Saline-alkali-tolerant growth-promoting bacillus subtilis and application thereof

Abstract

The invention provides a bacillus subtilis strain KY559 capable of resisting saline and alkaline and promoting growth and application of the bacillus subtilis strain KY559, and relates to the technical field of microorganisms and application of the microorganisms. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.35395. The bacillus subtilis KY559 strain disclosed by the invention has the capabilities of resisting salt and alkali and promoting plant growth, and also has the functions of decomposing fat, starch, cellulose and the like. The strain is excellent in performance and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbiology and its application technology, and in particular to a salt-tolerant and alkali-resistant Bacillus subtilis strain and its application. Background Technology

[0002] Saline-alkali land refers to soil where the electrical conductivity of saturated extracts from the root zone of plants exceeds 4 mS / cm and the content of soluble salt ions such as sodium, potassium, sulfate, and carbonate exceeds the standards stipulated in land use regulations, thus affecting the normal growth of crops. Improving and utilizing saline-alkali land can improve low- and medium-yield fields, increase arable land area, and reduce land abandonment and resource waste, which is of great significance for maintaining ecological security and food supply security.

[0003] Increased soluble salt content in soil affects soil structure (Rengasamy P et al., 1991). Salinized soils have poor aggregate structure, poor water permeability and aeration, and are prone to soil compaction. Furthermore, soil salinization not only increases soil pH and reduces soil organic matter (WenWZ et al., 2018), but also reduces internal porosity, decreases air contact area, slows microbial metabolic activity, and reduces soil nutrient activation. Consequently, the activities of sucrase, urease, and alkaline phosphatase, which are involved in nutrient cycling and metabolism, decrease with increasing salinity (Slimane M, 2020). It also reduces soil water content, organic carbon, nitrogen content, exchangeable potassium content, and the number of soil microorganisms. This leads to decreased soil fertility, restricts plant growth, and causes crop yield reduction. The effects on plant growth under high osmotic pressure are called salt stress (one of the most severe abiotic stresses experienced by plants during growth and development). Salt stress severely affects plant growth and development through multiple mechanisms, including osmotic stress, ion toxicity, and soil structure deterioration. Excessive salt content reduces the activity of PEP and RuBP carboxylases in plants, destroying chlorophyll and hindering its biosynthesis. Stomatal closure reduces photosynthetic rate, leading to difficulty in root water absorption, cell dehydration, and physiological drought. Even with sufficient soil moisture, plants may still wilt, producing large amounts of abscisic acid (ABA), which negatively impacts plant growth and development, ultimately affecting crop yield (Li Baozhu et al., 2012). Salt stress also significantly affects seed germination rate, germination time, and emergence rate. Studies have shown that as NaCl concentration increases, seed germination rate and emergence rate decrease. Furthermore, high NaCl concentrations delay embryo emergence and inhibit embryo growth; the higher the concentration, the more pronounced this inhibitory effect (Xin Longfei et al., 2022). Excessive soluble salts in the soil compete with essential mineral nutrients for plants. Excessive absorption of salt ions can cause ion toxicity, reducing the absorption of phosphorus, potassium, calcium, and other nutrients. Because of the high NaCl concentration in the soil... +If the concentration is too high, excessive absorption by plants can lead to a reaction with potassium. + Competitive binding site disrupts K + / Na + It balances and interferes with enzyme activity, affecting photosynthesis and inhibiting NO3. - Absorption leads to nitrogen metabolism disorders, etc. (Ahanger MA et al., 2017). It can be seen that saline soil has an extremely serious impact on plant growth, greatly reducing crop quality and yield.

[0004] As an important usable land resource, the improvement and management of saline-alkali soil has become a focus of attention. Improvement measures include water conservancy, agriculture, chemicals, and biology. Traditional methods of improving saline-alkali land, such as topsoil improvement, irrigation and drainage, and the addition of soil conditioners, are costly, ineffective, and prone to secondary soil pollution. Deep plowing and crop rotation / fallow can break up soil compaction, loosen the soil layer, and promote the even distribution of salts; however, if the soil is not covered afterward, salts may rise again due to evaporation. Furthermore, crop rotation cycles are long, with low short-term economic benefits, impacting land use. Chemical improvement methods, such as applying soil conditioners and acid-base neutralization, have rapid effects, but long-term use can damage soil structure, produce harmful gases, easily lead to soil acidification, and are costly, potentially disrupting the ecological balance. Microbial improvement of saline-alkali land utilizes salt-tolerant microorganisms to decompose organic matter, reduce soil salinity, and improve the soil environment. Because microorganisms can activate nutrients and improve soil structure through their own life activities, they can also enhance plant resistance and disease resistance, decompose and accelerate the utilization of nutrients that plants cannot directly absorb. Under salt stress, microorganisms can produce antioxidant enzymes and secrete plant hormones to promote ion homeostasis and maintain a relative balance of osmotic pressure. Microbial metabolites typically contain a large amount of organic acids, whose secretion can lower the pH of the rhizosphere soil, promote plant growth, and improve the soil environment. In addition, the secondary metabolites of microorganisms can enhance the salt tolerance of crops, acting as immunizing agents to improve crop resistance to salinity and alkali. Microbial remediation technology combined with other soil improvement techniques can very effectively protect crop yields under saline-alkali conditions. Microbial remediation is characterized by its green, long-lasting, energy-saving, excellent improvement effect, and strong persistence, and is currently the most widely used method. Therefore, screening for salt-tolerant strains is crucial for the improvement of saline-alkali land.

