A biological agent that promotes microbial colonization and its application

Abstract

This disclosure provides a safe, efficient, and low-cost biological agent comprising a composition of chitosan oligosaccharide and alginate oligosaccharide, wherein the mass ratio of chitosan oligosaccharide to alginate oligosaccharide is 1:(0.3-1.5). The biological agent specifically promotes the colonization of *Bacillus amyloliquefaciens* in the rhizosphere of tobacco, and is environmentally friendly, highly compatible with the strain, and enhances the strain's attachment and reproductive capacity in the rhizosphere. It not only effectively solves the application problems of *Bacillus amyloliquefaciens* in tobacco cultivation, but also provides a reference for the colonization and regulation of biocontrol bacteria in other crops, and has significant practical implications for promoting the large-scale application of agricultural biological control technologies.

Description

Technical Field

[0001] This disclosure belongs to the field of plant disease and pest control technology, specifically relating to a biological agent that promotes microbial colonization and its application. Background Technology

[0002] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens As a recognized plant growth-promoting and biocontrol agent, *Bacillus amyloliquefaciens* can produce a variety of antimicrobial substances and induce systemic resistance in plants, demonstrating great potential in the control of soil-borne plant diseases. However, in practical applications, biocontrol agents generally suffer from the bottleneck problem of low rhizosphere colonization efficiency. Exogenously applied *Bacillus amyloliquefaciens* often struggles to effectively compete with indigenous microorganisms, resulting in limited survival and reproduction capacity in the plant rhizosphere, leading to unstable biocontrol effects and short-term field efficacy.

[0003] Currently, conventional methods for improving the colonization efficiency of Bacillus amyloliquefaciens have significant limitations. For example, while strain screening and genetic modification can yield mutant strains with stronger colonization capabilities, they require substantial time for safety evaluation and field adaptability testing, and the ecological risks of the modified strains remain controversial. Optimizing application methods (such as seed coating, root irrigation, and furrow application) can increase rhizosphere inoculum in the short term, but it cannot fundamentally enhance the strains' competitiveness. While developing carrier materials (such as biochar, inoculum residue, and organic fertilizer composite carriers) can protect strains through physical adsorption, these carriers are costly, and some may alter the original soil microbial community structure. These methods are either complex to operate, costly, or have unstable effects, making them difficult to promote on a large scale in plant cultivation.

[0004] Tobacco, as an important economic crop, has a complex rhizosphere environment and is prone to soil-borne diseases, thus requiring higher colonization capabilities from biocontrol agents. Therefore, developing a safe, efficient, and low-cost adjuvant to specifically promote the colonization of Bacillus amyloliquefaciens in the tobacco rhizosphere can not only solve a key bottleneck in the current application of biocontrol agents but also provide a new technical pathway for green pest control in tobacco. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this disclosure provides a safe, efficient, and low-cost biological agent that specifically promotes the colonization of Bacillus amyloliquefaciens in the rhizosphere of tobacco. It is also environmentally friendly, highly compatible with the strain, and enhances the strain's attachment and reproductive capacity in the rhizosphere. This not only effectively solves the application problems of Bacillus amyloliquefaciens in tobacco cultivation but also provides a reference for the colonization and regulation of biocontrol bacteria in other crops, and has significant practical implications for promoting the large-scale application of agricultural biological control technologies.

[0006] Therefore, the present disclosure provides the following technical solution.

[0007] As a first aspect of this disclosure, a biological agent is provided.

[0008] As a second aspect of this disclosure, a method for preparing the biological agent described in the first aspect is provided.

[0009] As a third aspect of this disclosure, the use of the biological agents described in the first aspect in promoting plant rhizosphere colonization in microorganisms is provided.

[0010] As a fourth aspect of this disclosure, the use of the biological agents described in the first aspect in the prevention and control of plant diseases and / or the promotion of plant growth is provided.

[0011] As a fifth aspect of this disclosure, a method for promoting microbial colonization in the plant rhizosphere is provided.

[0012] As a sixth aspect of this disclosure, a method for preventing and controlling plant diseases and / or promoting plant growth is provided.

[0013] According to a first aspect of this disclosure, a biological agent is disclosed comprising a composition of chitosan oligosaccharide and alginate oligosaccharide, wherein the mass ratio of chitosan oligosaccharide to alginate oligosaccharide is 1:(0.3-1.5).

[0014] In some embodiments, the mass ratio of chitosan oligosaccharide to alginate oligosaccharide can be 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 or any value between them.

[0015] In some embodiments, the molecular weight of the chitosan oligosaccharide is ≤3000 Da, and the molecular weight can be 3000 Da, 2900 Da, 2800 Da, 2700 Da, 2600 Da, 2500 Da, 2400 Da, 2300 Da, 2200 Da, 2100 Da, 2000 Da, 1900 Da, 1800 Da, 1700 Da, 1600 Da, 1500 Da, 1400 Da, 1300 Da, 1200 Da, 1100 Da, 1000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, or any value between them. In some embodiments, it is 1000-2500 Da.

[0016] In some embodiments, the degree of polymerization of the chitosan oligosaccharide is 2-10, and the degree of polymerization can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or any value between them. In some embodiments, it is 3-8.

