Saline-alkali tolerant microbial seed coating agent and application thereof

Abstract

The invention relates to a saline-alkaline tolerant microbial seed coating agent and application thereof. The seed coating agent comprises a growth-promoting rhizobacteria inoculant and an auxiliary agent, the volume ratio of the growth-promoting rhizobacteria inoculant to the auxiliary agent is 1: (1-100); in each gram of the seed coating agent, the viable count of the growth-promoting rhizosphere bacteria is (1-100) * 10 < 10 > CFU; the growth-promoting rhizobacteria is selected from one or more of pseudomonas stutzeri A1501, pseudomonas fluorescens ATCC 13525, bacillus amyloliquefaciens FZB42 and azotobacter chroococcum BNCC336703, and the growth-promoting rhizobacteria is selected from one or more of bacillus amyloliquefaciens FZB42 and azotobacter chroococcum BNCC336703; on the basis of the total weight of the auxiliary agent, the auxiliary agent is prepared from the following components in percentage by weight: 1 to 9 percent of film forming agent, 1 to 9 percent of protective agent, 0.0001 to 0.001 percent of regulator, 0.0001 to 0.001 percent of active agent, 0 to 25 percent of filler and 58 to 97 percent of water. The seed coating agent can improve the rhizosphere colonization ability and survival rate of rhizosphere growth-promoting bacteria, can also improve the saline-alkaline tolerance of plant seeds, and effectively promotes plant growth.

Description

Technical Field

[0001] The present disclosure relates to the technical field of seed coating agents, and in particular, to a salt-alkali resistant microbial seed coating agent and applications thereof. Background Art

[0002] At present, a large amount of soil in the world is affected by salt damage. Excessive salt in the soil will inhibit the growth of crops and reduce their yields. Soil salinization is an important problem facing global agricultural development. Seed coating technology can standardize seeds, so that precision sowing and mechanized sowing technologies can be promoted and applied, saving manpower and greatly improving work efficiency. By coating the seed surface with a seed coating agent, the seed germination rate can be increased and the seedling resistance can be enhanced. Therefore, seed coating is an important measure to ensure crop yields, improve economic benefits, and realize agricultural modernization.

[0003] Plant rhizosphere growth-promoting bacteria refer to a type of probiotic bacteria that colonizes in the root system of plants and can promote plant growth and improve stress resistance. Rhizosphere growth-promoting bacteria have broad application prospects. Some strains are used in the field as inoculants and have achieved commercial products, but in practical applications, there are still problems such as insufficient stability and low efficiency in adverse environments. After the microbial seed coating agent wraps the seeds with rhizosphere growth-promoting bacteria, it can form a biological protective layer membrane with a certain strength and permeability on the surface of the seeds. As the seed seedlings grow, it colonizes on the surface and inside, and expands and colonizes in the root system and soil as the plants grow. At the same time, adding protective agents and active substances to microbial seed coating agents can effectively improve the tolerance of strains under drought and saline-alkali stress. Therefore, the development of microbial seed coating agents can improve the microenvironment of inoculated microorganisms on the surface of seeds, thereby improving the growth-promoting effect of microorganisms on plants.

[0004] Most of the seed dressing agents currently used are chemical pesticide seed dressing agents, which not only kill harmful organisms but also harm beneficial organisms in the field and even damage crop seedlings. Biological seed dressing agents use bio-based materials and microorganisms as their main ingredients. They are not only safe for humans and animals, pollution-free to the environment, and have nutritional functions for crops themselves, but also low in cost and high in benefits. Bio-based materials such as plant and animal proteins, alginates, etc. are ideal materials for microbial seed dressing agents.

[0005] There are many reports on the use of growth-promoting microorganisms as seed dressing agents, but most of them cannot be used in saline-alkali environments, and there are few good biological seed dressing agents for corn. Therefore, it is urgent to improve the low survival rate and poor stress resistance of rhizosphere growth-promoting bacteria during field application, and to improve the salt-alkali tolerance of plant seeds. Summary of the invention

[0006] The purpose of the present disclosure is to provide a salt-alkali tolerant microbial seed coating agent and its application, which can improve the rhizosphere colonization ability and survival rate of rhizosphere growth-promoting bacteria, and can also improve the salt-alkali tolerance of plant seeds, effectively promoting plant growth.

[0007] In order to achieve the above-mentioned object, the first aspect of the present disclosure provides a salt-alkali tolerant microbial seed coating agent, wherein the seed coating agent comprises: a rhizosphere growth-promoting bacterial agent and an adjuvant; The volume ratio of the rhizosphere growth-promoting bacteria agent to the auxiliary agent is 1: (1-100); The volume ratio of the rhizosphere growth-promoting bacteria agent to the auxiliary agent is 1: (1-100); The number of viable bacteria of the rhizosphere growth-promoting bacteria in each gram of the seed coating agent is (1-100)×10 10 CFU; the rhizosphere growth-promoting bacteria are selected from one or more of Pseudomonas stutzeri A1501, Pseudomonas fluorescens ATCC 13525, Bacillus amyloliquefaciens FZB42 and Azotobacter rotundifolia BNCC336703; Based on the total weight of the auxiliary agent, the auxiliary agent includes: 1-9 weight % of a film former, 1-9 weight % of a protective agent, 0.0001-0.001 weight % of a regulator, 0.0001-0.001 weight % of an active agent, 0-25 weight % of a filler and 58-97 weight % of water.

[0008] Optionally, the volume ratio of the rhizosphere growth-promoting bacteria agent to the auxiliary agent is 1: (10-100); The number of viable bacteria of the rhizosphere growth-promoting bacteria in each gram of the seed coating agent is (10-100)×10 10 CFU.

