Bacillus subtilis microbial agent for accelerating the decay of corn stalks and its application

Abstract

The present invention discloses a Bacillus subtilis microbial agent for promoting the decay of corn stalks and its application. The deposit date of Bacillus subtilis K15 is May 21, 2024. The deposit unit is the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC No. 30716. The present invention screened out Bacillus subtilis K15 from the soil in the North China Plain. Experiments have shown that this strain produces carboxymethyl cellulase, which can effectively promote the decomposition of corn stalks. The decomposed stalks provide a fertilizer source for wheat growth, promote the growth of wheat plants and roots, and effectively increase wheat yields. The present invention explores the function of Bacillus subtilis in promoting the decay of stalks under low temperature conditions in winter in the North China Plain, and provides a new strain resource for microbial fertilizers.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Bacillus subtilis microbial agent for promoting the decay of corn stalks and an application thereof. Background Art

[0002] Studies have shown that when returning straw to the field, the application of microbial agents containing lignocellulose-degrading enzymes can promote the degradation of straw, convert it into organic fertilizer and release nutrients in a short period of time. However, the survival rate and functional performance of exogenous microorganisms in this environment are currently poor, making it difficult to fully exert their expected effects, especially under low temperature conditions.

[0003] Bacillus subtilis has gradually attracted attention in the research of straw degradation. For example, prior art 1: Zhang Qian et al. Screening of corn straw decomposition strains in yellow-brown soil and study on their ability to promote decomposition [J], Biotechnology Bulletin, 2022, 38(12): 233-243. A Bacillus subtilis (XJ3) was screened from a long-term positioning experiment of wheat-corn rotation in yellow-brown soil, and it was found that the strain increased the degradation rate of corn straw by 6.33% within 28 days under 25°C culture conditions. Prior art 2: Patent application number 202210597069.1 provides a Bacillus subtilis (TXB2) that can be used for the degradation of potato, oat, buckwheat, corn, quinoa and wheat straw. Although these studies and patent results show that Bacillus subtilis has a certain ability to degrade straw under indoor culture conditions, its effectiveness in low temperature environment and its actual field application, especially its performance under the specific climate and soil conditions of the North China Plain, has not been systematically studied and verified. Therefore, how to screen out suitable Bacillus subtilis strains based on the climate and soil characteristics of the North China Plain to significantly improve the decomposition effect of corn straw and increase the yield of winter wheat has become an important technical issue that needs to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to screen out strains that can adapt to the environment and are easy to survive from the soil of the North China Plain according to the climate and soil characteristics of the North China Plain, and prepare them into bacterial agents for promoting the decomposition of corn straw.

[0005] The present invention is achieved through the following technical solutions:

[0006] A Bacillus subtilis K15, classified and named Bacillus subtilis, with a preservation date of May 21, 2024, a preservation unit of the General Microbiology Center of the China Microbiological Culture Collection Administration, a preservation number of CGMCC No.30716, and a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The strain K15 was screened from a long-term straw return test site and litter layer soil in the North China Plain tidal soil and sand ginger black soil. It is a Gram-positive bacterium with spores. The colonies formed after culturing on LB solid medium for 24 hours are white, round or irregular, and the surface is moist. The strain K15 was preliminarily identified as Bacillus subtilis by comparing the 16S rDNA gene sequence and constructing an evolutionary system tree using Mega software. The 16S rDNA gene sequence determination result of the strain is shown in the sequence table SEQ ID NO: 1.

[0008] The Bacillus subtilis K15 has carboxymethyl cellulase activity, and the enzyme activity value reaches 5.37 U / mL after two days of fermentation.

[0009] An application of Bacillus subtilis K15 in low-temperature degradation of straw.

[0010] The invention discloses an application of Bacillus subtilis K15 in preparing a low-temperature degradable straw product.

[0011] An application of Bacillus subtilis K15 in promoting wheat yield.

[0012] A Bacillus subtilis microbial agent, wherein the active ingredient contains Bacillus subtilis K15.

[0013] Furthermore, the content of Bacillus subtilis K15 in 1g of microbial agent is 2×10 8 ~2×10 10 cfu·mL -1 .

[0014] The invention discloses an application of a Bacillus subtilis microbial agent in straw fermentation and composting.

[0015] The invention discloses an application of a Bacillus subtilis microbial agent in promoting wheat yield increase.

