Bacillus subtilis KC-1, composition and application thereof

Abstract

The application provides a bacillus subtilis KC-1 and a composition and application thereof, and belongs to the technical field of microorganisms. The preservation number of the bacillus subtilis KC-1 is CGMCC No. 35237. The bacillus subtilis KC-1 can secrete IAA, has nitrogen fixation ability, and can generate an iron carrier. The bacillus subtilis KC-1 and whey protein are used as a composition, and the two are combined to prepare a seed treatment material, can play a synergistic effect, and can significantly promote corn seed germination and seedling root development. Experiments show that, compared with the use of whey protein and the bacillus subtilis KC-1 alone, the use of the two in combination can significantly improve seed germination rate, the length of an aerial bud, the length of a main root, total root volume, total root surface area, total root length and the concentration of auxin of seedlings, can significantly reduce the concentration of abscisic acid in seedlings, can promote seed germination and seedling growth under drought conditions, and can improve the drought resistance of plants.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a bacillus subtilis KC-1, a composition and application thereof. BACKGROUND

[0002] Under the background of increasingly complex and severe global ecological environment, drought problems are becoming more and more prominent, which poses a serious threat to agricultural production and the stability of the ecological system. Seed germination, as the key stage of the plant life cycle, is extremely sensitive to environmental changes. Under drought stress, seed germination faces many obstacles, which greatly restricts the establishment and population renewal of plants.

[0003] Traditional strategies to cope with drought stress and promote seed germination, such as breeding drought-resistant varieties, optimizing irrigation techniques, and using chemical regulators, have played a role to some extent, but also have many limitations. Breeding drought-resistant varieties is a long cycle and is limited by genetic resources; irrigation techniques are difficult to be widely applied due to water resource conditions; and chemical regulators may pose potential harm to the soil and environment. Therefore, it is urgent to explore new green, efficient and sustainable seed germination promotion technologies.

[0004] In recent years, with the deepening of the research on the interaction between microorganisms and plants, the potential of microbial agents in improving plant stress resistance has gradually emerged. Plant growth-promoting rhizobacteria (PGPR) is a typical representative among them. They can actively participate in the growth regulation and stress response process of plants through various mechanisms. Some PGPR can secrete plant hormones such as auxins, cytokinins and gibberellins, which can effectively break seed dormancy, enhance seed vigor, and then promote seed germination and seedling growth. Some PGPR also have the ability to decompose phosphorus, potassium and nitrogen, which can significantly improve the soil nutrient conditions and provide more adequate nutrient support for seed germination and seedling growth. In addition, PGPR can induce plants to produce a series of physiological and biochemical responses under drought stress, such as increasing antioxidant enzyme activity, enhancing the antioxidant defense system of plants, effectively removing active oxygen, and reducing the degree of membrane lipid peroxidation, thereby reducing the damage of drought stress to plant cells; they can also promote the accumulation of osmotic adjustment substances such as proline and betaine, regulate cell osmotic pressure, maintain normal cell turgor pressure, and improve the water retention capacity of plants. Related studies have shown that under drought stress conditions, the germination rate of wheat seeds inoculated with PGPR is significantly higher than that of the non-inoculated group, and the root length, seedling height and biomass of the seedlings are also significantly increased.

[0005] Although plant growth-promoting rhizobacteria can play a certain effect in crop drought resistance, its effect is often limited. In practical application, the plant growth-promoting rhizobacteria has poor regulation effect on crop growth. Therefore, it is of great significance for high yield and stable yield of corn to develop a microbial agent capable of enhancing drought resistance of crops while maintaining the effectiveness of the microbial agent. SUMMARY

[0006] In view of the defects in the prior art, the purpose of the present application is to provide a bacillus subtilis KC-1 and a composition thereof, which can synergistically act with whey protein to efficiently improve the drought resistance of corn.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] The present application provides a bacillus subtilis KC-1, and the preservation number of the bacillus subtilis KC-1 is CGMCC No.35237.

[0009] The present application provides a composition comprising the bacillus subtilis KC-1 and whey protein according to the above technical solution.

[0010] The present application provides a preparation method of the composition according to the above technical solution, comprising:

[0011] The bacillus subtilis KC-1 is cultured to a bacterial activity of greater than or equal to 10 8 CFU / mL to obtain a bacillus subtilis KC-1 bacterial solution.

[0012] The bacillus subtilis KC-1 bacterial solution is mixed with a mixed system containing whey protein to obtain a composition.

[0013] Preferably, the mixed system containing whey protein comprises: 10-15 g / L of whey protein, 7.0-8.0 g / L of glucose, 1.5-2.5 g / L of yeast powder, 0.08-0.12 g / L of magnesium sulfate heptahydrate, and 15-25 g / L of sodium chloride; and the pH value of the mixed system is 7.0-8.0.

