Microbial seed coating agent and application thereof

Abstract

The invention discloses a microbial seed coating agent, in the microbial seed coating agent, saline-alkali-resistant phosphorus-dissolving compost bacillus subtilis C007 is adopted, seeds coated by the microbial seed coating agent have a saline-alkali-resistant environment, and meanwhile, the microbial seed coating agent has a promoting effect on the growth of crops. The microbial seed coating agent disclosed by the invention further comprises saline-alkali-resistant phosphorus-solubilizing composting bacillus subtilis C007 and a bacillus velezensis complex microbial inoculant with the preservation number of CCTCC (China Center For Type Culture Collection) NO: M 20221937; the microbial seed coating agent adopting the complex microbial inoculants can promote the growth of wheat seedlings in saline-alkali soil and saline-containing aqueous solution.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a microbial seed coating agent and application thereof. Background Art

[0002] Seed coating agent is a special pesticide preparation, which is made of active ingredients (such as microorganisms, insecticides, fungicides, etc.), film-forming agents and other adjuvants. It can be directly or after dilution wrapped on the surface of seeds to form a protective layer with certain strength and permeability.

[0003] Compared with traditional seed soaking or seed dressing pesticide emulsions and powders, seed coating agents have significant advantages. Traditional pesticides have poor adhesion and are easy to fall off, which not only affects the efficacy but also may pollute the environment and pose a threat to the safety of humans and animals. Seed coating agents can not only disinfect seeds before sowing, but also continuously release active ingredients during seed germination and seedling growth, play a continuous role in preventing and controlling seed bacteria and soil pests and diseases, and can also promote seedling growth and increase yields.

[0004] However, existing seed coating agents also have some disadvantages. First, the permeability of the film-forming agent is poor, which may affect the germination rate of seeds. Secondly, some seed coating agents have poor adhesion, which causes the active ingredients to fall off easily, making them no different from uncoated seeds. Finally, the vast majority of seed coating agents use pesticides as their main ingredients, among which highly toxic and highly residual pesticides such as furadan and chloramphenicol are widely used, which is inconsistent with the development trend of pesticides and seed coating agents to be highly efficient, low-toxic, or even non-toxic.

[0005] By utilizing the antagonism between biological fungi, biological seed coating agents selectively cultivate beneficial antagonistic bacteria to effectively resist the invasion and reproduction of harmful pathogens, thereby achieving the purpose of disease prevention. Its characteristics are that it is completely pollution-free and non-toxic, representing the future trend of seed coating technology and pesticides.

[0006] Bacillus stercoris is a subspecies of Bacillus subtilis. It is a type of Gram-positive bacteria that can produce resistant endospores. Its cells are rod-shaped and covered with a large amount of calcium pyridine dicarboxylate. It can produce a large amount of secondary metabolites. Currently, it is widely used in sludge harmless treatment, food, microbial deodorant, microbial composting agent and biological water-soluble organic fertilizer.

[0007] Currently, there is no technical solution for preparing seed coating agents using a composite bacterial agent containing composting Bacillus stercoris to promote crop growth. Summary of the invention

[0008] The object of the present invention is to overcome the shortcomings of the prior art and provide a microbial seed coating agent. The microbial seed coating agent adopts a composite microbial agent, and the composite microbial agent further comprises a salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 and a Velez Bacillus with a preservation number of CCTCC NO: M 20221937. The microbial seed coating agent using the composite agent promotes the growth of wheat seedlings in saline-alkali soil and saline solution.

[0009] The present invention is achieved through the following technical solutions:

[0010] The present invention provides a microbial seed coating agent, wherein the microbial seed coating agent comprises at least one of the following:

[0011] Salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007; or

[0012] The culture of salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 or its processed product; or

[0013] A bacterial agent comprising the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007; or,

[0014] A composite microbial agent, wherein the composite microbial agent is an agent comprising the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007;

[0015] The salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 was deposited in the China Center for Type Culture Collection on August 28, 2024, and its preservation number is: CCTCC NO:M 20241868.

[0016] In the above technical solution, the composite microbial agent also includes a Bacillus Velez subtilis agent, and the Bacillus Velez subtilis agent is selected from the Bacillus Velez subtilis with a deposit number of CCTCC NO: M 20221937.