[0016] A method for preparing a Bacillus subtilis microbial agent comprises the following steps:

[0017] Step 1, streak the K15 strain on LB solid medium and culture overnight at 37°C for 18-36 hours;

[0018] Step 2, seed liquid culture: pick an activated single colony from the LB solid culture medium and transfer it to the LB liquid culture medium, and culture it in a shaking incubator at 37°C and 180 rpm for 10-12 hours to obtain a seed liquid;

[0019] Step 3, fermentation culture: transfer the seed solution obtained in step 2 to a new LB liquid culture medium at a 1% inoculation rate and culture in a shaker at 37° C. and 180 rpm for 10-14 h to expand the culture and obtain a culture solution;

[0020] Step 4: Peat, collagen, sodium tripolyphosphate, diatomaceous earth and starch are mixed to obtain auxiliary materials, the auxiliary materials are mixed with culture solution, and the mixed materials are dried to obtain Bacillus subtilis microbial agent.

[0021] The application method of Bacillus subtilis microbial agent in straw fermentation and composting is as follows: the straw is mechanically crushed and returned to the field and then covered on the ground surface; after the Bacillus subtilis microbial agent is evenly spread on the surface of the straw, the straw is rotary tilled and returned to the field.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention screens out Bacillus subtilis K15 from the soil in the North China Plain. Tests show that this strain produces carboxymethyl cellulase, which can effectively promote the decomposition of corn stalks. The decomposed stalks provide a fertilizer source for wheat growth, promote the growth of wheat plants and roots, and effectively increase wheat yield. The present invention explores the function of Bacillus subtilis in promoting the decomposition of stalks under low temperature conditions in winter in the North China Plain, and provides a new strain resource for microbial fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The colonies and microscopic morphology of K15 bacterial agent cultured in LB solid medium for 2 days, in which a is the colony morphology, b is the Gram staining microscopic morphology, and c is the spore staining microscopic morphology;

[0025] Figure 2 It is the transparent hydrolysis zone formed by Bacillus subtilis K15 strain on CMC-Na plate;

[0026] Figure 3 is the phylogenetic tree of Bacillus subtilis K15;

[0027] Figure 4 is the growth curve of Bacillus subtilis K15 at different pH and different NaCl concentrations, where a is the growth curve at different pH and b is the growth curve at different NaCl concentrations;

[0028] Figure 5 The results of the indoor test on promoting corn straw degradation by Bacillus subtilis K15;

[0029] Figure 6 Figure a shows the effect of K15 bacterial agent on the growth of wheat plants in fluvo-aquic soil, and figure b shows the effect of K15 bacterial agent on the growth of wheat plants in sandy black soil.

[0030] Figure 7 Comparison of the degradation rate of corn stalks in Example 7;

[0031] Figure 8The straw degradation rate in the field decomposition test of K15 bacterial agent is shown in Figure 1, where a is the straw degradation rate in fluvo-aquic soil and b is the straw degradation rate in sandy ginger black soil.

[0032] Fig. 9 Statistics of chemical components of straw in the field decomposition test of K15 microbial agent, where a is the chemical components of straw in fluvo-aquic soil, and b is the chemical components of straw in sandy ginger black soil;

[0033] Fig.10 These are the yield test results of the K15 bacterial agent applied to wheat field trials. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should belong to the model books protected by the present invention.

[0035] Example 1: Isolation and screening of strains

[0036] (1) Strain source

[0037] The Bacillus subtilis K15 of the present invention is separated and screened from soil of a long-term straw return to field location test site of tidal soil and sand ginger black soil in the North China Plain, and the sampling depth is about 10 cm.

[0038] (2) Strain isolation

[0039] Take 10g of soil sample and add it to a 250mL glass beaded triangular bottle with 90mL sterile water. Oscillate on a shaker at 180 rpm at 30℃ for 30min, let it stand for 30s, take 2mL of the supernatant, heat it in a 90℃ water bath for 10min, and then dilute it continuously with sterile water. The dilution factor is 10. -3 Up to 10 -5 100 μL of the dilution was evenly spread on the screening medium until the screening medium was dry. After culturing in an incubator at 37°C for 10-12 hours, a single colony grew on the plate.

[0040] Only Bacillus can survive the 90°C temperature, and this will kill other bacteria.

