Bacillus subtilis strain and application thereof

Abstract

The invention discloses a bacillus subtilis strain and application thereof, the bacillus subtilis strain is bacillus subtilis X-NY1 and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20242038. Bacillus subtilis X-NY1, bacillus velezensis and bacillus amyloliquefaciens are compounded into a microbial agent, a film-forming agent, a dispersing agent, a coloring agent, a stabilizing agent, a preservative, a conditioning agent and a nutritional agent are further added to prepare the compound microbial seed coating agent, the effective viable count of the microbial agent is 108-1011, and the quality guarantee period is longer than 6 months. The compound microbial seed coating agent can improve the plant height, root length, fresh weight and the like in the growth process of pennisetum alopecuroides, can effectively improve the stress resistance of the pennisetum alopecuroides under the salinization condition, and is small in addition amount, low in cost, safe, reliable, obvious in growth promoting effect and suitable for application and popularization.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Bacillus subtilis strain and application thereof. Background Art

[0002] Soil salinization is a global ecological problem. When land is affected by salinization, it will lead to land degradation and a decline in agricultural productivity. Soil salinization not only harms the growth and development of crops, but also deteriorates the physical properties of the soil, increases the mineralization of groundwater, makes the water bitter, and makes groundwater sources unable to be fully utilized, which in turn leads to large tracts of land being barren, a reduction in arable land, and the lack of coordination between water and fertilizer in the soil, which affects crop yields. For example, soil compaction and decreased fertility are not conducive to the absorption of nutrients by crops and hinder crop growth. As an important reserve land resource in my country, salinized soil has great development potential and high availability. With the continuous increase in population and the continuous reduction in cultivated area in my country, it is of great significance to strengthen the improvement of saline soil, effectively improve the quality of arable land, and increase crop yields.

[0003] Planting salt-tolerant plants in saline-alkali land is a common method for managing saline-alkali land. Salt-tolerant plants can act as biological pumps to absorb salt in the soil and reduce the evaporation of water on the soil surface. Their underground roots can improve the physical structure of the soil, and the root secretions can provide soil fertility. In addition, salt-tolerant crops such as Pennisetum are selected for planting. They have a well-developed root system, strong stems, excellent regeneration, large biomass, and high yield. They are currently widely used in animal husbandry as high-quality forage resources, which can improve the soil and generate certain economic benefits. Therefore, the current research direction of the academic community is to develop saline-alkali land into a cultivation site for some salt-tolerant plants, in order to achieve the transformation and diversified utilization of coastal tidal flat saline-alkali land. However, high concentrations of salt environment and pH value will inhibit the absorption of nutrients such as potassium, phosphorus, and iron by salt-tolerant plants, resulting in "malnutrition" of plants and endangering their normal growth and fruiting.

[0004] Current research shows that salt-tolerant growth-promoting microorganisms can promote the growth of salt-tolerant plants. They can mutually coexist with plants by secreting some substances. At the same time, they can also stimulate plants to produce plant hormones, produce immune responses, and produce a large amount of soil organic matter, thereby improving plant resistance and promoting plant growth. They can also improve the ability of plants to withstand biological stress. Compared with the application of traditional fertilizers and pesticides, inoculating salt-tolerant growth-promoting bacteria into saline-alkali land to promote plant growth has a better sustainable effect and is harmless and non-toxic to the soil environment and crop products. Adding salt-tolerant growth-promoting bacteria to solve soil problems from the root, increase organic matter content, and improve the soil environment is a very promising development direction. However, the current method of adding salt-tolerant growth-promoting microorganisms is mostly root irrigation or granular bacterial fertilizers. The transportation cost of such an operation process is high, which to a certain extent limits the application of salt-tolerant growth-promoting bacteria.

[0005] Seed coating agent is a kind of artificial chemical preparation used to treat seeds. It is usually made of pesticide original medicine, micro-fertilizer, growth regulator and supporting auxiliary agents such as film-forming agent, colorant and dispersant in a specific proportion. It can be wrapped on the surface of seeds directly or after dilution, which plays a protective role on seed growth and forms a permeable and tough film that firmly adheres to the surface of seeds, thereby improving the growth environment of plants, enhancing plant resistance, and increasing crop yield and quality. It can not only provide the nutrients needed for seeds to germinate, but also protect crops from biological and non-biological stresses, thereby increasing yield and quality. The use of seed coating agents can reduce agricultural production costs, reduce the phenomenon of plant seed rot, increase seedling rate, and avoid the waste caused by excessive sowing to ensure seedling rate. However, there are several major problems in the research on seed coating agents in my country: (1) The average film-forming time is long, the coating coverage is low, the seed coating is easy to fall off, and the coating film is highly soluble in water and unstable, which can easily cause the loss of active ingredients; (2) The product effect is single. Most seed coating agents on the market are for killing insects, and there are relatively few types of seed coating agents that can improve plant resistance. In addition, most coatings are chemical, while biological types are mostly for disease prevention. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a Bacillus subtilis strain and an application thereof. The strain is made into a composite microbial seed coating agent, which can improve the plant height, root length, vitality index and germination rate of Pennisetum during its growth. The added amount is small, the cost is low, the effect is obvious, the action time is long, the degree of harmlessness is high, and the strain is suitable for popularization and application.