[0017] In some embodiments, the degree of deacetylation of the chitosan oligosaccharide is ≥90%, for example, it can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any value between them. In some embodiments, it is 90%-95%.

[0018] In some embodiments, the molecular weight of the alginate oligosaccharide is ≤5000 Da, and the molecular weight can be 5000 Da, 4800 Da, 4600 Da, 4400 Da, 4200 Da, 3800 Da, 3600 Da, 3400 Da, 3200 Da, 3000 Da, 2800 Da, 2600 Da, 2400 Da, 2200 Da, 2000 Da, 1800 Da, 1600 Da, 1400 Da, 1200 Da, 1000 Da, 800 Da, 600 Da, 400 Da, 200 Da, or any value between them. In some embodiments, it is 3000-4000 Da.

[0019] In some embodiments, the degree of polymerization of the alginate oligosaccharide is 2-15, and the degree of polymerization can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or any value between them. In some embodiments, it is 5-10.

[0020] In some embodiments, the biological agent further includes additional active agents and / or excipients.

[0021] In some embodiments, the adjuvant activator is selected from one or more biocontrol agents, one or more chemical agents, one or more fertilizers, one or more herbicides, one or more growth promoters, or any combination thereof.

[0022] In some embodiments, the excipients are selected from agriculturally or horticulturally acceptable carriers, diluents, stabilizers, fillers, wetting agents, colorants, solvents, co-solvents, film-forming agents, spontaneous growth promoters, emulsifiers, dispersants, preservatives, antifreeze agents, thickeners, adjuvants, pH adjusters, or any combination thereof.

[0023] In some embodiments, the dispersant is selected from at least one of carboxymethyl cellulose, sodium polythiophosphate, xanthan gum, and Morwet D-500.

[0024] In some embodiments, the biological agent is in solid form, liquid form, powder form, or any combination thereof.

[0025] In some embodiments, the biological agent is selected from solutions, powders, granules, emulsions, emulsions, suspensions, tablets, microparticles, and wettable powders.

[0026] According to a second aspect of this disclosure, a method for preparing the biological agent described in the first aspect of this disclosure is provided, comprising the following steps: 1) Dissolving: Add chitosan oligosaccharide and alginate oligosaccharide to water and stir until completely dissolved; 2) Compound preparation: Add excipients and mix thoroughly; 3) Adjust pH: Adjust the pH of the mixture to near neutral; 4) Homogenization: The mixture is homogenized to form a stable homogeneous liquid.

[0027] In some embodiments, the method for preparing the biological agent includes the following steps: 1) Dissolving: Add the prescribed amounts of chitosan oligosaccharide and alginate oligosaccharide to an appropriate amount of water and stir until completely dissolved; 2) Compounding: Add dispersant and protective agent in sequence, and continue stirring to mix evenly; 3) Adjustment: Use a pH adjuster to adjust the pH of the mixture to 6.5-7.5; 4) Homogenization: The mixture is homogenized using a high-pressure homogenizer to form a stable homogeneous liquid, thus obtaining the biological agent.

[0028] In some embodiments, the pH of the mixed solution is adjusted to a range of values ​​such as 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, and 7.5 using a pH adjuster.

[0029] According to a third aspect of this disclosure, the application of the biological agents described in the first aspect of this disclosure in promoting plant rhizosphere colonization in microorganisms is disclosed.

[0030] In some embodiments, the microorganism is selected from one or more of bacteria, fungi, and viruses.

[0031] In some embodiments, the microorganism is selected from Bacillus subtilis (Bacillus subtilis). Bacillus subtilis Rennograf Holder ( Burkholderia rhinojensis ), Bacillus pumilus ( Bacillus pumilus ), Bacillus belesi ( Bacillus velezensis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Methylobacterium roximatei Methylobacterium rhodesianum ), Methylobacterium tumefaciens ( Methylobacterium extorting ) and Pirere's Bacillus ( Paenibacillus peoriae At least one of the following.

[0032] In some embodiments, the plant is selected from one or more of food crops, cash crops, and root and tuber crops.

[0033] In some embodiments, the food crop is selected from at least one of cereal crops, tuber crops, and legume crops.

[0034] In some embodiments, the cereal crop includes at least one of the following: rice, wheat, corn, barley, oats, rye, sorghum, millet, foxtail millet, barnyard millet, and buckwheat.

[0035] In some embodiments, the tuber crop includes at least one of the following: sweet potato, yam, or potato.

[0036] In some embodiments, the legume crop includes, but is not limited to, at least one of the following: soybeans, broad beans, peas, mung beans, and peanuts.

[0037] In some embodiments, the economic crop includes at least one of the following: plants from the Solanaceae, Rosaceae, Rutaceae, Musaceae, Cucurbitaceae, Brassicaceae, Orchidaceae, Fabaceae, Asteraceae, Liliaceae, Zingiberaceae, Passifloraceae, Bromeliaceae, Araliaceae, and Cactaceae families.

[0038] In some embodiments, the nightshade plants include tobacco, tomato, pepper, potato, and eggplant.

[0039] In some embodiments, the Rosaceae plants include strawberries and papayas.

[0040] In some embodiments, the Rutaceae plant includes citrus.

[0041] In some embodiments, the Musaceae plant includes banana.