[0041] (3) Strain screening

[0042] Pick a single colony on the screening medium and inoculate it on the identification medium. When the colony grows, stain it with 1 g / L Congo red solution, pour off the stain, and then decolorize and fix it with 1 mol / L NaCl aqueous solution for 30 minutes to observe whether there is an obvious transparent hydrolysis zone. Figure 2 As shown. The absence of a transparent hydrolysis zone indicates that the strain has no function of promoting decay, and the strain is eliminated. Use a vernier caliper to measure the diameter of the transparent hydrolysis zone (D) and the colony diameter (d) of the colony, calculate the ratio of the hydrolysis zone diameter to the colony diameter, and preliminarily judge the cellulase activity of the isolated strain according to the size of the ratio. The larger the ratio, the stronger the enzyme production ability and the stronger the decomposition ability. It is concluded that the strain with the largest ratio is K15.

[0043] Screening medium: CMC-Na 15g, NH 4 NO 3 1g, KH 2 PO 4 1g, MgSO 4 • 7H 2 O 0.5g, peptone 1g, agar 20g, distilled water 1L, pH 7.0.

[0044] Identification medium: CMC-Na 10 g, yeast extract 0.5 g, K 2 HPO 4 1.5 g MgSO 4 7H 2 O 0.25g, agar 15g, distilled water 1L, pH 7.0.

[0045] Unless otherwise specified, the above culture media were sterilized at 121°C for 20 min before use.

[0046] Example 2: Strain Identification

[0047] The K15 strain obtained in Example 1 was cultured on LB solid medium at 30°C for 2 days. The morphology, color, smoothness and edge characteristics of the generated single colonies were observed, and the Gram staining and spore staining preparation process was performed according to the steps of the solid medium kit. The results of Gram staining and spore staining of the strain were observed under an oil microscope. Figure 1 As shown in a, b, and c.

[0048] The species was identified by morphological microscopic observation and 16S rDNA sequencing results. The K15 strain was identified as Bacillus subtilis. Figure 3 .

[0049] Through whole genome sequencing, the total length of the genome was 4082766 bp, with a GC content of 43.52%. The whole genome predicted 4309 coding genes, with a total length of 3660378 bp and an average length of 849 bp; there were 67 tRNAs, 4 5s rRNAs, 1 16s rRNA, and 1 23S rRNA.

[0050] In order to preserve the Bacillus subtilis for a long time, the Bacillus subtilis can be stored in a glycerol storage solution at a storage temperature of -85°C to -20°C.

[0051] Example 3: Optimal growth conditions for strains

[0052] (1) Optimal pH for strain growth

[0053] Set the LB liquid culture medium with an initial pH of 4-12 (gradient of 1), transfer the bacterial solution with OD600=0.4 to the LB liquid culture medium at different pH with an inoculum volume of 1%, and culture at 37°C and 180 rpm on a shaker for 24 h. Measure the growth of the strain and the OD600 value.

[0054] (2) Optimal salinity for strain growth

[0055] Prepare LB liquid culture medium containing 1%, 3%, 5%, 7%, 9%, 11%, and 13% NaCl. Transfer the bacterial solution with OD600 = 0.4 to LB liquid culture medium with different salinities at a 1% inoculation amount, and culture at 37°C, 180 rpm shaking for 24 hours to measure the growth of the strain and the OD600 value.

[0056] The results of experiments showed that the optimal pH for the growth of Bacillus subtilis K15 is 5. Figure 4 As shown in a, the NaCl concentration is 1%. Figure 4 As shown in b.

[0057] Example 4: Determination of enzyme production capacity of strains

[0058] (1) Preparation of crude enzyme solution

[0059] The strains were inoculated into sodium carboxymethyl cellulose liquid enzyme-producing medium, and each strain was repeated 3 times. The culture was placed in a constant temperature shaker at 25°C and 120 r / min for 2 days. The samples were then centrifuged at 4°C and 12000 r / min for 10 minutes. The supernatant was the crude enzyme solution.

[0060] (2) Determination of carboxymethyl cellulase activity

[0061] Prepare 1% CMC-Na solution: weigh 10 g CMC-Na and dissolve it in 990 mL citric acid-sodium citrate buffer.

