in the above technical solution.

[0021] Preferably, the film-forming agent includes one or more of polyvinyl alcohol, carboxymethyl cellulose, and starch.

[0022] This invention provides the application of the probiotic microcapsules prepared by the preparation method described above, or the seed coating agent described above, in at least one of the following:

[0023] 1) Plant seed coating;

[0024] 2) Promotes plant seed development;

[0025] 3) Improve the encapsulation rate of probiotics;

[0026] 4) Improve plant drought resistance;

[0027] 5) Improve the salt tolerance of plants.

[0028] This invention provides a method for improving the drought resistance and / or salt tolerance of plants, comprising the following steps:

[0029] Plant seeds are coated with the seed coating agent described in the above technical solution before sowing.

[0030] The beneficial effects of this invention: This invention provides a strain of *Pseudoxanthomonas suwonensis* GM-4, with accession number CGMCC No. 34616. *Pseudoxanthomonas suwonensis* GM-4 possesses the ability to produce indoleacetic acid (IAA), catalase, and siderophores, promoting plant growth and alleviating drought and salt tolerance in plants.

[0031] This invention provides a method for preparing probiotic microcapsules. The method utilizes potassium alginate, pectin, and calcium carbonate to microencapsulate *Xanthomonas suis* GM-4. Calcium carbonate (CaCO3) slowly decomposes, releasing calcium ions, providing a continuous source of calcium ions for the cross-linking of potassium alginate and pectin, promoting the gradual formation of microcapsules. Potassium alginate and pectin undergo chain-to-chain association through hydrogen bonding and hydrophobic interactions, enhancing the stability of the gel network. When potassium alginate comes into contact with calcium ions, an ion exchange reaction occurs, generating insoluble calcium alginate gel, which further cross-links to form a composite gel structure. Furthermore, under acidic conditions, the carboxyl groups in pectin molecules dissociate into carboxylic acid anions. These anions combine with calcium ions in calcium carbonate through electrostatic interactions to form microcapsule structures, improving the encapsulation efficiency of probiotics, thus achieving the microencapsulation of probiotics using potassium alginate, pectin, and calcium carbonate.

[0032] In this invention, an oil phase is added as the dispersed phase to the probiotic microcapsules, forming tiny particles through emulsification, thus controlling the particle size and distribution of the microcapsules. After settling, the microcapsules settle in the lower water layer due to gravity, allowing for easy separation. Simultaneously, the potassium alginate and pectin in this invention provide a carbon source for microbial survival, extending their lifespan. The potassium and calcium sources in the probiotic microcapsules can regulate the osmotic pressure of plants under abiotic stress, increasing their drought and / or salt tolerance; pectin has strong water absorption capacity, which can alleviate drought stress.

[0033] This invention provides a seed coating agent that, through the combined action of *Pseudomonas suis* GM-4, potassium alginate, pectin, and calcium carbonate, increases the drought and / or salt tolerance of plants. Attached Figure Description

[0034] Figure 1 Functional identification of Xanthomonas suis GM-4; where a is indoleacetic acid (IAA) produced by the bacterium in tryptophan, b is catalase positive, and c is siderophore produced by the bacterium on CAS medium.

[0035] Figure 2 This is a flowchart of the microcapsule preparation process in Example 1;

[0036] Figure 3 The images show optical and electron microscopy scanning images of the microcapsules prepared in Example 1; where a is an electron microscopy image of Xanthomonas suis GM-4, b is an optical microscopy image of the microcapsules, and c is an electron microscopy image of the microcapsules.

[0037] Figure 4 The diagram shows the effects of different treatments on wheat growth and root system in Example 2 and Comparative Example 5.

[0038] Figure 5 The graph shows the effects of different treatments on wheat physiological indicators in Example 2 and Comparative Example 5.

[0039] Figure 6 The graph shows the effect of different treatments on soybean growth in Example 3 and Comparative Example 6.

[0040] Figure 7 The diagram shows the effects of different treatments on maize seeds in Example 4 and Comparative Example 7. Figures 4 to 7 In the middle, "coated" indicates seeds coated with a seed coating agent, while "CK" indicates uncoated seeds.

