Bacillus subtilis KC-1 as well as composition and application thereof

Abstract

The invention provides bacillus subtilis KC-1 as well as a composition and application thereof, and belongs to the technical field of microorganisms. The preservation number of the bacillus subtilis KC-1 is CGMCC (China General Microbiological Culture Collection Center) No.35237. The bacillus subtilis KC-1 can secrete IAA, has nitrogen fixation capacity and can generate siderophores. The bacillus subtilis KC-1 and whey protein are used as a composition, and the bacillus subtilis KC-1 and the whey protein jointly act to prepare the seed treatment material, so that a synergistic effect can be achieved, and corn seed germination and seedling root development are remarkably promoted. Experiments show that compared with single use of the whey protein and the bacillus subtilis KC-1, the combined use of the whey protein and the bacillus subtilis KC-1 can significantly improve the seed germination rate, the length of buds on the overground part, the length of main roots on the underground part, the total root volume, the total root surface area, the total root length and the seedling auxin concentration, and also can significantly reduce the concentration of abscisic acid in seedlings, so that the survival rate of the seedlings is improved. The germination of seeds and the growth of seedlings under drought conditions are promoted, and the drought resistance of plants is improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus subtilis KC-1, its composition, and its applications. Background Technology

[0002] Against the backdrop of an increasingly complex and severe global ecological environment, drought has become a more prominent issue, posing a serious threat to agricultural production and the stability of ecosystems. Seed germination, as a crucial stage at the beginning of a plant's life cycle, is extremely sensitive to environmental changes. Under drought stress, seed germination faces numerous obstacles, which in turn greatly restricts plant establishment and population regeneration.

[0003] Traditional strategies for promoting seed germination under drought stress, such as breeding drought-resistant varieties, optimizing irrigation techniques, and using chemical regulators, have played a role to some extent, but have also revealed many limitations. Breeding drought-resistant varieties is a lengthy process and limited by genetic resources; irrigation techniques are constrained by water resource conditions and cannot be widely applied; and chemical regulators may pose potential harm to soil and the environment. Therefore, exploring new green, efficient, and sustainable germination-promoting technologies is urgently needed.

[0004] In recent years, with the deepening research on the interaction between microorganisms and plants, the potential of microbial inoculants in enhancing plant stress resistance has gradually emerged. Plant rhizosphere growth-promoting bacteria (PGPRs) are a typical example, actively participating in plant growth regulation and stress response processes through multiple mechanisms. Some PGPRs can secrete plant hormones, such as auxins, cytokinins, and gibberellins, which can effectively break seed dormancy, enhance seed vigor, and thus promote seed germination and seedling growth. Some PGPRs also possess the ability to solubilize phosphorus and potassium, as well as fix nitrogen, significantly improving soil nutrient status and providing more sufficient nutritional support for seed germination and seedling growth. In addition, under drought stress, PGPRs can induce a series of physiological and biochemical responses in plants, such as increasing the activity of antioxidant enzymes, enhancing the plant's antioxidant defense system, effectively scavenging reactive oxygen species, and reducing the degree of membrane lipid peroxidation, thereby mitigating the damage of drought stress to plant cells; they can also promote the accumulation of osmotic regulatory substances, such as proline and betaine, regulating cell osmotic pressure, maintaining normal cell turgor pressure, and improving the plant's water retention capacity. Related studies have shown that under drought stress, wheat seeds inoculated with PGPR have a significantly higher germination rate than uninoculated seeds, and seedling root length, seedling height, and biomass also increase significantly.

[0005] While plant rhizosphere growth-promoting bacteria can play a role in crop drought resistance, their effects are often limited. In practical applications, their regulatory effect on crop growth is relatively poor. Therefore, developing a microbial agent that can enhance crop drought resistance while maintaining its high efficacy is of great significance for achieving high and stable yields of maize. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention aims to provide Bacillus subtilis KC-1 and its composition, wherein Bacillus subtilis KC-1 can synergistically work with whey protein to effectively improve the drought resistance of maize.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides a strain of Bacillus subtilis KC-1, the preservation number of which is CGMCC No.35237.

[0008] The present invention provides a composition comprising Bacillus subtilis KC-1 and whey protein as described in the above technical solution.