[0009] Optionally, based on the total weight of the auxiliary agent, the auxiliary agent includes: 1-6 wt % of a film former, 3-6 wt % of a protective agent, 0.0005-0.001 wt % of a regulator, 0.0005-0.001 wt % of an active agent, 0-25 wt % of a filler and 63-90 wt % of water.

[0010] Optionally, in the auxiliary agent, the weight ratio of the film-forming agent, protective agent, regulator and active agent is (1-3): (1-5): (0.0001-0.001): (0.0001-0.001), preferably (1-2): (3-5): (0.0005-0.001): (0.0005-0.001).

[0011] Optionally, the number of viable bacteria of the rhizosphere growth-promoting bacteria agent is (1-100)×10 12 CFU / mL, preferably (10-100)×10 12CFU / mL.

[0012] Optionally, the preparation method of the rhizosphere growth-promoting bacteria agent comprises: S1, inoculating the isolated and purified rhizosphere growth-promoting bacteria strain into an activation medium for activation treatment to obtain an activated strain; S2, inoculating the activated strain into a liquid culture medium for cultivation, and then centrifuging and removing the supernatant to obtain a cultured strain; S3. Resuspend the cultured strain 2-3 times using phosphate buffer, and then centrifuge to remove the supernatant to obtain the rhizosphere growth-promoting bacteria agent.

[0013] Optionally, in step S1, the activation medium includes LB liquid medium and / or K medium; In step S2, the liquid culture medium includes LB liquid culture medium and / or K culture medium.

[0014] Optionally, the film-forming agent is selected from one or more of sodium carboxymethyl cellulose, polyvinyl alcohol, sodium alginate, gum arabic and pectin; The protective agent is selected from one or more of whey protein, silk fibroin, corn protein, soy protein isolate, skim milk powder, trehalose and betaine; The regulator is selected from one or more of nano iron oxide, nano silicon dioxide, nano zinc dioxide and nano titanium dioxide; The active agent is selected from one or more of apigenin, luteolin, quercetin and epigallocatechin; The filler is selected from one or more of bentonite, diatomaceous earth and perlite.

[0015] The second aspect of the present disclosure provides the use of the seed coating agent described in the first aspect in improving the salt-alkali tolerance of plant seeds and plants and in promoting plant growth.

[0016] Optionally, the plant is selected from at least one of corn, wheat, cucumber, rapeseed and tomato; preferably corn.

[0017] Through the above technical scheme, the present disclosure provides a salt-alkali tolerant microbial seed coating agent and its application, which can improve the rhizosphere colonization ability and survival rate of rhizosphere growth-promoting bacteria, and can also improve the salt-alkali tolerance of plant seeds, effectively promoting plant growth.

[0018] Other features and advantages of the present disclosure will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0019] The specific embodiments of the present disclosure are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

[0020] The first aspect of the present disclosure provides a salt-alkali tolerant microbial seed coating agent, the seed coating agent comprising: a rhizosphere growth-promoting bacterial agent and an adjuvant; The volume ratio of the rhizosphere growth-promoting bacteria agent to the auxiliary agent is 1: (1-100); The number of viable bacteria of the rhizosphere growth-promoting bacteria in each gram of the seed coating agent is (1-100)×10 10 CFU; the rhizosphere growth-promoting bacteria are selected from one or more of Pseudomonas stutzeri A1501, Pseudomonas fluorescens ATCC 13525, Bacillus amyloliquefaciens FZB42 and Azotobacter rotundifolia BNCC336703; Based on the total weight of the auxiliary agent, the auxiliary agent includes: 1-9 weight % of a film former, 1-9 weight % of a protective agent, 0.0001-0.001 weight % of a regulator, 0.0001-0.001 weight % of an active agent, 0-25 weight % of a filler and 58-97 weight % of water.

[0021] Among them, the classification of Pseudomonas stutzeri A1501 is named Pseudomonas stutzeri The Pseudomonas stutzeri A1501 is a strain disclosed in patent number CN98102764.4; the classification name of Pseudomonas fluorescens ATCC 13525 is Pseudomonas fluorescens The product number of the fluorescein Pseudomonas ATCC 13525 is ATCC 13525; the classification name of Bacillus amyloliquefaciens FZB42 is Bacillus amyloliquefaciens The product number of the Bacillus amyloliquefaciens FZB42 is DSM 23117; the classification name of the round brown nitrogen-fixing bacteria BNCC336703 is Azotobacter chroococcum The product number of the round brown nitrogen-fixing bacteria BNCC336703 is BNCC336703.

[0022] In the present disclosure, a film-forming agent, a protective agent, a regulator, an active agent and a filler are used to form an adjuvant. On the one hand, a cross-linked structure can be formed to immobilize rhizosphere growth-promoting bacteria, thereby improving the survival rate of rhizosphere growth-promoting bacteria on the surface of seeds and improving the microenvironment on the surface of seeds; on the other hand, the adjuvant can activate the stress resistance system of rhizosphere growth-promoting bacteria and plants, improve the performance of rhizosphere growth-promoting bacteria and seeds under stress conditions, and enhance the interaction between plants and rhizosphere growth-promoting bacteria, provide an opportunity for rhizosphere growth-promoting bacteria to effectively colonize in the root system of plants, improve the structure of root microbial communities, and effectively resist diseases. The seed coating agent disclosed in the present disclosure can significantly improve the survival rate and rhizosphere colonization ability of rhizosphere growth-promoting bacteria, and the seed coating agent has slow release and degradability, improves the stability and stress resistance of rhizosphere growth-promoting bacteria in field application, and effectively promotes the germination rate and growth of plant seeds under salt stress conditions, especially the application of the microbial seed coating agent to corn seeds, which has a significant growth-promoting effect on the growth and development of corn, and can significantly improve the salt-alkali tolerance of corn.