[0007] The present invention is achieved through the following technical solutions:

[0008] A Bacillus subtilis strain is Bacillus subtilis X-NY1, which has been deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242038.

[0009] Preferably, the nucleotide sequence of 16S rDNA of the strain is as shown in SEQ ID NO.1.

[0010] Application of the above-mentioned Bacillus subtilis strain in the preparation of seed coating agents.

[0011] A composite microbial seed coating agent comprises the following components by mass percentage: 0.3% to 5% of a film former, 1% to 5% of a dispersant, 0.2% to 2% of a colorant, 0.1% to 5% of a stabilizer, 0.1% to 5% of a preservative, 0.1% to 5% of a regulator, 0.04% to 2% of a nutrient, and the balance is supplemented by microbial inoculant fermentation liquid; wherein the microbial inoculant fermentation liquid comprises the above-mentioned Bacillus subtilis strain, as well as Bacillus velezii and Bacillus amyloliquefaciens.

[0012] Preferably, the volume ratio of the Bacillus subtilis strain, Bacillus Velez and Bacillus amyloliquefaciens is 2-5:1:1.

[0013] Preferably, the film-forming agent includes sodium alginate and polyvinyl alcohol, and the mass ratio of the two is 1:0.2-0.8; the regulator includes salicylic acid and polyaspartic acid, and the mass ratio of the two is 1:1-3; the nutrient includes urea and dipotassium hydrogen phosphate, and the mass ratio of the two is 1:0.2-0.6.

[0014] Preferably, the dispersant is sodium lignin sulfonate; the colorant is acid fuchsin; the stabilizer is calcium carbonate; and the preservative is sodium benzoate.

[0015] The preparation method of the composite microbial seed coating agent comprises the following steps:

[0016] Step 1) Inoculate Bacillus subtilis, Bacillus Velezii and Bacillus amyloliquefaciens into LB liquid culture medium respectively, shake and culture at 30-37°C for 12-24h to obtain seed solution of each strain; resuspend by centrifugation and adjust OD 600 =1.0 of bacterial solution, and then mixed according to the proportion to obtain a composite liquid microbial agent;

[0017] Step 2) weighing the raw materials in proportion, adding the dispersant, colorant, stabilizer, preservative, regulator, and nutrient into the composite liquid microbial agent prepared in step 1), and mixing and stirring thoroughly;

[0018] Step 3) Finally, add the film-forming agent, heat and stir in a water bath at 30° C. until the solution is dissolved and becomes a viscous liquid, and mix and stir thoroughly to obtain the product.

[0019] The above-mentioned composite microbial seed coating agent, or the composite microbial seed coating agent prepared by the above-mentioned preparation method, is used in promoting the growth of Pennisetum under salinization conditions.

[0020] Preferably, the composite microbial seed coating agent and Pennisetum seeds are weighed at a volume / mass ratio of 50 mL:1 kg, poured into a coating machine, and fully stirred for coating until the seed surface is uniform in color.

[0021] The beneficial effects of the present invention are as follows:

[0022] (1) The present invention adopts microbial agents to replace the traditional coating active ingredients mainly composed of pesticides. The main functions of Bacillus subtilis X-NY1 are salt tolerance, IAA production, extracellular polysaccharide, ACC deaminase and other growth-promoting abilities. The main function of Bacillus Velezii is phosphorus and potassium solubilization. The main function of Bacillus amyloliquefaciens is nitrogen fixation. The three strains act synergistically and can effectively promote the growth of Pennisetum seeds under salt stress environment.

[0023] (2) The film-forming agent added to the composite microbial seed coating agent of the present invention has a short film-forming time, moderate viscosity, high coating uniformity, and low coating shedding rate. Dispersants, stabilizers and preservatives make its properties stable, and the bacterial activity remains ≥2×10 within six months. 8 . Adding regulators and nutrients increases its salt resistance and promotes seed germination and plant growth. The composite microbial seed coating agent has a good growth-promoting effect on the growth of Pennisetum in saline-alkali land.