[0009] This invention provides a method for preparing the composition described in the above technical solution, comprising: Bacillus subtilis KC-1 was cultured until the bacterial count was ≥10. 8 After CFU / mL, Bacillus subtilis KC-1 bacterial culture was obtained; The Bacillus subtilis KC-1 bacterial culture was mixed with a mixture containing whey protein to obtain a composition.

[0010] Preferably, the mixture containing whey protein comprises: 10-15 g / L whey protein, 7.0-8.0 g / L glucose, 1.5-2.5 g / L yeast extract, 0.08-0.12 g / L magnesium sulfate heptahydrate, and 15-25 g / L sodium chloride; the pH of the mixture is 7.0-8.0.

[0011] This invention provides a microbial inoculant, comprising Bacillus subtilis KC-1 as described in the above technical solution.

[0012] Preferably, the viability of Bacillus subtilis KC-1 in the microbial agent is ≥10. 8 CFU / mL.

[0013] This invention provides a method for preparing the microbial inoculant described in the above technical solution, comprising: The Bacillus subtilis KC-1 was cultured in a culture medium to obtain a microbial inoculum.

[0014] This invention provides the application of Bacillus subtilis KC-1 described in the above technical solution, the composition described in the above technical solution, or the composition prepared by the preparation method described in the above technical solution in improving plant drought resistance; the plant is corn.

[0015] This invention provides a method for improving the drought resistance of maize, comprising: Corn seeds were coated with a composition containing Bacillus subtilis KC-1 and whey protein before sowing.

[0016] The beneficial effects of this invention are: This invention provides a strain of Bacillus subtilis KC-1, whose preservation number is CGMCC No. 35237. The Bacillus subtilis KC-1 provided by this invention can secrete IAA, has nitrogen-fixing ability, and can generate siderophores. This invention combines Bacillus subtilis KC-1 with whey protein to prepare a composition, which, when used as a seed treatment material, can synergistically promote maize seed germination and maize seedling root development. Specifically, whey protein provides a rich nutrient source for Bacillus subtilis KC-1, promoting its colonization and reproduction on the seed surface and in the rhizosphere soil, and enhancing the effect of the microbial agent; Bacillus subtilis KC-1, by improving plant nutritional status and physiological and biochemical characteristics, improves the absorption and utilization efficiency of whey protein nutrients by plants. The synergistic effect of the two significantly improves seed germination rate and seedling root indicators under drought conditions. The results of the embodiments of the present invention show that, compared with the use of whey protein and Bacillus subtilis KC-1 alone, the combined use of the two can significantly increase the germination rate of maize seeds, the length of aboveground buds, the length of underground taproots, the total root volume, the total root surface area, the total root length, and the concentration of auxin in seedlings. At the same time, it can also significantly reduce the concentration of abscisic acid in seedlings, which is beneficial to promoting the germination of maize seeds and the growth of seedlings under drought conditions and improving the drought resistance of maize.

[0017] Biological Preservation Instructions Bacillus subtilis KC-1 ( Bacillus subtilis It was deposited on July 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35237. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Phylogenetic tree diagram of Bacillus subtilis KC-1; Figure 2 The transparent halo produced by Bacillus subtilis KC-1 in the decomposition of whey protein; Figure 3 Functional effect diagram of Bacillus subtilis KC-1; Figure 4 Comparison of maize seedlings from different treatment groups after 10 days of growth in a dry soil environment; Figure 5 Figures showing the aboveground and belowground lengths of maize seedlings in different treatment groups under drought conditions. Figure 6 Root scans of maize seedlings from different treatment groups. Detailed Implementation

[0020] This invention provides a strain of Bacillus subtilis KC-1, the preservation number of which is CGMCC No.35237.

[0021] The *Bacillus subtilis* KC-1 provided by this invention was isolated from maize root soil in the arid region of Xing'an League, Inner Mongolia. The 16S rDNA sequence of *Bacillus subtilis* KC-1 is shown in SEQ ID NO.1. *Bacillus subtilis* KC-1 can secrete IAA, has nitrogen-fixing ability, and can generate siderophores. This invention combines *Bacillus subtilis* KC-1 with whey protein to prepare seed treatment materials. The two can synergistically promote maize seed germination and maize seedling root development, significantly improving seed germination rate and seedling root indicators under arid conditions. The results of the examples show that compared with using whey protein or *Bacillus subtilis* KC-1 alone, the combined use of the two can significantly increase seed germination rate, aboveground bud length, underground taproot length, total root volume, total root surface area, total root length, and seedling auxin concentration, while also significantly reducing the concentration of abscisic acid in seedlings. This is beneficial for promoting maize seed germination and seedling growth under arid conditions, and improving the drought resistance of maize.