[0023] In a preferred embodiment of the present disclosure, the volume ratio of the rhizosphere growth-promoting bacteria agent to the auxiliary agent is 1: (10-100); the number of viable bacteria of the rhizosphere growth-promoting bacteria per gram of the seed coating agent is (10-100)×10 10 In the above embodiment, the survival rate of the rhizosphere growth-promoting strains on the seed surface can be greatly improved, while the seed germination rate and plant growth-promoting effect can be improved.

[0024] In a preferred embodiment of the present disclosure, based on the total weight of the auxiliary agent, the auxiliary agent includes: 1-6 weight % of a film former, 3-6 weight % of a protective agent, 0.0005-0.001 weight % of a regulator, 0.0005-0.001 weight % of an active agent, 0-25 weight % of a filler and 63-90 weight % of water.

[0025] In the present disclosure, the combination of film-forming agent and protective agent can effectively improve the survival rate of rhizosphere growth-promoting bacteria on the surface of plant seeds; at the same time, the present disclosure can further improve the germination rate and seedling growth of plant seeds under saline-alkali stress conditions by further adding regulators and active agents, and expand the application scope of seed coating agents in adverse environments. In particular, when the weight ratio of the film-forming agent, protective agent, regulator and active agent is (1-3): (1-5): (0.0001-0.001): (0.0001-0.001), preferably (1-2): (3-5): (0.0005-0.001): (0.0005-0.001); within the above ratio, the interaction between the film-forming agent, protective agent, regulator and active agent can be further enhanced to obtain a seed coating agent suitable for saline-alkali environment, having the characteristics of high survival rate of strains, good stability and sustained release.

[0026] In one embodiment of the present disclosure, the number of viable bacteria of the rhizosphere growth-promoting bacteria agent is (1-100)×10 12 CFU / mL, preferably (10-100)×10 12 CFU / mL.

[0027] In one embodiment of the present disclosure, the preparation method of the rhizosphere growth-promoting bacteria agent comprises: S1, inoculating the isolated and purified rhizosphere growth-promoting bacteria strain into an activation medium for activation treatment to obtain an activated strain; S2, inoculating the activated strain into a liquid culture medium for cultivation, and then centrifuging and removing the supernatant to obtain a cultured strain; S3. Resuspend the cultured strain 2-3 times using phosphate buffer, and then centrifuge to remove the supernatant to obtain the rhizosphere growth-promoting bacteria agent.

[0028] In one embodiment of the present disclosure, in step S1, the activation culture medium includes LB liquid culture medium and / or K culture medium; in step S2, the liquid culture medium includes LB liquid culture medium and / or K culture medium.

[0029] In one embodiment of the present disclosure, the film-forming agent is selected from one or more of sodium carboxymethyl cellulose, polyvinyl alcohol, sodium alginate, gum arabic and pectin; preferably sodium carboxymethyl cellulose; the protective agent is selected from one or more of whey protein, silk fibroin, corn protein, soy protein isolate, skim milk powder, trehalose and betaine; preferably whey protein; the regulator is selected from one or more of nano iron oxide, nano silicon dioxide, nano zinc dioxide and nano titanium dioxide; the active agent is selected from one or more of apigenin, luteolin, quercetin and epigallocatechin; the filler is selected from one or more of bentonite, diatomaceous earth and perlite.

[0030] In the present disclosure, the film-forming agent can be sodium carboxymethyl cellulose, which has good film-forming properties and can effectively improve the survival rate of rhizosphere growth-promoting bacteria on the seed surface when combined with whey protein; adding regulators and activators on this basis can further improve the germination rate and seedling growth of plant seeds under saline-alkali stress conditions.

[0031] The second aspect of the present disclosure provides the use of the seed coating agent described in the first aspect in improving the salt-alkali tolerance of plant seeds and plants and in promoting plant growth.

[0032] In one embodiment of the present disclosure, the plant is selected from at least one of corn, wheat, cucumber, rapeseed and tomato; preferably corn.

[0033] The present disclosure is further described in detail by way of examples. The raw materials used in the examples can all be obtained through commercial sources.

[0034] The LB solid culture medium used in the present disclosure includes: 10 g tryptone, 5 g yeast extract and 10 g sodium chloride.

[0035] The K culture medium used in the present disclosure includes: 0.4 g potassium dihydrogen phosphate, 0.1 g potassium hydrogen phosphate, 0.01 g manganese sulfate, 0.01 g magnesium sulfate, 0.01 g sodium molybdate monohydrate, 0.1 g sodium chloride, and 0.01 g ferric sulfate monohydrate.

[0036] Example 1 (1) Add sodium carboxymethyl cellulose to water, heat and stir with a magnetic stirrer until fully dissolved, and prepare a 5 (w / v)% sodium carboxymethyl cellulose aqueous solution; prepare a 15 (w / v)% whey protein solution, a 25 (w / v)% bentonite suspension, a 0.0025 (w / v)% nano-iron oxide solution, and a 0.005 (w / v)% apigenin solution; (2) Adding whey protein solution, bentonite suspension, nano iron oxide solution and apigenin solution to sodium carboxymethyl cellulose aqueous solution in sequence, wherein the weight ratio of sodium carboxymethyl cellulose, whey protein nano iron oxide and apigenin is 1:3:0.0005:0.001; stirring with a magnetic stirrer at room temperature until fully dissolved to prepare an auxiliary agent; based on the total weight of the auxiliary agent, the content of sodium carboxymethyl cellulose is 1 weight%, the content of whey protein is 3 weight%, the content of bentonite is 20 weight%, the content of nano iron oxide is 0.0005 weight% and the content of apigenin is 0.001 weight%; (3) The isolated and purified Pseudomonas stutzeri A1501 strain was inoculated into an activation medium for activation treatment to obtain an activated strain; the activated strain was inoculated into a liquid culture medium for culture (30°C, 24h), and then centrifuged (5000rpm, 10min), and the supernatant was discarded to obtain a cultured strain; the cultured strain was resuspended in a phosphate buffer for the cultured strain, and the supernatant was discarded after centrifugation at 5000rpm for 10min, and the resuspension step was repeated twice to obtain a Pseudomonas stutzeri A1501 bacterial agent; (4) The Pseudomonas stutzeri A1501 strain was resuspended in a volume ratio of 1:1 with the auxiliary agent, and the supernatant was removed after centrifugation at 5000 rpm for 10 min to obtain the seed coating agent S-1. The number of viable bacteria of Pseudomonas stutzeri A1501 in the seed coating agent S-1 was 2.0×10 11 CFU / mL.