[0017] In the above technical solution, the composite microbial agent, wherein the spore content of salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 is not less than 100 billion cfu / g;

[0018] and / or,

[0019] The Bacillus Velez spore content in the Bacillus Velez inoculant is not less than 130 billion cfu / g.

[0020] In the above technical solution, the microbial seed coating agent comprises the following raw materials in parts by weight:

[0021] 100-200 parts of compound microbial agent;

[0022] 3-5 parts of film-forming agent;

[0023] 1-3 parts of silk fibroin;

[0024] 3-5 parts of trehalose.

[0025] In the above technical solution, the composite microbial agent comprises: 45 to 55 parts by weight of the microbial composting Bacillus stercoris C007 agent; and 45 to 55 parts by weight of Bacillus velezii agent.

[0026] In the above technical solution, the film-forming agent is at least one of carboxymethyl cellulose, methyl cellulose, ethyl cellulose, polyvinyl acetate cellulose salt or polyvinyl alcohol.

[0027] In another aspect of the present invention, the present invention provides a method for preparing a microbial seed coating agent, comprising the following steps:

[0028] Cultivating beneficial microorganisms, collecting cell bodies, and obtaining a composite microbial agent; mixing the composite microbial agent, a film-forming agent, silk fibroin, trehalose, and distilled water to obtain the microbial seed coating agent;

[0029] The preparation process of the composite microbial agent is as follows: the culture medium used for the microbial composting Bacillus stercoris C007 is 8 g / L corn starch, 8.5 g / L bean cake powder, 2 g / L K2HPO4, 1 g / L KH2PO4, 0.1 g / L ZnSO4, 2 mg / L MgSO4, 2 mg / L CuSO4, and 1 mg / L Na2MoO4;

[0030] The culture medium used for Bacillus Velezii was 6 g / L corn starch, 7 g / L bean cake powder, 1.5 g / L rice powder, 1.5 g / L bran, 1 g / L corn steep liquor, 0.5 g / L calcium carbonate, 0.2 g / L potassium nitrate, 0.3 g / L magnesium sulfate, 0.2 g / L K2HPO4, and 0.2 g / L Na2HPO4. Both strains were fermented in a 20L fermenter, with sampling every 4 hours and the tank was stopped when the spore maturity was 50%.

[0031] The two bacterial liquids are respectively subjected to high-temperature centrifugal drying to be directly sprayed to obtain microbial composting Bacillus stercoris C007 and Bacillus velezii, and the two are mixed to obtain a composite microbial agent; the composite microbial agent comprises: 45 to 55 parts by weight of the microbial composting Bacillus stercoris C007 and 45 to 55 parts by weight of Bacillus velezii;

[0032] The Bacillus Velez subtilis is selected from the Bacillus Velez subtilis with a deposit number of CCTCC NO: M 20221937.

[0033] In another aspect of the present invention, the present invention provides a seed coating method, using the microbial seed coating agent described in the above technical solution for seed coating, and the mass ratio of the microbial seed coating agent to the seed is 1:10-40;

[0034] Furthermore, the microbial seed coating agent described in the above technical solution is used for seed coating, and the mass ratio of composite microbial seed coating agent: seeds: solvent = 1:10 to 40:10; further, the solvent is distilled water.

[0035] In the above technical solution, after the seeds are coated, the spore content in the microbial seed coating agent wrapped in each seed is 1×10 6 ~1×10 8 cfu / seed.

[0036] In another aspect of the present invention, the present invention proposes the use of the microbial seed coating agent described in the above technical solution in at least one of the following:

[0037] Improve crop resistance to stress, especially resistance to salinity and alkali;

[0038] Promote crop growth.

[0039] The present invention discloses a salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 isolated and purified from compost. The strain has been deposited in China Center for Type Culture Collection (CCTCC), with a deposit address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on August 28, 2024, and its deposit number is CCTCC NO: M 20241868.

[0040] The salt-alkali resistant phosphate-dissolving composting bacillus Bacillus stercoris C007 has the ability to activate inorganic phosphorus (P), can grow with sparingly soluble phosphate as a phosphorus source, and convert it into soluble phosphate. Under laboratory shaking flask conditions, the composting bacillus Bacillus stercoris C007 can convert tricalcium phosphate to 189.17 mg / L.

