A seed coating agent for promoting plant stress resistance and a preparation method and application thereof

Abstract

The application discloses a seed coating agent for promoting plant stress resistance and a preparation method and application thereof. The coating agent comprises an A agent, a B agent and a C agent. The A agent comprises phosphate rock powder, potash powder and biochar powder. The B agent comprises Massilia bacteria and / or Brevibacillus brevis. The C agent comprises malt dextrin. The Massilia bacteria has a preservation number of CCTCC NO:M2023996, and the Brevibacillus brevis has a preservation number of CCTCC NO:M2023995. The coating agent can improve the drought resistance or salt stress resistance of plants.

Description

Technical Field

[0001] This invention belongs to the field of microbial resource utilization and ecological agriculture technology, and relates to a seed coating agent that promotes plant stress resistance, its preparation method and application. Background Technology

[0002] Wheatgrass (Leymus chinensis) is an easy-to-grow and high-quality forage grass in deserts, with good feed value. Livestock readily consume wheatgrass during its tillering stage. However, drought and soil infertility reduce vegetation cover, further leading to low yields of edible grasses. This not only hinders restoration efforts in severely desertified areas but also limits related production. In recent decades, the increasing population and socio-economic development in pastoral areas have increased pressure on grasslands, leading to increasingly severe overgrazing and accelerated grassland degradation. Severe grassland degradation can result in a sharp decline in biodiversity, endangered species face extinction, natural grasslands are subjected to uncontrolled exploitation, forage grasses have no opportunity for growth and regeneration, the conflict between livestock and forage grasses is becoming increasingly prominent, and the balance of grassland ecosystems is severely disrupted. Improving plant nutrient supply, especially increasing potassium and silicate levels, helps plants resist drought and salt stress. Microorganisms can promote plant growth, yield, and quality under abiotic stress through a series of functions and actions: producing various plant hormones such as auxins to promote root development and formation, increasing nutrient and water absorption; stimulating the synthesis of soluble sugars and proline, and increasing the activity of antioxidant enzymes such as SOD to reduce drought and other abiotic stresses; improving photosynthetic performance and enhancing plant productivity; producing ACC deaminase to inhibit premature aging or apoptosis; suppressing the occurrence and development of diseases; and so on. Nitrogen, phosphorus, and potassium are macronutrients required for plant growth, and microorganisms can increase their supply through phosphorus and potassium solubilization and nitrogen fixation. Drought and other abiotic stresses have the greatest impact on plant emergence and seedling growth, and seed and root or stem coating is an important means to promote emergence and seedling growth.

[0003] Currently, some patents related to seed coating can be found. The majority of these involve chemical agents for controlling pests and diseases. A small number also involve coating agents combining chemical substances and microorganisms. For example, a patent titled "Coating Composition for Pathogen Control in Vegetables" (application number "201280030540.3") describes combining wax and some microorganisms to form a particulate carrier for controlling pathogens; a patent titled "Disease-resistant, Insect-repellent, Bio-type Seed Coating Agent and its Preparation Method" (application number "201710206032.0") describes mixing Pseudomonas fluorescens, Bacillus subtilis, and chitin to form a liquid coating agent for controlling pests and diseases; and a patent titled "A Microbial Seed Coating..." The patent titled "Preparation Method and Application of Trichoderma" (application number "201910299546.4") emphasizes the use of coating technology that binds Trichoderma spores with diatomaceous earth, cyclodextrin, etc., mainly to solve the problem of short preservation time of Trichoderma spores; the patent titled "A Microbial Seed Granulation Coating Composition" (application number "201910124557.9") describes the formation of a microbial coating composition by combining calcium peroxide with various Bacillus and molds to achieve disease prevention and control and improve seed germination in barren land; and so on. Summary of the Invention

[0004] Based on the technical problems existing in the prior art, the first objective of this invention is to provide a microorganism that promotes plant stress resistance; the second objective of this invention is to provide a coating agent that promotes plant stress resistance; the third objective of this invention is to provide a method for preparing the coating agent that promotes plant stress resistance; and the fourth objective of this invention is to provide the application of a composite coating agent in promoting plant stress resistance.