[0037] Example 2 (1) Pectin was added into water, heated and stirred with a magnetic stirrer until fully dissolved, and a 5 (w / v)% pectin aqueous solution was prepared; a 15 (w / v)% silk fibroin solution, a 45 (w / v)% diatomaceous earth suspension, a 0.0025 (w / v)% nano-silica solution, and a 0.005 (w / v)% luteolin solution were prepared; (2) Adding whey protein solution, bentonite suspension, nano iron oxide solution and apigenin solution to pectin aqueous solution, wherein the weight ratio of pectin, silk fibroin, nano silicon dioxide and luteolin is 1:3:0.0005:0.001; stirring with a magnetic stirrer at room temperature until fully dissolved to prepare an auxiliary agent; based on the total weight of the auxiliary agent, the content of pectin is 1 weight%, the content of silk fibroin is 3 weight%, the content of diatomaceous earth is 15 weight%, the content of nano silicon dioxide is 0.0005 weight% and the content of luteolin is 0.001 weight%; (3) The isolated and purified Pseudomonas stutzeri A1501 strain is inoculated into an activation medium for activation treatment to obtain an activated strain; the activated strain is inoculated into a liquid culture medium for culture (30°C, 24h), and then centrifuged (5000rpm, 10min), and the supernatant is discarded to obtain a cultured strain; the cultured strain is resuspended in a phosphate buffer for the cultured strain, and the supernatant is discarded after centrifugation at 5000rpm for 10min, and the resuspension step is repeated twice to obtain a Pseudomonas stutzeri A1501 bacterial agent; (4) The Pseudomonas stutzeri A1501 strain was resuspended in a volume ratio of 1:1 with the auxiliary agent, and the supernatant was removed after centrifugation at 5000 rpm for 10 min to obtain the seed coating agent S-2. The number of viable bacteria of Pseudomonas stutzeri A1501 in the seed coating agent S-2 was 2.0×10 11 CFU / mL.

[0038] Example 3 (1) Add pectin to water, heat and stir with a magnetic stirrer until fully dissolved, and prepare a 5 (w / v)% sodium alginate aqueous solution; prepare a 25 (w / v)% corn protein solution, a 125 (w / v)% perlite suspension, a 0.005 (w / v)% nano zinc dioxide solution, and a 0.005 (w / v)% quercetin solution; (2) Adding whey protein solution, bentonite suspension, nano iron oxide solution and apigenin solution to pectin aqueous solution, wherein the weight ratio of sodium alginate, zein, nano zinc dioxide and quercetin is 1:5:0.001:0.001; stirring with a magnetic stirrer at room temperature until fully dissolved to prepare an auxiliary agent; based on the total weight of the auxiliary agent, the content of sodium alginate is 1 weight%, the content of zein is 5 weight, the content of perlite is 25 weight, the content of nano zinc dioxide is 0.0001 weight, and the content of quercetin is 0.001 weight; (3) The isolated and purified Pseudomonas stutzeri A1501 strain is inoculated into an activation medium for activation treatment to obtain an activated strain; the activated strain is inoculated into a liquid culture medium for culture (30°C, 24h), and then centrifuged (5000rpm, 10min), and the supernatant is discarded to obtain a cultured strain; the cultured strain is resuspended in a phosphate buffer for the cultured strain, and the supernatant is discarded after centrifugation at 5000rpm for 10min, and the resuspension step is repeated twice to obtain a Pseudomonas stutzeri A1501 bacterial agent; (4) The Pseudomonas stutzeri A1501 strain was resuspended in a volume ratio of 1:1 between the Pseudomonas stutzeri A1501 agent and the auxiliary agent. The supernatant was removed after centrifugation at 5000 rpm for 10 min to obtain the seed coating agent S-3. The number of viable bacteria of Pseudomonas stutzeri A1501 in the seed coating agent S-3 was 2.0×10 11 CFU / mL.

[0039] Example 4 The seed coating agent was prepared by the same method as in Example 1, except that the weight ratio of sodium carboxymethyl cellulose, whey protein, nano-iron oxide and apigenin was 1:9:0.001:0.005; based on the total weight of the auxiliary agent, the content of sodium carboxymethyl cellulose was 1% by weight, the content of whey protein was 9% by weight, the content of nano-iron oxide was 0.001% by weight, the content of apigenin was 0.001% by weight, and the content of bentonite was 20% by weight.

[0040] The seed coating agent S-4 was obtained. In the seed coating agent S-4, the number of viable bacteria of Pseudomonas stutzeri A1501 was 2.0×10 11 CFU / mL.

