Soybean composite growth-promoting microbial coating agent and coating method thereof

Abstract

The invention provides a soybean composite growth-promoting microbial coating agent and a coating method thereof, bacterial strains with a soybean growth-promoting effect are selected and compounded into a mixed bacterial agent according to a certain variety and proportion, biochar is innovatively used for seed coating, and the composite coating agent comprises an adsorbent, a nutritional agent, a binder and the bacterial composition. According to the invention, strains with a plant growth promoting effect are selected and compounded into a mixed microbial inoculum according to a certain variety and proportion, and the biochar is innovatively used for seed coating, so that plant growth is promoted by using the coating agent, and the abiotic stress resistance is improved. The coating agent is efficient, low in toxicity, safe and reliable, the preparation method is simple and feasible, and the coating agent has important practical value.

Description

Technical Field

[0001] The invention relates to the technical field of plant planting, and in particular to a soybean composite growth-promoting microbial coating agent and a coating method thereof. Background Art

[0002] At present, most of the domestic patent applications related to biochar and microbial coating agents only involve the use of biochar or microbial agents alone, or the application of combined pesticides and fertilizers; there are relatively few patent applications using biochar as an adsorbent for microbial seed coating.

[0003] The patent document with application number CN202010776820.5 and titled "A saline-alkali soil soybean fertilizer and a fertilization method for improving the nutrient efficiency of saline-alkali soil soybeans" describes that by spreading a base fertilizer composed of biochar and soil conditioners before soybean planting and then cultivating the land, the physical and chemical properties of the soil are improved, soil productivity is increased, and pollution to the environment is reduced; the application number is CN 202211465818.1, the patent document named "A soybean seed coating agent and its application" describes the problem of preparing a composite bacterial liquid in a specific proportion for seed coating to prevent and control soybean Phytophthora root rot; the patent document with application number CN202222011105.X and name "A planting seed packaging structure" describes a method of using biochar as an antifreeze layer for seed coating; the patent document with application number CN202211620591.3 and name "A soybean rhizobium biological protective agent and its preparation method and application" describes a method of using Bacillus brevis and rhizobia to mix naked seeds or coated seeds of plants, which can improve the seed coating efficiency and the survival rate of rhizobia during seed mixing and storage.

[0004] Based on the existing technology, the coating products on the market are mainly pesticide coatings, and there are few reports on composite coatings constructed using biochar-non-rhizobium soybean growth-promoting bacteria. Although the use of pesticide coatings can effectively inhibit the occurrence of soil-borne diseases, it also brings about problems such as pesticide residues in the soil and inhibition of soybean rhizosphere microbial activity, causing environmental pollution and being detrimental to the development of agricultural ecological circular economy. Therefore, it is particularly important to develop a seed coating agent that increases soybean yield. Summary of the invention

[0005] The inventors collected soybean roots from arid areas and used plate coating and streaking technology to isolate rhizosphere strains with plant growth-promoting properties such as nitrogen fixation, phosphorus solubilization, potassium solubilization, plant hormone production, iron carrier production, and ACC deaminase production; a series of strains that can promote soybean stress-resistant growth were obtained through separation and purification, and through combined inoculation verification, strains A and B with excellent effects were finally selected to construct a composite microbial agent. After species identification, they were Chryseobacterium ( Chryseobacterium daecheongenseGraJ1), Marseille bacteria ( Massilia phyllostachyos JC3), and the above-mentioned strains were deposited for the two strains respectively. Among them, the deposit date of the Chryseobacterium is: January 12, 2024, the deposit unit is: China Center for Type Culture Collection (CCTCC), the deposit unit address is: Wuhan University, Wuhan, China, the classification name is: Chryseobacterium daecheongense GraJ1, the deposit number is CCTCC NO: M 2024089. The deposit date of the Marseille bacteria is January 12, 2024, the deposit unit is China Center for Type Culture Collection (CCTCC), the deposit unit address is Wuhan University, Wuhan, China, the classification name is: Massilia phyllostachyos JC3, the deposit number is CCTCCNO: M 2024087.