[0042] In some embodiments, the cucurbitaceous plants include cucumber, winter melon, pumpkin, bitter melon, loofah, watermelon, and monk fruit.

[0043] In some embodiments, the cruciferous plants include Chinese cabbage, rapeseed, kale, radish, and cauliflower.

[0044] In some embodiments, the orchid family includes orchids.

[0045] In some embodiments, the Fabaceae plant includes soybean.

[0046] In some embodiments, the Asteraceae plant includes lettuce.

[0047] In some embodiments, the lily family plant includes garlic.

[0048] In some embodiments, the ginger family plant includes ginger.

[0049] In some embodiments, the passionflower plant includes passion fruit.

[0050] In some embodiments, the bromeliad includes golden pineapple.

[0051] In some embodiments, the Araliaceae plant includes Panax notoginseng.

[0052] In some embodiments, the cactus plant includes dragon fruit.

[0053] In some embodiments, the root and tuber crops include potatoes, carrots, leafy vegetables, solanaceous vegetables, strawberries, grapes, citrus fruits, bananas, kiwifruit, dragon fruit, tomatoes, peppers, beans, ginger, Panax notoginseng, and ginseng.

[0054] According to the fourth aspect of this disclosure, the use of the biological agents described in the first aspect in the prevention and control of plant diseases and / or the promotion of plant growth is disclosed.

[0055] In some embodiments, the plant diseases and pests are selected from plant replanting diseases, plant bacterial diseases, plant fungal diseases, plant viral diseases, plant soil-borne diseases, plant insect pests and / or plant oomycete diseases.

[0056] In some embodiments, the plant diseases and pests are caused by one or more of plant pathogenic bacteria, fungi, viruses, insects, insect eggs, and nematodes.

[0057] In some embodiments, the control of plant diseases and pests is achieved by generating siderophores, inducing systemic resistance of plants or their seeds to plant pathogens, and / or reducing the chemotaxis of plants to plant pathogens.

[0058] In some embodiments, the promotion of plant growth is selected from one or more of increasing plant height, increasing stem diameter, and promoting plant root development.

[0059] In some embodiments, promoting plant growth includes promoting the growth of plant organs and / or tissues.

[0060] In some embodiments, the promotion of plant organ growth is selected from promoting the growth of plant roots, stems, leaves, or any combination thereof.

[0061] In some embodiments, the plant organ comprises one or more of the following: root, stem, leaf, flower, fruit, and seed.

[0062] In some embodiments, the plant tissue comprises one or more of meristematic tissue, protective tissue, ground tissue, and vascular tissue.

[0063] In some embodiments, the promotion of plant growth is achieved by providing at least one of nitrogen fixation, phosphorus solubilization, indoleacetic acid production, gibberellin production, and weed removal.

[0064] In some embodiments, the plant is selected from one or more of food crops, cash crops, and root and tuber crops.

[0065] In some embodiments, the food crop is selected from at least one of cereal crops, tuber crops, and legume crops.

[0066] In some embodiments, the cereal crop includes at least one of the following: rice, wheat, corn, barley, oats, rye, sorghum, millet, foxtail millet, barnyard millet, and buckwheat.

[0067] In some embodiments, the tuber crop includes at least one of the following: sweet potato, yam, or potato.

[0068] In some embodiments, the legume crop includes, but is not limited to, at least one of the following: soybeans, broad beans, peas, mung beans, and peanuts.

[0069] In some embodiments, the economic crop includes at least one of the following: plants from the Solanaceae, Rosaceae, Rutaceae, Musaceae, Cucurbitaceae, Brassicaceae, Orchidaceae, Fabaceae, Asteraceae, Liliaceae, Zingiberaceae, Passifloraceae, Bromeliaceae, Araliaceae, and Cactaceae families.

[0070] In some embodiments, the nightshade plants include tobacco, tomato, pepper, potato, and eggplant.

[0071] In some embodiments, the Rosaceae plants include strawberries and papayas.

[0072] In some embodiments, the Rutaceae plant includes citrus.

[0073] In some embodiments, the Musaceae plant includes banana.

[0074] In some embodiments, the cucurbitaceous plants include cucumber, winter melon, pumpkin, bitter melon, loofah, watermelon, and monk fruit.

[0075] In some embodiments, the cruciferous plants include Chinese cabbage, rapeseed, kale, radish, and cauliflower.

[0076] In some embodiments, the orchid family includes orchids.

[0077] In some embodiments, the Fabaceae plant includes soybean.

[0078] In some embodiments, the Asteraceae plant includes lettuce.

[0079] In some embodiments, the lily family plant includes garlic.

[0080] In some embodiments, the ginger family plant includes ginger.

[0081] In some embodiments, the passionflower plant includes passion fruit.

[0082] In some embodiments, the bromeliad includes golden pineapple.

[0083] In some embodiments, the Araliaceae plant includes Panax notoginseng.

[0084] In some embodiments, the cactus plant includes dragon fruit.

[0085] In some embodiments, the root and tuber crops include potatoes, carrots, leafy vegetables, solanaceous vegetables, strawberries, grapes, citrus fruits, bananas, kiwifruit, dragon fruit, tomatoes, peppers, beans, ginger, Panax notoginseng, and ginseng.