[0014] The present application provides a microbial agent comprising the bacillus subtilis KC-1 according to the above technical solution.

[0015] Preferably, the bacterial activity of the bacillus subtilis KC-1 in the microbial agent is greater than or equal to 10 8 CFU / mL.

[0016] The present application provides a preparation method of the microbial agent according to the above technical solution, comprising:

[0017] The Bacillus subtilis KC-1 is cultured in a culture medium to obtain a microbial inoculum.

[0018] The application provides application of the Bacillus subtilis KC-1, the composition or the composition prepared by the preparation method in improving drought resistance of plants.

[0019] The application provides a method for improving drought resistance of corn, comprising the following steps:

[0020] The corn seeds are coated by using the composition containing the Bacillus subtilis KC-1 and whey protein, and then sowed.

[0021] The application has the following beneficial effects:

[0022] The application provides a Bacillus subtilis KC-1, and the preservation number of the Bacillus subtilis KC-1 is CGMCC No.35237. The Bacillus subtilis KC-1 provided by the application can secrete IAA, has nitrogen fixation ability and can generate an iron carrier. The Bacillus subtilis KC-1 is combined with whey protein to prepare a composition, and the composition is used as seed treatment material, and can synergistically promote corn seed germination and corn seedling root development. The whey protein can provide rich nutrient sources for the Bacillus subtilis KC-1, promote colonization and reproduction of the Bacillus subtilis KC-1 on the seed surface and in rhizosphere soil, and enhance the effect of the microbial inoculum; the Bacillus subtilis KC-1 can improve plant nutrition status and physiological and biochemical characteristics, improve the absorption and utilization efficiency of the plant to the whey protein nutrient components, and the two synergistically improve the seed germination rate and seedling root indexes under a drought environment. The results of the examples of the application show that, compared with the whey protein and the Bacillus subtilis KC-1 used alone, the combination of the two can significantly improve the corn seed germination rate, the length of the above-ground bud, the length of the main root of the under-ground part, the total root volume, the total root surface area, the total root length and the auxin concentration of the seedling, and can also significantly reduce the concentration of abscisic acid in the seedling, thereby being beneficial to promoting corn seed germination and seedling growth under a drought condition and improving drought resistance of the corn.

[0023] Biological preservation

[0024] The Bacillus subtilis KC-1 (CGMCC No.35237) is preserved in the China General Microbiological Culture Collection Center on July 15, 2025, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No.1, Yihuan West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.35237. Bacillus subtilis BRIEF DESCRIPTION OF DRAWINGS

[0025] ​In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed in the embodiments will be briefly introduced as follows. Obviously, the accompanying drawings in the following description only only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0026] Figure 1 A phylogenetic tree of Bacillus subtilis KC-1;

[0027] Figure 2 A transparent halo produced by Bacillus subtilis KC-1 in decomposing whey protein;

[0028] Figure 3 A functional effect diagram of Bacillus subtilis KC-1;

[0029] Figure 4 A comparison diagram of corn seedlings of different treatment groups after growing for 10 days in a soil drought environment;

[0030] Figure 5 A length diagram of the aboveground and underground parts of corn seedlings of different treatment groups in a soil drought environment;

[0031] Figure 6 A root scanning diagram of corn seedlings of different treatment groups. DETAILED DESCRIPTION

[0032] The present application provides a Bacillus subtilis KC-1, the preservation number of the Bacillus subtilis KC-1 is CGMCC No.35237.

[0033] The Bacillus subtilis KC-1 provided by the present application is separated from the corn root soil in a drought area of Xing'an Meng, Inner Mongolia; the 16s rDNA sequence of the Bacillus subtilis KC-1 is shown as SEQ ID NO.1; the Bacillus subtilis KC-1 can secrete IAA, has nitrogen fixation ability, and can generate iron carrier. The present application prepares seed treatment material by combining the Bacillus subtilis KC-1 with whey protein, and the two can synergistically promote corn seed germination and corn seedling root development, and significantly improve the seed germination rate and seedling root index in a drought environment. The results of the embodiments show that, compared with whey protein and Bacillus subtilis KC-1 alone, the combination of the two can significantly improve the seed germination rate, length of aboveground bud, length of underground main root, total root volume, total root surface area, total root length and auxin concentration of seedlings, and also can significantly reduce the concentration of abscisic acid in seedlings, thereby being beneficial to promoting corn seed germination and seedling growth in a drought condition and improving the drought resistance of corn.