[0041] Biological Preservation Instructions

[0042] Pseudoxanthomonas suwonensis GM-4 was deposited on May 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34616. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0043] This invention provides a strain of *Pseudoxanthomonas suwonensis* GM-4, with accession number CGMCC No. 34616. The *Pseudoxanthomonas suwonensis* GM-4 described in this invention was isolated from the rhizosphere of plants in saline-alkali soils. *Pseudoxanthomonas suwonensis* GM-4 exhibits both salt and drought tolerance. The colonies of *Pseudoxanthomonas suwonensis* GM-4 are spherical, with a smooth, moist surface and regular edges. The *Pseudoxanthomonas suwonensis* GM-4 described in this invention is capable of producing indoleacetic acid (IAA), catalase, and siderophore production.

[0044] The Pseudoxanthomonas hydrophila of the present invention was isolated from the rhizosphere of wheat in Dongying City, Shandong Province, China, and was identified as Pseudoxanthomonas hydrophila by 16S rDNA sequence identification. Specifically: SEQ ID NO.1: 5’-CCCGAAGGTTAAGCTACCTGCTTCTGGTGCAACAAACTCCCATGGTGTG ACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTGAGATGGGGTTTCTGGGATTGGCTCACCGTCGCCGGCTTGCAGCCCTCTGTCCCCACCATTGTAGTACGTGTGTAGCCCTGGTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCGGTCTCCTTAGAGTTCCCACCATTACGTGCTGGCAACTAAGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACGGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCCGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATACTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGCGAACTTAACGCGTTAGCTTCGATACTGGGTTCCAAGTTGAACCCAACATCCAGTTCGCATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCCTCAGTGTCAGTGTTGGCCCAGGTGGCCGCCTTCGCCACGGATGTTCCTCCCGATCTCTACGCATTTCACTGCTACACCGGGAATTCCGCCACCCTCTACCACACTCTAGTGACCCAGTATCCACTGCAATTCCCAGGTTGAGCCCAGGGCTTTCACAACAGACTTAAATCACCACCTACGCACGCTTTA-3’。

[0045] This invention provides a method for preparing probiotic microcapsules, comprising:

[0046] A mixture of potassium alginate and pectin, the *Xanthomonas suis* GM-4 strain described in the above technical solution, and calcium carbonate are mixed to obtain a first mixture; the volume-to-mass ratio of the potassium alginate and pectin mixture to calcium carbonate is 50-200 mL: 0.5-2 g.

[0047] The potassium alginate and pectin mixture has a potassium alginate concentration of 10-55 g / L and a pectin concentration of 5-55 g / L; and the mass ratio of potassium alginate to pectin in the mixture is (2.5-3.5):1.

[0048] The first mixture is emulsified with the first oil phase, then mixed with the second oil phase and allowed to stand to obtain an aqueous phase substance;

[0049] The aqueous phase material was centrifuged and dried to obtain probiotic microcapsules. This invention utilizes potassium alginate, pectin, and calcium carbonate to microencapsulate *Xanthomonas suis*, resulting in high encapsulation efficiency of probiotics and extending the survival time of microorganisms.

[0050] As an optional implementation, this invention first prepares a potassium alginate solution and a pectin solution, then mixes the potassium alginate solution and the pectin solution to obtain a mixture of potassium alginate and pectin. Both the potassium alginate solution and the pectin solution in this invention are sterile solutions after sterilization. The solvent for the potassium alginate solution is preferably water. The solvent for the pectin solution is preferably water. This invention does not have special requirements for the preparation method of the potassium alginate solution and the pectin solution; techniques well known in the art can be used.

[0051] The potassium alginate concentration in the potassium alginate and pectin mixture of the present invention is 10-55 g / L, or 15-50 g / L; the pectin concentration in the mixture is 5-55 g / L, or 5-30 g / L. As an optional embodiment, the mass ratio of potassium alginate to pectin in the mixture of the present invention is (2.5-3.5):1; more preferably 3:1.