[0086] According to a fifth aspect of this disclosure, a method for promoting microbial colonization in the rhizosphere of plants is disclosed, the method comprising applying to the plant or seeds the biological agent described in the first aspect of this disclosure.

[0087] In some embodiments, the microorganism is selected from one or more of bacteria, fungi, and viruses.

[0088] In some embodiments, the microorganism is selected from Bacillus subtilis (Bacillus subtilis). Bacillus subtilis Rennograf Holder ( Burkholderia rhinojensis ), Bacillus pumilus ( Bacillus pumilus ), Bacillus belesi ( Bacillus velezensis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Methylobacterium roximatei Methylobacterium rhodesianum ), Methylobacterium tumefaciens ( Methylobacterium extorting ) and Pirere's Bacillus ( Paenibacillus peoriae At least one of the following.

[0089] In some embodiments, the plant is selected from one or more of food crops, cash crops, and root and tuber crops.

[0090] In some embodiments, the food crop is selected from at least one of cereal crops, tuber crops, and legume crops.

[0091] In some embodiments, the cereal crop includes at least one of the following: rice, wheat, corn, barley, oats, rye, sorghum, millet, foxtail millet, barnyard millet, and buckwheat.

[0092] In some embodiments, the tuber crop includes at least one of the following: sweet potato, yam, and potato. In some embodiments, the legume crop includes, but is not limited to, at least one of the following: soybean, broad bean, pea, mung bean, and peanut.

[0093] In some embodiments, the economic crop includes at least one of the following: plants from the Solanaceae, Rosaceae, Rutaceae, Musaceae, Cucurbitaceae, Brassicaceae, Orchidaceae, Fabaceae, Asteraceae, Liliaceae, Zingiberaceae, Passifloraceae, Bromeliaceae, Araliaceae, and Cactaceae families.

[0094] In some embodiments, the nightshade plants include tobacco, tomato, pepper, potato, and eggplant.

[0095] In some embodiments, the Rosaceae plants include strawberries and papayas.

[0096] In some embodiments, the Rutaceae plant includes citrus.

[0097] In some embodiments, the Musaceae plant includes banana.

[0098] In some embodiments, the cucurbitaceous plants include cucumber, winter melon, pumpkin, bitter melon, loofah, watermelon, and monk fruit.

[0099] In some embodiments, the cruciferous plants include Chinese cabbage, rapeseed, kale, radish, and cauliflower.

[0100] In some embodiments, the orchid family includes orchids.

[0101] In some embodiments, the Fabaceae plant includes soybean.

[0102] In some embodiments, the Asteraceae plant includes lettuce.

[0103] In some embodiments, the lily family plant includes garlic.

[0104] In some embodiments, the ginger family plant includes ginger.

[0105] In some embodiments, the passionflower plant includes passion fruit.

[0106] In some embodiments, the bromeliad includes golden pineapple.

[0107] In some embodiments, the Araliaceae plant includes Panax notoginseng.

[0108] In some embodiments, the cactus plant includes dragon fruit.

[0109] In some embodiments, the root and tuber crops include potatoes, carrots, leafy vegetables, solanaceous vegetables, strawberries, grapes, citrus fruits, bananas, kiwifruit, dragon fruit, tomatoes, peppers, beans, ginger, Panax notoginseng, and ginseng.

[0110] In some embodiments, the application includes one or more of the following: root soaking, spraying, composting, seed soaking, coating, flood irrigation, drip irrigation of plants or plant organs, smearing of plants or plant organs, and drip application of plants or plant organs.

[0111] In some embodiments, the application includes one or more of root irrigation, seed soaking, and seed coating.

[0112] According to a sixth aspect of this disclosure, a method for controlling plant diseases and pests and / or promoting plant growth is disclosed, the method comprising applying the biological agent described in the first aspect of this disclosure to the plant or seeds.

[0113] In some embodiments, the plant diseases and pests are selected from plant replanting diseases, plant bacterial diseases, plant fungal diseases, plant viral diseases, plant soil-borne diseases, plant insect pests and / or plant oomycete diseases.

[0114] In some embodiments, the plant diseases and pests are caused by one or more of plant pathogenic bacteria, fungi, viruses, insects, insect eggs, and nematodes.

[0115] In some embodiments, the control of plant diseases and pests is achieved by generating siderophores, inducing systemic resistance of plants or their seeds to plant pathogens, and / or reducing the chemotaxis of plants to plant pathogens.

[0116] In some embodiments, the promotion of plant growth is selected from one or more of increasing plant height, increasing stem diameter, and promoting plant root development.

[0117] In some embodiments, promoting plant growth includes promoting the growth of plant organs and / or tissues.

[0118] In some embodiments, the promotion of plant organ growth is selected from promoting the growth of plant roots, stems, leaves, or any combination thereof.

[0119] In some embodiments, the plant organ comprises one or more of the following: root, stem, leaf, flower, fruit, and seed.

[0120] In some embodiments, the plant tissue comprises one or more of meristematic tissue, protective tissue, ground tissue, and vascular tissue.

[0121] In some embodiments, the promotion of plant growth is achieved by providing at least one of nitrogen fixation, phosphorus solubilization, indoleacetic acid production, gibberellin production, and weed removal.