[0022] This invention provides a microbial inoculant comprising Bacillus subtilis KC-1 as described in the above-mentioned technical solution. As an optional embodiment of this invention, the viability of Bacillus subtilis KC-1 in the microbial inoculant is ≥10⁻⁶. 8 CFU / mL, or 10 8 CFU / mL.

[0023] This invention provides a method for preparing the microbial inoculant described in the above-mentioned technical solution, comprising: culturing Bacillus subtilis KC-1 on a culture medium to obtain the microbial inoculant. This invention does not specifically limit the type of culture medium; any conventional culture medium in the art that can promote the good growth of Bacillus subtilis KC-1 can be used. As an optional embodiment of this invention, the culture medium can be LB medium, TSA medium, or whey protein medium. As an optional embodiment of this invention, the composition of the whey protein medium can be: whey protein 10-15 g / L, glucose 7.0-8.0 g / L, yeast extract 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 whey protein medium can be 7.0-7.5. In this invention, 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.

[0024] The present invention provides a composition comprising Bacillus subtilis KC-1 and whey protein as described in the above technical solution.

[0025] This invention provides a method for preparing the composition described in the above technical solution, comprising: Bacillus subtilis KC-1 was cultured until the bacterial count was ≥10. 8 After CFU / mL, Bacillus subtilis KC-1 bacterial culture was obtained; The composition was obtained by mixing the Bacillus subtilis KC-1 bacterial culture with a mixture containing whey protein.

[0026] This invention does not specifically limit the cultivation method of Bacillus subtilis KC-1; any conventional cultivation method in the art can be used. As an optional embodiment of this invention, the cultivation method of Bacillus subtilis KC-1 can be: culturing Bacillus subtilis KC-1 in a culture medium to obtain a Bacillus subtilis KC-1 bacterial suspension. As an optional embodiment of this invention, the culture medium can be LB medium or TSA medium; the cultivation time can be 1-3 days, or 1, 2, or 3 days; the cultivation temperature can be 25℃-30℃, or 30℃; the cultivation process preferably involves shaking; the shaking speed can be 180 rpm. This invention preferably cultivates the bacteria to an OD value of 180 rpm in the fermentation system. 600nm The concentration was set to 0.8~1.0 to obtain a Bacillus subtilis KC-1 culture medium. After obtaining the Bacillus subtilis KC-1 culture medium, the present invention preferably adjusts the effective bacterial activity of the Bacillus subtilis KC-1 culture medium to ≥10⁻⁶. 8CFU / mL, or 10 8 ~10 9 CFU / mL was used to obtain Bacillus subtilis KC-1 bacterial suspension.

[0027] After obtaining the Bacillus subtilis KC-1 bacterial culture, the present invention mixes the Bacillus subtilis KC-1 bacterial culture with a mixture containing whey protein to obtain a composition. As an optional embodiment of the present invention, the mixture containing whey protein comprises: 10-15 g / L whey protein, 7.0-8.0 g / L glucose, 1.5-2.5 g / L yeast extract, 0.08-0.12 g / L magnesium sulfate heptahydrate, and 15-25 g / L sodium chloride; the pH value of the mixture is 7.0-8.0. In the present invention, the composition of the mixture containing whey protein can also be 10.0 g / L whey protein, 7.5 g / L glucose, 2.0 g / L yeast extract, 0.1 g / L magnesium sulfate heptahydrate, and 20.0 g / L sodium chloride; the pH value of the mixture is 7.0. The present invention does not specifically limit the mixing method; conventional mixing methods in the art can be used. This invention combines Bacillus subtilis KC-1 with whey protein to prepare a composition, which, when used as a seed treatment material, synergistically promotes maize seed germination and maize seedling root development. Whey protein provides a rich nutrient source for Bacillus subtilis KC-1, promoting its colonization and reproduction on the seed surface and in the rhizosphere soil, thus enhancing the effectiveness of the microbial agent. Bacillus subtilis KC-1, on the other hand, improves plant nutrition and physiological and biochemical characteristics, increasing the efficiency of plant absorption and utilization of whey protein nutrients. The synergistic effect of these two components significantly improves the germination rate of maize seeds and the root indicators of seedlings under drought conditions.