[0052] Potassium alginate is a non-toxic macromolecular polysaccharide with good biocompatibility and biodegradability, and is widely used for encapsulating microorganisms. Furthermore, potassium ions play an important role in alleviating osmotic stress in plant cells. Pectin is an important structural component of the cell wall of higher plants, linked by α-1,4-glycosidic bonds. Pectin is commonly used as a film-forming agent because it is non-toxic, biocompatible, and readily biodegradable. As a polysaccharide, pectin can also provide energy for microbial growth. Alginate and pectin, at specific mass ratios, are linked through chain-chain association and contain divalent cations (such as Ca2+). 2+Afterwards, a strong complex gel is formed. The gel has high viscosity. Pectin forms microcapsules through the electrostatic interaction between calcium ions and carboxylic acid anions, and reacts synergistically with alginate, which helps to improve the encapsulation rate of probiotics.

[0053] As an optional implementation, the application form of *Xanthomonas sugaru* GM-4 of the present invention includes a *Xanthomonas sugaru* bacterial suspension, wherein the viable count of *Xanthomonas sugaru* GM-4 in the bacterial suspension is ≥10. 8 CFU / mL. This invention does not have specific requirements for the preparation method of the *Xanthomonas suis* bacterial suspension; techniques well-known in the art can be used. The *Xanthomonas suis* GM-4 exhibits salt and drought tolerance.

[0054] As an optional implementation, this invention mixes a mixture of potassium alginate and pectin, a suspension of *Xanthomonas suis* GM-4, and calcium carbonate to obtain a first mixture. The technical solution of this invention can be achieved by any solid substance containing calcium ions; calcium carbonate was chosen because it is inexpensive and readily available.

[0055] The volume-to-mass ratio of the potassium alginate and pectin mixture to calcium carbonate in this invention is 50-200 mL: 0.5-2 g, or 80-150 mL: 0.8-1.5 g, more preferably 100 mL: 1 g. The volume-to-mass ratio of the potassium alginate and pectin mixture, the *Xanthomonas suis* suspension, and calcium carbonate in this invention is 100-300 mL: 10-30 mL: 0.5-2 g, or 100-200 mL: 10-20 mL: 0.5-1.5 g, more preferably 100 mL: 10 mL: 1 g. This invention does not have special requirements for the mixing method; uniform mixing is sufficient.

[0056] This invention involves emulsifying the first mixture with a first oil phase to obtain an emulsion. The emulsion of this invention is a water-in-oil mixture. As an optional embodiment, the first oil phase preferably comprises soybean oil or paraffin oil, more preferably soybean oil; the first oil phase preferably contains Span80; the concentration of Span80 in the first oil phase is preferably 0.5–2 g / mL, or 0.8–1.5 g / mL, more preferably 1 g / mL; the volume ratio of the first mixture to the first oil phase is 50–150:100–300, or 80–120:130–200, more preferably 111:200. The emulsification of this invention is preferably carried out under stirring conditions, the stirring speed is preferably 300–650 rpm, more preferably 600 rpm, and the emulsification time is preferably 5–20 min, more preferably 10 min. The obtained emulsion is then mixed with a second oil phase to obtain a water-in-oil mixture, and then allowed to stand to obtain an aqueous phase.

[0057] As an optional embodiment, the volume ratio of the first oil phase to the second oil phase of the present invention is preferably 100-400:50-100, more preferably 200:100. The second oil phase of the present invention preferably includes soybean oil or paraffin oil, more preferably soybean oil; the types of substances in the second oil phase are preferably the same as those in the first oil phase. As an optional embodiment, the second oil phase of the present invention contains glacial acetic acid; the volume ratio of the second oil phase to glacial acetic acid is preferably 50-100:0.2-0.6, more preferably 100:0.5. The mixing of the emulsion and the second oil phase is preferably carried out under stirring conditions; the stirring speed is preferably 300-620 rpm, more preferably 600 rpm; the stirring time is preferably 5-15 min, more preferably 10 min. Adding glacial acetic acid helps release carbonate ions from calcium carbonate, allowing the separated calcium ions to fully react with potassium alginate and pectin. The resulting calcium alginate and calcium pectin can form a tightly cross-linked network composite material, encapsulating probiotic microorganisms within microcapsules.

[0058] In this invention, the obtained emulsion is mixed with a second oil phase and then allowed to stand to obtain an aqueous phase. As an optional embodiment, the standing time is preferably 0.5–2 hours, more preferably 1 hour; this standing facilitates the separation of the second mixed substance, and the resulting lower layer is the aqueous phase. As an optional embodiment, the aqueous phase is centrifuged to obtain a precipitate. The centrifugation speed is preferably 300–630 rpm, more preferably 600 rpm; the centrifugation time is preferably 5–20 minutes, more preferably 10 minutes.