[0122] In some embodiments, the plant is selected from one or more of food crops, cash crops, and root and tuber crops.

[0123] In some embodiments, the food crop is selected from at least one of cereal crops, tuber crops, and legume crops.

[0124] In some embodiments, the cereal crop includes at least one of the following: rice, wheat, corn, barley, oats, rye, sorghum, millet, foxtail millet, barnyard millet, and buckwheat.

[0125] In some embodiments, the tuber crop includes at least one of the following: sweet potato, yam, or potato.

[0126] In some embodiments, the legume crop includes, but is not limited to, at least one of the following: soybeans, broad beans, peas, mung beans, and peanuts.

[0127] In some embodiments, the economic crop includes at least one of the following: plants from the Solanaceae, Rosaceae, Rutaceae, Musaceae, Cucurbitaceae, Brassicaceae, Orchidaceae, Fabaceae, Asteraceae, Liliaceae, Zingiberaceae, Passifloraceae, Bromeliaceae, Araliaceae, and Cactaceae families.

[0128] In some embodiments, the nightshade plants include tobacco, tomato, pepper, potato, and eggplant.

[0129] In some embodiments, the Rosaceae plants include strawberries and papayas.

[0130] In some embodiments, the Rutaceae plant includes citrus.

[0131] In some embodiments, the Musaceae plant includes banana.

[0132] In some embodiments, the cucurbitaceous plants include cucumber, winter melon, pumpkin, bitter melon, loofah, watermelon, and monk fruit.

[0133] In some embodiments, the cruciferous plants include Chinese cabbage, rapeseed, kale, radish, and cauliflower.

[0134] In some embodiments, the orchid family includes orchids.

[0135] In some embodiments, the Fabaceae plant includes soybean.

[0136] In some embodiments, the Asteraceae plant includes lettuce.

[0137] In some embodiments, the lily family plant includes garlic.

[0138] In some embodiments, the ginger family plant includes ginger.

[0139] In some embodiments, the passionflower plant includes passion fruit.

[0140] In some embodiments, the bromeliad includes golden pineapple.

[0141] In some embodiments, the Araliaceae plant includes Panax notoginseng.

[0142] In some embodiments, the cactus plant includes dragon fruit.

[0143] In some embodiments, the root and tuber crops include potatoes, carrots, leafy vegetables, solanaceous vegetables, strawberries, grapes, citrus fruits, bananas, kiwifruit, dragon fruit, tomatoes, peppers, beans, ginger, Panax notoginseng, and ginseng. Detailed Implementation

[0144] The technical solutions in the embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this disclosure, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without inventive effort are within the scope of protection of this disclosure.

[0145] Unless otherwise stated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise stated, the practice of this disclosure employs conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology, and pharmacology known to those skilled in the art.

[0146] It should be noted that all headings and subheadings used herein are for convenience only and should not be construed as limiting this disclosure in any way. Unless otherwise stated, the use of any and all examples or exemplary wording provided herein (e.g., “such”) is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention.

[0147] define The term "alginate oligosaccharides" used in this article, also known as seaweed oligosaccharides or alginate oligosaccharides, is a linear long-chain molecule produced from cold-water brown algae using low-temperature directional biolysis technology. It consists of irregularly linked 1,4-mannuronic acid (M) and 1,4-guluronic acid (G), representing the most active fragment in brown algae raw materials. It can rapidly promote the synthesis of endogenous growth hormones in plants, accelerate root growth, and improve nutrient absorption efficiency. Studies have shown that alginate oligosaccharides can mitigate the harmful effects of abiotic stresses such as high salt and heavy metals. Alginate oligosaccharides degraded by *Streptomyces alginate* have been used as effective plant growth promoters. Its structural formula is as follows: The term "chitosan oligosaccharide" used in this article, also known as amino oligosaccharide or chitosan oligosaccharide, is an oligosaccharide product with a degree of polymerization between 2 and 20, obtained by degrading chitosan using a special bioenzymatic technique. Chitosan oligosaccharide is a polysaccharide composed of N-acetyl-D-glucosamine linked by β-1,4 glycosidic bonds. It is the only oligosaccharide in nature with positively charged cationic basic amino groups. It possesses many unique functions not found in chitosan, such as high solubility (completely soluble in water) and easy absorption and utilization by organisms; its effects are 14 times greater than those of chitosan. Registered and classified as a plant inducer by the Ministry of Agriculture and Rural Affairs, chitosan oligosaccharide is now widely used in agriculture, food, chemical industry, energy, environmental protection, and medicine. Its structural formula is as follows: As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0148] The terms “comprising” and “including” as used in the specification and claims of this application mean the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or combinations thereof.

[0149] It should be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. Unless the context clearly indicates otherwise, the terms "a," "an," "the," or "the" as used in this specification and claims may mean one or more. Unless the context clearly indicates otherwise, terms presented in the singular also include plural cases.

[0150] It should also be understood that the term "and / or" as used in this specification and claims refers to any combination of one or more of the associated listed items, and all possible combinations thereof. Furthermore, in some embodiments of the invention, features or combinations of features set forth herein may be excluded or omitted.

[0151] The various aspects and specific embodiments of the present invention will now be described in more detail through non-limiting embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0152] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.