[0041] Comparative Example 1 The seed coating agent was prepared by the same method as in Example 1, except that sodium carboxymethyl cellulose was not added, the weight ratio of whey protein, nano iron oxide and apigenin was 3:0.0005:0.001; based on the total weight of the adjuvant, the content of whey protein was 3% by weight, the content of bentonite was 20% by weight, the content of nano iron oxide was 0.0005% by weight, and the content of apigenin was 0.001% by weight. The seed coating agent D-1 was obtained, and the number of viable bacteria of Pseudomonas stutzeri A1501 in the seed coating agent D-1 was 2×10 11 CFU / mL.

[0042] Comparative Example 2 The seed coating agent was prepared by the same method as in Example 1, except that whey protein was not added, the weight ratio of sodium carboxymethyl cellulose, nano iron oxide and apigenin was 1:0.0005:0.001; based on the total weight of the adjuvant, the content of sodium carboxymethyl cellulose was 1% by weight, the content of bentonite was 20% by weight, the content of nano iron oxide was 0.0005% by weight, and the content of apigenin was 0.001% by weight. Seed coating agent D-2 was obtained, and the number of viable bacteria of Pseudomonas stutzeri A1501 in seed coating agent D-2 was 2.0×10 11 CFU / mL.

[0043] Test Example 1 The seed coating agent obtained in Examples 1-4 and the seed coating agent obtained in Comparative Examples 1-2 were subjected to the following tests: (1) Film-forming properties The microbial seed coating agent was cast on a flat plate of 15cm×15cm to form a film with a thickness of 0.05mm, and then placed in an oven at 35±1℃ for drying. The film-forming property was examined in three levels: uniform film formation and the film that could be completely scraped off the glass plate indicated good film-forming property, which was marked as level Ⅰ; uniform film formation, but the film could not be completely scraped off, indicated medium film-forming property, which was marked as level Ⅱ; and the film that could not be uniformly formed indicated poor film-forming property, which was marked as level Ⅲ. Levels Ⅰ and Ⅱ were qualified, and level Ⅲ was unqualified.

[0044] (2) Film formation time After the microbial seed coating agent is cast into a film on a dry glass plate, the glass plate with the film attached is placed in a 35ºC oven until dry, and the time taken for the film to be completely dry is recorded.

[0045] (3) Determination of coating shedding rate Immerse 50g corn seeds in the microbial seed coating agent and take them out after 2 hours. After the coating film is dry, weigh two portions of 10g of coated seeds and place them in conical flasks respectively. Add 100mL of ethanol solution to one portion and place it in a CNC ultrasonic cleaner for 10 minutes to fully dissolve the coating film on the surface of the seeds. After taking it out, let it stand for 10 minutes, take 10mL of the upper clear liquid in a 50mL volumetric flask, make up to volume with ethanol, shake well, and mark it as solution A; the other portion of coated seeds is placed in an oscillator first, shake for 10 minutes, and then obtain solution B according to the treatment method of A. The absorbance of A and B is measured at a wavelength of 550nm, with ethanol as the reference (550nm is the absorption wavelength when rhodamine B is used as the dye). Calculate according to the following formula: Coating shedding rate (%) = (A0 / m0-A1 / m1) / A0m0×100= (A0m1-A1m0) / A0m1×100; Where: m0 is the mass of coated seeds weighed to prepare solution A; m1 is the mass of coated seeds weighed to prepare solution B; A0 is the absorbance of solution A; A1 is the absorbance of solution B; (4) Determination of coating uniformity Randomly select 20 coated seeds for film-forming property determination and place them in 20 10mL centrifuge tubes with caps. Use a pipette to accurately add 2mL of ethanol to each centrifuge tube, cover it, shake and extract for 15min, let it stand and centrifuge to obtain a clear red liquid. Use ethanol as a reference and determine its absorbance A at a wavelength of 550nm. (550nm is the detection wavelength when rhodamine B is used as the dye. If other components are used as dyes, it can be selected according to its components).

[0046] Arrange the 20 measured absorbance data from small to large, and calculate the average absorbance value A a The coating uniformity of the sample (%) is calculated as follows: Coating uniformity (%) = n / 20 × 100 = 5n; Where: n is the measured absorbance A in the range of 0.7~1.3A a Number of coated seeds within the range; 20 is the number of coated seeds tested.

[0047] (5) Suspension rate Take two seed samples A and B of the same mass, 5.0g each, and place them in a 200mL beaker. Add 50mL of distilled water to each and shake by hand at a speed of 100r / min for about 2min. Transfer the suspension to a 250mL ground-mouth quartz tube with a stopper, and use 100mL of distilled water to wash all the remaining contents in the beaker into the quartz tube, and dilute it to the scale with distilled water, cover the stopper, and use the bottom of the quartz tube as the axis to turn the quartz tube upside down 30 times within 1min (inversion and reset is counted as 1 time, each time is about 2s). Take the A sample solution and immediately use a pipette to quickly transfer 225mL of the suspension of the contents, making sure that the opening of the pipette is always a few millimeters below the liquid surface of the contents. Transfer the remaining 25mL of the residue to a 100mL dried beaker, remove water to about 2mL in a constant temperature water bath, add 1mL of ethanol, continue to remove water in a water bath until constant weight, weigh, and obtain the residue mass m a Place the quartz tube of sample B vertically in a constant temperature water bath without vibration, open the glass stopper, leave it for 30 minutes, and repeat the treatment of A to obtain the residue mass m b The calculation is as follows: Suspension rate (%) = [(10m a -m b )×10 / 10m a ×9]×100=(10 m a -m b ) / m a ×11.1; Where: m a is the mass of the residue left in quartz tube A (g); m b is the mass (g) of the residue remaining in quartz tube B.