[0041] The salt-alkali resistant and phosphate-dissolving composting Bacillus stercoris C007 can secrete indoleacetic acid (IAA) under the induction of L-tryptophan, and the secretion amount can reach 5.29 mg / L.

[0042] The salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 can grow in a saline-alkali environment, and its growth-tolerant NaCl concentration is greater than 14% (w / v, 140 g / L), and the tolerable pH value is 8.0-10.5.

[0043] The salt-alkali resistant and phosphate-dissolving composting Bacillus stercoris C007 has the ability to degrade cellulose. When sodium carboxymethyl cellulose is used as the sole carbon source, the cellulase activity of this strain is 6.12 U / mL.

[0044] The advantages and beneficial effects of the present invention are:

[0045] The microbial seed coating agent disclosed in the present invention, which contains the above-mentioned composite bacterial agent, can not only enhance the survival and colonization of beneficial microorganisms in saline-alkali farmland soil and plant root system, but also has the functions of nitrogen fixation, phosphorus solubilization and secretion of plant growth hormone (IAA), which promotes the growth of plants. The microbial seed coating agent can not only provide necessary nutrients and protection for the seeds of crops, but also improve the germination rate of seeds, improve the quality of seedlings, enhance the stress resistance of seedlings, and play the role of insecticide, disease prevention and growth promotion. In addition, the living microorganisms in the biological seed coating agent can reproduce and colonize rapidly and in large quantities, can secrete a variety of protein and hormone active substances, are natural and non-toxic, and play a role continuously, which can affect the entire growth period of plants.

[0046] The composite bacterial agent prepared by combining salt-alkali resistant phosphate-dissolving composting Bacillus stercoris and Bacillus velezii (deposit number is CCTCC NO: M 20221937, for details, see the disclosed content in the patent application with application number 2023113257516) can cooperate with each other to achieve the effect of 1+1 greater than 2.

[0047] Collection Information

[0048] Composting Bacillus stercoris C007, the strain has been deposited, the depository unit: China Center for Type Culture Collection (CCTCC), the deposit address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit date: August 28, 2024, the deposit number: CCTCC NO:M 20241868. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a colony observation diagram of the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 of the present invention;

[0050] Figure 2 It is a phylogenetic tree of the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 of the present invention based on 16S rDNA sequence analysis;

[0051] Figure 3 This is a schematic diagram of wheat growth with composite microbial seed coating agent under soil culture conditions;

[0052] Figure 4 This is a schematic diagram of wheat growth with composite microbial seed coating agent under hydroponic conditions;

[0053] Figure 5 This is a comparison chart of wheat plant height and root length grown with composite microbial seed coating agent under hydroponic conditions;

[0054] Figure 6 It is a statistical chart of wheat plant height grown with composite microbial seed coating agent under hydroponic conditions.

[0055] For ordinary technicians in this field, other relevant drawings can be obtained based on the above drawings without any creative work. DETAILED DESCRIPTION

[0056] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with specific embodiments.

[0057] In this document, the terms “salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007”, “composting Bacillus stercoris C007”, “Bacillus stercoris C007” and “C007” are interchangeable.

[0058] Example 1 Functional determination of composite microbial agent

[0059] In one aspect of the present invention, the present invention provides a composite microbial agent. According to an embodiment of the present invention, the composite agent includes the microbial composting Bacillus stercoris C007 agent as described in the above technical solution. The composite agent of the present invention has great application value in improving crop stress resistance, especially salt-alkali resistance; and promoting crop seedling growth.

[0060] According to an embodiment of the present invention, the composite microbial agent further comprises a Bacillus Velez subtilis agent, and the Bacillus Velez subtilis agent is selected from Bacillus Velez subtilis with a deposit number of CCTCC NO: M 20221937. According to this composite agent, composting Bacillus stercoris C007 and Bacillus Velez subtilis have great application value for the growth of wheat seedlings, and the synergistic effect of the two bacteria on the improvement of wheat plant height reaches 8-10% compared with the single application.

[0061] Among them, the separation and purification of salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 is completed by the following methods:

[0062] Sample source: Compost samples from Nanpi pig farm in Cangzhou, Hebei Province, sampling time: August 2017.