[0028] This invention provides the application of Bacillus subtilis KC-1 described in the above-described technical solutions, the composition described in the above-described technical solutions, or the composition prepared by the preparation method described in the above-described technical solutions in improving plant drought resistance. As an optional embodiment of this invention, the plant can be corn.

[0029] The present invention provides a method for improving the drought resistance of maize, comprising: coating maize seeds with a composition containing Bacillus subtilis KC-1 and whey protein before sowing.

[0030] As an optional embodiment of the present invention, the composition can be the composition described in the above technical solution. Before coating the seeds, the present invention preferably disinfects the seeds. The present invention does not specifically limit the disinfection method; any conventional disinfection method in the art can be used. As an optional embodiment of the present invention, the disinfection method can be: mixing the seeds with a 1% sodium hypochlorite solution for disinfection; the disinfection time can be 10 minutes. After disinfection, the present invention rinses the obtained seeds and then coats them. In the present invention, the rinsing is preferably done with distilled water; the rinsing can be repeated 3 times. After rinsing, the present invention obtains disinfected seeds. After obtaining the disinfected seeds, the present invention coats the obtained seeds. The present invention does not specifically limit the coating method; any conventional coating method in the art can be used. As an optional embodiment of the present invention, the coating method can be to completely immerse the seeds in the seed treatment material for coating. In the present invention, the coating temperature can be 20℃~30℃, or 30℃; the coating time can be 1h~2h, or 2h. After the coating process is completed, the coated seeds can be planted according to this invention. This invention does not impose any particular limitation on the planting method; any conventional planting method in the art can be used.

[0031] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1 Strain screening and identification 1. Experimental Materials Soil samples: Maize rhizosphere soil samples from the arid region of Xing'an League, Inner Mongolia, were stored at 4℃ and transported back to the laboratory.

[0033] Whey protein: Maclean's reagent.

[0034] Test culture medium: 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. Sterilize at 121℃ for 15 min, adjust pH to 7.0. Add 18 g of agar to the solid medium.

[0035] LB medium (g / L): tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, adjust pH to 7.4, sterilize at 121℃ for 20 min. For solid medium, add 18 g of agar.

[0036] TSA medium (g / L): tryptone 15.0 g / L, soybean peptone 5.0 g / L, sodium chloride 5.0 g / L, adjust pH to 7.3, sterilize at 121℃ for 20 min. For solid medium, add 18 g of agar.

[0037] 0.9% physiological saline: NaCl 0.9g, distilled water 99.1mL, autoclaved at 121℃ for 20 min.

[0038] 2. Screening of strains (1) Take 10 g of soil from the rhizosphere of maize in the arid region of Xingan League, Inner Mongolia, put it into 90 mL of sterile physiological saline, place it in a constant temperature shaker to mix, and let it stand for 30 min to obtain a preliminarily activated bacterial solution.

[0039] (2) Take the preliminarily activated bacterial solution from step (1) and inoculate it into 100 mL of new whey protein medium at a volume ratio of 10% and incubate it at 180 rpm and 30℃ for 48 h to obtain activated and enriched bacterial solution. (3) Take the bacterial culture from step (2) and inoculate it into 100 mL of new whey protein medium at a volume ratio of 10% and incubate it at 180 rpm and 30°C for 72 h. (4) Take the bacterial culture from step (3) and inoculate it into 100 mL of new whey protein medium at a volume ratio of 10% and incubate it at 180 rpm and 30℃ for 24 h. (5) Take 10 mL of the cultured bacterial solution from step (4), add glass beads and shake, then dilute with sterile water to 10 mL. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 100 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. (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.

[0040] (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.

[0041] 3. Strain identification 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.

[0042] The 16S rDNA of Bacillus subtilis KC-1 is shown in SEQ ID NO.1.

[0043] SEQ ID NO.1:

[0044] Example 2 Functional testing of Bacillus subtilis KC-1 Test materials: Siderophore medium: CAS solution: 60 mg Chrome Azurol S and 100 mg cetyltrimethylammonium bromide (CTAB) dissolved in 100 mL distilled water, stored protected from light. Basal medium: (NH4)2SO4 2 g·L -1 0.5 g·L⁻¹ KH₂PO₄ -1 MgSO4·7H2O 0.2 g·L -1 NaCl 0.1 g·L -1 10 g·L glucose -1 15 g·L agar -1 Sterilize at pH 7.0-7.2 at 121℃ for 20 min. Cool the basal medium to about 50℃, slowly add CAS solution (avoid precipitation), shake well, and pour into plates.