[0062] Add 200 μL of crude enzyme solution to the prepared 200 μL 1% CMC-Na solution, mix well and place in a 50°C water bath for 30 min, add 600 μL of DNS reagent, shake well and place in a boiling water bath for 10 min, cool quickly to room temperature in an ice water bath, and dilute to 5 mL with distilled water. Take 200 μL and add it to a 96-well plate. Use an ELISA reader to measure its absorbance at 540 nm. According to the glucose standard curve (y=1.9924x-0.013, R 2 =0.994) to calculate the activity of carboxymethyl cellulose. The method for calculating the activity of the enzyme is as follows:

[0063] Enzyme activity (U / mL) =

[0064] Where: W is the amount of glucose produced by the enzymatic reaction, which is obtained from the glucose standard curve; represents the dilution, that is, the ratio of the total volume of the test tube to the volume of the added enzyme solution; 10 3 represents unit conversion; 180.16 represents the molecular weight of glucose (180.16 g / mol); 30 represents the reaction time between the enzyme and the substrate (30 min).

[0065] The results showed that strain K15 had good enzyme production ability, and the carboxymethyl cellulase activity value reached 5.37U / mL after 2 days of cultivation.

[0066] Example 5: Microbial agent containing Bacillus subtilis K15 fermentation broth

[0067] The microbial agent contains 2×10 8 ~2×10 10 cfu·mL -1 .

[0068] The preparation method of the microbial agent is as follows:

[0069] Step 1: Pick the K15 strain from the -80℃ glycerol freeze-dried tube and streak it on LB solid medium, and culture it at 37℃ overnight for 18~36h.

[0070] Step 2, seed liquid culture: pick an activated single colony from the LB solid culture medium and transfer it to the LB liquid culture medium, and culture it in a shaking incubator at 37°C and 180 rpm for 10-12 hours to obtain a seed liquid;

[0071] Step 3, fermentation culture: transfer the seed solution obtained in step 2 to new LB liquid culture medium at a 1% inoculation rate and culture in a shaking incubator at 37°C and 180 rpm for 10-14 h until OD600 = 1.0 to obtain culture solution.

[0072] Step 4: Weigh the auxiliary materials for biological inoculants, which contain turf carbon, collagen, sodium tripolyphosphate, diatomaceous earth and starch; relative to 1×10 9 cfu of the Bacillus subtilis K15, 0.1-1g of grass carbon, 0.05-0.5g of collagen, 0.005-0.05g of sodium tripolyphosphate, 0.3-3g of diatomaceous earth, and 0.5-5g of starch. Then the auxiliary materials are evenly mixed with the culture solution, and the mixed materials are dried to obtain the microbial agent containing the fermentation liquid of Bacillus subtilis K15 (hereinafter referred to as K15 agent) of the present invention.

[0073] LB solid medium: peptone 10.0 g, yeast extract powder 5.0 g, NaCl 10.0 g, agar 20.0 g, distilled water 1 L, pH 7.0~7.2.

[0074] LB liquid medium: peptone 10.0 g, yeast extract powder 5.0 g, NaCl 10.0 g, distilled water 1 L, pH 7.0~7.2.

[0075] Example 6: Experiment on promoting corn stalk degradation with K15 bacterial agent indoors

[0076] The effect of K15 bacterial agent on promoting corn straw degradation was determined in the form of PVC ring knife filling using corn straw, tidal soil and sand ginger black soil. Two treatment groups were set up: CK group (without K15 bacterial agent) and group with K15 bacterial agent.

[0077] Prepare a PVC ring knife (5 cm in diameter, 5 cm in height). Tie the bottom of the ring knife with a rubber band and a 300-mesh nylon net, and record its weight. Put 100 g of air-dried soil with a particle diameter of less than 2 mm and 0.72 g of air-dried straw (2-3 cm in length) in a nylon bag into a small plastic basin, mix well, and put it into the ring knife. Sterilize them before use.

[0078] Wherein: the bulk density of sandy ginger black soil and tidal soil is 1.3 g / cm 3 Calculate the soil layer thickness as 15 cm and assume 7500 kg·hm per season. -2The amount of straw returned to the field is 0.72% per year; 10 kg of urea is recommended per mu, and the annual urea input is 0.014%. 5 mL of K15 culture solution from step 3 of Example 5 was applied, and the balanced soil column was placed in an incubator and cultured at 15°C for 30 days. During the entire decomposition process, water was added regularly, and the water content was ensured to be 60% of the field water holding capacity after each water addition by weighing method.

[0079] Method for determining straw degradation rate: pick out the uncomposted and soft straw in the nylon bag, rinse it clean, and dry it in a 70°C oven to constant weight. Straw degradation rate = (straw mass before cultivation - straw mass on day n of cultivation) / straw mass before cultivation × 100%.