[0063] Separation and purification process and steps:

[0064] Take 10 grams of the sample and put it in 90 ml of sterile water with glass beads, place it on a 55℃ shaker and shake it for 30 minutes to break up the bacterial mass as much as possible. After the sample is processed, it is immediately diluted with sterile water to obtain bacterial solutions of different concentrations. Use a sterile pipette to draw 100 microliters of diluted bacterial solution, and drip it on a sodium carboxymethyl cellulose medium (CMC-Na 20g, Na2HPO42.5g, KH2PO41.5g, Agar Powder20g, peptone 2.5g, deionized water 1000ml, pH 8.0) plate with aseptic operation, and spread it evenly with a sterile spreader. After inoculation, place the plate upside down in a constant temperature incubator and culture at 55℃. According to the morphological characteristics of the colony, pick a single colony with good growth, moderate size, and obvious appearance. Use an inoculation loop to pick a single colony and streak it on the plate for further separation and purification. The composition of the plate streaking medium is the same as that of the medium used for coating the plate. After repeated plate streaking, single colony C007 was purified.

[0065] Identification of salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007

[0066] 1) Identification of biological characteristics

[0067] Bacillus stercoris C007, a salt-alkali resistant phosphate-dissolving composting bacillus, has large, white, raised colonies with neat edges, a smooth and moist surface, is opaque, and produces spores. It has relatively loose requirements for growth conditions and light conditions, and can use a variety of organic and inorganic substances as carbon and nitrogen sources for growth and reproduction. (See Figure 1 ).

[0068] 2) Identification of physiological and biochemical characteristics

[0069] The physiological and biochemical characteristics of the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 were studied using a bacterial microbiochemical identification tube. The physiological and biochemical characteristics of the composting Bacillus stercoris C007 are shown in Table 1.

[0070] Table 1 Physiological and biochemical characteristics of Bacillus stercoris C007

[0071]

[0072] 3) Systematic classification and identification

[0073] Primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R

[0074] (5'-TACGGYTACCTTGTTACGACTT-3') amplified the rRNA gene sequence of strain C00716S. The PCR reaction system was 25 μL, which contained 12.5 μL 2×GoldStar Best MasterMix, 0.5 μL primer 27F, 0.5 μL primer 1492R, 3 μL bacterial solution template, and finally added sterile ultrapure water to 25 μL. The PCR reaction program was: (i) 94℃5min pre-denaturation; (ii) 34 cycles of 94℃1min denaturation, 55℃1min annealing, 72℃1.5min extension; (iii) the final extension condition was 72℃10min. The PCR amplification product was submitted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0075] SeqMan 7.1.0.44 was used to splice the measured sequences to obtain the 16S rRNA gene sequence of strain C007. The spliced ​​sequence file was compared with the data in the NCBI 16S database using the NCBI Blast program to obtain the species information with the greatest similarity to the sequence of the species to be tested, which was the identification result. The obtained sequence was Bacillus composting.

[0076] The 16S rRNA gene sequence of stercoris C007 is shown in SEQ ID NO:1.

[0077] The 16S rRNA gene sequence of Bacillus stercoris C007 was compared with the NCBI (National Center for Biotechnology Information) database. The results showed that the sequence similarity between the 16S rRNA gene sequence of Bacillus stercoris C007 and that of Bacillus stercoris N6 was as high as 99.86%. The phylogenetic tree was constructed using MEGA7.1 (see Figure 2 ), forming a stable evolutionary branch with the sequence of Bacillus stercorisN6.

[0078] The Bacillus Velezii agent has a deposit number of CCTCC NO: M 20221937. The specific isolation, purification and identification process can be found in the disclosure of patent application No. 2023113257516.

[0079] According to an embodiment of the present invention, the composite microbial agent includes: 45 to 55 parts by weight (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or any two of the values ​​as the range of endpoint values) of the microbial composting Bacillus stercoris C007 agent; 45 to 55 parts by weight (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or any two of the values ​​as the range of endpoint values) of the Bacillus velez agent. Thus, the synergistic effect of promoting the growth of wheat seedlings can be better exerted.

[0080] The composting Bacillus stercoris C007 inoculant contains no less than 100 billion cfu / g of spores of Bacillus stercoris C007. The composting Bacillus stercoris C007 inoculant contains no less than 130 billion cfu / g of spores of Bacillus berlez.