[0024] (3) The composite microbial seed coating agent of the present invention uses biological agents as active ingredients, contains microorganisms that are beneficial to crops, does not contain fungicides, is environmentally friendly, has a simple preparation method, is easy to use, safe and reliable, and has good application potential and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The colony morphology of Bacillus subtilis X-NY1 on the plate in Example 1;

[0026] Figure 2 is the phylogenetic tree of Bacillus subtilis X-NY1 in Example 1;

[0027] Figure 3 The germination effect of Pennisetum seeds after being coated with the composite microbial seed coating agent in Example 4;

[0028] Figure 4 This is the growth effect of Pennisetum after coating with the composite microbial seed coating agent in Example 5. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] Example 1 Screening of Bacillus subtilis X-NY1

[0033] 1. Sample source

[0034] In 2021, soil was collected from Dongtaijinaier Salt Lake in Qinghai Province (located in Golmud City, Haixi Mongolian and Tibetan Autonomous Prefecture), placed in sterile bags and stored at 4°C.

[0035] 2. Strain screening

[0036] Grind the soil sample into fine powder and bag it for later use. Weigh 1g of soil sample into a 50mL centrifuge tube, add 9mL of sterile water, shake on a shaker at 180rpm for 30min, take 100μL of the shaking liquid into a centrifuge tube containing 10mL LB liquid culture medium, and enrich and culture it at 37℃ and 180rpm for 24h. -3 , 10 -4 , 10 -5 , 10 -6 Gradient dilution, use a pipette to draw 100 μL into a beef extract peptone medium plate containing 50 g / L sodium chloride, evenly spread and mark, and culture at 37°C for 72 hours. Observe the colonies on the plate, pick out single colonies with different appearances on the plate, and perform colony purification and streaking until a pure single colony is obtained. Inoculate the selected single colony into LB liquid culture medium and wait for its OD 600 When the value is between 0.6 and 0.8, the bacteria are preserved with glycerol and temporarily stored in a -20°C refrigerator.

[0037] LB medium: peptone 10 g, yeast powder 5 g, NaCl 10 g, agar powder 20 g, water 1000 mL.

[0038] Beef extract peptone culture medium: peptone 1.0g, beef extract 0.3g, NaCl 0.5g, pH = 7.0 ~ 7.2.

[0039] 3. Observation of colony morphology of X-NY1 strain

[0040] The selected X-NY1 strain was cultured on LB solid medium at 37°C for 3 days and then Gram-stained. The vegetative cells were observed to be single cells and spherical under a microscope. After culturing in the above medium at 37°C for 12 hours, the bacteria could grow in large quantities, such as Figure 1 As shown, the colonies are round, dirty white or slightly yellow, with a rough, opaque and wrinkled surface.

[0041] 4. Physiological and biochemical characteristics

[0042] (1) Culture temperature: 20-50°C;

[0043] (2) Growth in the pH range of 4 to 10;

[0044] (3) Salt tolerance 80 g / L growth;

[0045] (4) Gram stain: positive;

[0046] (5) Catalase: positive;

[0047] (6) VP reaction: positive;

[0048] (7) Citrate utilization: positive;

[0049] (8) Glucose reaction: positive;

[0050] (9) Nitrate reduction reaction: positive;

[0051] (10) Starch hydrolysis reaction: positive;

[0052] (11) Methyl red reaction: negative.

[0053] 5. Bacterial species identification (16S rDNA sequence analysis)

[0054] (1) PCR amplification using bacterial 16S rDNA universal primers as follows:

[0055] 27F (SEQ ID NO.2): 5'-AGAGTTTGATCMTGGCTCAG-3';

[0056] 1492R (SEQ ID NO. 3): 5'-GGTTACCTTGTTACGACTT-3'.

[0057] (2) PCR reaction system (20 μL): Taq DNA polymerase (10 μL), deionized water (8.5 μL), primer 27F (0.5 μL), primer 1492R (0.5 μL), genomic DNA (0.5 μL).

[0058] Reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 60 s, extension at 72°C for 30 s, for a total of 30 cycles, and full extension at 72°C for 7 min.

[0059] (3) Identification results

[0060] Most of the 16S rDNA sequence was measured, as shown in SEQ ID NO. 1. The measured sequence was compared with BLAST on the NCBI website to construct a phylogenetic tree based on the complete 16S rDNA sequence, as shown in Figure 2 As shown, the bacterium X-NY1 was identified as Bacillus subtilis. The strain has been deposited in the China Center for Type Culture Collection (CCTCC), No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and is classified as Bacillus subtilis X-NY1, with a deposit number of CCTCC NO: M 20242038 and a deposit date of September 23, 2024.

[0061] Example 2 Growth-promoting properties of Bacillus subtilis X-NY1

[0062] The Bacillus subtilis X-NY1 screened in Example 1 was subjected to the following experiment:

[0063] 1. Determination of salt tolerance of strains

[0064] The strains were inoculated into LB liquid medium containing 60 g / L, 70 g / L, 80 g / L, 90 g / L, and 100 g / L NaCl, respectively, and cultured in a shaker at 37 °C and 180 rpm for 24 h. The OD 600 The salt tolerance of strain X-NY1 was measured to be as high as 80g / L.