[0059] As an optional implementation, the present invention dries the precipitated material to obtain the probiotic microcapsules. The drying temperature of the present invention is ≤30℃; the present invention has no special requirements for the drying equipment and time, and the drying is based on the removal of moisture from the surface of the microcapsules. The probiotic microcapsules prepared by the present invention have a high survival rate of probiotics within the microcapsules and a high encapsulation rate of probiotics.

[0060] This invention provides the application of probiotic microcapsules obtained by the preparation method described above in the preparation of seed coating agents.

[0061] This invention provides a seed coating agent, comprising, by volume of solvent: 1-20 g / L probiotic microcapsules and 5-100 g / L film-forming agent; the probiotic microcapsules are probiotic microcapsules prepared by the preparation method described in the above technical solution. By volume of solvent, the concentration of probiotic microcapsules in the seed coating agent of this invention is 1-20 g / L. In specific embodiments of this invention, the concentration of the microcapsules can be 1, 3, 5, 8, 10, 13, 15, or 20 g / L. The presence of probiotic microcapsules helps to extend the storage time of probiotics and improve seed germination rate. By volume of solvent, the seed coating agent of this invention includes 5-100 g / L of film-forming agent, but can also be 6-50 g / L, 8-12 g / L, and more preferably 10 g / L. In specific embodiments of the present invention, the concentration of the film-forming agent can be 5, 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 g / L. As an optional embodiment, the film-forming agent of the present invention includes one or more of polyvinyl alcohol, carboxymethyl cellulose, and starch, more preferably polyvinyl alcohol, starch, or carboxymethyl cellulose. The film-forming agent of the present invention forms a continuous and uniform film on the surface of probiotic microcapsules, adhering to and fixing them onto the probiotic microcapsules, thereby improving the storage time of probiotics and the seed germination rate.

[0062] The method for preparing the seed coating agent of this invention includes mixing the film-forming agent and probiotic microcapsules. The seed coating agent prepared by this invention has a higher number of live bacteria.

[0063] This invention provides the application of probiotic microcapsules prepared by the preparation method described above, or the application of the seed coating agent described above in plant seed coating.

[0064] This invention provides the application of probiotic microcapsules prepared by the preparation method described above, or the seed coating agent described above, in promoting plant seed development.

[0065] As an optional implementation, promoting plant seed development includes one or more of the following: increasing plant seed germination rate, promoting root growth, increasing biomass, chlorophyll content, antioxidant enzyme activity, and reducing the content of oxidative harmful substances.

[0066] This invention provides the application of probiotic microcapsules prepared by the preparation method described in the above-described technical solutions, or the seed coating agent described in the above-described technical solutions, in improving the encapsulation rate of probiotics. The probiotic microcapsules prepared by this invention have a high encapsulation rate of probiotics.

[0067] This invention provides the application of probiotic microcapsules prepared by the preparation method described above, or the seed coating agent described above, in improving plant drought resistance.

[0068] As an optional implementation, the present invention does not have specific requirements regarding the type of plant. In a specific embodiment of the present invention, the plant is one or more of wheat, corn, and soybean.

[0069] The improvement of plant drought resistance described in this invention includes increasing the seedling emergence rate, root growth indicators, content of plant growth-promoting hormones, content of phenolic plant hormones, and reducing one or more of plant growth-inhibiting hormones under drought stress. As an optional embodiment, the root growth indicators include radicle length. As an optional embodiment, the plant growth-promoting hormones include auxin and / or gibberellin; the phenolic plant hormones include salicylic acid; and the plant growth-inhibiting hormones include abscisic acid. In specific embodiments of this invention, the probiotic microcapsules or seed coating agents of this invention are beneficial for improving the seedling emergence rate, radicle length, auxin, gibberellin, and salicylic acid content of maize seeds under drought stress, while reducing abscisic acid content.

[0070] This invention provides the application of probiotic microcapsules prepared by the preparation method described above, or the seed coating agent described above, in improving the salt tolerance of plants.