[0081] Preparation of composite bacterial agent: The culture medium used for C007 is 8g / L corn starch, 8.5g / L bean cake powder, 2 g / L K2HPO4, 1 g / L KH2PO4, 0.1 g / L ZnSO4, 2 mg / L MgSO4, 2 mg / L CuSO4, 1 mg / L Na2MoO4. The culture medium used for Bacillus Velezii is 6g / L corn starch, 7g / L bean cake powder, 1.5g / L rice flour, 1.5g / L bran, 1g / L corn steep liquor, 0.5g / L calcium carbonate, 0.2g / L potassium nitrate, 0.3g / L magnesium sulfate, 0.2 g / L K2HPO4, 0.2 g / L Na2HPO4. Both strains are fermented in a 20L fermenter, sampling every 4h, and stopping the tank when the spore maturity is 50%. The obtained bacterial liquid is directly sprayed onto the bacterial powder by high-temperature centrifugal drying, and the two are mixed in a mass ratio of 1:1 to obtain a composite bacterial agent.

[0082] Preparation of wheat seeds: Select complete and disease-free wheat varieties "Jiemai 19" seeds, soak them in 75% ethanol for 3 minutes, then soak them in 5% (v / v) sodium hypochlorite solution for 3 minutes, and then rinse the wheat seeds three times with sterile distilled water to complete the surface disinfection and sterilization of the wheat seeds. After the surface disinfection and sterilization of the wheat seeds is completed, place the wheat seeds in a sterile culture dish, and fill the culture dish with sterile distilled water so that the wheat seeds are completely immersed in the sterile distilled water to break the dormancy of the wheat seeds.

[0083] Preparation of saline-alkali soil: The saline-alkali farmland soil (soil conductivity 1.9ms / cm) from Nandagang, Huanghua, Hebei Province was selected and sieved through a 2mm screen and placed in a 8cm x 8cm x 8cm pot. The amount of microbial agent applied was based on the recommended amount for field use (15kg / hm 2 ), convert the content of bacterial fertilizer in each kilogram of soil, and apply the biological agent at a dosage of 0.02g / kg. Mix the soil and biological agent evenly, then pot and water.

[0084] Ten wheat seeds were planted in each pot (as the first day of planting); after the seedlings emerged more evenly, thinning was performed (as the fifth day of planting) and 25 mL of the prepared bacterial solution was poured around the roots of the wheat seedlings (as the first inoculation), and the negative control group was poured with the same volume of sterile 1x phosphate buffer (PBS), and the potted plants were grown at room temperature.

[0085] After 28 days of cultivation, the wheat potted plants were observed, and various indicators of the potted plants were measured and analyzed. The growth of the wheat potted plants was observed, and the plant height, root length and other indicators of the potted wheat seedlings were measured. It was found that the growth of the wheat seedlings inoculated with the composite microbial agent was better than that of the negative control group and the wheat seedlings inoculated with a single microbial agent (see Table 2).

[0086] Table 2 C007 and Bacillus Velezii synergistically promote wheat growth

[0087]

[0088] Experimental data show that compound microbial agents can better enhance the synergistic effect of beneficial microorganisms and promote plant growth better.

[0089] Example 2 Functional determination of composite microbial seed coating agent

[0090] In another aspect of the present invention, the present invention provides a microbial seed coating agent. According to an embodiment of the present invention, the microbial seed coating agent comprises at least one of the following: the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007, its culture or its processed product, the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 bacterial agent, and the composite microbial bacterial agent. According to an embodiment of the present invention, the composite microbial seed coating agent comprises the following raw materials in parts by weight:

[0091] 100-200 parts of compound microbial agent;

[0092] 3-5 parts of film-forming agent;

[0093] 1-3 parts of silk fibroin;

[0094] 3-5 parts of trehalose;

[0095] The composite microbial agent includes a Bacillus Velez subtilis agent, and the Bacillus Velez subtilis agent is selected from the Bacillus Velez subtilis with a deposit number of CCTCC NO: M 20221937. According to the composite agent, the composting Bacillus stercoris C007 and the Bacillus Velez subtilis agent have great application value in promoting the growth of wheat seedlings.