[0048] (6) Air permeability measurement method Take 2g of microbial seed coating agent. Use a brush to evenly apply it on one surface of a circular filter paper (9cm in diameter). After drying, cover the filter paper on the mouth of a beaker containing quantitative distilled water and seal it on all sides. Place the beaker in a warm water bath at 80℃. After 3 hours, take out the beaker, measure the mass of the remaining water in the beaker, and calculate the evaporation amount. Set up 3 replicates for each treatment and take the average value.

[0049] (7) Water permeability measurement method Take 2g of microbial seed coating agent and apply it evenly on one surface of a circular filter paper (9cm in diameter) with a brush. After drying, cover the filter paper on a clean petri dish cover that has been weighed in advance (the side coated with the film-forming agent faces downward). Use a pipette to draw 10mL of distilled water and drop it on the surface of the filter paper. After 3 hours, weigh the weight of the petri dish cover and calculate the weight of the water that seeps into the filter paper. Repeat each treatment 3 times and take the average value.

[0050] (8) Swelling determination method The microbial seed coating agent was cast into a film on a clean glass slide (2×8 cm), weighed on an electronic balance after drying, and then immersed in distilled water. After 6 hours, it was weighed again and the swelling rate was calculated. Each treatment was repeated 3 times and the average value was taken.

[0051] Swelling rate (%) = (WW o ) / W o ×100; Note: W represents the wet weight of the membrane; W o It represents the dry weight of the membrane.

[0052] (9) Water solubility determination method The microbial seed coating agent was cast into a film on a clean glass slide (2×8 cm), dried and immersed in distilled water, and the film dissolution area on the slide was calculated after 18 hours. Each treatment was repeated 3 times and the average value was taken.

[0053] (10) Water absorption determination method The prepared membrane was dried in an oven to constant weight (about 4 h), and a 4 cm long and 1.7 cm wide dry membrane was taken out with a blade, and its mass was weighed in a Petri dish. Then, it was immersed in deionized water, and taken out after 6 hours. The solution on the membrane surface was gently wiped off with absorbent paper, and the membrane was placed in a Petri dish and weighed. B(%)=(W2-W0) / (W1-W0)×100; Where: B = membrane water absorption rate; W0 = culture dish mass; W1 = first weighing mass; W2 = second weighing mass.

[0054] (11) Determination of viscosity After the sample to be tested is fully shaken, it is placed in a constant temperature water bath. When the temperature of the sample to be tested reaches 25°C, about 400 mL of the sample to be tested is placed in a beaker and placed in a constant temperature water bath at 25°C for 1 hour. After the viscometer is debugged normally, the rotor is installed, and the rotor speed is adjusted to (30±1) r / min, the rotor is slowly inserted into the sample so that the liquid level just submerges the groove on the rotor, the engine is started, and the viscosity value (mPa·s) is immediately read after 1 minute.

[0055] (12) Sustained release determination method The seeds were weighed in advance (W i The samples were placed in tea bags with a pore size of 100 mm and buried in soil with a humidity of 25%. The soil moisture was tested with a hydrometer and deionized water was added every 5 days to maintain a moisture content of 25%. The samples were extracted on the 28th day, quickly washed in a PBS solution, and then air-dried at room temperature for 24 h before their weight (W) was measured. d ).

[0056] Sustained release (%) = (W i -Wd ) / W i ×100; Among them, W i is the initial dry weight of the sample, W d It is the dry weight of the sample after a period of time in the soil.

[0057] The experimental results are shown in Table 1: Table 1

[0058] It can be seen from the above table that the microbial seed coating agent disclosed in the present invention has higher swelling and water absorption, which can provide a more suitable microenvironment for the survival of rhizosphere growth-promoting bacteria; it also has better sustained release, which can allow the plant rhizosphere growth-promoting bacteria to be slowly released into the root system.

[0059] Test Example 2 Determination of the survival rate of rhizosphere growth-promoting bacteria in microbial seed coating agents: (1) Place the microbial seed coating agent in an incubator at 28°C; (2) Take 100 μL of the mixture on day 0, 2, 5, 10, 20, and 30 and dilute it to 10 with PBS solution. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 100 μL of each was spread on the culture medium plate, with 3 parallels in each group; (3) Cultivate in a 30°C incubator, count the number of colonies on the plate after 24 hours, and calculate the number of rhizosphere growth-promoting bacteria in the seed coating agent. The experimental results are shown in Table 2: Table 2

[0060] As shown in Table 2, the rhizosphere growth-promoting bacteria disclosed in the present invention can survive in the microbial seed coating agent for more than 30 days, and the number of viable bacteria can reach 10 10 The above results show that the microbial seed coating agent can well preserve the rhizosphere growth-promoting bacteria, and the shelf life is more than 30 days.

[0061] Test Example 3 Determination of the survival rate of rhizosphere growth-promoting bacteria on the surface of corn seeds under the treatment of microbial seed coating agents: (1) Select corn seeds of uniform size and full grains, rinse them three times with sterile water, soak them in 75% ethanol for 3 minutes, soak them in 5% sodium hypochlorite for 10 minutes, then rinse them five times with 1000-fold diluted mold inhibitor ppm, and finally rinse them five times with sterile water; (2) Remove the sterilized corn seeds and immerse them in the microbial seed coating agent for 2 hours; (3) Take out the corn seeds, spread them out flat and blow dry; (4) On the 0th, 10th, 20th, 30th, 60th, and 90th day, take out 10 coated corn seeds and put them into a 50-mL centrifuge tube; (5) Add 10 mL of PBS solution, shake thoroughly for 3 min, place in an incubator at 30 °C, 220 rpm for 1 h to allow it to fully dissolve, and finally shake thoroughly for 1 min; (6) Take 100 μL of the mixture and dilute it to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 100 μL of each was spread on the culture medium plate, with 3 parallels in each group; (7) Cultivate in a 30°C incubator, count the number of colonies on the plate after 24 hours, and calculate the number of rhizosphere growth-promoting bacteria on the seed surface. The experimental results are shown in Table 3: Table 3

[0062] It can be seen from Table 3 that the microbial seed coating agent disclosed in the present invention significantly increases the colony count of rhizosphere growth-promoting bacteria on the seed surface.