[0005] In recent years, the application of microbial technology in saline-alkali soil remediation has received increasing attention and strengthening, mainly focusing on research into microbial enhancement of plant salt tolerance. Studies have shown that salt-tolerant microorganisms can improve the rhizosphere environment of plants, reduce the inhibitory effect of salt on crop growth in saline-alkali soils, and thus improve saline-alkali soils. Utilizing salt-tolerant microorganisms to produce inoculants as plant protectants in saline-alkali soils reduces the inhibitory effect of saline-alkali soil on plants, allowing plants to easily absorb nutrients from the soil and promote growth, which is of great significance for improving saline-alkali soils and increasing grain yields. Currently, the main salt-tolerant strains being screened include those from the genera *Rhizobium*, *Pseudomonas*, and *Bacillus*. Furthermore, *Bacillus* strains possess characteristics such as growth promotion, high temperature tolerance, and acid and alkali tolerance, making them dominant species in soil ecosystems and possessing great potential for agricultural applications. Currently, there are few reports on salt-tolerant Bacillus species. Li Liyan et al. (2022) isolated a Bacillus curvatureus YP2 from the roots of halophytes, which can promote oat growth and improve its salt resistance under salt stress conditions with a maximum tolerance of 10% NaCl. Zhang Xueyan et al. (patent number: CN202310090748.4) screened a Bacillus subtilis strain from saline-alkali soil in Ningxia, a salt-tolerant bacterium B21 with a maximum salt concentration tolerance of 14%, which can significantly improve the salt tolerance and growth promotion ability of cucumber under salt stress and has an inhibitory effect on pathogenic fungi. Liang Xuejie et al. (patent number: CN202311772611.3) screened a Bacillus subtilis strain from corn farmland soil, a salt-tolerant bacterium GN504 with a maximum salt concentration tolerance of 12%. Therefore, the maximum salt tolerance of the currently reported growth-promoting Bacillus is between 10% and 14%. However, there are no reports to date on Bacillus subtilis that can tolerate higher salt concentrations and promote growth.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] This disclosure aims to at least partially address one of the technical problems in the related art. Therefore, the object of the present invention is to provide a novel microbial strain, namely Bacillus subtilis strain KY559 and its applications, and to provide a method for promoting crop growth under environmental stress.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides a salt- and alkali-tolerant Bacillus subtilis strain KY559, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 35395 and deposit date of July 25, 2025.

[0010] The inventors of this application selected 575 Bacillus strains from their own microbial library. By testing their salt tolerance, they found that 306 strains were tolerant to 5% (w / v%) NaCl, 183 strains were tolerant to 10% NaCl, and 8 strains were tolerant to 15% NaCl. However, only one strain, Bacillus subtilis KY559, showed the best plant protection effect when planted in saline-alkali soil. Further research on this strain revealed that it could tolerate a maximum salt concentration of 17% and a maximum alkali concentration of pH 12, and effectively promoted plant seed germination and growth. Furthermore, this strain also had the ability to degrade starch, cellulose, and lipids.

[0011] In one embodiment, the 16S rDNA sequence of the Bacillus subtilis strain KY559 is the sequence shown in SEQ ID No. 1. The inventors of this application performed 16S DNA sequencing on the above-mentioned strain KY559 and compared it with the NCBI database. The results showed that Bacillus subtilis has 100% high homology, confirming it as a salt-tolerant, alkali-tolerant, growth-promoting Bacillus subtilis strain KY559.

[0012] In a second aspect, the present invention provides a microbial composition containing the aforementioned Bacillus subtilis strain KY559 or a culture of Bacillus subtilis strain KY559.

[0013] In one specific implementation, the microbial composition is in liquid or solid form.

[0014] In one specific embodiment, the total viable count of the Bacillus subtilis strain KY559 contained in the microbial composition is at least 1 × 10⁻⁶. 7 cfu·mL -1 Or 1×10 7 cfu·g -1 Preferably, it is at least 1×10 8 cfu·mL -1 Or 1×10 8 cfu·g -1 .