[0080] The results show that: Figure 5 As shown in the data, in the fluvo-aquic soil, the degradation rate of K15 treatment reached 50.0% after 30 days, which was 18.1% higher than that of CK; in the sandy black soil, the degradation rate of K15 treatment also reached 49.3% after 30 days, which was 10.1% higher than that of CK.

[0081] Example 7: Effects of K15 bacterial agent on corn stalk degradation and wheat plant growth

[0082] Corn stalks, tidal soil and sandy black soil were used, and the wheat variety for the test was Jimai 22. Two treatment groups were set up: CK group (without K15 inoculant) and group with K15 inoculant. Both groups selected round flower pots with an inner diameter of 12.5 cm and a height of 16.5 cm, and each pot contained about 1.8 kg of soil. The surface of the wheat seeds was disinfected with 2% sodium hypochlorite for 30 min, rinsed with tap water, and then washed with distilled water several times. Prepare a culture dish, place soaked filter paper in the culture dish, separate the wheat seeds evenly according to the number of treatments and place them on the soaked filter paper, and germinate in the dark at 25°C for 48 h. Assuming 7500 kg·hm per season -2 The amount of corn straw returned to the field is 0.385% (i.e., 0.385 g straw / 100 g soil, 3.85 g straw / 1 kg soil). About 6.9 g of straw is needed for each pot, which is put into 3 mesh bags (7×9 cm) and buried in the soil in layers. The top mesh bag is buried at a depth of about 10-15 cm. 20 mL of K15 culture solution from step 3 of Example 5 was applied to the flower pots of the K15 bacterial agent group, and an equal amount of distilled water was applied to the CK group to adjust the water content to 60% of the field water holding capacity. The biomass and straw degradation rate were measured after 30 days.

[0083] The results showed that in fluvo-aquic soil, compared with the CK group, the wheat plant height, aboveground dry weight, and underground dry weight of the K15 bacterial agent treatment group increased by 22.2%, 14.8%, and 7.7%, respectively. Figure 6As shown in a, compared with the CK group, the bacterial treatment made the wheat plants grow better and the root system grow more vigorously. In the sandy black soil, compared with the CK group, the wheat plant height, aboveground dry weight, and underground dry weight of the K15 bacterial agent treatment group increased by 16.9%, 25.0%, and 15.4%, respectively. Figure 6 As shown in middle b, compared with the CK group, the bacterial treatment made the wheat plants grow taller and better, and the root system grew more vigorously.

[0084] The corn stalks were taken out and dried, and the degradation rate was measured. The results showed that in the moist soil, the degradation rate of K15 treatment reached 41.7% after 30 days of pot planting, which was 11.23% higher than that of the CK group; in the sand ginger black soil, the degradation rate of K15 treatment reached 47.9% after 30 days of pot planting, which was 12.7% higher than that of the CK group. Figure 7 shown.

[0085] Example 8: Field decomposition test of K15 bacterial agent

[0086] Corn stalks were used for experiments in two areas: Linyi, Shandong, a typical tidal soil area, and Huaiyuan, Anhui, a typical sandy black soil area. Two treatment groups were set up: CK group (without K15 inoculant) and group with K15 inoculant. The harvested corn stalks were washed, dried, cut into 5 cm segments, and dried at 65 ° C to constant weight. 20 g of stalks were quantitatively weighed and put into a 300-mesh decomposition bag (15×20 cm). 10 g of the inoculant of Example 5 was applied to the surface of the corn stalks in the decomposition bag and mixed evenly with the corn stalks. The decomposition bag was buried at a depth of 10 cm and marked with a sign on the ground. Samples were taken at 15d, 30d, 60d, 90d, and 150d after the bags were buried, and the stalks were washed, dried, and weighed for the determination of the decomposition rate of corn stalks and the chemical components of the stalks.

[0087] The results showed that the straw degradation rate in both soil types increased significantly with the extension of degradation time. Figure 8 As shown in a and b, the degradation rates of fluvo-aquic soil and shajiang black soil were 37.4% and 48.9%, respectively, which were increased by 15.54% and 7.54%, respectively; after 150 days of degradation, the degradation rates of fluvo-aquic soil and shajiang black soil were 71.93% and 60.26%, respectively, which were increased by 24.04% and 5.16%, respectively.