[0005] In one aspect, the present invention provides an isolated microorganism comprising *Massilia sp.* and / or *Brevibacillus parabrevis*, wherein the *Massilia sp.* has the accession number CCTCC NO: M2023996 and the *Brevibacillus parabrevis* has the accession number CCTCC NO: M2023995. In some preferred embodiments, the microorganism is a group consisting of *Massilia sp.* and *Brevibacillus parabrevis*.

[0006] The *Massilia* sp. strain (*Massilia sp.* EPRRC13) and the *Brevibacillus parabrevis* strain (*Brevibacillus parabrevis* EPRRN17) used in this invention were deposited at the China Center for Type Culture Collection (CCTCC) on June 12, 2023. The accession number for *Massilia* sp.* EPRRC13 is CCTCC NO: M2023996, and the accession number for *Brevibacillus parabrevis* EPRRN17 is CCTCC NO: M2023995.

[0007] In this invention, the functions of the microorganisms include, but are not limited to, the production of plant hormones such as auxins, gibberellins, cytokinins, or jasmonic acid; the solubilization of calcium phosphate-phosphorus (phosphate rock powder-phosphorus); the solubilization of potassium (potassium rock powder-potassium); autotrophic nitrogen fixation; the production of ACC (p-2,4-diacetylgammourne) deaminase; the production of siderophores; and the production of HCN (hydrogen cyanide) or DAPG (p-2,4-diacetylgammourne). One or more of these microorganisms may produce extracellular polysaccharides and form biofilms. There is no significant antagonism between the microorganisms.

[0008] Another aspect of the present invention provides a composition comprising agent A, agent B, and agent C, wherein agent A comprises phosphate rock powder, potassium rock powder, and biochar powder, agent B comprises the aforementioned microorganisms, and agent C comprises maltodextrin.

[0009] In this invention, the biochar powder is obtained by crushing and decomposing waste plant material from various plants at high temperature. Together with the polysaccharides produced by the microorganisms and other materials, it forms a well-permeable membrane that can effectively adsorb microorganisms by utilizing its own pore size, thereby achieving the effect of storing microorganisms. It can be sterilized at high temperature before use.

[0010] In some preferred embodiments, the microorganism is a combination of *Machilus* and *Bacillus brevis*. In some particularly preferred embodiments, the effective viable count ratio of *Machilus* to *Bacillus brevis* is (1-3):(1-3). More preferably, the effective viable count ratio of *Machilus* to *Bacillus brevis* is 1:1.

[0011] In some preferred embodiments, the number of viable microorganisms in the composition is 10-1. 9 -10 12 CFU / g.

[0012] In some preferred embodiments, the weight ratio of agent A, agent B, and agent C is (1000-1500):(1-5):(1000-2500). More preferably, the weight ratio of agent A, agent B, and agent C is 1050:4:1300.

[0013] In some preferred embodiments, the weight ratio of the phosphate rock powder, potash powder, and biochar powder is (5-10):(5-10):(200-400). In some more preferred embodiments, the weight ratio of the phosphate rock powder, potash powder, and biochar powder is 1:1:40.

[0014] In some embodiments, agent A further includes EDTA and / or fulvic acid.

[0015] In some embodiments, agent C further includes trehalose and / or molasses.

[0016] In an embodiment of the present invention, the concentration of maltodextrin is 30% of the total amount of agent C.

[0017] In a preferred embodiment, agent C comprises maltodextrin and molasses. More preferably, the ratio of maltodextrin to molasses is 5:1. More preferably, the total mass percentage concentration of maltodextrin and molasses in agent C is 30-60%.

[0018] In a specific embodiment of the present invention, agent C comprises 1000 mL of sterile water, 50 g of molasses, and 250 g of maltodextrin.

[0019] In some embodiments, the composition may be prepared as one or more of the following formulations: emulsion, colloid, powder, granule, pellet, powder, spray, emulsion, and solution.

[0020] In some preferred embodiments, the composition is a coating agent.

[0021] In another aspect, the present invention provides a method for promoting plant resistance to stress or preventing plant diseases, wherein the method comprises applying the aforementioned microorganisms or the aforementioned composition to the plant or its seeds, or to the environment surrounding the plant or its seeds.

[0022] In some preferred embodiments, the method includes mixing the composition with the seeds of the plant until they are thoroughly mixed. More preferably, after each mixing, the mixture should be dried for at least 10-12 hours to ensure full absorption. After drying, the seeds should be sown promptly, or they can be sealed in a clean packaging bag and stored in a cool place for no more than 2 days before sowing.