[0034] The present application provides a microbial agent, comprising the Bacillus subtilis KC-1 described in the above technical solution. 8 The microbial agent can comprise the Bacillus subtilis KC-1 with a bacterial activity of 10 8 CFU / mL.

[0035] The present application provides a preparation method of the microbial agent described in the above technical solution, comprising: culturing the Bacillus subtilis KC-1 in a culture medium to obtain the microbial agent. The present application does not have special limitations on the type of culture medium, and any conventional culture medium that can make the Bacillus subtilis KC-1 grow well can be used. As an optional embodiment of the present application, the type of culture medium can be LB culture medium, TSA culture medium or whey protein culture medium. As an optional embodiment of the present application, the whey protein culture medium can comprise 10-15 g / L of whey protein, 7.0-8.0 g / L of glucose, 1.5-2.5 g / L of yeast powder, 0.08-0.12 g / L of magnesium sulfate heptahydrate and 15-25 g / L of sodium chloride. The pH value of the whey protein culture medium can be 7.0-7.5. In the present application, the culture temperature can be 25-30℃, or 25, 26, 27, 28, 29 or 30℃. The culture time can be 1-3 days, or 1, 2 or 3 days. The culture process is preferably accompanied by shaking. The shaking speed can be 180 rpm.

[0036] The present application provides a composition comprising the Bacillus subtilis KC-1 described in the above technical solution and whey protein.

[0037] The present application provides a preparation method of the composition described in the above technical solution, comprising:

[0038] The Bacillus subtilis KC-1 is cultured to a bacterial activity of 10 8 CFU / mL to obtain a Bacillus subtilis KC-1 bacterial solution.

[0039] The Bacillus subtilis KC-1 bacterial solution is mixed with a mixed system containing whey protein to obtain a composition.

[0040] The present application does not have special restrictions on the culture method of the Bacillus subtilis KC-1, and the conventional culture method in the art can be used. As an optional embodiment of the present application, the culture method of the Bacillus subtilis KC-1 can be: culturing the Bacillus subtilis KC-1 in a culture medium to obtain a Bacillus subtilis KC-1 bacterial solution. As an optional embodiment of the present application, the culture medium can be LB culture or TSA culture medium; the culture time can be 1-3 days, and can also be 1, 2 or 3 days; the culture temperature can be 25-30°C, and can also be 30°C; preferably, the culture process is accompanied by shaking; the shaking speed can be 180 rpm. The present application preferably cultures to OD 600nm The present application preferably adjusts the effective bacteria activity of the Bacillus subtilis KC-1 culture solution to be ≥10 8 CFU / mL, and can also be 10 8 ~10 9 CFU / mL, to obtain a Bacillus subtilis KC-1 bacterial solution.

[0041] After obtaining the Bacillus subtilis KC-1 bacterial solution, the present application mixes the Bacillus subtilis KC-1 bacterial solution with a mixed system containing whey protein to obtain a composition. As an optional embodiment of the present application, the mixed system containing whey protein comprises: whey protein 10-15 g / L, glucose 7.0-8.0 g / L, yeast powder 1.5-2.5 g / L, magnesium sulfate heptahydrate 0.08-0.12 g / L, and sodium chloride 15-25 g / L; the pH value of the mixed system is 7.0-8.0. In the present application, the composition of the mixed system containing whey protein can also be whey protein 10.0 g / L, glucose 7.5 g / L, yeast powder 2.0 g / L, magnesium sulfate heptahydrate 0.1 g / L, and sodium chloride 20.0 g / L; the pH value of the mixed system is 7.0. The present application does not have special restrictions on the mixing method, and the conventional mixing method in the art can be used. The present application prepares a composition by the combined action of the Bacillus subtilis KC-1 and whey protein, and uses the composition as seed treatment material, which can synergistically promote the germination of corn seeds and the root development of corn seedlings. Among them, whey protein can provide a rich source of nutrients for Bacillus subtilis KC-1, promote its colonization and reproduction on the seed surface and in the rhizosphere soil, and enhance the effect of microbial inoculants; Bacillus subtilis KC-1 can improve the nutritional status and physiological and biochemical characteristics of plants, improve the absorption and utilization efficiency of plants to whey protein nutrients, and the synergistic effect of the two can significantly improve the germination rate of corn seeds and the root index of seedlings in a drought environment.

[0042] The application provides application of the Bacillus subtilis KC-1, the composition or the composition prepared by the preparation method in improving drought resistance of plants.

[0043] The application provides a method for improving drought resistance of corn, which comprises the following steps: coating corn seeds with a composition containing the Bacillus subtilis KC-1 and whey protein, and then sowing the seeds.