[0088] From the perspective of chemical components of straw, compared with 0-day straw, after 60 days of degradation, the content of each chemical component decreased by about 50% overall. Fig. 9As shown in Figure a, in the fluvo-aquic soil, compared with the CK group, the application of K15 bacterial agent can significantly reduce the content of various chemical components, among which the proportions of soluble sugar, lignin, hemicellulose and cellulose decreased by 0.49%, 0.89%, 2.16% and 1.90%, respectively. Fig. 9 As shown in middle b, in the sand ginger black soil, compared with the CK group, after applying the K15 bacterial agent, except that the proportion of cellulose was slightly higher than that of the CK group, the proportions of soluble sugar, hemicellulose and lignin were 1.19%, 2.67% and 1.25% lower than that of the CK group, respectively.

[0089] Example 9: Wheat field test of K15 microbial agent

[0090] The test sites were located in the tidal soil area of ​​Dezhou, Shandong and the black soil area of ​​Longkang, Anhui. The test area was 12m long and 6m wide, with 2m protection rows. Two treatment groups were set: CK group (without K15 microbial agent) and K15 microbial agent group. After the corn was harvested in the test area, the straw was mechanically crushed and returned to the field. The straw after returning to the field was basically evenly covered on the surface; the microbial agent of Example 5 was evenly applied on the surface of the straw, at a rate of 7500 kg·hm per season. -2 The amount of corn stalks returned to the field is calculated, and the amount of microbial agent applied is 2 kg / mu; rotary tillage is returned to the field, and the depth of the return is about 15 cm. The next wheat crop is sown and field management is carried out routinely, and the wheat is harvested. The wheat plot with a length and width of 1 meter is harvested and the yield is measured. The 5-point sampling method is used, and finally the 1m 2 The yield per mu is obtained by multiplying the yield by 666.67 and taking the average value.

[0091] The results show that if Fig.10 As shown in the data, the application of K15 microbial agent increased the yield significantly in both soil types. According to the data of Dezhou Chao soil in Shandong, the application of K15 microbial agent increased the wheat yield by about 12.4% compared with the CK group; according to the data of Longkang Shajiang black soil in Anhui, the application of K15 microbial agent increased the yield by about 16.2% compared with the CK group.

[0092] Although the present invention has been described in detail by reference to the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A Bacillus subtilis K15, characterized in that: The Bacillus subtilis ( Bacillus subtilis ) The K15 preservation date is May 21, 2024. The preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation number is CGMCC No.30716.

2. Use of the Bacillus subtilis K15 according to claim 1 in low-temperature degradation of straw, wherein the low temperature is 15°C.

3. Use of the Bacillus subtilis K15 according to claim 1 in preparing low-temperature degradable straw products, wherein the low temperature is 15°C.

4. Use of the Bacillus subtilis K15 according to claim 1 in promoting wheat yield.

5. The Bacillus subtilis microbial agent is characterized in that: The active ingredient contains the Bacillus subtilis K15 according to claim 1.

6. The Bacillus subtilis microbial agent according to claim 5, characterized in that: The content of Bacillus subtilis K15 in 1g of microbial agent is 2×10 8 ~2×10 10 cfu·mL -1 .

7. Use of the Bacillus subtilis microbial agent according to claim 5 in straw fermentation and composting.

8. Use of the Bacillus subtilis microbial agent according to claim 5 in promoting wheat yield.

9. The method for preparing the Bacillus subtilis microbial agent according to claim 5, characterized in that: The steps include: Step 1, streak the K15 strain on LB solid medium and culture overnight at 37°C for 18-36 hours; Step 2, seed liquid culture: pick an activated single colony from the LB solid culture medium and transfer it to the LB liquid culture medium, and culture it in a shaking incubator at 37°C and 180 rpm for 10-12 hours to obtain a seed liquid; Step 3, fermentation culture: transfer the seed solution obtained in step 2 to a new LB liquid culture medium at a 1% inoculation rate and culture in a shaker at 37° C. and 180 rpm for 10-14 h to expand the culture and obtain a culture solution; Step 4: Peat, collagen, sodium tripolyphosphate, diatomaceous earth and starch are mixed to obtain auxiliary materials, the auxiliary materials are mixed with culture solution, and the mixed materials are dried to obtain Bacillus subtilis microbial agent.

10. The method for applying the Bacillus subtilis microbial agent in straw fermentation and decomposition according to claim 5, characterized in that: The straw is mechanically crushed and returned to the field and then covered on the ground. Bacillus subtilis microbial agent is evenly spread on the surface of the straw and then the straw is rotary tilled and returned to the field.

Images