[0023] In some preferred embodiments, the weight and volume ratio of the plant seeds to the composition is (50-100 kg):(1-5 L). More preferably, the weight and volume ratio of the plant seeds to the composition is 50 kg:1 L.

[0024] In some preferred embodiments, the promotion of plant stress resistance includes enhancing the plant's ability to resist drought or salt stress.

[0025] In some preferred embodiments, the plant is *Elymus sibiricum*.

[0026] Another aspect of the present invention provides a method for preparing a composite coating agent, which includes the following steps:

[0027] The microorganisms mentioned above are activated, cultured, and collected by centrifugation to obtain Agent B mentioned above.

[0028] Prepare the aforementioned agent C into a solvent;

[0029] Add agent B to agent C and stir until homogeneous; add agent A (mentioned above) to the mixture of agent B and agent C to obtain the composite coating agent.

[0030] In some preferred embodiments, the microorganisms are a combination of Massilia sp. and Brevibacillus parabrevis. These two microorganisms can be cultured separately or in combination.

[0031] In some preferred embodiments, the weight-to-volume ratio of agent A to the mixture is (1-1.5 kg): (1-2.5 L).

[0032] In some preferred embodiments, the effective viable count of the microorganisms in the composite coating agent is 10. 9 -10 13 CFU / mL. In some preferred embodiments, the culture includes shake flask culture or fermenter scale-up culture.

[0033] In some embodiments, the composite coating agent described in this invention can be prepared and used immediately.

[0034] Another aspect of the present invention provides the following applications:

[0035] (1) The use of the microorganisms or the compositions described above in the preparation of products that promote plant resistance to stress or prevent plant diseases;

[0036] (2) The application of the aforementioned microorganisms or the aforementioned compositions in promoting plant resistance to stress or preventing plant diseases.

[0037] In some preferred embodiments, the product includes a coating agent or a fertilizer.

[0038] In some preferred embodiments, the promotion of plant stress resistance includes enhancing the plant's ability to resist drought or salt stress.

[0039] In some preferred embodiments, the plant is *Elymus sibiricum*.

[0040] Terminology Definition

[0041] Winged wheatgrass (scientific name: *Elymus dahuricus* Turcz.) is a loosely tufted, erect plant belonging to the genus *Elymus* in the family Poaceae. It grows to a height of 70-140 cm, with loosely tufted stems, smooth, hairless leaf sheaths, and relatively dense spike-like inflorescences. This plant mainly grows on grassy slopes or roadsides. It is drought-tolerant, cold-tolerant, alkali-tolerant, and wind-resistant, flowering and fruiting from July to September, and is primarily used as forage.

[0042] "Promoting plant stress resistance" includes, but is not limited to, promoting plant growth, yield and quality under adverse stress, producing a variety of plant hormones such as auxin to promote root development and formation, increasing nutrient and water absorption, stimulating plants to synthesize soluble sugars and proline, and increasing the activity of antioxidant enzymes such as SOD to reduce adverse stress such as drought, improving photosynthetic performance, enhancing plant productivity, producing ACC deaminase, inhibiting premature aging or premature apoptosis of plants, and inhibiting the occurrence and development of diseases.

[0043] Beneficial effects:

[0044] This invention is the first to screen and obtain strains with strong plant growth-promoting functions, including Massiliasp. and Brevibacillus parabrevis. This invention specifically clarifies the functions that functional microorganisms should possess, facilitating the tailored combination of strains according to local conditions and functions in production, leading to a more open approach and wider applications.

[0045] This invention discloses a coating agent that, in addition to preventing and controlling diseases, also enhances the plant's resistance to drought and salt stress. The coating agent of this invention differs from existing composite coating agents in its material composition, particularly in its use of inexpensive mineral powders, such as silicate mineral powder; no composite coating agent currently contains all the same materials. In this patented coating agent, different functional components are packaged separately and mixed in proportion before use, which is more conducive to ensuring microbial survival, extending shelf life, and guaranteeing coating efficacy. The material composition and proportions of the coating agent of this invention are suitable for the cultivation of *Leymus chinensis*. Attached Figure Description

[0046] Figure 1 The effects of different treatments on the plant height of Leymus chinensis;

[0047] Figure 2 The effects of different treatments on the fresh weight of crested wheatgrass.