[0044] As an optional embodiment of the application, the composition can be the composition described in the above technical solution. Before coating the seeds, the application preferably performs a disinfection treatment on the seeds. The disinfection treatment method is not particularly limited in the application, and a conventional disinfection treatment method in the art can be used. As an optional embodiment of the application, the disinfection method can be that the seeds are mixed with a 1% sodium hypochlorite solution for disinfection treatment, and the disinfection treatment time can be 10 min. After the disinfection treatment is completed, the application rinses the obtained seeds and then coats the seeds. In the application, the rinsing is preferably performed with distilled water, and the rinsing can be performed for 3 times. After the rinsing is completed, the application obtains the seeds after the disinfection treatment. After obtaining the seeds after the disinfection treatment, the application coats the seeds. The coating method is not particularly limited in the application, and a conventional coating method in the art can be used. As an optional embodiment of the application, the coating method can be that the seeds are completely immersed in the seed treatment material for coating. In the application, the coating temperature can be 20-30 DEG C or 30 DEG C, and the coating time can be 1-2 h or 2 h. After the coating treatment is completed, the application plants the obtained coated seeds. The planting method is not particularly limited in the application, and a conventional planting method in the art can be used.

[0045] In order to further illustrate the application, the technical solutions provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0046] Example 1

[0047] Strain screening and identification

[0048] 1. Experimental materials

[0049] Soil sample: corn rhizosphere soil sample in a dry region of Xing'an Meng, Inner Mongolia, stored at 4 DEG C, and transported back to the laboratory.

[0050] Whey protein: McLean reagent.

[0051] Test medium:

[0052] Whey protein medium (g / L): whey protein 10.0 g / L, glucose 7.5 g / L, yeast extract 2.0 g / L, magnesium sulfate heptahydrate 0.1 g / L, sodium chloride 20.0 g / L, sterilized at 121 ℃ for 15 min, and adjusted to pH 7.0. Add agar 18 g for solid medium.

[0053] LB medium (g / L): tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, adjusted to pH 7.4, sterilized at 121 ℃ for 20 min. Add agar 18 g for solid medium.

[0054] TSA medium (g / L): tryptone 15.0 g / L, soybean peptone 5.0 g / L, sodium chloride 5.0 g / L, adjusted to pH 7.3, sterilized at 121 ℃ for 20 min. Add agar 18 g for solid medium.

[0055] 0.9% normal saline: NaCl 0.9 g, distilled water 99.1 mL, sterilized at 121 ℃ for 20 min.

[0056] 2. Strain screening

[0057] (1) Take 10 g of soil from the rhizosphere of corn in the arid region of Inner Mongolia Xing'an League and place it in 90 mL of sterile normal saline. Shake well in a constant temperature shaker and let stand for 30 min to obtain a preliminary activated bacterial solution.

[0058] (2) Take the preliminary activated bacterial solution in step (1) and inoculate it into a new 100 mL whey protein medium at a volume ratio of 10% inoculation amount. Incubate at 180 rpm and 30 ℃ for 48 h to obtain an activated and enriched bacterial solution.

[0059] (3) Take the bacterial solution in step (2) and inoculate it into a new 100 mL whey protein medium at a volume ratio of 10% inoculation amount. Incubate at 180 rpm and 30 ℃ for 72 h.

[0060] (4) Take the bacterial solution in step (3) and inoculate it into a new 100 mL whey protein medium at a volume ratio of 10% inoculation amount. Incubate at 180 rpm and 30 ℃ for 24 h.

[0061] (5) Take 10 mL of the bacterial solution after incubation in step (4), add glass beads and dilute with sterile water to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7100 μL of bacterial culture was taken from each sample and spread onto a whey protein solid culture medium, which was then incubated at 30°C for 48 h.

[0062] (6) Observe the growth of colonies and pick out single colonies with obvious hydrolysis zones from step (5), streak them onto new LB solid medium, and incubate at 30°C for 48 h.

[0063] (7) Pick the single colony obtained in step (6) and place it on LB liquid medium. Incubate it at 180 rpm and 30℃ for 48 h and measure the OD of the bacterial solution. 600 When the value reaches the range of 0.8 to 1.0, the effective viable count is ≥10. 8 CFU / mL was used to obtain the desired bacterial culture.

[0064] 3. Strain identification

[0065] The strain was identified as Bacillus subtilis by Beijing Ruiboxingke Biotechnology Co., Ltd. using 16S rDNA. Bacillus subtilis The phylogenetic tree of Bacillus subtilis KC-1 is as follows: Figure 1 As shown in the image. The effect of Bacillus subtilis KC-1 on the degradation of whey protein is illustrated in the image. Figure 2 As shown. The preservation number of Bacillus subtilis KC-1 is CGMCC No. 35237. The colony morphology of Bacillus subtilis KC-1 is mainly characterized by being milky white with irregular edges.