[0006] On the other hand, the present invention provides a composite microbial agent, comprising the above-mentioned bacterial composition, wherein the composite microbial agent is a liquid agent or a solid powdered agent. The above-mentioned microbial agent is preferably in a ratio of 1:9 to 9:1, preferably (1-5): (1-5), and more preferably, in a ratio of (0.5-2): (0.5-2).

[0007] In the above-mentioned composite microbial agent, preferably, the liquid agent or solid powdered agent comprises a protective agent and the above-mentioned bacterial composition; preferably, the effective viable bacterial count concentration of the bacterial composition in the liquid agent is 10 6 -10 13 CFU / mL; preferably, the effective viable bacterial count concentration of the bacterial composition in the solid powdered bacterial agent is 10 6 -10 13 CFU / g. Preferably, the above effective viable count concentration is not less than 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 CFU / g.

[0008] In the above-mentioned composite microbial agent, preferably, the protective agent includes K2HPO4-KH2PO4 buffer with a pH of 7.2 and / or glycerol.

[0009] On the other hand, the present invention provides a composite coating agent, which includes: the above-mentioned bacterial composition or composite microbial agent, an adsorbent, and a binder.

[0010] In the above-mentioned composite coating agent, preferably, the composite coating agent also includes a nutrient, and the nutrient includes a combination of one or more of crystalline trehalose, molasses, phosphate rock powder, potassium rock powder, boron rock powder, iron silicate ore powder, composite trace elements, fulvic acid, and humic acid.

[0011] In the above-mentioned composite coating agent, preferably, the binder comprises carboxymethyl cellulose and maltodextrin; wherein the weight ratio of the carboxymethyl cellulose to the maltodextrin is (40-60):(60-40); preferably, the weight ratio of the carboxymethyl cellulose to the maltodextrin is (0.5-2):1.

[0012] In the above-mentioned composite coating agent, preferably, the adsorbent includes a combination of one or more of biochar, diatomaceous earth, and straw powder. More preferably, the adsorbent consists of biochar and diatomaceous earth; further preferably, the weight ratio of the biochar to the diatomaceous earth is (1-9): (9-1); the weight ratio of diatomaceous earth to biochar is (1-4): (1-4); further preferably, the weight ratio of the biochar to the diatomaceous earth is (0.5-2): 1.

[0013] In the above-mentioned composite coating agent, preferably, the weight ratio of the bacterial composition or composite microbial agent, adsorbent, binder and nutrient is (1-100): (200-500): (20-500): (100-200).

[0014] On the other hand, the present invention provides a method for preparing a composite coating agent, specifically, using a modified R2A medium to activate the bacteria in the above bacterial composition, shake flask culture and fermentation amplification culture, centrifugally collect the cells, freeze-dry and add freeze-dried additives to make a solid bacterial agent, or directly add a liquid protective agent to make a liquid bacterial agent, and mix according to the above proportions to prepare the above composite microbial agent. According to the above formulas of adsorbent, nutrient and binder, prepare adsorbent, nutrient and binder respectively; adsorbent, nutrient, binder and composite microbial agent are independently packaged to obtain a composite coating agent.

[0015] In the above-mentioned preparation method of the composite coating agent, preferably, the composite coating agent also includes the following preparation steps during use: mixing the adsorbent, the nutrient agent and the binder, adding sterile water and stirring evenly to obtain a mixed matrix solution; then adding the composite microbial agent to the mixed matrix solution and mixing evenly to prepare a composite coating agent liquid solution. Preferably, in the composite coating agent liquid solution, the amount ratio of the binder to the sterile water is (20-100) g: 1000 mL; the effective live bacteria concentration of the composite microbial agent in the composite coating agent liquid solution is 10 6 -10 9 CFU / mL; preferably not less than 106 , 10 7 , 10 8 , 10 9 CFU / mL.