[0048] Microbial preservation for patented procedures:

[0049] 1. The Massilia sp. EPRRC13 strain of the present invention

[0050] Date of deposit: June 12, 2023;

[0051] Preservation institution: China Center for Type Culture Collection (CCTCC);

[0052] Accession number: CCTCC NO:M2023996;

[0053] Classification and nomenclature: Massilia sp.;

[0054] Address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0055] 2. The *Brevibacillus parabrevis* strain of the present invention (EPRRN17)

[0056] Date of deposit: June 12, 2023;

[0057] Preservation institution: China Center for Type Culture Collection (CCTCC);

[0058] Accession number: CCTCC NO:M2023995;

[0059] Classification and nomenclature: Brevibacillus parabrevis

[0060] Address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Detailed Implementation

[0061] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0062] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0063] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0064] Example 1: Isolation and Identification of Bacterial Strains

[0065] Wild *Leymus chinensis* plants were collected, and the rhizosphere strains exhibiting plant growth-promoting traits such as nitrogen fixation, phosphorus solubilization, potassium solubilization, plant hormone production, siderophore production, and ACC deaminase production were isolated using streak plating. Using the purified genomic DNA of the isolated strains as templates, and with 27F and 1492R primers as primers, the 16S rRNA gene of the strains was amplified by PCR. The amplified products were sequenced, and the obtained sequences were classified using online EzTaxon (strains with 16S rRNA gene sequence similarity greater than 97% were preliminarily identified as belonging to the same species). The 16S rRNA gene sequences of the nine obtained bacteria showed 98%–100% similarity to the type species. The results are shown in Table 1, which presents the 16S rRNA gene similarity analysis of strains with strong plant growth-promoting functions.

[0066] Table 1. Strain Isolation Information

[0067]

[0068] Example 2: Bacterial Activation, Cultivation, and Collection

[0069] Activation culture of strains with strong plant growth-promoting ability: Spread 100 mL of glycerol preservation solution onto a solid medium with corresponding capabilities, such as nitrogen fixation or phosphorus and potassium solubilization, and incubate at 25°C. Pick single colonies with typical colony characteristics and rapid growth, place them on a modified liquid medium, and incubate at 25°C. When the culture reaches the mid-to-late logarithmic phase, centrifuge at 8000 g, wash three times repeatedly with K2HPO4-KH2PO4 buffer (pH 7.2), and resuspend in the same buffer to a cell concentration of 10. 13 CFU / mL.

[0070] Example 3: Determination of basic biological characteristics of the strain and its plant growth-promoting ability

[0071] The *Massilia* sp. strain (*Massilia sp.* EPRRC13) and the *Brevibacillus parabrevis* strain (*Brevibacillus parabrevis* EPRRN17) used in this invention were deposited at the China Center for Type Culture Collection (CCTCC) on June 12, 2023. The accession number for *Massilia* sp.* EPRRC13 is CCTCC NO: M2023996, and the accession number for *Brevibacillus parabrevis* EPRRN17 is CCTCC NO: M2023995.

[0072] The effects of salinity, temperature, and pH on the growth of the strains were determined, and the plant growth-promoting abilities were qualitatively or quantitatively measured using nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing culture media. Some results are shown in Table 2. It is evident that *Massilia sp.* (MC) and *Brevibacillus parabrevis* (BP) possess multiple growth-promoting abilities and are well-adapted to the climatic or geological conditions (large temperature differences, drought, and salinity) of arid northern regions. Subsequent plate confrontation analysis revealed no antagonism between the strains, and mixed inoculation showed a better growth-promoting effect on *Elymus sibiricum* than single-coating inoculation.

[0073] Table 2 Single-strain inoculation experiments

[0074]

[0075] Example 4: Preparation and Application of Coating Agent

[0076] Step 1: Microbial culture. Microbial strains with corresponding functions were activated, cultured in shake flasks, and scaled up in fermenters using phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing media, respectively. Cells were collected by centrifugation. The cells of each strain were mixed, freeze-dried, or stored at low temperature with a preservative such as glycerol for later use, or used directly. This yields Agent B.

[0077] Step 2: Preparation of Agents A and C. First, sterilize the biochar powder at high temperature. Then, weigh out each substance according to its weight or volume ratio and mix them thoroughly according to the components of Agents A and C. Seal and store at room temperature for later use, or use directly. This prepares Agents A and C.