[0066] The 16S rDNA of Bacillus subtilis KC-1 is shown in SEQ ID NO.1.

[0067] SEQ ID NO.1:

[0068]

[0069] Example 2 Function test of Bacillus subtilis KC-1

[0070] Test materials:

[0071] Iron carrier medium: CAS solution: Chrome Azurol S 60 mg, cetyltrimethylammonium bromide (CTAB) 100 mg were dissolved in 100 mL distilled water, and stored in the dark. Base medium: (NH4)2SO4 2 g·L -1 , KH2PO4 0.5 g·L -1 , MgSO4·7H2O 0.2 g·L -1 , NaCl 0.1 g·L -1 , glucose 10 g·L -1 , agar 15 g·L -1 , pH 7.0-7.2, sterilized at 121°C for 20 min. The base medium was cooled to about 50°C, and the CAS solution was slowly added (to avoid precipitation), and then poured into a flat plate after shaking.

[0072] Ashby medium: mannitol 10 g·L -1 , KH2PO4 0.2 g·L -1 , MgSO4·7H2O 0.2 g·L -1 , NaCl 0.2 g·L -1 , CaSO4·2H2O 0.1 g·L -1 , CaCO3 5 g·L -1 , agar 15 g·L -1 , pH 7.0-7.2, sterilized at 121°C for 20 min.

[0073] Function tests were performed on Bacillus subtilis KC-1 to test their ability to secrete auxins, fix nitrogen, and produce iron carriers, respectively, as follows:

[0074] IAA function test: The strain was inoculated into LB liquid medium containing L-tryptophan at 0.1 g·L -1 , with 3 replicates per strain, and cultured at 30°C for 1 day on a 180 rpm shaker. The bacterial suspension was then added to a test tube, and an equal volume of Salkowski colorimetric solution was added. The mixture of uninfected LB liquid medium and an equal volume of colorimetric solution was used as a control. The test tubes were observed after being placed in the dark at room temperature for 30 min. A pink color indicated a positive result, indicating the ability to secrete IAA, with a deeper color indicating a greater secretion intensity. No color change indicated a negative result, indicating the inability to secrete IAA.

[0075] Inoculate the strain with an inoculating loop on the Ashby nitrogen fixation medium plate, and observe after constant temperature culture at 28℃ for 4-7 days. Large convex mucous colonies are strains with nitrogen fixation ability.

[0076] Inoculate the strain with an inoculating loop on the siderophore production medium plate, and observe after constant temperature culture at 28℃ for 4-7 days. Whether there is an orange iron precipitation zone around the target colony of the medium proves that the strain has the ability to produce siderophores.

[0077] The functional effect diagram of Bacillus subtilis KC-1 is shown in Figure 3 . A is the IAA function test result diagram in Figure 3 . The left test tube in A is the control, and the right test tube is the IAA production effect diagram of the strain; B is the siderophore production ability diagram of the strain; and C is the nitrogen fixation ability diagram of the strain. Figure 3 The results show that in the IAA production test, the pink color indicates that Bacillus subtilis KC-1 can secrete IAA; in the Ashby nitrogen fixation medium, the strain of Bacillus subtilis KC-1 shows large convex mucous colonies, which are strains with nitrogen fixation ability; in the siderophore production medium, there is an orange iron precipitation zone around the colony of Bacillus subtilis KC-1, which proves that the strain has the ability to produce siderophores.

[0078] Example 3 Seed coating with microbial seed promotes seed germination in drought environment

[0079] Test soil: taken from the Shunyi Agricultural Environment Test Base of the Chinese Academy of Agricultural Sciences (N40°10', E116°92'). The test soil is loam, with pH 8.1, organic matter 13.18 g / kg, total nitrogen 1.03 g / kg, available phosphorus 13.69 mg / kg, and available potassium 171.82 mg / kg. After natural air drying, pass through a 10 mm sieve.

[0080] Test crop: corn (Zea mays L.), variety Zhengdan 958.

[0081] Coating material: whey protein medium, Bacillus subtilis KC-1.

[0082] Steps:

[0083] Put the soil into a flowerpot with an upper diameter x lower bottom diameter x height of 16 cm x 13 cm x 17.5 cm, and water to keep the soil volume water content at 12%-15%, reaching the level of light drought, and place it in the greenhouse of the Institute of Environmental and Agricultural Research of the Chinese Academy of Agricultural Sciences.