[0016] In the above-mentioned method for preparing the composite coating agent, preferably, the modified R2A medium has the following components in the formula: 1.3 g glucose, 1.3 g bacteriological peptone, 1.3 g acid hydrolyzed casein, 1.3 g yeast extract, 0.8 g K2HPO3·3H2O, 0.8 g sodium pyruvate, 0.15 g MgSO4·7H2O, and 1000 mL of distilled water; for agar solid culture medium, 15 g of agar can be added for every 1000 mL of culture medium.

[0017] In the above-mentioned method for preparing the composite coating agent, preferably, the additive is an additive for maintaining the survival of the bacterial strain.

[0018] On the other hand, the present invention provides the use of the above-mentioned bacterial composition, composite microbial agent, and composite coating agent in promoting plant stress-resistant growth.

[0019] In another aspect, the present invention provides the use of the above-mentioned bacterial composition, composite microbial agent, and composite coating agent in improving the biological performance of plants. Preferably, improving the biological performance of plants includes but is not limited to increasing the plant grain weight, increasing the number of grains per area, increasing the number of grains per plant and / or thousand-grain weight, increasing the yield per mu, and / or increasing the biomass.

[0020] Preferably, the plant is a dicotyledonous plant, preferably a leguminous plant, preferably a leguminous plant, more preferably a plant of the order Fabaceae; more preferably a leguminous plant; the leguminous plant is alfalfa, lotus root, soybean, pea, peanut, kidney bean, mung bean, adzuki bean, broad bean, cowpea, astragalus, licorice or astragalus; more preferably a plant of the genus Glycine; more preferably soybean.

[0021] The beneficial effects of the present invention include but are not limited to: In the present invention, diatomaceous earth / biochar is used to adsorb the bacteria in the composite microbial agent and provide a certain habitat for them; the coating agent is tightly attached to the plant seeds and is more suitable for providing beneficial microbial communities for the plants in the early stage of plant germination; the composite microbial community can promote plant growth, and at the same time, the competition generated by the pre-occupancy effect can reduce the occurrence of soil-borne diseases to a certain extent.

[0022] The present invention selects strains with plant growth-promoting effects, mixes them into mixed bacterial agents according to certain types and proportions, and innovatively uses biochar for seed coating, thereby realizing the use of coating agents to promote plant growth and improve their resistance to abiotic stress. The coating agent is highly efficient, low-toxic, safe and reliable, and the preparation method is simple and easy, and has important practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The growth status of soybeans treated with composite coating agents at the seedling stage compared with those without coating. CK is naked seeds planted directly; BC is seeds treated with bacterial composite coating agents.

[0024] Figure 2 Comparison of soybean development status after treatment with composite coating agent at jointing stage. CK is naked seeds; BC is treated with coating agent with bacteria; NK is treated with coating agent without bacteria.

[0025] Figure 3 Soybean coating drying effect.

[0026] Figure 4 The effect of coating on the jointing stage in the field environment. The left picture shows the root system of V6 soybean without coating, and the right picture shows the root system of V6 soybean with coating. DETAILED DESCRIPTION

[0027] Before further describing the 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 terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0028] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0029] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the technical field.

[0030] The modified R2A medium involved in the present invention has the following components in the formula: 1.3 g glucose, 1.3 g bacteriological peptone, 1.3 g acid hydrolyzed casein, 1.3 g yeast extract, 0.8 g K2HPO3·3H2O, 0.8 g sodium pyruvate, 0.15 g MgSO4·7H2O, and 1000 mL of distilled water; for agar solid medium, 15 g of agar can be added for every 1000 mL of medium.

[0031] Example 1 Screening, Isolation and Identification of Bacteria The roots of soybeans planted in arid soil were collected, the rhizosphere soil was brushed off with a sterilized brush, 1 g of the test soil was added to a test tube with glass beads and 9 mL of sterile water, and the soil was shaken on a vortex shaker for 10 min, and then diluted in sequence.