[0153] Example Example 1 Take 30g of chitosan oligosaccharide and 20g of fucoidan oligosaccharide and add them to deionized water, stirring until completely dissolved; then add 5g of dispersant carboxymethyl cellulose and 5g of protectant in sequence to form mixed solution A; adjust the pH of mixed solution A to 6.5-7.5 with a pH adjuster to obtain mixed solution B; homogenize mixed solution B through a high-pressure homogenizer to form a stable homogeneous liquid, thus obtaining the biological agent.

[0154] Example 2 Take 60g of chitosan oligosaccharide and 40g of fucoidan oligosaccharide and add them to deionized water, stirring until completely dissolved; then add 10g of dispersant sodium polythiophosphate and 10g of protectant in sequence to form mixed solution A; adjust the pH of mixed solution A to 6.5-7.5 with a pH adjuster to obtain mixed solution B; homogenize mixed solution B through a high-pressure homogenizer to form a stable homogeneous liquid, thus obtaining the biological agent.

[0155] Example 3 Add 40g of chitosan oligosaccharide and 30g of fucoidan oligosaccharide to deionized water and stir until completely dissolved. Then add 7g of dispersant xanthan gum and 8g of protectant to form mixed solution A. Adjust the pH of mixed solution A to 6.5-7.5 with a pH adjuster to obtain mixed solution B. Homogenize mixed solution B using a high-pressure homogenizer to form a stable homogeneous liquid, thus obtaining the biological agent.

[0156] Example 4 Add 50g of chitosan oligosaccharide and 30g of fucoidan oligosaccharide to deionized water and stir until completely dissolved. Then add 8g of dispersant MorwetD-500 and 7g of protectant to form mixed solution A. Adjust the pH of mixed solution A to 6.5-7.5 with a pH adjuster to obtain mixed solution B. Homogenize mixed solution B using a high-pressure homogenizer to form a stable homogeneous liquid, thus obtaining the biological agent.

[0157] Example 5: Colony Quantity Test of Bacillus amyloliquefaciens Treatment groups were set up as follows: 1: Water control group (CK); 2: Single bacterial agent application group; which was treated with Bacillus amyloliquefaciens bacterial solution (bacterial solution concentration 1×10⁻⁶). 8CFU / mL); 3-6: Biological agents from Examples 1-4 diluted 300 times + Bacillus amyloliquefaciens bacterial suspension (bacterial suspension concentration 1×10⁻⁶). 8 (CFU / mL).

[0158] Methods: K326 flue-cured tobacco seedlings with uniform growth were selected and transplanted into pots filled with sterilized soil. Different treatment groups were treated with root irrigation at transplanting, with 50 mL applied to each seedling. Day 0 was designated as the day of root irrigation. Rhizosphere soil samples were collected from the seedlings on days 7, 14, and 21 post-treatment. The number of Bacillus amyloliquefaciens was determined using the dilution plating method. Gently brush off the soil adhering to the tobacco root surface, accurately weigh 1g of soil sample, add it to an Erlenmeyer flask containing 9mL of sterile water, and shake to mix thoroughly. Place the soil suspension in an 80℃ water bath for 20 minutes to kill most non-spore-forming bacteria; then perform a series of 10-fold serial dilutions of the soil suspension, typically requiring the preparation of 10... 5 -10 8 Bacterial suspensions at different dilutions were prepared, with three replicates for each dilution to ensure accurate counting. 100 µL of each dilution was evenly spread onto LB agar plates. The plates were inverted and incubated at 37°C for 24-48 hours. After colonies appeared, plates with 30-300 colonies were selected for counting. For *Bacillus amyloliquefaciens*, the characteristic rough, wrinkled colonies provide high distinguishability.

[0159] Result calculation: Total colony count (CFU / g) = Average colony count / Soil sample volume × Dilution factor The colonization numbers of Bacillus amyloliquefaciens in the tobacco rhizosphere soil of each treatment group at different time points are shown in Table 1.

[0160] Table 1 (Unit: ×10) 6 CFU / g dry soil) Table 1 shows that the number of rhizosphere colonies in tobacco after applying *Bacillus amyloliquefaciens* initially increased and then decreased. Compared with the single application of the inoculant (No. 2), the colonization of *Bacillus amyloliquefaciens* in the rhizosphere was significantly increased after adding the biological inoculants prepared in Examples 1-4 (Nos. 3-6). All combined treatments reached their colonization peak on day 14, with treatment No. 5 showing the best effect. On days 7, 14, and 21, the rhizosphere colony counts of the biological agent + inoculant treatment in Example 3 were 4.05 times, 5.18 times, and 5.77 times that of the single application treatment, respectively, with significant differences (P < 0.05). These results demonstrate that the biological inoculant of the present invention can significantly promote the colonization of *Bacillus amyloliquefaciens* in the tobacco rhizosphere, especially in the later stages of colonization.