[0084] Microbial inoculant preparation: pick a single colony of Bacillus subtilis KC-1 into LB liquid medium, and culture the bacterial solution to OD600nm 0.8-1.0, adjust the effective viable count of the fermentation bacterial solution to 10 8CFU / mL, and the Bacillus subtilis KC-1 bacterial solution was obtained, that is, the microbial inoculant.

[0085] The whey protein medium was mixed with the prepared microbial inoculant Bacillus subtilis KC-1 bacterial solution at a volume ratio of 10:1 to obtain a seed treatment material, which was referred to as a composition in the following experiments.

[0086] Before sowing, the corn seeds needed to be disinfected by soaking them in a 1% sodium hypochlorite solution for 10 min, and then rinsing them with sterilized distilled water for 3 times to obtain disinfected seeds. The experiment consisted of 4 treatment groups, namely CK: control group, disinfected seeds treated with distilled water; R: disinfected seeds treated with whey protein medium; J: disinfected seeds treated with Bacillus subtilis KC-1 bacterial solution with an effective viable count of 10 8 CFU / mL; J+R: disinfected seeds treated with the composition. When treating the seeds, the amount of distilled water or solution used should be enough to completely immerse the seeds in the distilled water or the corresponding solution. The temperature for treating the seeds of each treatment group was 20-30°C, and the time for treating the seeds was 2 h.

[0087] Seeds with plump grains were selected for planting in each treatment group, and 50 seeds were sown in each pot. During the planting period, the soil volume water content was maintained at 12%-15% (the conventional water content during corn planting is 60%-80% of the field water holding capacity) by watering. Three replicates were set up, and all the replicate pots were randomly placed in the greenhouse and periodically rotated to minimize the effects of environmental differences. During the entire experiment, the temperature in the greenhouse was controlled according to day and night, with the temperature in the greenhouse maintained at 25°C during the day and 20°C at night, the light period was 12 h, and the light intensity was 240 μmol·m -2 ·s -1 .

[0088] Results determination:

[0089] On the 3rd and 7th day after sowing, the seed germination rate was counted. On the 10th day after sowing, 5 corn seedlings were taken from each replicate, and the length of the aboveground and underground parts of the corn seedlings was measured. The complete corn root system was scanned and analyzed using WinRHIZO (Canada Regent), and the root morphology was scanned and analyzed according to the method provided by the manufacturer. The corn seedling hormones were determined as follows:

[0090] Ten days after sowing, samples of corn seedlings were collected, rapidly frozen in liquid nitrogen, and ground into powder. The phytohormones (IAA, ABA) in the seedlings were detected by UPLC-MS. 50 mg of sample powder was accurately weighed, loaded into a 2 mL centrifuge tube, 500 μL of extraction solution (IPA:H2O:HCl = 2:1:0.002) was added, vortexed for 10 s, and oscillated at 4°C and 900 rpm for 30 min. 1 mL of extraction solution (CHCl3) was added, vortexed for 10 s, and oscillated at 4°C and 900 rpm for 30 min. Then centrifuged at 4°C and 14000 rpm for 5 min, and two phases were formed. 1.2 mL of lower liquid was transferred and blown dry with nitrogen at room temperature. 0.1 mL of MeOH was added, oscillated at 4°C and 900 rpm for 20 min, centrifuged at 14000 rpm for 5 min, and the supernatant was taken, filtered through a 0.1 μm filter membrane, loaded into a sample injection vial, and detected by UPLC-MS. An ultra-high pressure liquid chromatography method was used. The mobile phase A was water (0.05% formic acid), and the mobile phase B was acetonitrile (0.05% formic acid). The column temperature was 35°C, the sample temperature was 15°C, and the flow rate of the mobile phase was 0.3 mL / min. The sample gradient elution program is shown in Table 1.

[0091] Table 1 Sample gradient elution program

[0092]

[0093] Mass spectrometry was performed using a 5500 Qtrap-MS system (AB SCIEX). Ion source: HESl, spray voltage (-): 3000V, capillary temperature: 320°C, sheath gas: 30 arb, auxiliary gas: 10 arb, reserve gas: 5 arb, probe heating temperature: 350°C, S lens radio frequency level: 55%.

[0094] FULL MS-SIM: resolution: 70,000@ m / z 200, automatic gain control target value: 3e 6 ion counts, maximum ion injection time: 100 ms, scan range: 50~750 m / z.

[0095] The growth of the aboveground and underground parts of the seedlings of each treatment group during the corn seedling period is shown in Figures 4-6 and Table 2. Figure 4 is a comparison chart of corn seedlings of different treatment groups after growing in a drought environment for 10 days. Figure 5 is a chart of the aboveground and underground lengths of corn seedlings of different treatment groups in a drought environment, where indicates that the difference between different treatment groups is significant, and ns indicates that the difference between different treatment groups is not significant. Figure 6 is a root system scanning chart of corn seedlings of different treatment groups.