[0096] According to an embodiment of the present invention, the composite microbial agent includes: 45 to 55 parts by weight (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or any two of the values ​​as the range of endpoint values) of the microbial composting Bacillus stercoris C007 agent; 45 to 55 parts by weight (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or any two of the values ​​as the range of endpoint values) of the Bacillus velez agent. Thus, the synergistic effect of the binary composite agent can be better exerted.

[0097] The composting Bacillus stercoris C007 inoculant contains no less than 100 billion cfu / g of spores of Bacillus stercoris C007. The composting Bacillus stercoris C007 inoculant contains no less than 130 billion cfu / g of spores of Bacillus berlez.

[0098] According to the composite microbial seed coating agent described in an embodiment of the present invention, the film-forming agent is at least one of carboxymethyl cellulose, methyl cellulose, ethyl cellulose, polyvinyl acetate cellulose salt or polyvinyl alcohol.

[0099] In another aspect of the present invention, the present invention provides a method for preparing a composite microbial seed coating agent. According to an embodiment of the present invention,

[0100] Cultivating beneficial microorganisms, collecting cell bodies, and obtaining a composite microbial agent; mixing the composite microbial agent, a film-forming agent, silk fibroin, trehalose, and distilled water to obtain the microbial seed coating agent;

[0101] The preparation process of the composite microbial agent is as follows: the culture medium used for C007 is 8g / L corn starch, 8.5g / L bean cake powder, 2 g / L K2HPO4, 1 g / L KH2PO4, 0.1 g / L ZnSO4, 2 mg / L MgSO4, 2 mg / L CuSO4, and 1 mg / L Na2MoO4. The culture medium used for Bacillus Velezii is 6g / L corn starch, 7g / L bean cake powder, 1.5g / L rice flour, 1.5g / L bran, 1g / L corn steep liquor, 0.5g / L calcium carbonate, 0.2g / L potassium nitrate, 0.3g / L magnesium sulfate, 0.2 g / L K2HPO4, and 0.2g / L Na2HPO4. Both strains are fermented in a 20L fermentation tank, and samples are taken every 4 hours, and the tank is stopped when the spore maturity is 50%. The obtained bacterial liquid is directly sprayed onto bacterial powder by high-temperature centrifugal drying, and the two are mixed in a mass ratio of 1:1 to obtain a composite microbial agent.

[0102] In another aspect of the present invention, the present invention provides a seed coating method. According to an embodiment of the present invention, the composite microbial seed coating agent prepared by the above method is used for seed coating, and the mass ratio of the composite microbial seed coating agent:seed is 1:10 to 40. Further, the composite microbial seed coating agent prepared by the above method is used for seed coating, and the mass ratio of the composite microbial seed coating agent:seed:solvent is 1:10 to 40:10; further, the solvent is distilled water.

[0103] The seeds of the composite microbial seed coating agent prepared by the above method are mixed and stirred evenly until the surface of each seed is evenly coated with the coating agent. In some embodiments, after the seeds are coated, the spore content in the seed coating agent coated with each seed is 1×106 ~1×10 8 cfu / seed, and then dried in the shade at room temperature.

[0104] The seeds are wheat seeds, and the preparation ratios are:

[0105] Ratio ①: The mass ratio of the composite microbial seed coating agent, seeds, and distilled water is 1:20:10 (referred to as coating ①).

[0106] Ratio ②: The mass ratio of the composite microbial seed coating agent, seeds, and distilled water is 1:40:10 (referred to as coating ②).

[0107] Ratio ③: The mass ratio of the composite microbial seed coating agent, seeds, and distilled water is 1:15:10 (referred to as coating ③).

[0108] Ratio ④: The mass ratio of the composite microbial seed coating agent, seeds, and distilled water is 1:10:10 (referred to as coating ④).

[0109] The results showed that the germination rate of the control group (no coating) was 80.18%, the germination rate of coating ① was 84.95%, the germination rate of coating ② was 85.71%, the germination rate of coating ③ was 67.82%, and the germination rate of coating ④ was 79.62%. The germination rates of coating ① and coating ② were higher than those of the control group.