[0065] 2. Determination of the ability of strains to produce indoleacetic acid (IAA)

[0066] After the strain was activated, it was inoculated into LB liquid medium containing L-tryptophan (100 mg / L) at 1% and cultured at 37°C and 180 rpm for 3 days. The bacterial solution was centrifuged at 8000 rpm for 10 min, and 2 mL of the bacterial supernatant was taken. An equal volume was mixed with the Salkowski colorimetric reagent, and the reaction was carried out in the dark for 30 min. The OD was measured. 530 The IAA production of strain X-NY1 was measured to be 16.72 mg / L.

[0067] 3. Determination of the ability of strains to produce extracellular polysaccharides (EPS)

[0068] Centrifuge the LB culture medium inoculated with bacterial liquid at 8000rpm for 10min, and collect the supernatant. Take 5mL of the supernatant and add 3 times the volume of 95% ethanol, let it stand overnight in a refrigerator at 4℃, centrifuge at 8000rpm for 10min, and collect the precipitate; dissolve the precipitate with 10mL of distilled water, add 3 times the volume of 95% ethanol again, let it stand overnight in a refrigerator at 4℃, centrifuge at 8000rpm for 10min, remove the supernatant, dissolve the precipitate and make the volume to 100mL, and use the sulfuric acid-phenol method to determine the OD of EPS 490 The polysaccharide content was calculated based on the standard curve of glucose. The extracellular polysaccharide production capacity of the X-NY1 strain was measured to be 90.72 mg / L.

[0069] 4. Determination of the phosphate solubilization ability of strains

[0070] The strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of inorganic phosphorus (tricalcium phosphate) bacterial liquid culture medium, with an inoculation volume of 2%. No bacteria were used as a control, and three replicates were set for each group. The culture was cultured at 37°C and 180 rpm for 5 days. The culture solution was centrifuged at 8000 rpm for 20 min. The supernatant was taken and the OD was determined by molybdenum blue colorimetry. 700 The soluble phosphorus content was calculated according to the phosphorus standard curve. The ability of X-NY1 strain to decompose inorganic phosphorus was measured to be 22.77 mg / L.

[0071] 5. Determination of potassium solubilization ability of strains

[0072] The strain was inoculated into potassium-dissolving medium and cultured in a shaker at 37°C and 180 rpm for 7 days. The medium without bacteria was used as the control. Three replicates were set for each group. The culture was centrifuged at 8000 rpm for 20 min. The supernatant was appropriately diluted and the K of the supernatant was determined by atomic absorption spectrometry. + The potassium concentration of X-NY1 strain was measured to be 11.22 mg / L.

[0073] 6. Determination of nitrogen fixation ability of strains

[0074] The strain was inoculated into nitrogen-fixing medium and cultured in a shaker at 37°C and 180 rpm for 7 days. The medium without bacteria was used as the control. Three replicates were set for each group. The supernatant was appropriately diluted and the total nitrogen content of the supernatant was determined by a semi-automatic Kjeldahl nitrogen analyzer. The nitrogen fixation capacity of the X-NY1 strain was measured to be 8.17 mg / L.

[0075] 7. Determination of ACC deaminase activity produced by strains

[0076] The strain was inoculated into a centrifuge tube containing 20 mL of TSB liquid medium, cultured at 28°C and 200 rpm for 12 h, centrifuged at 4°C and 8000 rpm for 10 min, and then the obtained bacteria were resuspended in 7.5 mL of DF medium without nitrogen source, and then 0.22 μL of 0.5 mol / L 1-aminocyclopropane-1-carboxylic acid solution was added after sterilization by filtration, cultured at 28°C and 200 rpm for 24 h, centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was removed, and the bacterial precipitate was collected. The bacterial precipitate was resuspended in 5 mL of 0.5 mol / LTris-HCl buffer (pH=7.6), centrifuged at 4°C and 8000 rpm for 10 min to collect the precipitate, and this step was repeated 3 times to completely remove the DF medium. Add 1mL 0.1mol / L Tris-HCl buffer (pH=7.6) to the bacteria, centrifuge at 12000rpm for 10min, then resuspend the bacteria in 600μL 0.1mol / L Tris-HCl buffer (pH=8.5), add 30μL toluene, and oscillate on a vortex shaker for 0.5min to break the bacteria. Take 200μL of the broken bacterial liquid, add 20μL 0.5mol / L ACC solution, mix well and place in a water bath at 37℃ for 15min; then add 1mL 0.56mol / L HCl, centrifuge at room temperature and 12000rpm for 10min, take 1mL of the supernatant and add 800μL 0.56mol / L HCl, mix well and add 300μL 2,4-dinitrophenylhydrazine, place in a water bath at 30℃ for 30min, and finally add 2mL 2mol / L NaOH for color development and measure OD 540, with distilled water as a blank control. Then calculate the content of α-ketobutyric acid based on the standard curve, and determine the amount of protein in the remaining strain suspension at the same time, and finally calculate the ACC deaminase activity. The measured ACC deaminase activity of the X-NY1 strain was 1.20 U / mg.