[0078] Step 3: Preparation of the composite coating agent. Add B to C, then add an appropriate amount of sterile water that has been filtered or sterilized at high temperature, and stir evenly in a clean environment; add agent A to the mixture of B and C, with a weight-to-volume ratio of [missing information].

[0079] (1-1.5kg): (1-2.5L), and the effective viable count is 10. 9 -10 12 CFU / mL, prepare fresh before use.

[0080] Step 4: Application of the compound coating agent. The weight and volume ratio of the compound coating agent mixture prepared in Step 3 is 50 kg: 1 L. Mix the compound coating agent mixture with the wheatgrass seeds evenly. After each mixing, it is necessary to dry for at least 10-12 hours to ensure full absorption. Sow in time after drying, or seal in a clean packaging bag and store in a cool place for no more than 2 days before sowing.

[0081] The above-mentioned Agent B refers to a single microbial strain or combination thereof involved in this invention, with a dosage of 4g;

[0082] Agent A above consists of 25g of phosphate rock powder, 25g of potassium ore powder, and 1000g of biochar powder;

[0083] The above-mentioned agent C consists of 1000mL of sterile water, 50g of molasses, and 250g of maltodextrin;

[0084] In this embodiment, the weight ratio of agent A, agent B and agent C is 1050:4:1300.

[0085] Example 5: Optimization of Non-biological Coating Components

[0086] Using PEG6000 to simulate drought stress in potted *Elymus sibiricum*, based on single-factor studies, the optimal non-biological coating ratio was selected by comprehensively considering the total viable count of the compound bacteria MC and BP, the seedling emergence rate and size of *Elymus sibiricum* at 15 days, and adhesion (the shedding rate after drying and inverting and shaking at 100 rpm for 10 minutes, i.e., the weight of seed coating material falling off / the total weight of coating material before falling off). Some results are shown in Tables 3-5. Tables 3-5 show that combination 1 has excellent comprehensive effects in terms of coating effect and growth promotion, and is the optimal combination ratio.

[0087] Table 3 Optimization of single-strain coating of Massilia sp. EPRRC13

[0088]

[0089] The combination numbers represent: 1, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 10:10:400; 2, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 10:10:350; 3, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 10:10:200; 4, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 5:5:300; 5, the weight ratio of phosphate rock powder, potash powder, and biochar powder is... The weight ratio of powders is 10:5:300; 6, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 5:10:300; 7, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 7:7:400; 8, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 7:7:300; 9, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 7:7:200; 10, CK (uncoated).

[0090] Table 4. Optimization of single-strain coating of Brevibacillus parabrevis EPRRN17

[0091]

[0092]

[0093] The combination numbers represent: 1, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 10:10:400; 2, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 10:10:350; 3, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 10:10:200; 4, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 5:5:300; 5, the weight ratio of phosphate rock powder, potash powder, and biochar powder is... The weight ratio of powders is 10:5:300; 6, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 5:10:300; 7, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 7:7:400; 8, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 7:7:300; 9, the weight ratio of phosphate rock powder, potash powder, and biochar powder is 7:7:200; 10, CK (uncoated).

[0094] Table 5. Optimization Experiment of Coating of Composite Strains

[0095]

[0096] The combination numbers represent: 1, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 10:10:400; 2, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 10:10:350; 3, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 10:10:200; 4, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 5:5:300; 5, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 10:5:300; 6, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 5:10:300; 7, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 7:7:400; 8, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 7:7:300; 9, the weight ratio of phosphate rock powder, potash powder, and bio-carbon powder is 7:7:200; 10, CK (uncoated).

[0097] Example 6

[0098] Culture medium formulation for strains:

[0099] R2A complete nutrient medium: 2.5g soluble starch, 2.5g glucose, 2.5g bacteriological peptone, 2.5g acid-hydrolyzed casein, 2.5g yeast extract, 1.5g K2HPO3·3H2O, 1.5g sodium pyruvate, 0.25g MgSO4·7H2O, 1000mL distilled water.