[0071] The present invention describes improving plant salt tolerance by promoting root growth, increasing seedling emergence rate, biomass, stem length, and the content of soluble sugars and soluble proteins under salt stress. As an optional implementation, promoting root growth includes increasing one or more of the following: total root length, total root area, average root diameter, total root volume, and root hair length.

[0072] The results of the examples show that the seed coating agent of the present invention is beneficial to improving the germination rate, root length, stem length, and content of soluble sugars and soluble proteins in soybean seeds under salt stress. The seed coating agent of the present invention is beneficial to improving the germination rate, biomass, total root length, total root area, average root diameter, total root volume, and root hair length in wheat seeds under salt stress.

[0073] This invention provides a method for improving the drought resistance and / or salt tolerance of plants, comprising the following steps: coating plant seeds with the seed coating agent described in the above technical solution before sowing. As an optional embodiment, the volume-to-mass ratio of the seed coating agent to the plant seeds is (1-5):(50-150), or (1-2):(50-80), more preferably 1:50. As an optional embodiment, the coating method of this invention includes a roller method or a dipping method. This invention does not specifically limit the parameters of the roller; conventional parameters are acceptable. This invention does not specifically limit the sowing method; conventional methods are acceptable.

[0074] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0075] Example 1

[0076] I. Functional identification of *Xanthomonas suis* GM-4

[0077] The identification method was based on existing technology (Huo Q, Gong M, Jiang Y, et al. Microencapsulated Microbial Seed Coating Could Improve Soil Environment and Maize Grain Yield in Saline Soil[J]. Plants, 2024, 13(22): 3139-3139), and the identification results are as follows. Figure 1 As shown, *Xanthomonas suis* strain GM-4 appears pink in the supernatant of LB broth containing L-tryptophan and Salkowski colorimetric solution, indicating that the strain has the ability to produce IAA. The production of bubbles after adding the bacterial culture to hydrogen peroxide solution indicates that *Xanthomonas suis* GM-4 is catalase-positive. On CAS medium, the blue color around the colony turns orange-yellow, indicating that this strain can produce siderophores.

[0078] II. Preparation of Probiotic Microcapsules

[0079] The preparation process of probiotic microcapsules is as follows: Figure 2 .

[0080] A method for preparing probiotic microcapsules, comprising the following steps:

[0081] 1. *Pseudomonas sugarbha* GM-4 was isolated from the rhizosphere soil of wheat in saline-alkali farmland in Dongying. *Pseudomonas sugarbha* GM-4 is capable of producing indoleacetic acid (IAA), catalase, and siderophore production. A bacterial suspension of *Pseudomonas sugarbha* GM-4 was prepared, with a viable count of 10-1. 8 CFU / mL. The culture medium used to prepare the bacterial suspension consisted of water as the solvent, with each 100 mL of water containing 0.1 g peptone, 0.1 g yeast extract, 0.5 g (NH4)2SO4, 1.5 g NaCl and 1.5 g agar, and the initial pH of the culture medium was 7.5.

[0082] 2. 100 mL of sterile potassium alginate and pectin mixture, the concentration of the mixture is 2% (w / v), the mixture contains 1.5 g of potassium alginate and 0.5 g of pectin.

[0083] Take 100 mL of potassium alginate and pectin mixture, 10 mL of *Xanthomonas syringae* suspension obtained in step 1, and 1 g of calcium carbonate, mix and homogenize to obtain a mixture. Disperse the mixture into 200 mL of soybean oil phase containing 1% (w / v) Span 80, and then stir at 600 rpm for 10 min to emulsify. Then, add another 100 mL of soybean oil containing 0.5 mL of glacial acetic acid and 1% (w / v) Span 80, and continue stirring at 600 rpm for 10 min. After standing for 1 h to separate the layers, centrifuge the lower aqueous phase at 300–800 rpm. Dry the precipitate obtained by centrifugation at ≤30℃ to finally obtain probiotic microcapsules. Optical and electron microscopy scanning images of the probiotic microcapsules are shown below. Figure 3 From b and c, it can be seen that the microcapsules have a uniform particle size distribution, good sphericity, smooth surface, and no depressions. Figure 3 Image a is an electron microscope image of Xanthomonas suisinii GM-4.