[0045] Ashubei medium: mannitol 10 g·L -1 0.2 g·L⁻¹ KH₂PO₄ -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 15 g·L agar -1 Sterilize at pH 7.0~7.2 at 121℃ for 20 min.

[0046] Functional tests were performed on Bacillus subtilis KC-1 to determine its ability to secrete auxin, fix nitrogen, and produce siderophores. The methods are as follows: IAA Functional Test: The bacterial strain was inoculated with a solution containing 0.1 g·L⁻¹ L-tryptophan. -1 In LB broth, each strain was cultured in triplicate at 30°C and 180 rpm for one day. The bacterial suspension was then added to test tubes along with an equal volume of Salkowski colorimetric solution. A mixture of uninoculated LB broth and an equal volume of the colorimetric solution served as a control. The test tubes were incubated at room temperature in the dark for 30 minutes. A pink color indicated a positive result, suggesting the secretion of IAA; a darker color indicated stronger secretion. No color change indicated a negative result, suggesting the inability to secrete IAA.

[0047] Inoculate the bacterial strain onto Assumption nitrogen-fixing agar plates using an inoculation loop and incubate at 28°C for 4–7 days, then observe. Large, raised, slimy colonies indicate strains capable of nitrogen fixation.

[0048] Inoculate the bacterial strain onto a siderophore-producing medium plate using an inoculation loop, and incubate at 28°C for 4–7 days. Observe the medium for orange iron precipitation bands around the target colonies; the presence of such bands indicates that the strain has the ability to produce siderophores.

[0049] Functional effects of Bacillus subtilis KC-1 are shown in the figure. Figure 3 As shown. Figure 3 In the diagram, A represents the results of the IAA functional test, with the left tube in A being the control and the right tube representing the strain's IAA production effect; B represents the strain's siderophore production capacity; and C represents the strain's nitrogen fixation capacity. Figure 3 The results showed that Bacillus subtilis KC-1 was positive for IAA production when the color turned pink, indicating that it could secrete IAA. In Assumption nitrogen-fixing medium, Bacillus subtilis KC-1 strains appeared as large, raised, mucous-type colonies, indicating that they had nitrogen-fixing capabilities. In siderophore-producing medium, Bacillus subtilis KC-1 colonies had an orange iron precipitation band around them, proving that the strain had the ability to produce siderophores.

[0050] Example 3: Microbial seed coating promotes seed germination under drought conditions Test soil: Taken from the Shunyi Agricultural Environmental Experiment Base of the Chinese Academy of Agricultural Sciences (N40°10′, E116°92′). The test soil was loam with a pH of 8.1, organic matter of 13.18 g / kg, total nitrogen of 1.03 g / kg, available phosphorus of 13.69 mg / kg, and available potassium of 171.82 mg / kg. After air drying, it was sieved through a 10 mm sieve.

[0051] The test crop was maize (Zea mays L.), variety Zhengdan 958.

[0052] Coating materials: whey protein culture medium, Bacillus subtilis KC-1.

[0053] step: Fill a flowerpot with a top diameter × bottom diameter × height of 16 cm × 13 cm × 17.5 cm with soil, water it to keep the soil volume moisture content at 12%~15%, reaching a slightly dry level, and place it in the greenhouse of the Institute of Environmental Development, Chinese Academy of Agricultural Sciences.

[0054] Preparation of microbial inoculum: Single colonies of Bacillus subtilis KC-1 were picked and cultured on LB liquid medium until the OD600nm value reached 0.8-1.0. The effective viable count of the fermentation broth was then adjusted to 10-1. 8CFU / mL was used to obtain Bacillus subtilis KC-1 bacterial suspension, which is the microbial inoculant.

[0055] The whey protein culture medium was mixed with the prepared microbial agent Bacillus subtilis KC-1 bacterial solution at a volume ratio of 10:1 to obtain the seed treatment material, which will be referred to as the composition in the following experiments.