[0032] The dilution coating method was used to coat the strains on Montgena medium, Alexander Rove medium, Ashubei medium, combined nitrogen fixation medium, and PKO medium, respectively, and cultured at 30 °C. According to the growth of the strains on the selection medium, single colonies with faster growth and better growth were selected, repeatedly purified by the plate streaking method, and cultured in the corresponding liquid culture medium. After 3 days, the bacterial solution was centrifuged and stored at -80 °C for later use.

[0033] The bacteria were inoculated on Ashubei medium, combined nitrogen fixation medium, Montgena medium, and PKO medium, and their nitrogen fixation, phosphate solubilization, and phosphate dissolving activities were determined by the growth of the strains on the selection medium and the presence or absence of transparent circles. Potassium-solubilizing bacteria were inoculated on Alexander Rof medium (with bromothymol blue added), and after culturing in a 30 ℃ incubator for 3 to 5 days, the colonies were observed to see if they turned yellow to determine the potassium-solubilizing ability of the strains.

[0034] A series of strains that can promote soybean stress resistance growth were obtained through separation and purification, and the strains with excellent effects were finally selected to construct the bacterial composition through combined inoculation verification. The results are shown in Tables 1, 2 and 3.

[0035] Table 1 Effect of single strain inoculation on soybean seedling growth

[0036] Table 2 Synthetic community construction scheme

[0037] Table 3 Effects of synthetic communities on soybean seedling growth

[0038] Through consideration of various factors such as the results of plant experiments, it was determined that the artificial synthetic community SynCom-01 would be constructed with mr006 as the core strain and nr002 as the auxiliary strain.

[0039] After obtaining pure culture of bacteria, heat and lyse the bacteria using Chelex-100 resin method to extract bacterial genomic DNA. The specific operation is as follows: pick an appropriate amount of purified single bacterial colony into 100 μL 10% Chelex-100 resin suspension, vortex to mix the bacteria and resin suspension thoroughly, break the cells in boiling water bath to release bacterial genomic DNA. Centrifuge at 10000rpm for 10 min, and take the supernatant as PCR template.

[0040] According to the PCR reaction system in Table 4 and the amplification program in Table 5, the 16S rRNA gene was amplified.

[0041] Table 4 16S rRNA gene PCR reaction system

[0042] Table 5 16S rRNA gene PCR amplification procedure

[0043] The PCR products were sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. After obtaining the results, the sequence fragments with good sequencing quality (about 700 bp) were intercepted and the 16S sequence was compared with the NCBI rRNA / ITS database using the web version of BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Chryseobacterium daecheongense GraJ1), Marseille bacteria ( Massilia phyllostachyos JC3).

[0044] Example 2 Activation, cultivation, collection and preparation of microbial culture fluid 100 mL of glycerol bacterial culture solution (Chryseobacterium and Massimoylation isolated in Example 1) was applied to the modified R2A medium and cultured at 25°C. Single colonies with typical colony characteristics and rapid growth were selected and placed in the modified R2A corresponding liquid medium and cultured at 25°C. When the culture reached the middle and late logarithmic phase, the cells were centrifuged at 8000 g, washed three times with K2HPO4-KH2PO4 buffer (pH 7.2), and then resuspended with the same buffer to a cell concentration of 10 9 CFU / mL, that is, the bacterial liquids of Chryseobacterium tumefaciens and Masseilli were prepared respectively.

[0045] Example 3: Comparative test on the effect of different bacterial composition coatings on soybean growth 1. Preparation of the composite coating agent liquid solution used in this example a) Mixed matrix preparation: 200 g of adsorbent (100 g of diatomaceous earth + 100 g of biochar), 20 g of binder (10 g of carboxymethyl cellulose + 10 g of maltodextrin), and 100 g of nutrient (50 g of molasses + 50 g of humic acid) were mixed, and 1000 mL of sterile water was added and stirred to obtain a mixed matrix solution.