[0084] Comparative Example 1

[0085] Same as Example 1, except that in step 2, 100 mL of a mixture of 2% (w / v) potassium alginate and pectin is used, with 0.5 g of potassium alginate and 1.5 g of pectin in the mixture.

[0086] Comparative Example 2

[0087] Same as Example 1, except that in step 2, 100 mL of a mixture of 2% (w / v) potassium alginate and pectin is used, with 1.0 g of potassium alginate and 1.0 g of pectin in the mixture.

[0088] Comparative Example 3

[0089] Same as Example 1, except that step 2 is 100 mL of 2% (w / v) potassium alginate solution, and the potassium alginate content in the potassium alginate solution is 2.0 g.

[0090] Comparative Example 4

[0091] Same as Example 1, except that in step 2, there is 100 mL of 2% (w / v) pectin solution and the pectin content in the pectin solution is 2.0 g.

[0092] Microcapsule performance analysis:

[0093] The encapsulation efficiency of the microcapsules was determined using the dilution-spread plate method. 0.1 g of microcapsules were placed in a beaker, and 20 mL of 0.05 mol / L potassium dihydrogen phosphate solution (sterilized for 15 min) was added. The mixture was thoroughly mixed and then transferred to a constant-temperature shaker (180 rpm, 37 °C). The microcapsules were shaken for 1 h to dissolve, thus completely releasing the microorganisms embedded within them. The sample was diluted with distilled water to an appropriate concentration, inoculated onto a culture medium, and incubated at 37 °C for 48 hours for counting. Results are expressed as log CFU / g. The experiment was repeated three times. The encapsulation efficiency (E) was calculated as: E = N / N0 × 100%, where N is the amount of microorganisms encapsulated within the microcapsules, and N0 is the total amount of microorganisms before encapsulation.

[0094] The particle size distribution of the microcapsules was measured using a dynamic laser light scattering experimental system (Zetasizer Nano ZS, Bettersize Ltd., Dandong, China). The particle size span (SPAN) value represents the particle size dispersion, calculated according to the equation SPAN = (D... 90 -D 10 ) / D 50 D 90 D 50 and D 10 These represent the average particle size at 90%, 50%, and 10% of the cumulative volume, respectively.

[0095] For viscosity determination, the prepared microcapsules were diluted with deionized water by 1:1 and mixed well. The viscosity value of the sample was measured using an NDJ-5S (Shanghai Jingtian Electronic Instruments Technology Co., Ltd., Shanghai, China, Rotor No. 2) rotational viscometer.

[0096] Table 1 Characterization of microcapsules

[0097] Name Viscosity (mPa-s) Particle size span SPAN Encapsulation efficiency (%) Example 1 1069.7±81.3b 1.3±0.003b 90.6±2.4a Comparative Example 1 605.0±40.5d 1.4±0.007b 65.3±2.8c Comparative Example 2 826.0±64.5c 1.4±0.001b 73.4±1.7b Comparative Example 3 1745.3±28.7a 1.0±0.002c 75.7±3.2b Comparative Example 4 239.7±34.1e 1.6±0.006a 64.2±1.3c

[0098] As shown in Table 1, the viscosity of the composite microcapsules gradually decreased with increasing pectin ratio, while the particle size distribution of the microcapsules was most uniform and consistent when the ratio of potassium alginate to pectin was 3:1. The encapsulation efficiency of the composite microcapsules initially increased and then decreased with increasing pectin ratio, reaching its maximum at a potassium alginate to pectin ratio of 3:1 (Example 1). Encapsulation efficiency is the most important indicator in probiotic microcapsules and plays a decisive role in their preparation. Therefore, in subsequent experiments, a potassium alginate to pectin ratio of 3:1 (Example 1) was used as the preparation scheme for probiotic microcapsules.

[0099] Example 2

[0100] A method to improve plant salt tolerance includes the following steps:

[0101] (1) Seed coating agent prepared using polyvinyl alcohol as a film-forming agent: 0.1 g of the probiotic microcapsules prepared in Example 1 and 1 g of polyvinyl alcohol were dissolved in 100 mL of water to obtain the seed coating agent. The concentration of the probiotic microcapsules in the seed coating agent was 1 g / L and the concentration of polyvinyl alcohol was 10 g / L, based on the volume of water. The ratio of the volume of the seed coating agent to the mass of wheat was 1:50.