[0077] 8. Determination of the siderophore activity of the strain

[0078] Inoculate 2% of the culture medium into MKB medium, culture at 37°C and 180 rpm for 48 h, centrifuge at 8000 rpm for 20 min, take the supernatant and CAS detection solution and mix them in a 1:1 volume ratio, let it stand for 1 h, and measure the OD with a UV photometer. 630 (A), distilled water was used as a control to adjust to zero, and uninoculated sterilized MKB medium was used as a reference value (Ar), and the A / Ar value was calculated to determine the ability of the strain to produce siderophores. The A / Ar value of the X-NY1 strain was measured to be 0.53.

[0079] Example 3 Preparation of composite microbial seed coating agent and performance measurement

[0080] 1. Preparation of composite microbial seed coating agent

[0081] (1) The Bacillus subtilis X-NY1, Bacillus Velez X-P18 and Bacillus amyloliquefaciens screened in Example 1 were inoculated into Luria Bertani (LB) liquid medium and fermented at 37°C for 24 h. The culture medium was centrifuged, resuspended and the OD was adjusted. 600 =1.0. Then, the fermentation broths of Bacillus subtilis X-NY1, Bacillus Velez X-P18 and Bacillus amyloliquefaciens were mixed at a volume ratio of 2:1:1 to obtain a liquid microbial agent.

[0082] Among them, Bacillus velezensis X-P18 was screened by the laboratory, disclosed in patent CN110184220B, and deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2019317. It has the function of solubilizing phosphorus and potassium; Bacillus amyloliquefaciens was purchased from the American Type Culture Collection (ATCC) with a deposit number of ATCC 23845, and has a nitrogen fixation function.

[0083] Luria Bertani liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, agar powder 20g / L.

[0084] (2) Mix 1% sodium alginate and 0.3% polyvinyl alcohol to prepare a film-forming agent, boil it in a water bath until it dissolves and becomes viscous, and cool it to 40-50°C for use. Add 0.2% acid fuchsin (colorant), 5% sodium lignin sulfonate (dispersant), 0.1% calcium carbonate (stabilizer), 0.1% sodium benzoate (preservative), 0.1% salicylic acid + 0.1% polyaspartic acid (regulator), 1% urea + 0.04% potassium dihydrogen phosphate (nutrient) to the microbial agent and stir until dissolved. Add the reserved film-forming agent last and stir for 30 minutes until the solution becomes viscous. At this point, the composite microbial seed coating agent is ready.

[0085] (3) The surface of the Pennisetum seeds was disinfected with 1.0% sodium hypochlorite solution (subject to the seeds being covered), then washed with sterile water for 3 times and dried naturally in the shade. The microbial seed coating agent and Pennisetum seeds were weighed at a volume / mass ratio of 50 mL:1 kg, poured into a coating machine, and stirred at a low speed for coating until the surface color of the seeds was uniform and non-sticky. At this point, the Pennisetum seeds were coated.

[0086] 2. Performance determination of composite microbial seed coating agent

[0087] The main performance indicators of microbial seed coating agents were tested in accordance with the national standard "GB / T 17768-1999 Specification for the Preparation of Suspension Seed Coating Agents" and the agricultural microbial agent standard "GB 20287-2006". The results are shown in Table 1 below.

[0088] Table 1 Test results of composite microbial seed coating agent indicators

[0089] project standard Test results Number of viable bacteria <![CDATA[≥2×10 8 cfu / mL]]> <![CDATA[2.5×10 9 cfu / mL]]> pH 5～8 7.78 Suspension rate ≥90％ 99.9±0.4％ Viscosity 100~600MPa·s 599.80MPa·s Film forming time ≤20min 4.10min Coating uniformity ≥90％ 96.38％ Coating shedding rate ≤8％ 4.30％ Film forming properties qualified qualified Low temperature stability qualified qualified Thermal storage stability qualified qualified

[0090] As shown in Table 1, the content of viable bacteria in the prepared composite microbial seed coating agent is greater than 2×10 8 cfu / mL, the suspension rate was greater than 90%, the viscosity was 599.8MPa·s, the film-forming time was 4.1min, the coating uniformity was 96.38%, the coating shedding rate was 4.3%, and the low-temperature stability and heat storage stability were qualified, indicating that the prepared composite microbial seed coating agent met the requirements of relevant national standards.