[0063] Test Example 4 Determination of germination rate of corn seeds treated with microbial seed coating agents under salt stress conditions: (1) Select corn seeds of uniform size and full grains, rinse them three times with sterile water, soak them in 75% ethanol for 3 minutes, soak them in 5% sodium hypochlorite for 10 minutes, then rinse them five times with 1000-fold diluted mold inhibitor ppm, and finally rinse them five times with sterile water; (2) Remove the sterilized corn seeds and immerse them in the microbial seed coating agent for 2 hours; (3) Take out the corn seeds, spread them out flat and blow dry; (4) Place sterile uncoated control, uncoated control and coated corn seeds in a Petri dish lined with two layers of filter paper and fully moisten them with 0 mM NaCl and 300 mM NaCl, respectively. Place 50 seeds in each Petri dish and set up 3 parallel replicates for each inoculation treatment. (6) Place in a 30°C climate chamber incubated in the dark with relative humidity of 50%, 25°C, 16 h of light and 8 h of darkness; (7) The germination rate was calculated on the 7th day.

[0064] Germination rate = number of germinations / number of seeds sown × 100%.

[0065] The experimental results are shown in Table 4: Table 4

[0066] It can be seen from Table 4 that the microbial seed coating agent promoted seed germination under the salt stress conditions of 0 mM NaCl and 300 mM NaCl, among which the seed coating agent prepared in Example 1 had the best effect on promoting corn seed germination under salt stress.

[0067] Test Example 5 Effects of microbial seed coating agents on root colonization ability of growth-promoting bacteria under salt stress conditions: (1) Select corn seeds of uniform size and full grains, rinse them three times with sterile water, soak them in 75% ethanol for 3 minutes, soak them in 5% sodium hypochlorite for 10 minutes, then rinse them five times with 1000-fold diluted mold inhibitor ppm, and finally rinse them five times with sterile water; (2) Remove the sterilized corn seeds and immerse them in the microbial seed coating agent for 2 hours; (3) Take out the corn seeds, spread them out flat and blow dry; (4) After 0 and 30 days of storage, maize seeds were placed with the embryo facing upward in pots filled with vermiculite. Three seeds were sown in each pot. Five pots were grouped as a treatment, and three parallel replicates were set for each inoculation treatment. (5) Soak the soil with 300 mM NaCl solution; (6) Place in a 30°C climate chamber incubated in the dark with relative humidity of 50%, 25°C, 16 h of light and 8 h of darkness; (7) Add 50 mL of Hoagland medium to the flower pot every 7 days; (8) Take out the corn seedlings on the 7th, 14th, and 21st day after sowing and rinse off the vermiculite with PBS solution; (9) Cut the roots, dry them with filter paper, weigh them, and place them in 50 mL centrifuge tubes; (10) Add 10 mL of PBS solution, shake thoroughly for 3 min, place in an incubator at 30 °C, 220 rpm for 1 h to allow it to fully dissolve, and finally shake thoroughly for 1 min; (11) Take 100 μL of the mixture and dilute it to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 100 μL of each was spread on the culture medium plate, with 3 parallels in each group; (12) Cultured in a 30°C incubator, counted the number of colonies on the plate after 24 hours, and calculated the number of rhizosphere growth-promoting bacteria colonized on the root surface. The experimental results are shown in Table 5: Table 5

[0068] As can be seen from Table 5, the microbial seed coating agent disclosed in the present invention significantly improves the colonization ability of rhizosphere growth-promoting bacteria in the root system of corn seedlings, among which the rhizosphere growth-promoting bacteria in the seed coating agent prepared in Example 1 has the strongest colonization ability in the root system.

[0069] Test Example 6 Evaluation of the growth-promoting effect of microbial seed coating agents on corn under salt stress conditions: (1) Select corn seeds of uniform size and full grains, rinse them three times with sterile water, soak them in 75% ethanol for 3 minutes, soak them in 5% sodium hypochlorite for 10 minutes, then rinse them five times with 1000-fold diluted mold inhibitor ppm, and finally rinse them five times with sterile water; (2) Remove the sterilized corn seeds and immerse them in the microbial seed coating agent for 2 hours; (3) Take out the corn seeds, spread them out flat and blow dry; (4) Place the sterile uncoated control, uncoated control and coated corn seeds with the embryo facing upward in a pot filled with vermiculite. Sow three seeds in each pot. Five pots form a group of treatments. Set up three parallel replicates for each inoculation treatment. (5) Soak the soil with 300 mM NaCl solution and use the same amount of sterile water as the control; (6) Place in a 30°C climate chamber incubated in the dark with relative humidity of 50%, 25°C, 16 h of light and 8 h of darkness; (7) Every 7 days, add 50 mL of 0 mM NaCl and 300 mM NaCl Hoagland medium to the flower pots respectively; (8) On the 30th day after sowing, the corn seedlings were taken out and the chlorophyll content, plant height, root length, plant fresh weight, and plant dry weight of the corn plants were measured.

[0070] The experimental results are shown in Table 6: Table 6

[0071] It can be seen from Table 6 that under salt stress conditions, the microbial seed coating agent significantly promoted the growth of corn, among which the seed coating agent of Example 1 had the best effect on promoting the growth of corn under salt stress.