[0100] The specific implementation steps are as follows: Activate strains Massilia sp. and Brevibacillus parabrevis in R2A liquid medium, culture for 2-3 days, collect the bacterial cells by centrifugation, and mix the two bacterial strains into a bacterial slurry at a 1:1 ratio. The total viable bacterial count in the slurry is approximately 10. 11 CFU / g. After obtaining the bacterial sludge, it was diluted with sterile water to prepare a bacterial suspension with a concentration of approximately 10. 9 CFU / mL. Mix agent A and agent B in a certain proportion, and then mix with agent C to obtain a composite coating agent.

[0101] Agent B mentioned above is a bacterial sludge.

[0102] The above-mentioned Agent A consists of 25g of phosphate rock powder, 25g of potassium rock powder, and 1000g of biochar powder.

[0103] The above-mentioned agent C consists of 1000mL of sterile water, 50g of molasses, and 250g of maltodextrin.

[0104] The weight ratio of Agent A, Agent B and Agent C is 1050:4:1300.

[0105] In addition, single-strain coating, aseptic coating, and blank treatment were prepared. The preparation method for single-strain coating is the same as that for the above-mentioned compound strain coating, the difference being that agent B contains only a single strain, and the viable count of the single strain in the agent B sludge is approximately 10. 11CFU / g.

[0106] The experimental results are shown in Table 6. Figure 1 , Figure 2 As shown, mixed inoculation with Massilia sp. strain (MC) and Brevibacillus parabrevis strain (BP) showed a better growth-promoting effect on crested wheatgrass than single coating inoculation.

[0107] Table 6

[0108]

[0109] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A composition comprising agent A, agent B, and agent C, wherein, Agent A includes phosphate rock powder, potash rock powder, and biochar powder. Agent B includes microorganisms. The C agent mentioned above includes maltodextrin; The microorganism mentioned is *Cyperus maseriae* ( Massilia sp . ) and Bacillus shortbacterium ( Brevibacillus parabrevis The combination of the above-mentioned *C. maseriae* has the accession number CCTCC NO:M2023996, and the accession number of *Bacillus breviculatus* has the accession number CCTCC NO:M2023995; the effective viable count ratio of *C. maseriae* and *Bacillus breviculatus* is 1:

1. The weight ratio of agent A, agent B and agent C is (1000-1500):(1-5):(1000-2500). The weight ratio of the phosphate rock powder, potassium rock powder, and bio-carbon powder is (5-10):(5-10):(200-400).

2. The composition according to claim 1, wherein, The weight ratio of Agent A, Agent B and Agent C is 1050:4:1300.

3. The composition according to claim 1, wherein, The weight ratio of the phosphate rock powder, potassium ore powder, and bio-carbon powder is 1:1:

40.

4. The composition according to claim 1, wherein, Agent A also includes EDTA and / or fulvic acid.

5. The composition according to claim 1, wherein, Agent C also includes trehalose and / or molasses.

6. The composition according to any one of claims 1-5, wherein, The composition is a coating agent.

7. A method for promoting plant stress resistance, wherein, The method comprises applying the composition of any one of claims 1-6 to the plant or its seeds, or to the environment surrounding the plant or its seeds; the promotion of plant stress resistance includes enhancing the plant's ability to resist drought; the plant is *Leymus chinensis*.

8. The method according to claim 7, wherein, The method includes mixing the composition with the seeds of the plant until homogeneous.

9. The method according to claim 8, wherein, The weight and volume ratio of the plant seeds to the composition is (50-100 kg): (1-5 L).

10. A method for preparing a composite coating agent, comprising the following steps: The microorganism described in claim 1 is activated, cultured, and collected by centrifugation to obtain agent B as described in any one of claims 1-6; The agent C described in any one of claims 1-6 is formulated into a solvent; Add agent B to agent C and stir until homogeneous; add agent A as described in any one of claims 1-6 to the mixture of agent B and agent C to obtain the composite coating agent.

11. The preparation method according to claim 10, wherein, The weight-to-volume ratio of agent A to the mixture is (1-1.5 kg): (1-2.5 L).

12. The preparation method according to claim 10, wherein, The effective viable count of microorganisms in the composite coating agent is 10. 9 -10 13 CFU / mL.

13. The following applications: (1) The use of the composition according to any one of claims 1-6 in the preparation of products that promote plant stress resistance; (2) The use of the composition according to any one of claims 1-6 in promoting plant stress resistance; The aforementioned promotion of plant stress resistance includes enhancing the plant's ability to resist drought; The plant in question is *Elymus sibiricum*.

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