[0102] Using a roller, the seed coating agent from step (1) was evenly coated onto the surface of wheat (Jimai 22) seeds. The coated wheat seeds were then planted in potting soil containing sodium chloride (saturated conductivity EC = 8 ds / m). 20 seeds were planted in each pot, and each pot was considered a treatment. Each treatment was tested in three parallel experiments, and routine management was carried out.

[0103] Comparative Example 5

[0104] Wheat seeds (Jimai 22) were sown in potting soil containing sodium chloride (saturated conductivity EC = 8 ds / m), with 20 seeds planted in each pot. Each pot was considered a treatment, and each treatment was tested in triplicate. Routine management was performed.

[0105] Results and Analysis:

[0106] Seven days after planting, the growth and physiological indicators of the wheat in Example 2 and Comparative Example 5 were statistically analyzed, and the results are shown in Tables 2 and 3. Figure 4 and Figure 5 .

[0107] Table 2 Effects of different treatments on wheat growth

[0108]

[0109] Note: Different letters in the table represent significant differences (P < 0.05), and the same applies below.

[0110] Table 3 Effects of different treatments on wheat physiological indicators

[0111]

[0112] From Tables 2-3, Figures 4 to 5 As can be seen, after adopting the technical solution provided in this application, the wheat emergence rate, root growth indicators, and biomass were significantly improved. In terms of physiological indicators, the chlorophyll and antioxidant enzyme activities (CAT, SOD, and POD) of wheat were significantly increased, while the content of oxidative harmful substances (H2O2 and MDA) was significantly reduced. Microencapsulated wheat exhibited enhanced photosynthesis and salt tolerance. Figure 5 Different letters in the text represent significant differences (P < 0.05).

[0113] Example 3

[0114] A method to improve plant salt tolerance includes the following steps:

[0115] (1) Using starch as a film-forming agent, a seed coating agent was prepared: 1 g of microcapsules prepared in Example 1 and 10 g of starch were dissolved in 100 mL of aqueous solution to obtain the seed coating agent. Based on the volume of water, the concentration of microcapsules in the seed coating agent was 10 g / L and the concentration of starch was 100 g / L.

[0116] (2) Using a roller, the seed coating agent from step (1) was evenly coated onto the surface of soybean seeds (Zhonghuang 13). The coated seeds were then sown in potting soil containing sodium chloride (saturated conductivity EC = 6 ds / m). Five seeds were planted in each pot, with each pot constituting one treatment. Each treatment was repeated three times in parallel, and routine management was performed. The ratio of seed coating agent volume to soybean seed mass was 1:50.

[0117] Comparative Example 6

[0118] Soybean seeds were sown in potting soil containing sodium chloride (saturated conductivity EC = 6 ds / m), with 5 seeds planted in each pot. Each pot was considered a treatment, and each treatment was tested in triplicate. Routine management was performed.

[0119] Results and Analysis:

[0120] Fifteen days after planting, the growth of soybeans in Example 3 and Comparative Example 6 was statistically analyzed, and the results are shown in Table 4. The soybean growth was observed... Figure 6 .

[0121] Table 4. Effects of different treatments on soybean germination rate and seedling growth under salt stress.

[0122]

[0123] Note: Different letters in the table represent significant differences (P < 0.05).

[0124] From Table 4 and Figure 6 It can be seen that after adopting the technical solution provided in this application, the emergence rate of soybeans under salt stress is significantly improved, the root length and stem length are significantly improved, and the content of soluble sugar and soluble protein is significantly improved.

[0125] Example 4

[0126] A method to improve plant drought resistance, the steps are as follows:

[0127] (1) Seed coating agent prepared using carboxymethyl cellulose as a film-forming agent: 1g of microcapsules and 10g of carboxymethyl cellulose prepared in Example 2 were dissolved in 100mL of aqueous solution to obtain the seed coating agent. The concentration of microcapsules in the seed coating agent was 10g / L and the concentration of carboxymethyl cellulose was 100g / L, based on the volume of water.

[0128] (2) Using a roller, the seed coating agent from step (1) was evenly coated onto the surface of corn (Zhengdan 958) seeds. Then, the corn seeds were placed in a solution containing PEG6000 (15%) to simulate a drought environment. Each treatment was repeated in triplicate for germination tests. The ratio of seed coating agent volume to corn seed mass was 1:50.