[0096] Table 2 Indexes of corn seedlings in different treatment groups under drought environment

[0097]

[0098] Note: The data in the table are mean ± standard deviation. The same row in the table indicates significant difference; the same row indicates no significant difference.

[0099] As shown in Table 2, the statistical results of seed germination rate show that on the 3rd day, the seed germination rate of the seed treated with the composition of the application is 62%, the seed germination rate of the seed treated with whey protein alone is 30%, the seed germination rate of the seed treated with microbial inoculant alone is 18%, and the seed germination rate of the control group is 12%. On the 7th day, the seed germination rate of the seed treated with the composition of the application is 96%, the seed germination rate of the seed treated with whey protein alone is 92%, the seed germination rate of the seed treated with microbial inoculant alone is 82%, and the seed germination rate of the control group is 60%.

[0100] As shown in Table 2 and Figures 4-6 It can be seen that the length of the aboveground and underground parts of the corn seedlings treated with the composition of the application is significantly higher than that of the control group, the length of the aboveground part is 42.5% higher than that of the control group on average (p<0.01), and the length of the underground part is 65.3% higher than that of the control group on average (p<0.01); the length of the aboveground and underground parts of the corn seedlings treated with microbial inoculant alone is significantly higher than that of the control group, the length of the aboveground part is 34.4% higher than that of the control group on average (p<0.05), and the length of the underground part is 55.3% higher than that of the control group on average (p<0.01); the length of the underground part of the corn seedlings treated with whey protein alone is significantly higher than that of the control group, the length of the underground part is 45.5% higher than that of the control group on average (p<0.01), and the length of the aboveground part has no significant difference with the control group, but is 13.5% higher than that of the control group.

[0101] As shown in Table 2 and Figures 4-6 The results show that the total root length, total root surface area and total root volume of the corn seedlings treated with the composition of the application under drought environment are significantly higher than those of the CK treatment, which promotes the development of corn root system; compared with the CK, the average content of auxin of the corn seedlings treated with J+R and J is significantly increased by 105.16% and 96.77% respectively, and the average content of abscisic acid is significantly reduced by 83.42% and 53.41% respectively. The reason is that Bacillus subtilis KC-1 activates the biosynthesis of auxin in the host, which leads to the change of root structure by affecting the length, volume and surface area of the root, thereby increasing the water absorption capacity during drought.

[0102] Example 4 Comparison of the application of Bacillus subtilis KC-1 and other microorganisms in seed coating agent

[0103] Tested crop: corn (Zea mays L.), variety Zhengdan 958. Zea mays L. ), variety Zhengdan 958.

[0104] Coating material: whey protein medium, Bacillus subtilis KC-1, Bacillus subtilis (ACCC 19742, China General Microbiological Culture Collection Center).

[0105] Microbial culture medium: LB liquid medium.

[0106] Drought stress simulation reagent: PEG-6000.

[0107] Steps:

[0108] Microbial agent preparation: single colonies of Bacillus subtilis KC-1 and Bacillus subtilis ACCC 19742 were picked into LB liquid medium, and the bacterial liquid was cultured to OD600nm 0.8-1.0. The effective viable cell number of the fermentation bacterial liquid was adjusted to 10 8 CFU / mL, respectively, to obtain Bacillus subtilis KC-1 bacterial liquid and Bacillus subtilis ACCC 19742 bacterial liquid.

[0109] The whey protein medium was mixed with the prepared microbial agents Bacillus subtilis KC-1 bacterial liquid and Bacillus subtilis ACCC 19742 bacterial liquid according to a volume ratio of 10:1 to obtain seed treatment materials, which were referred to as Bacillus subtilis KC-1 and whey protein composition and Bacillus subtilis ACCC 19742 and whey protein composition in the following experiments.

[0110] Corn seeds were subjected to disinfection treatment, soaked in 1% sodium hypochlorite solution for 10 min, and then rinsed with sterilized distilled water for 3 times to obtain disinfected seeds. The experiment consisted of 4 treatment groups, which were J: disinfected seeds treated with Bacillus subtilis KC-1 bacterial liquid with an effective viable cell number of 10 8 CFU / mL; J+R: disinfected seeds treated with Bacillus subtilis KC-1 and whey protein composition; S: disinfected seeds treated with Bacillus subtilis ACCC 19742 bacterial liquid with an effective viable cell number of 10 8 CFU / mL; S+R: disinfected seeds treated with Bacillus subtilis ACCC 19742 and whey protein composition. The reagent dosage for treating seeds in each treatment group can be used to completely immerse the seeds in the solution. The temperature for treating seeds in each treatment group was 20-30°C, and the time for treating seeds was 2h.