[0161] Example 6: Test on the promoting effect on plant growth After disinfecting the tobacco seeds, place them in a sterilized nutrient substrate for seedling cultivation. When the seedlings have grown to 4-5 leaves, transplant them into flowerpots (about 10cm in diameter) containing about 180g of natural soil. The seedlings are cultivated in a greenhouse (light conditions: 16h, 28℃; darkness conditions: 8h, 20℃; relative humidity 60%), and watered with 60mL of sterilized water per plant per week. When the seedlings have grown to 6-7 leaves, different treatments are applied to the potted seedlings, with 5 replicates for each treatment. Treatment 1 consisted of 50 mL of sterile water; Treatment 2 consisted of 50 mL of chitosan oligosaccharide (concentration 5 g / L); Treatment 3 consisted of 50 mL of fucoidan oligosaccharide (concentration 5 g / L); Treatment 4 consisted of 50 mL of Bacillus amyloliquefaciens bacterial solution (OD600 = 0.3); Treatment 5 consisted of 50 mL of fucoidan oligosaccharide (concentration 5 g / L) + Bacillus amyloliquefaciens bacterial solution (OD600 = 0.3); Treatment 6 consisted of 50 mL of the biological agent from Example 1 + Bacillus amyloliquefaciens bacterial solution; Treatment 7 consisted of 50 mL of the biological agent from Example 2 + Bacillus amyloliquefaciens bacterial solution; Treatment 8 consisted of 50 mL of the biological agent from Example 3 + Bacillus amyloliquefaciens bacterial solution; and Treatment 9 consisted of 50 mL of the biological agent from Example 4 + Bacillus amyloliquefaciens bacterial solution. The solution was evenly applied around the plants, once every 7 days, for two consecutive treatments. The effect of Bacillus on the growth-promoting effect of tobacco seedlings was tested 7 days after the second application. The experimental results are shown in Table 2.

[0162] Table 2 Agronomic traits under different experimental treatments As shown in Table 2, the combined application of the biological agent of this disclosure with Bacillus (treatments 6-9) significantly increased tobacco leaf length, leaf width, leaf area, aboveground fresh weight, and root fresh weight compared with the application of chitosan oligosaccharide, fucoidan oligosaccharide alone, and the application of Bacillus amyloliquefaciens alone. The increase in aboveground fresh weight and root fresh weight was the most significant.

[0163] Among them, the fresh weight of the aboveground parts increased by 34.0%-50.5%, 37.1%-53.9%, and 48.0%-66.2% respectively compared with the application of chitosan oligosaccharide alone, fucoidan oligosaccharide alone, and Bacillus amyloliquefaciens alone. The fresh weight of the roots increased by 36.2%-105.8%, 42.4%-115.2%, and 44.6%-118.5% respectively. Compared with treatments 2, 3, and 4, treatment 8 showed a 2.06-fold, 2.15-fold, and 2.18-fold increase in root fresh weight, respectively; leaf area increased by 10.2%-28.3%, 2.2%-19.0%, and 4.0%-21.1%, respectively; and leaf length and width increased by an average of 11.5%, 5.9%, and 6.1%, and 10.4%, 5.9%, and 7.6%, respectively, significantly promoting tobacco seedling growth. Compared with treatment 5, treatments 6-9 showed a larger variation in the promotion of underground parts (root fresh weight) by the combined application of biological agents and Bacillus, ranging from 2.2% to 54.3%; the promotion of aboveground fresh weight was more uniform, ranging from 15.2% to 29.4%; the maximum increase in leaf area was 16.6%; and the maximum increases in leaf length and width were 8.9% and 7.1%, respectively. Therefore, the combined application of biological agents and Bacillus can significantly promote plant growth, especially showing a clear advantage in biomass accumulation.

[0164] While some exemplary embodiments of this application have been described and illustrated, this application is not limited to the disclosed embodiments. Rather, those skilled in the art will recognize that modifications and changes may be made to the described embodiments without departing from the spirit and scope of this application as described in the appended claims.

Claims

1. A biological agent comprising a composition of chitosan oligosaccharide and alginate oligosaccharide, characterized in that, The mass ratio of chitosan oligosaccharide to alginate oligosaccharide is 1:(0.3-1.5).

2. The biological agent according to claim 1, characterized in that, The molecular weight of the chitosan oligosaccharide is ≤3000 Da, preferably 1000-2500 Da; Preferably, the degree of polymerization of the chitosan oligosaccharide is 2-10, more preferably 3-8; Preferably, the degree of deacetylation of the chitosan oligosaccharide is ≥90%, more preferably 90%-95%; Preferably, the molecular weight of the alginate oligosaccharide is ≤5000 Da, and more preferably 3000-4000 Da; Preferably, the degree of polymerization of the alginate oligosaccharide is 2-15, more preferably 5-10.

3. The biological agent according to claim 1, characterized in that, The biological agent also includes additional active agents and / or excipients; Preferably, the adjuvant activator is selected from one or more biological control agents, one or more chemical agents, one or more fertilizers, one or more herbicides, one or more growth promoters, or any combination thereof; Preferably, the excipients are selected from agriculturally or horticulturally acceptable carriers, diluents, stabilizers, fillers, wetting agents, colorants, solvents, co-solvents, film-forming agents, spontaneous growth promoters, emulsifiers, dispersants, preservatives, antifreeze agents, thickeners, adjuvants, pH adjusters, or any combination thereof; Preferably, the biological agent is in solid form, liquid form, powder form, or any combination thereof; Preferably, the biological agent is selected from solutions, powders, granules, emulsions, emulsions, suspensions, tablets, microparticles, and wettable powders.