[0046] b) Preparation of composite coating agent liquid solution: Then, according to the method in Example 2, Chryseobacterium daecheongense GraJ1, Massilia phyllostachyos JC3 were prepared into bacterial solutions, and the effective live bacteria concentration in the two bacterial solutions was 10 9 CFU / mL, then Chryseobacterium daecheongense GraJ1, Massilia phyllostachyos 5 mL of JC3 bacterial solution was added to the mixed matrix solution and mixed evenly to prepare a composite coating agent, wherein the effective live bacteria concentration in the liquid solution of the composite coating agent was not less than 10 7 CFU / mL.

[0047] 2. Soybean seed coating and planting Add the dried wheat seeds into a seed coating machine / mixer, add the composite coating agent after starting to stir, wherein the weight ratio of the wheat seeds to the liquid solution of the composite coating agent is 1 kg: 25 mL; stir until the coating agent is evenly coated on the surface of the seeds, and sow after drying for 120 minutes.

[0048] This example continues to explore the effect of different bacterial composition coatings on soybean growth. A fixed adsorbent combination, in which diatomaceous earth: biochar weight ratio of 5:5 was used as an adsorbent for seed coating. After adding or not adding different bacterial composition strain combinations, soybean growth indicators were measured. The results are shown in Table 6.

[0049] Table 6 Effects of different bacterial composition coatings on soybean growth

[0050] Diatomaceous earth: biochar weight ratio of 5:5 was used as adsorbent for seed coating. After adding growth-promoting microbial strains, the dry weight of the above-ground part and the dry weight of the underground part of soybean increased compared with the coating treatment without adding strains. Chryseobacterium daecheongense GraJ1 and Massilia phyllostachyos The JC3 bacterial composition for microbial coating has a further enhanced growth-promoting effect on soybeans compared to adding the above strains alone for coating.

[0051] Example 4: Optimization of non-biological coating components This example further optimizes the non-biological coating components. Different adsorbent formulations were prepared as shown in Table 7, and soybean growth indicators were further measured.

[0052] 1. Preparation of the composite coating agent liquid solution used in this example a) Mixed matrix preparation: 200 g of adsorbent (prepared as in Table 7), 20 g of binder (10 g of carboxymethyl cellulose + 10 g of maltodextrin), and 100 g of nutrient (50 g of molasses + 50 g of humic acid) were mixed, and 1000 mL of sterile water was added and stirred to obtain a mixed matrix solution.

[0053] b) Preparation of composite coating agent liquid solution: Then, according to the method in Example 2, Chryseobacterium daecheongense GraJ1, Massilia phyllostachyos JC3 were prepared into bacterial solutions, and the effective live bacteria concentration in the two bacterial solutions was 10 9 CFU / mL, then Chryseobacterium daecheongense GraJ1, Massilia phyllostachyos 5 mL of JC3 bacterial solution was added to the mixed matrix solution and mixed evenly to prepare a composite coating agent, wherein the effective live bacteria concentration in the liquid solution of the composite coating agent was not less than 10 7 CFU / mL.

[0054] 2. Soybean seed coating and planting After adding the seed coating agent to the dried soybean seeds, place them in a 1.5 kw feed mixer, add the composite coating agent after starting stirring, wherein the ratio of the weight of the soybean seeds to the liquid solution of the composite coating agent is 1 kg: 25 mL; stir until the coating agent is evenly coated on the surface of the seeds, and sow after drying for 120 minutes.

[0055] Table 7 Effects of adsorbents on soybean growth

[0056] Soybean straw powder and sawdust powder that had passed through a 1 mm sieve were unable to adhere well to the surface of soybean seeds. However, when diatomaceous earth and biochar were mixed in different proportions as adsorbents, the biomass of soybeans in the seedling stage was increased, with the increase ranging from about 3% to 12-14%. The best effect was achieved when diatomaceous earth and biochar were mixed in equal proportions.

[0057] Example 5: Effect of coating on soybean growth under stress conditions 1. Preparation of the composite coating agent liquid solution used in this example a) Mixed matrix preparation: 200 g of adsorbent (100 g of diatomaceous earth + 100 g of biochar), 20 g of binder (10 g of carboxymethyl cellulose + 10 g of maltodextrin), and 100 g of nutrient (50 g of molasses + 50 g of humic acid) were mixed, and 1000 mL of sterile water was added and stirred to obtain a mixed matrix solution.