[0072] Test Example 7 Evaluation of the effect of microbial seed coating agent on corn growth in saline-alkali land: (1) Select corn seeds of uniform size and full grains, rinse them three times with sterile water, soak them in 75% ethanol for 3 minutes, soak them in 5% sodium hypochlorite for 10 minutes, then rinse them five times with 1000-fold diluted mold inhibitor ppm, and finally rinse them five times with sterile water; (2) Remove the sterilized corn seeds and immerse them in the microbial seed coating agent for 2 hours; (3) Take out the corn seeds, spread them out flat and blow dry; (4) The sterile uncoated control, coated control and uncoated corn seeds were sown in saline-alkali land. The saline-alkali land in this experiment is located in Dongying City, Shandong Province (37°08´28´´N, 118°32´16´´E); the soil properties in this area are pH 8.48, total nitrogen 0.68g / kg, total phosphorus 0.68g / kg, total potassium 19.5g / kg, organic matter 8.68g / kg, ammonium nitrogen 0.37g / kg, nitrate nitrogen 5.20g / kg; soil particle composition: 2-0.2mm accounts for 0.62%, 0.2-0.02mm accounts for 45.41%, 0.02-0.002mm accounts for 27.11%, and <0.002mm accounts for 26.86%.

[0073] (5) All treatments were set up with 3 replicates, arranged in random blocks, each plot was 5 m long and 3 m wide, and the sowing density was 75,000 plants / hm2. 2 ; (6) On the 60th day after sowing, the chlorophyll content, plant height, root length, plant fresh weight, and plant dry weight of the corn plants were measured.

[0074] The experimental results are shown in Table 7: Table 7

[0075] It can be seen from Table 7 that the microbial seed coating agent disclosed in the present invention significantly promotes the growth of corn in saline-alkali land, among which the seed coating agent of Example 1 has the best effect on promoting the growth of corn in saline-alkali land.

[0076] The preferred embodiments of the present disclosure are described in detail above; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0077] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0078] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A salt-alkali tolerant microbial seed coating agent, characterized in that: The seed coating agent includes: rhizosphere growth-promoting bacteria agent and auxiliary agent; The volume ratio of the rhizosphere growth-promoting bacteria agent to the auxiliary agent is 1: (1-100); The number of viable bacteria of the rhizosphere growth-promoting bacteria in each gram of the seed coating agent is (1-100)×10 10 CFU; the rhizosphere growth-promoting bacteria are selected from one or more of Pseudomonas stutzeri A1501, Pseudomonas fluorescens ATCC 13525, Bacillus amyloliquefaciens FZB42 and Azotobacter rotundifolia BNCC336703; Based on the total weight of the auxiliary agent, the auxiliary agent includes: 1-9 weight % of a film former, 1-9 weight % of a protective agent, 0.0001-0.001 weight % of a regulator, 0.0001-0.001 weight % of an active agent, 0-25 weight % of a filler and 58-97 weight % of water.

2. The seed coating agent according to claim 1, wherein The volume ratio of the rhizosphere growth-promoting bacteria agent to the auxiliary agent is 1: (10-100); The number of viable bacteria of the rhizosphere growth-promoting bacteria in each gram of the seed coating agent is (10-100)×10 10 CFU.

3. The seed coating agent according to claim 1, wherein Based on the total weight of the auxiliary agent, the auxiliary agent includes: 1-6 weight % of a film former, 3-6 weight % of a protective agent, 0.0005-0.001 weight % of a regulator, 0.0005-0.001 weight % of an active agent, 0-25 weight % of a filler and 63-90 weight % of water.

4. The seed coating agent according to claim 3, wherein In the auxiliary agent, the weight ratio of the film former, protective agent, regulator and active agent is (1-3): (1-5): (0.0001-0.001): (0.0001-0.001), preferably (1-2): (3-5): (0.0005-0.001): (0.0005-0.001).

5. The seed coating agent according to claim 1, wherein The number of live bacteria of the rhizosphere growth-promoting bacteria agent is (1-100)×10 12 CFU / mL, preferably (10-100)×10 12 CFU / mL.

6. The seed coating agent according to claim 5, wherein The preparation method of the rhizosphere growth-promoting bacteria agent comprises: S1, inoculating the isolated and purified rhizosphere growth-promoting bacteria strain into an activation medium for activation treatment to obtain an activated strain; S2, inoculating the activated strain into a liquid culture medium for cultivation, and then centrifuging and removing the supernatant to obtain a cultured strain; S3. Resuspend the cultured strain 2-3 times using phosphate buffer, and then centrifuge to remove the supernatant to obtain the rhizosphere growth-promoting bacteria agent.

7. The seed coating agent according to claim 6, wherein In step S1, the activation medium includes LB liquid medium and / or K medium; In step S2, the liquid culture medium includes LB liquid culture medium and / or K culture medium.

8. The seed coating agent according to claim 4, wherein The film-forming agent is selected from one or more of sodium carboxymethyl cellulose, polyvinyl alcohol, sodium alginate, gum arabic and pectin; The protective agent is selected from one or more of whey protein, silk fibroin, corn protein, soy protein isolate, skim milk powder, trehalose and betaine; The regulator is selected from one or more of nano iron oxide, nano silicon dioxide, nano zinc dioxide and nano titanium dioxide; The active agent is selected from one or more of apigenin, luteolin, quercetin and epigallocatechin; The filler is selected from one or more of bentonite, diatomaceous earth and perlite.

9. Use of the seed coating agent according to any one of claims 1 to 8 in improving the salt-alkali tolerance of plant seeds and plants and in promoting plant growth.

10. The use according to claim 9, wherein: The plant is selected from at least one of corn, wheat, cucumber, rapeseed and tomato; preferably corn.

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