[0129] Comparative Example 7

[0130] A germination test was conducted by placing corn seeds in a solution containing PEG6000 at a mass concentration of 15%.

[0131] Seven days after germination, the growth of corn seeds in Example 4 and Comparative Example 7 was statistically analyzed, and the results are shown in Table 5. The germination status of the corn seeds is as follows: Figure 7 As shown.

[0132] Table 5 Effects of different treatments on maize seeds

[0133]

[0134]

[0135] Note: Different letters in the table represent significant differences (P < 0.05).

[0136] From Table 5 and Figure 7 It can be seen that after adopting the technical solution provided in this application, the seedling emergence rate of maize seeds under drought stress for 7 days was significantly increased compared with Comparative Example 7, the radicle length was significantly increased, the content of auxin, gibberellin and salicylic acid plant hormones was significantly increased, and the content of abscisic acid was significantly reduced, showing significant effects.

[0137] In summary, the seed coating agent prepared using the microcapsules of this invention can significantly improve the drought resistance and salt tolerance of plants, and can not only promote plant growth but also improve plant quality.

[0138] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of *Pseudoxanthomonas suwonensis* GM-4, with accession number CGMCCNo.34616.

2. A method for preparing probiotic microcapsules, characterized in that, include: A first mixture is obtained by mixing a mixture of potassium alginate and pectin, the *Xanthomonas suis* GM-4 as described in claim 1, and calcium carbonate; the volume-to-mass ratio of the mixture of potassium alginate and pectin to calcium carbonate is 50-200 mL: 0.5-2 g. The mass concentration of potassium alginate in the mixture of potassium alginate and pectin is 10-55 g / L, and the mass concentration of pectin is 5-55 g / L; and the mass ratio of potassium alginate to pectin in the mixture is (2.5-3.5):

1. The first mixture is emulsified with the first oil phase, then mixed with the second oil phase and allowed to stand to obtain an aqueous phase substance. The aqueous phase material was centrifuged and dried to obtain probiotic microcapsules.

3. The preparation method according to claim 2, characterized in that, The application form of *Xanthomonas suis* includes a bacterial suspension, wherein the viable count of *Xanthomonas suis* in the bacterial suspension is ≥10. 8 CFU / mL.

4. The preparation method according to claim 3, characterized in that, The volume ratio of the bacterial suspension, the potassium alginate and pectin mixture is (10-30):(100-300).

5. The preparation method according to claim 2, characterized in that, The first oil phase comprises soybean oil or paraffin oil; the second oil phase comprises soybean oil or paraffin oil. The first oil phase contains Span80, and the concentration of Span80 in the first oil phase is 0.5-2 g / mL; The second oil phase contains Span80 and glacial acetic acid; the concentration of Span80 in the second oil phase is 0.5-2 g / mL; the volume ratio of the second oil phase to glacial acetic acid is 50-100:0.2-0.6; the volume ratio of the first oil phase to the second oil phase is 100-400:50-100.

6. The use of the *Xanthomonas suis* GM-4 as described in claim 1 or the probiotic microcapsules prepared by any one of claims 2 to 5 in the preparation of seed coating agents.

7. A seed coating agent, characterized in that, Based on the volume of the solvent, it comprises: 1-20 g / L probiotic microcapsules and 5-100 g / L film-forming agent; wherein the probiotic microcapsules are probiotic microcapsules obtained by any one of claims 2-5.

8. The seed coating agent according to claim 7, characterized in that, The film-forming agent includes one or more of polyvinyl alcohol, carboxymethyl cellulose, and starch.

9. The use of the probiotic microcapsules prepared by the preparation method according to any one of claims 2 to 5, or the seed coating agent according to claim 7 or 8, in at least one of the following: 1) Plant seed coating; 2) Promotes plant seed development; 3) Improve the encapsulation rate of probiotics; 4) Improve plant drought resistance; 5) Improve the salt tolerance of plants.

10. A method for improving the drought resistance and / or salt tolerance of plants, characterized in that, The process includes the following steps: coating plant seeds with the seed coating agent described in claim 7 or 8 before sowing.

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