[0111] Culture dish filter paper method was used, 3 replicates were set for each treatment, and 50 corn seeds were used for each replicate:

[0112] Petri dish preparation: 2 layers of sterile filter paper were laid in a sterile Petri dish, and 10-15 mL of PEG-6000 solution with a concentration of 10% was added (keep the filter paper moist but not waterlogged);

[0113] Seed placement: evenly place the corn seeds treated with different treatments on the filter paper, avoiding overlapping of the seeds;

[0114] Cultivation conditions: place in an artificial climate chamber, set the temperature to 25℃, light / dark 12h / 12h (240 μmol·m -2 ·s -1 ), relative humidity 60%, and continuously cultivate for 7 days;

[0115] Index determination: record the number of germinated seeds every day (germination standard: radicle breaks through seed coat ≥2 mm), and after 7 days of cultivation, determine the following indexes:

[0116] Germination rate (%) = (number of germinated seeds within 7 days / number of tested seeds) x 100;

[0117] Germination potential (%) = (number of germinated seeds within 3 days / number of tested seeds) x 100;

[0118] Radicle length (cm): after 7 days, randomly select 10 germinated seedlings, measure the radicle length with a vernier caliper, and determine the average value;

[0119] The results are shown in Table 3.

[0120] Table 3 Effect of Bacillus subtilis KC-1 and other microorganisms as seed treatment materials on the growth indexes of corn seedlings in drought environment

[0121]

[0122] Note: the data in the table are mean ± standard deviation. The same row in the table indicates significant difference; the same row in the table indicates no significant difference.

[0123] As shown in Table 3, the statistical results of seed germination energy and germination rate show that there is no significant difference in germination energy between Bacillus subtilis KC-1 and ACCC 19742 treatment groups on the 3rd day. Compared with the use of microbial inoculants alone, the use of Bacillus subtilis KC-1 combined with whey protein composition (J+R) can significantly improve the seed germination energy, but Bacillus subtilis ACCC 19742 combined with whey protein composition (S+R) does not have significant effect, and the seed germination energy of whey protein composition (J+R) treatment is 2.14 times that of (S+R) treatment. The selected Bacillus subtilis KC-1 and whey protein show significant combination advantage. On the 7th day, in terms of seed germination rate, (J+R) significantly improves the germination rate by 22.03%, 13.38% and 24.14% compared with (S+R), J and S treatments.

[0124] After adding whey protein, Bacillus subtilis KC-1 has a significantly better effect on promoting the growth of corn radicle than the corresponding treatment group using microbial alone; however, Bacillus subtilis ACCC 19742 does not show this advantage. Moreover, the germination rate of (J+R) combination is the most significant, which has the best advantage. In summary, from the aspects of radicle length, germination energy and germination rate of corn, the combination of Bacillus subtilis KC-1 strain and whey protein (J+R) has the best effect, and the selected Bacillus subtilis KC-1 has significant advantage.

[0125] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the protection scope of the present application.

Claims

1. A composition characterized in that, Bacillus subtilis (Bacillus subtilis) Bacillus subtilis KC-1 and a mixed system containing whey protein; the preservation number of the Bacillus subtilis KC-1 is CGMCC NO. 35237; the mixed system containing whey protein comprises: whey protein 10-15 g / L, glucose 7.0-8.0 g / L, yeast powder 1.5-2.5 g / L, magnesium sulfate heptahydrate 0.08-0.12 g / L and sodium chloride 15-25 g / L; the pH value of the mixed system is 7.0-8.

0.

2. A process for the preparation of a composition according to claim 1, characterized in that, comprising: Bacillus subtilis KC-1 was cultured to a bacterial activity of > 10 8 CFU / mL, and then a Bacillus subtilis KC-1 bacterial solution was obtained. mixing the Bacillus subtilis KC-1 bacterial solution with a mixed system containing whey protein to obtain a composition.

3. A microbial inoculant, characterized in that, comprising the composition of claim 1.

4. The microbial inoculant of claim 3, wherein, The microbial inoculant has a bacterial activity of ≥ 10 8 CFU / mL.

5. The use of the composition of claim 1, the composition prepared by the method of claim 2, or the microbial inoculant of claim 3 or 4 in improving the drought resistance of plants; the plants are corn.

6. A method of increasing drought tolerance in maize, comprising, comprising: sowing corn seeds coated with the composition of claim 1.

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