[0056] Before sowing, corn seeds need to be disinfected by soaking them in a 1% sodium hypochlorite solution for 10 minutes, followed by rinsing them three times with sterile distilled water to obtain disinfected seeds. The experiment consisted of four treatments: CK: control group, seeds disinfected with distilled water; R: seeds disinfected with whey protein culture medium; J: seeds with an effective viable count of 10-1. 8 Seeds were disinfected by treatment with CFU / mL Bacillus subtilis KC-1 bacterial solution; J+R: seeds were disinfected by treatment with the combined solution. For each treatment group, the amount of distilled water or solution used should be sufficient to completely submerge the seeds. The treatment temperature for each treatment group was 20–30°C, and the treatment time was 2 hours.

[0057] Each treatment group selected plump seeds for planting, sowing 50 seeds per pot. During the planting period, each treatment group was watered to maintain a soil moisture content of 12%–15% (the conventional moisture content for maize planting is 60%–80% of field capacity). Three replicates were set up, with all pots randomly placed in the greenhouse and rotated periodically to minimize the impact of environmental differences. Throughout the experiment, the greenhouse temperature was controlled diurnally, maintaining a daytime temperature of 25°C and a nighttime temperature of 20°C, a photoperiod of 12 h, and a light intensity of 240 μmol·m⁻¹. -2 ·s -1 .

[0058] Result determination: Seed germination rates were recorded on days 3 and 7 after sowing. On day 10 after sowing, five corn plants were collected from each replicate, and the lengths of the aboveground and underground parts of the corn seedlings were measured. The complete corn root system was scanned and analyzed using a WinRHIZO (Regent, Canada) scanner, following the manufacturer's recommended methods for root morphology analysis. Hormone levels in the corn seedlings were determined using the following methods: After different seed treatments, maize seedlings were collected 10 days after sowing, rapidly frozen in liquid nitrogen, and ground into powder. Plant hormones (IAA, ABA) in the seedlings were detected by UPLC-MS. 50 mg of sample powder was accurately weighed and placed in a 2 mL centrifuge tube. 500 μL of extraction buffer (IPA:H₂O:HCl = 2:1:0.002) was added, vortexed for 10 s, and then shaken at 4℃ and 900 rpm for 30 min. 1 mL of extraction buffer (CHCl₃) was added, vortexed for 10 s, and then shaken at 4℃ and 900 rpm for 30 min. The mixture was then centrifuged at 4℃ and 14000 rpm for 5 min, resulting in two phases. 1.2 mL of the lower layer was transferred and dried under nitrogen at room temperature. The mixture was reconstituted with 0.1 mL of MeOH, shaken at 4℃ and 900 rpm for 20 min, centrifuged at 14000 rpm for 5 min, and the supernatant was collected, filtered through a 0.1 μm filter membrane, and placed in a vial for analysis. Ultra-high pressure liquid chromatography (UHPLC) was used. Mobile phase A was water (0.05% formic acid), and mobile phase B was acetonitrile (0.05% formic acid). Column temperature was 35℃, sample temperature was 15℃, and mobile phase flow rate was 0.3 mL / min. The sample gradient elution program is shown in Table 1 below.

[0059] Table 1 Sample gradient elution procedure

[0060] Mass spectrometry analysis was performed using a 5500 Qtrap-MS system (AB SCIEX). Ion source: HESl, spray voltage (-): 3000V, capillary temperature: 320℃, sheath gas: 30arb, auxiliary gas: 10arb, spare gas: 5arb, probe heating temperature: 350℃, S-lens RF level: 55%.

[0061] FULL MS-SIM: Resolution: 70,000 @ m / z 200, Automatic Gain Control Target Value: 3e 6 Ion counts, maximum ion implantation time: 100ms, scan range: 50~750m / z.

[0062] The aboveground and belowground growth of corn seedlings in each treatment group during the seedling stage is as follows: Figures 4-6 As shown in Table 2. Figure 4 Comparison of maize seedlings from different treatment groups after 10 days of growth in a drought environment. Figure 5 The figures show the aboveground and belowground lengths of maize seedlings in different treatment groups under drought conditions. ns indicates that the differences between different treatment groups are significant, while ns indicates that the differences between different treatment groups are not significant. Figure 6 Root scans of maize seedlings from different treatment groups.

[0063] Table 2. Indicators of maize seedlings under different treatment groups under drought conditions

[0064] Note: Data in the table are mean ± standard deviation. Different letters in the same row indicate significant differences; the same letter in the same row indicates no significant differences.