[0091] Example 4 Effect of composite microbial seed coating agent on germination of Pennisetum seeds

[0092] 1. Experimental steps

[0093] Prepare a culture dish (9 cm), lay two layers of filter paper (9 cm) flat on the bottom of the culture dish, cover and sterilize, and cool for use; adopt a culture dish hydroponic method, with sterile water (CK) and sterilized sodium chloride solution (CK-4‰) as controls, and the experimental groups are: non-salinized control group CK and experimental group ZYJ (group with composite microbial seed coating agent added); salinized (4‰) control group CK-4‰ and experimental group ZYJ-4‰ (group with composite microbial seed coating agent added). The composite microbial seed coating agent is obtained by preparing Example 3, and the surface of the Pennisetum seeds is disinfected with 1.0% sodium hypochlorite solution (based on the seeds not being covered), and then washed with sterile water 3 times, and dried naturally in the shade. The seeds are placed in a culture dish until the filter paper is saturated with water (about 15 mL), 3 repetitions are set for each group, and 30 seeds are placed in each culture dish. After the seeds germinate, germination records are made at the same time every day, with the embryo breaking through the seed coat as the standard, and germination is completed after 7 days. Take the seeds that have completed germination, randomly select 5 plants from each treatment, and measure the root length and sprout length with a vernier caliper.

[0094] 2. Experimental results

[0095] The experimental results are as follows Figure 3 As shown in Tables 2 and 3 below. Figure 3 It can be seen that the composite microbial seed coating agent has no adverse effect on the germination rate and vitality index of Pennisetum seeds, and the composite microbial seed coating agent significantly improves the germination rate and seed vitality index of Pennisetum seeds.

[0096] Table 2 Effects of seed coating agents on germination of Pennisetum seeds

[0097] Group Germination rate (%) Radicle (cm) Germ(cm) Germination Index Vitality Index CK 75±4 9.87±0.28 5.15±0.38 13.15±0.27 54.08±0.83 JZ 100±2 10.96±0.58 5.24±0.74 17.72±0.92 105.60±0.26

[0098] As shown in Table 2, under non-salinization conditions, the germination rate of the composite microbial seed coating agent increased significantly by 34% compared with the CK group, and the seed vigor index increased significantly by 94%. In addition, the coating had a certain growth-promoting effect on the germination and growth of Pennisetum seeds, and the radicle length of Pennisetum treated with ZYJ was significantly increased compared with the CK group.

[0099] Table 3 Effects of seed coating agents on germination of Pennisetum seeds under salt stress

[0100] Group Germination rate (%) Radicle (cm) Germ(cm) Germination Index Vitality Index CK-4‰ 60±4 5.88±0.27 4.10±0.36 10.58±0.28 25.14±0.02 ZYJ-4‰ 85±2 9.81±0.17 5.51±0.28 14.93±0.10 94.27±0.39

[0101] As shown in Table 3, under salinization, the composite microbial seed coating agent significantly increased the germination rate and seed vigor index of Pennisetum seeds, among which the germination rate was significantly increased by 41% compared with the CK-4‰ group, and the seed vigor index was significantly increased by 276%.

[0102] Example 5 Effect of composite microbial seed coating agent on the growth of Pennisetum

[0103] 1. Experimental steps

[0104] (1) The experiment was carried out in a greenhouse in the form of potted plants, with 450g of soil in each pot. The experiment set up four treatment groups, namely: CK, CK-4‰, ZYJ, and ZYJ-4‰, and each potted experimental group was set up with three replicates. Among them, the treatment group CK was a blank control group, CK-4‰ was a control group under 4‰ saline soil, ZYJ was an experimental group in non-salinized soil (a group with the addition of a composite microbial seed coating agent), and ZYJ-4‰ was an experimental group under 4‰ saline soil (a group with the addition of a composite microbial seed coating agent). The composite microbial seed coating agent was prepared in Example 3. After the soil was added to the potted plant, an equal and appropriate amount of distilled water was poured into the bottom tray to keep the soil moist. After 3 days, the Pennisetum planting experiment was carried out. Twenty seeds of Pennisetum were planted in each pot and cultured in a greenhouse with 12h light / 12h dark conditions (light intensity of 4000Lux) and temperature of 25±2℃. An equal amount of distilled water (50mL) was watered every day before germination, and watered every 3 days after germination. The seeds were planted for 30 days, and various physical and chemical properties of the plants were measured after the end of the planting.

[0105] (2) Determination of plant biomass and physiological indicators

[0106] ① Plant height: After the growth cycle (30 days), plants were randomly selected to measure the distance (cm) from the root base to the highest growth point, with 15 replicates for each treatment.

[0107] ②Root length: Use a vernier caliper to measure the length of the cleaned taproot, with 15 replicates for each treatment.

[0108] ③ Aboveground biomass: After potting, separate the aboveground part from the roots, weigh the weight (g) of all fresh plants aboveground, and store them in a -80℃ refrigerator for later use.