[0110] a. Effects of composite microbial seed coating agent on the growth of wheat seedlings in saline-alkali soils with different salinity levels

[0111] Preparation of wheat seeds: Select intact and disease-free wheat seeds, soak them in 75% ethanol for 3 minutes, and then transfer them to 5% (v / v) sodium hypochlorite solution for 3 minutes to complete the surface disinfection of the wheat seeds. Finally, rinse the wheat seeds with sterile distilled water for 3 to 5 times to wash away the ethanol and sodium hypochlorite remaining on the surface of the seeds. The wheat seeds that have completed surface disinfection are mixed with microbial seed coating agents (coating ① and coating ②, respectively) and then dried in the shade. Wheat seeds without the addition of composite microbial seed coating agents are used as controls.

[0112] Soil preparation: Select mild, moderate and severe (mild, moderate and severe soil conductivity are 1.9ms / cm, 4.1ms / cm and 7.9ms / cm respectively) saline-alkali farmland soil from Nandagang, Huanghua, Hebei, sieve through 2mm and place in 8cm x 8cm x 8cm pots. Soil was drenched with sterile distilled water and wheat was planted. Five seeds were planted in each pot (as the first day of planting), and 5 replicates were used for each treatment; after the seedlings emerged more uniformly, thinning was performed (as the fifth day of planting), and the potted plants were placed at room temperature for growth. After 14 days of cultivation, the plant pots were observed, and the various result indicators of the potted plants were measured and analyzed.

[0113] 1) Observe the growth of wheat potted plants and find that the growth of wheat seedlings treated with the composite microbial seed coating agent is better than that of the negative control group, and the seedling height is significantly higher than that of the negative control group ( Figure 3 );

[0114] 2) The plant height and root length of potted wheat seedlings were measured. The measurement results and analysis showed that the application of microbial seed coating agents promoted the growth of wheat seedlings, which was mainly manifested in the increase of plant height and root length of wheat seedlings. Based on the statistical method, the single-factor ANOVA test of SPSS software (P<0.05 indicated significant differences; P<0.01 indicated extremely significant differences) was used to conduct statistical analysis on various measurement indicators of plant height of potted wheat seedlings. The analysis results showed that the plant height of wheat seedlings coated with composite microbial seed coating agents ① and ② in mild saline-alkali soil was significantly increased compared with the negative control group, with an increase of 11.13%-17.38%; the plant height of wheat seedlings coated with microbial seed coating agents ② in moderate saline-alkali soil was 2.41% higher than that of the negative control group, and the root length was 24.92% higher than that of the negative control group.

[0115] b. Effects of microbial seed coating agents on the growth of wheat seedlings in aqueous solutions with different salt contents

[0116] The wheat seeds with microbial seed coating agents and the control treatment were transferred to 50ml centrifuge tubes containing aqueous solutions with different salt contents after germination in the culture dishes, and the plants were fixed with sponges. The treatments were: 75mM salt solution + conventional seeds, 75mM salt solution + coated seeds ①, 75mM salt solution + coated seeds ②; 150mM salt solution + conventional seeds, 150mM salt solution + coated seeds ①, 150mM salt solution + coated seeds ②; 300mM salt solution + conventional seeds, 300mM salt solution + coated seeds ①, 300mM salt solution + coated seeds ②. The hydroponic wheat was grown at room temperature. After 14 days of cultivation, the plant pots were observed, and the various result indicators of hydroponic wheat were measured and analyzed.

[0117] 1) Observe the growth of wheat and find that the growth of wheat seedlings treated with microbial seed coating is better than that of the negative control group (no coating) under the same salinity, and the seedling height is significantly higher than that of the negative control group ( Figure 4 , Figure 5 );

[0118] 2) The plant height of wheat seedlings was measured and it was found that the application of microbial seed coating promoted the growth of wheat seedlings ( Figure 6 );

[0119] 3) After measuring the height of wheat seedlings, the measurement results were statistically analyzed using SPSS.

[0120] Data collection and analysis results: Based on statistical methods, the SPSS software's one-way ANOVA test (P<0.05 indicates a significant difference; P<0.01 indicates an extremely significant difference) was used to conduct a statistical analysis of the various measurement indicators of the height of potted wheat seedlings. The analysis results showed that the height of wheat seedlings treated with microbial seed coating agents was higher than that of the negative control group, with the increase ranging from 7.05% to 23.57%, all of which were significantly different.

[0121] The seed coating agent containing composite microorganisms can promote the growth of wheat seedlings in saline-alkali soil and saline solution.