[0058] b) Preparation of composite coating agent liquid solution: Then, according to the method in Example 2, Chryseobacterium daecheongense GraJ1 and Massilia phyllostachyos JC3 were prepared into bacterial solutions, and the effective live bacteria concentration in the two bacterial solutions was 10 9 CFU / mL, then Massilia phyllostachyos JC3 bacterial solution Chryseobacterium daecheongense 5 mL of GraJ1 bacterial solution was added to the mixed matrix solution and mixed evenly to prepare a composite coating agent, wherein the effective live bacteria concentration in the liquid solution of the composite coating agent was 10 7 CFU / mL.

[0059] 2. Soybean seed coating and planting Add the dried soybean seeds into a seed coating machine / mixer, add the composite coating agent after starting to stir, wherein the weight ratio of the soybean seeds to the liquid solution of the composite coating agent is 1 kg: 25 mL; stir until the coating agent is evenly coated on the surface of the seeds, and sow after drying for 120 minutes.

[0060] (1) Results of strain pot culture experiment: The pot experiments were all completed under drought stress conditions. The simulated drought stress conditions were irrigated once a week with 60 ml of 15% peg-6000 solution. The growth index of soybeans at stage V6 was measured, and the results are shown in Table 8 Table 8 Effects of coating on growth of V6 soybean under stress conditions

[0061] Compared with the uncoated treatment, the germination rate of soybeans treated with the composite coating agent increased by 6%, the dry weight of the above-ground part increased by 69%, and the dry weight of the underground part increased by 82%.

[0062] (2) Field demonstration experiment results: In the 2024 field trial, the root system of soybeans treated with the composite coating agent was more developed than that of soybeans not treated with the coating agent, with more lateral fibrous roots and a significantly increased number of nodules. Figure 4The grain weight per unit area and the number of grains per unit area increased by 10.47% and 16.15% respectively, the number of grains per plant and the thousand-grain weight increased by 7.26% and 3.74% respectively; the yield per mu increased from 473 kg to 540 kg, an increase of 14.16%.

[0063] From the above data, it can be seen that the physiological indicators of soybeans coated with the composite coating agent are significantly improved compared with those of soybeans not coated.

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chryseobacterium Chryseobacterium daecheongense GraJ1), characterized in that The strain has the function of protecting plant roots and is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 2024089.

2. A type of Marseilles mushroom ( Massilia phyllostachyos JC3), characterized in that This strain has the function of protecting plant roots and is deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC NO: M 2024087.

3. A composite bacterial agent, characterized in that: It comprises the Chryseobacterium as claimed in claim 1 and / or the Massillaria as claimed in claim 2.

4. A composite coating agent, characterized in that: The composite bacterial agent is prepared according to claim 3.

5. The composite coating agent according to claim 4, characterized in that It also contains adsorbents, binders and / or nutrients.

6. The composite coating agent according to claim 4 or 5, characterized in that: The effective live bacterial concentration of the strain is not less than 10 7 CFU / ml.

7. The composite coating agent according to claim 6, characterized in that The adsorbent comprises diatomaceous earth and biochar in a weight ratio of (1-4): (1-4); the binder is carboxymethyl cellulose and / or maltodextrin; and the nutrient is molasses and / or humic acid.

8. Use of the strain according to claim 1 or 2, the composite bacterial agent according to claim 3 or the composite coating agent according to any one of claims 4 to 7 in improving the biological performance of plants.

9. The use according to claim 8, characterized in that The improvement of the biological performance of the plant includes but is not limited to increasing the grain weight of the plant, increasing the number of grains per area, increasing the number of grains per plant and / or the thousand-grain weight, increasing the yield per mu and / or increasing the biomass.

10. The use according to claim 8 or 9, characterized in that: The plant is a leguminous plant.

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