[0109] ④Underground biomass: Wash the roots with water, wipe them dry and weigh their total weight.

[0110] ⑤Soluble sugar: Weigh 0.5g of dried broken leaves on an analytical balance, add 15mL of distilled water to a test tube, extract in boiling water for 20min, filter the extract after cooling, pour the filtrate into a 100mL volumetric flask, rinse the remaining residue with distilled water several times, and make up the volume. After the sample is diluted 100 times, take a glass tube, add 1mL of the test solution and 5mL of anthrone reagent, mix well, react in a boiling water bath for 10min, take out and cool quickly, measure the absorbance at 620nm, and repeat three times. Calculate the soluble sugar content according to the following formula:

[0111]

[0112] ⑥Soluble protein: Weigh 0.5 g of fresh leaves on an analytical balance, mix with 5 mL of distilled water, grind into slurry, centrifuge at 1000 r / min for 10 min, collect the supernatant, dilute appropriately, react with 5 mL of Coomassie Brilliant Blue solution for 5 min, and measure the absorbance at 595 nm.

[0113] ⑦ Malondialdehyde (MDA): 250mL of 5% trichloroacetic acid (TCA), 10% trichloroacetic acid (TCA), 100mL of 0.67% thiobarbituric acid (TBA, dissolved with a small amount of 1mol / L sodium hydroxide), and 10% TCA to a constant volume of 50mL. Take about 0.5g of plant sample (leaves), add 5mL of 5% TCA, grind and centrifuge the resulting homogenate at 3000r / min for 10min. Use a pipette to slowly take 2mL of supernatant (not too fast to avoid inhalation of the plant body), add 2mL of 0.67% TBA, mix and boil in a 100℃ water bath for 15min, cool and centrifuge again (10min). Measure the absorbance values ​​of the supernatant at 450nm, 532nm and 600nm respectively, and use 2mL of distilled water plus 2mL of 0.67% TBA as a control. Calculate the concentration of malondialdehyde (MDA) according to the following formula:

[0114]

[0115] Where: A 532 , A 600 , A 450 Indicates the absorbance value at the corresponding wavelength.

[0116] ⑧ Determination of relative chlorophyll content: Weigh 0.1g of fresh cabbage leaves on an analytical balance, cut them into pieces, put them into a 15mL centrifuge tube with a lid, add 5mL of 95% ethanol, store in a dark place until the leaves turn white, take the supernatant, dilute it 10 times, measure the absorbance at 649nm and 665nm, repeat three times, and calculate the chlorophyll content according to the following formula:

[0117] Chlorophyll a pigment concentration = 13.95 × A 665 -6.88×A 649

[0118] Chlorophyll b pigment concentration = 24.96 × A 665 -7.32×A 649

[0119]

[0120] Where: A 649 , A 665 Indicates the absorbance value at the corresponding wavelength.

[0121] ⑨ Vitamin C content determination refers to the 2.6-dichloroindophenol titration method in the national standard GB 5009.86-2016. Weigh 0.5g of fresh sample on an analytical balance, grind it into a homogenate, and transfer the homogenate to a 25mL volumetric flask with oxalic acid solution (20g / L) to mix. Accurately pipette 10mL of the above solution into a 50mL conical flask, and titrate with a calibrated 2.6-dichloroindophenol sodium solution until the endpoint is pink, keep it without fading for 15s, record the data, repeat three times, and perform a blank test at the same time. Vitamin C content is calculated according to the following formula:

[0122] X=(V-V0)×T×A / m×100

[0123] Where: X is the vitamin C content in the sample, in mg / 100g; V is the amount of 2,6-dichloroindophenol solution consumed in the titration of the sample, in mL; V0 is the amount of 2,6-dichloroindophenol solution consumed in the titration of the blank, in mL; T is the titer of the 2,6-dichloroindophenol solution; A is the dilution multiple; M is the sample mass, in g.

[0124] 2. Experimental results

[0125] The experimental results are as follows Figure 4 As shown in Table 4 below.

[0126] Table 4 Effect of composite microbial seed coating agent on the growth of Pennisetum

[0127]

[0128]

[0129] Depend on Figure 4 As shown in Table 4, for Pennisetum, under non-salinization conditions, the plant height of ZYJ group increased by 8%, the root length increased by 27%, the fresh weight increased by 23.9%, the aboveground biomass increased by 23.3%, and the underground biomass increased by 61.5% compared with the CK group. Under salinization conditions, the plant height of ZYJ-4‰ group increased by 14.4%, the root length increased by 45.7%, the fresh weight increased by 25.3%, the aboveground biomass increased by 6.5%, and the underground biomass increased by 114.1% compared with the CK-4‰ group. This shows that the addition of composite microbial seed coating agent can effectively promote the growth of Pennisetum, and the growth-promoting effect will be more obvious under salinization.