[0122] SEQ ID NO:1

[0123] >C007_16S rDNA

[0124]

[0125] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A microbial seed coating agent, characterized in that: The microbial seed coating agent comprises: A composite microbial agent, wherein the composite microbial agent is an agent comprising the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007; The composite microbial agent also includes a Bacillus Velez subtilis agent, and the Bacillus Velez subtilis is selected from the Bacillus Velez subtilis with a deposit number of CCTCCNO: M20221937; The salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 was deposited in the China Center for Type Culture Collection on August 28, 2024, and its preservation number is: CCTCCNO: M20241868.

2. The microbial seed coating agent according to claim 1, characterized in that The composite microbial agent has a spore content of no less than 100 billion cfu / g of salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007.

3. The microbial seed coating agent according to claim 1, characterized in that In the composite microbial agent, the content of Bacillus Velez spores in the Bacillus Velez spore agent is not less than 130 billion cfu / g.

4. The microbial seed coating agent according to claim 1, characterized in that It includes the following raw materials in parts by weight: 100-200 parts of compound microbial agent; 3-5 parts of film-forming agent; 1-3 parts of silk fibroin; 3-5 parts of trehalose.

5. The microbial seed coating agent according to claim 1, characterized in that The composite microbial agent comprises: 45 to 55 parts by weight of the microbial composting Bacillus stercoris C007 agent; and 45 to 55 parts by weight of Bacillus velezii agent.

6. The microbial seed coating agent according to claim 4, characterized in that The film-forming agent is at least one of carboxymethyl cellulose, methyl cellulose, ethyl cellulose, polyvinyl acetate cellulose salt or polyvinyl alcohol.

7. A method for preparing a microbial seed coating agent, characterized in that: The following steps are involved: Cultivating beneficial microorganisms, collecting cell bodies, and obtaining a composite microbial agent; mixing the composite microbial agent, a film-forming agent, silk fibroin, trehalose, and distilled water to obtain the microbial seed coating agent; The preparation process of the composite microbial agent is as follows: the culture medium used for microbial composting Bacillus stercoris C007 is 8 g / L corn starch, 8.5 g / L bean cake powder, 2 g / L K2HPO4, 1 g / L KH2PO4, 0.1 g / L ZnSO4, 2 mg / L MgSO4, 2 mg / L CuSO4, and 1 mg / L Na2MoO4; The culture medium used for Bacillus Velezii was 6 g / L corn starch, 7 g / L bean cake powder, 1.5 g / L rice powder, 1.5 g / L bran, 1 g / L corn steep liquor, 0.5 g / L calcium carbonate, 0.2 g / L potassium nitrate, 0.3 g / L magnesium sulfate, 0.2 g / L K2HPO4, and 0.2 g / L Na2HPO4. Both strains were fermented in a 20 L fermenter, with sampling every 4 hours, and the tank was stopped when the spore maturity was 50%. The two bacterial liquids were respectively subjected to high temperature centrifugal drying and direct spraying to obtain microbial composting Bacillus stercoris C007 and Bacillus velezii respectively, and the composite microbial agent was obtained by mixing the two. The composite microbial agent comprises: 45 to 55 parts by weight of the microbial composting Bacillus stercoris C007 agent; 45-55 parts by weight of Bacillus Velez bacteria agent; The salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 was deposited in the China Center for Type Culture Collection on August 28, 2024, and its deposit number is: CCTCC NO:M 20241868; The Bacillus Velez subtilis is selected from the Bacillus Velez subtilis with a deposit number of CCTCC NO: M 20221937.

8. A seed coating method, characterized in that: The microbial seed coating agent according to any one of claims 1 to 6 is used for seed coating, and the mass ratio of the microbial seed coating agent to seeds is 1:10 to 40; Furthermore, the microbial seed coating agent according to any one of claims 1 to 6 is used for seed coating, and the mass ratio of composite microbial seed coating agent:seed:solvent=1:10-40:10; further, the solvent is distilled water.

9. The seed coating method according to claim 8, characterized in that: After the seeds are coated, the spore content in the microbial seed coating agent coated on each seed is 1×10 6 ~1×10 8 cfu / seed.

10. Use of the microbial seed coating agent according to any one of claims 1 to 6 in at least one of the following: Improve crop resistance to stress, especially resistance to salinity and alkali; Promote crop growth.

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