4. A method for preparing a biological agent according to any one of claims 1-3, characterized in that, Includes the following steps: 1) Dissolving: Add chitosan oligosaccharide and alginate oligosaccharide to water and stir until completely dissolved; 2) Compound preparation: Add excipients and mix thoroughly; 3) Adjust pH: Adjust the pH of the mixture to neutral; 4) Homogenization: The mixture is homogenized to form a stable homogeneous liquid.

5. The use of a biological agent according to any one of claims 1-3 in promoting plant rhizosphere colonization in microorganisms.

6. The application according to claim 5, characterized in that, The microorganisms are selected from one or more of bacteria, fungi, and viruses; Preferably, the microorganism is selected from Bacillus subtilis (Bacillus subtilis). Bacillus subtilis Rennograf Holder ( Burkholderiarinojensis ), Bacillus pumilus ( Bacillus pumilus ), Bacillus belesi ( Bacillus velezensis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Methylobacterium roximatei Methylobacterium rhodesianum ), Methylobacterium tumefaciens ( Methylobacterium exorquens ) and Pirere's Bacillus ( Paenibacilluspeoriae At least one of the following; Preferably, the plant is selected from one or more of food crops, cash crops, and root and tuber crops; Preferably, the food crop is selected from at least one of cereal crops, tuber crops, and legume crops; Preferably, the cereal crop includes at least one of the following: rice, wheat, corn, barley, oats, rye, sorghum, millet, foxtail millet, barnyard millet, and buckwheat; Preferably, the tuber crop includes at least one of the following: sweet potato, yam, and potato; preferably, the legume crop includes, but is not limited to, at least one of the following: soybean, broad bean, pea, mung bean, and peanut; Preferably, the economic crop includes at least one of the following: plants from the Solanaceae, Rosaceae, Rutaceae, Musaceae, Cucurbitaceae, Brassicaceae, Orchidaceae, Fabaceae, Asteraceae, Liliaceae, Zingiberaceae, Passifloraceae, Bromeliaceae, Araliaceae, and Cactaceae families; Preferably, the Solanaceae plants include tobacco, tomato, pepper, potato, and eggplant; Preferably, the Rosaceae plants include strawberry and papaya; Preferably, the Rutaceae plant includes citrus; Preferably, the Musaceae plant includes banana; Preferably, the cucurbitaceous plants include cucumber, winter melon, pumpkin, bitter melon, loofah, watermelon, and monk fruit; Preferably, the cruciferous plants include Chinese cabbage, rapeseed, kale, radish, and cauliflower; Preferably, the orchid species includes orchids; Preferably, the Fabaceae plant includes soybean; Preferably, the Asteraceae plant includes lettuce; Preferably, the lily family plant includes garlic; Preferably, the ginger family plant includes ginger; Preferably, the passionflower plant includes passion fruit; Preferably, the bromeliad includes golden pineapple; Preferably, the Araliaceae plant includes Panax notoginseng; Preferably, the cactus plant includes dragon fruit; Preferably, the root and tuber crops include potatoes, carrots, leafy vegetables, solanaceous vegetables, strawberries, grapes, citrus fruits, bananas, kiwifruit, dragon fruit, tomatoes, peppers, beans, ginger, Panax notoginseng, and ginseng.

7. The use of the biological agent according to any one of claims 1-3 in the prevention and control of plant diseases and / or the promotion of plant growth.

8. The application according to claim 7, characterized in that, The plant diseases and pests mentioned are selected from plant replanting diseases, plant bacterial diseases, plant fungal diseases, plant viral diseases, plant soil-borne diseases, plant insect pests and / or plant oomycete diseases. Preferably, the plant diseases and pests are caused by one or more of the following: plant pathogenic bacteria, fungi, viruses, insects, insect eggs, and nematodes; Preferably, the control of plant diseases and pests is achieved by generating siderophores, inducing systemic resistance of plants or their seeds to plant pathogens, and / or reducing the chemotaxis of plant pathogens to plants; Preferably, the plant growth promotion is selected from one or more of increasing plant height, increasing stem diameter, and promoting plant root development; Preferably, promoting plant growth includes promoting the growth of plant organs and / or tissues; Preferably, the promotion of plant organ growth is selected from promoting the growth of plant roots, stems, leaves, or any combination thereof; Preferably, the plant organ comprises one or more of the following: root, stem, leaf, flower, fruit, and seed; Preferably, the plant tissue comprises one or more of meristematic tissue, protective tissue, ground tissue, and vascular tissue; Preferably, the promotion of plant growth is achieved by providing at least one of nitrogen fixation, phosphorus solubilization, indoleacetic acid production, gibberellin production, and weed removal.

9. A method for promoting microbial colonization in the plant rhizosphere, characterized in that, The method includes applying the biological agent according to any one of claims 1-3 to the plant or seed.

10. The method according to claim 9, characterized in that, The application includes one or more of the following: root soaking, spraying, composting, seed soaking, coating, flood irrigation, drip irrigation of plants or plant organs, smearing on plants or plant organs, and drip application to plants or plant organs. Preferably, the application includes one or more of root irrigation, seed soaking, and seed coating.

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