[0130] The physical and chemical properties of Pennisetum were further determined, and the results are shown in Table 5 below.

[0131] Table 5 Effects of composite microbial seed coating agent on the physical and chemical properties of Pennisetum

[0132]

[0133] As shown in Table 5, for Pennisetum, under non-salinization conditions, the chlorophyll content of ZYJ group increased by 7.9%, the malondialdehyde content decreased by 31.5%, the soluble sugar content increased by 19%, the soluble protein increased by 94.6%, and the vitamin C content increased by 13.58% compared with the CK group. Under salinization conditions, the chlorophyll content of ZYJ-4‰ group increased by 8.24%, the malondialdehyde content decreased by 27.52%, the soluble sugar content increased by 102.9%, the soluble protein increased by 218.75%, and the vitamin C content increased by 9.61% compared with the CK-4‰ group.

[0134] The higher the chlorophyll content, the stronger the photosynthesis of the plant. The decrease in malondialdehyde content is often related to the increase in proline content. Proline can improve the cold resistance of plants, stabilize the structure of biological macromolecules, reduce cell acidity, relieve ammonia toxicity, and play an important role as an energy reservoir to regulate cell redox. The lower the malondialdehyde content, the better the plant's stress resistance. Soluble sugar and soluble protein are one of the important indicators of plant stress resistance, and are also important osmotic regulating substances and nutrients in plants. The higher the content of soluble sugar and soluble protein, the better the plant's stress resistance. The higher the vitamin C content, the better the plant's antioxidant properties. It can be seen from the results shown in Table 5 that the application of the composite microbial seed coating agent promoted the growth and development of Pennisetum and improved its stress resistance.

[0135] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A Bacillus subtilis strain, characterized in that The strain is Bacillus subtilis X-NY1, which has been deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242038.

2. A Bacillus subtilis strain according to claim 1, characterized in that The nucleotide sequence of 16SrDNA of the strain is shown in SEQ ID NO.

1.

3. Use of the Bacillus subtilis strain according to claim 1 or 2 in the preparation of a seed coating agent.

4. A composite microbial seed coating agent, characterized in that: The invention comprises the following components by mass percentage: 0.3% to 5% of film former, 1% to 5% of dispersant, 0.2% to 2% of colorant, 0.1% to 5% of stabilizer, 0.1% to 5% of preservative, 0.1% to 5% of regulator, 0.04% to 2% of nutrient, and the balance is made up of microbial inoculant fermentation liquid; wherein the microbial inoculant fermentation liquid comprises the Bacillus subtilis strain as claimed in claim 1 or 2, as well as Bacillus Velezii and Bacillus amyloliquefaciens.

5. A composite microbial seed coating agent according to claim 4, characterized in that: The volume ratio of the Bacillus subtilis strain, Bacillus Velez and Bacillus amyloliquefaciens is 2-5:1:

1.

6. A composite microbial seed coating agent according to claim 4, characterized in that: The film-forming agent includes sodium alginate and polyvinyl alcohol, and the mass ratio of the two is 1:0.2-0.8; the regulating agent includes salicylic acid and polyaspartic acid, and the mass ratio of the two is 1:1-3; the nutrient includes urea and dipotassium hydrogen phosphate, and the mass ratio of the two is 1:0.2-0.

6.

7. A composite microbial seed coating agent according to claim 3, characterized in that: The dispersant is sodium lignin sulfonate; the colorant is acid fuchsin; the stabilizer is calcium carbonate; and the preservative is sodium benzoate.

8. The method for preparing a composite microbial seed coating agent according to any one of claims 4 to 7, characterized in that: The following steps are involved: Step 1) Inoculate Bacillus subtilis, Bacillus Velezii and Bacillus amyloliquefaciens into LB liquid culture medium respectively, shake and culture at 30-37°C for 12-24h to obtain seed solution of each strain; resuspend by centrifugation and adjust OD 600 =1.0 of bacterial solution, and then mixed according to the proportion to obtain a composite liquid microbial agent; Step 2) weighing the raw materials in proportion, adding the dispersant, colorant, stabilizer, preservative, regulator, and nutrient into the composite liquid microbial agent prepared in step 1), and mixing and stirring thoroughly; Step 3) Finally, add the film-forming agent, heat and stir in a water bath at 30° C. until the solution is dissolved and becomes a viscous liquid, and mix and stir thoroughly to obtain the product.

9. Use of the composite microbial seed coating agent according to any one of claims 4 to 7, or the composite microbial seed coating agent prepared by the preparation method according to claim 8 in promoting the growth of Pennisetum under salinization conditions.

10. The use according to claim 9, characterized in that: The composite microbial seed coating agent and Pennisetum seeds were weighed at a volume / mass ratio of 50 mL:1 kg, poured into a coating machine, and fully stirred for coating until the surface of the seeds had a uniform color.

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