[0065] Table 2 shows the seed germination rate statistics. On day 3, the germination rate of seeds treated with the composition of this invention was 62%, the germination rate of seeds treated with whey protein alone was 30%, the germination rate of seeds treated with microbial inoculant alone was 18%, and the germination rate of the control group was 12%. On day 7, the germination rate of seeds treated with the composition of this invention was 96%, the germination rate of seeds treated with whey protein alone was 92%, the germination rate of seeds treated with microbial inoculant alone was 82%, and the germination rate of the control group was 60%.

[0066] From Table 2 and Figures 4-6 It was found that the aboveground and underground lengths of maize seedlings treated with the composition of the present invention were significantly longer than those of the control group, with the aboveground length being 42.5% (p<0.01) and the underground length being 65.3% (p<0.01) longer. The aboveground and underground lengths of maize seedlings treated with microbial agents alone were also significantly longer than those of the control group, with the aboveground length being 34.4% (p<0.05) and the underground length being 55.3% (p<0.01) longer. The underground length of maize seedlings treated with whey protein alone was significantly longer than that of the control group, with the underground length being 45.5% (p<0.01) longer. The aboveground length was not significantly different from the control group, but was 13.5% longer.

[0067] From Table 2 and Figures 4-6 The results showed that, under drought conditions, the total root length, total root surface area, and total root volume of maize seedlings treated with the composition of this invention were significantly higher than those treated with the control (CK) treatment, promoting maize root development. Simultaneously, compared with the control (CK), the average auxin content of maize seedlings treated with J+R and J treatments significantly increased by 105.16% and 96.77%, respectively, while the average abscisic acid content significantly decreased by 83.42% and 53.41%, respectively. This is because Bacillus subtilis KC-1 activates the biosynthesis of auxin in the host, affecting root length, volume, and surface area, leading to changes in root structure and thus increasing water absorption capacity during drought.

[0068] Example 4: Comparison of the application of Bacillus subtilis KC-1 with other microorganisms in seed coating agents Test crop: maize ( Zea mays L.), the variety is Zhengdan 958.

[0069] Coating materials: whey protein culture medium, Bacillus subtilis KC-1, Bacillus subtilis (ACCC 19742, China Agricultural Microbial Culture Collection Center).

[0070] Microbial culture medium: LB liquid medium.

[0071] Drought stress simulation reagent: PEG-6000.

[0072] step: Preparation of microbial inoculants: Single colonies of Bacillus subtilis KC-1 and Bacillus subtilis ACCC 19742 were picked and cultured on LB liquid medium until the OD600nm value was 0.8-1.0. The effective viable count of the fermentation broth was then adjusted to 10-1. 8 CFU / mL, Bacillus subtilis KC-1 bacterial suspension and Bacillus subtilis ACCC 19742 bacterial suspension were obtained respectively.

[0073] The whey protein culture medium was mixed with the prepared microbial agents Bacillus subtilis KC-1 bacterial solution and Bacillus subtilis ACCC 19742 bacterial solution at a volume ratio of 10:1 to obtain seed treatment materials, which were referred to in the following experiments as the Bacillus subtilis KC-1 and whey protein composition and the Bacillus subtilis ACCC 19742 and whey protein composition.

[0074] Corn seeds were disinfected by soaking them in a 1% sodium hypochlorite solution for 10 minutes, followed by rinsing them three times with sterile distilled water to obtain disinfected seeds. The experiment consisted of four treatments, with J being an effective viable count of 10-1. 8 Seeds treated with Bacillus subtilis KC-1 bacterial solution at CFU / mL for disinfection; J+R: Seeds treated with a combination of Bacillus subtilis KC-1 and whey protein for disinfection; S: Effective viable count of 10-1 8 Seeds were disinfected by treatment with a CFU / mL solution of Bacillus subtilis ACCC 19742; seeds were also disinfected by a combination of Bacillus subtilis ACCC 19742 and whey protein (S+R). The amount of reagent used in each treatment group was sufficient to completely submerge the seeds in the solution. The treatment temperature for each group was 20–30°C, and the treatment time was 2 hours.

[0075] The petri dish filter paper method was used, with each treatment replicated three times, each replicate containing 50 corn seeds. Petri dish preparation: Line a sterile petri dish with two layers of sterile filter paper and add 10-15 mL of 10% PEG-6000 solution (keep the filter paper moist but without water accumulation). Seed placement: Place corn seeds from different treatments evenly on the filter paper, avoiding overlapping; Culture conditions: Placed in an artificial climate chamber, with a temperature of 25℃ and a light / dark cycle of 12h / 12h (240 μmol·m⁻¹). -2 ·s -1 ), relative humidity 60%, continuously cultured for 7 days; Indicator determination: Record the number of seed germinations daily (germination is defined as the radicle breaking through the seed coat ≥ 2 mm). After 7 days of culture, determine the following indicators: Germination rate (%) = (Number of seeds germinating within 7 days / Number of seeds tested) × 100; Germination potential (%) = (Number of seeds germinating within 3 days / Number of seeds tested) × 100; Radicle length (cm): After 7 days, 10 germinating seedlings were randomly selected, and the radicle length was measured using vernier calipers. The average value was determined. The results are shown in Table 3.

[0076] Table 3. Effects of Bacillus subtilis KC-1 and other microorganisms as seed treatment materials on growth indicators of maize seedlings under drought conditions.

[0077] Note: Data in the table are mean ± standard deviation. Different letters in the same row indicate significant differences; the same letter in the same row indicates no significant differences.

[0078] Table 3 shows that, based on the statistical results of seed germination potential and germination rate, on day 3, there was no significant difference in germination potential between the groups treated with Bacillus subtilis KC-1 and ACCC 19742 alone. Compared to using microbial agents alone, the combination of Bacillus subtilis KC-1 and whey protein (J+R) significantly improved seed germination potential, but the combination of Bacillus subtilis ACCC 19742 and whey protein (S+R) did not show a significant effect. The germination potential of seeds treated with the whey protein combination (J+R) was 2.14 times that of the (S+R) treatment. The screened Bacillus subtilis KC-1 and whey protein showed significant combined advantages. On day 7, in terms of seed germination rate, the (J+R) treatment significantly increased the germination rate by 22.03%, 13.38%, and 24.14% compared to the (S+R), J, and S treatments, respectively.

[0079] Bacillus subtilis KC-1, when supplemented with whey protein, significantly promoted maize radicle growth compared to the corresponding treatments using the microorganism alone; however, Bacillus subtilis ACCC 19742 did not exhibit this advantage. Furthermore, the (J+R) combination showed the most significant germination rate, demonstrating the best advantage. In summary, considering maize radicle length, germination potential, and germination rate, the combination of Bacillus subtilis KC-1 strain and whey protein (J+R) showed the best effect, and the screened Bacillus subtilis KC-1 strain exhibited significant advantages.

[0080] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis KC-1, characterized in that, The Bacillus subtilis KC-1 has the accession number CGMCC No. 35237.

2. A composition, characterized in that, It includes Bacillus subtilis KC-1 as described in claim 1 and whey protein.

3. A method for preparing the composition according to claim 2, characterized in that, include: Bacillus subtilis KC-1 was cultured until the bacterial count was ≥10. 8 After CFU / mL, Bacillus subtilis KC-1 bacterial culture was obtained; The Bacillus subtilis KC-1 bacterial culture was mixed with a mixture containing whey protein to obtain a composition.

4. The preparation method according to claim 3, characterized in that, The mixture containing whey protein comprises: 10-15 g / L whey protein, 7.0-8.0 g / L glucose, 1.5-2.5 g / L yeast extract, 0.08-0.12 g / L magnesium sulfate heptahydrate, and 15-25 g / L sodium chloride; the pH of the mixture is 7.0-8.

0.

5. A microbial inoculant, characterized in that, Includes Bacillus subtilis KC-1 as described in claim 1.

6. The microbial agent according to claim 5, characterized in that, The microbial agent contains ≥10% viable counts of Bacillus subtilis KC-1. 8 CFU / mL.

7. The method for preparing the microbial inoculant according to claim 6, characterized in that, include: The Bacillus subtilis KC-1 was cultured in a culture medium to obtain a microbial inoculum.

8. The application of the Bacillus subtilis KC-1 of claim 1, the composition of claim 2, or the composition prepared by the preparation method of claim 3 or 4 in improving plant drought resistance; wherein the plant is maize.

9. A method for improving the drought resistance of maize, characterized in that, include: Corn seeds were coated with a composition containing Bacillus subtilis KC-1 and whey protein before sowing.

Images