[0015] In a third aspect, the present invention provides the use of the aforementioned Bacillus subtilis strain KY559 or the aforementioned microbial composition in the following:

[0016] (a) for improving saline-alkali land; (b) for promoting plant growth; (c) for improving plant salt tolerance; and / or (d) for breaking down starch, cellulose and / or fats.

[0017] In one specific implementation, promoting plant growth includes promoting plant growth in saline-alkali or non-saline-alkali environments.

[0018] In one specific implementation, promoting plant growth includes promoting the growth of maize in a saline-alkali environment.

[0019] In a fourth aspect, the present invention provides a method for promoting crop growth under environmental stress, the method comprising applying the aforementioned Bacillus subtilis strain KY559 or the aforementioned microbial composition to the seeds of a plant or to a substrate in which the plant is grown.

[0020] In one specific implementation, the stress environment includes salt stress and / or alkali stress.

[0021] In one specific implementation, the plant includes at least one of corn, soybean, and wheat.

[0022] The Bacillus subtilis strain provided in this application, named KY559, is deposited 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; the deposit date is July 25, 2025, and the accession number is CGMCC No. 35395. It was confirmed as a viable strain by the collection center on July 25, 2025.

[0023] Compared with the prior art, the beneficial effects of this application are as follows:

[0024] (1) The strain of this application has excellent tolerance to high salt and high alkali, with a salt concentration of up to 17% (w / v%) and a maximum alkali concentration of pH=12. It can grow well in saline-alkali soil environment, and therefore can be used to reduce the soluble salt content in saline-alkali land or improve primary and secondary saline-alkali soils such as farmland and forest land.

[0025] (2) The strain of this application can effectively promote plant growth, not only promoting plant growth in non-saline-alkali environments, but also reducing the toxic effects of saline-alkali stress on plants. In particular, it can also promote plant growth and development in saline-alkali environments.

[0026] (3) The strains of the present invention or the microbial compositions containing them are of great significance for improving the stress resistance and growth of crops and improving saline-alkali soil, and have wide application value.

[0027] (4) This invention also provides a method for promoting crop growth under environmental stress. Currently, there are relatively few reports on the role of microorganisms in promoting plant growth in salt-alkali tolerant environments (especially high-salt and high-alkaline environments). This invention provides a new way to improve plant salt and alkali tolerance, significantly improve plant growth status, and increase yield and quality. Attached Figure Description

[0028] Figure 1 The germination of corn seeds after soaking them in bacterial solutions of different strains (KY559, KY53, KY40, KY207, KY151 and 92068) was compared with that of commercial multifunctional strain 92068 as a control.

[0029] Figure 2 The germination of corn seeds after soaking them in bacterial solutions of different strains (KY1178 and 92068) was compared with that of the commercial multifunctional strain 92068 as a control.

[0030] Figure 3 The germination of corn seeds after soaking them in bacterial solutions of different strains (KY1188 and 92068) was compared with that of commercial multifunctional strain 92068 as a control.

[0031] Figure 4 The germination of corn seeds after soaking them in bacterial solutions of different strains (KY1190 and 92068) was compared with that of commercial multifunctional strain 92068 as a control.

[0032] Figure 5 Germination and emergence of corn soaked in KY559 bacterial solution, 92068 bacterial solution and CK (water) in saline-alkali soil (EC value ≈ 6.95 ms / cm);

[0033] Figure 6 Germination and emergence of corn soaked in KY559 bacterial solution, 92068 bacterial solution and CK (water) in saline-alkali soil (EC value ≈ 10.3 ms / cm);

[0034] Figure 7 The growth of strain KY559 under different concentrations of NaCl;

[0035] Figure 8 The growth of strain KY559 at different pH values;

[0036] Figure 9 The ability of strain KY559 to hydrolyze starch was determined on plates with 15% NaCl and pH=9.

[0037] Figure 10 The ability of strain KY559 to hydrolyze lipids on plates with 15% NaCl and pH=9 was determined.

[0038] Figure 11 The ability of strain KY559 to decompose CMC on a 15% NaCl, pH=9 plate was determined. Detailed Implementation

[0039] This application aims to screen for highly salt-tolerant and growth-promoting Bacillus subtilis strains. 575 Bacillus strains were obtained from the rare microbial functional strain library of Kang Shengyuan (Zhaoqing) Biotechnology Co., Ltd. Through testing their salt tolerance, 306 strains were found to be tolerant to 5% NaCl, 183 strains to 10% NaCl, and 8 strains to 15% NaCl. Among these, only one strain, Bacillus subtilis KY559, showed the best plant protection effect when planted in saline-alkali soil. Furthermore, 16S DNA sequencing of this strain and comparison with the NCBI database showed that Bacillus subtilis has 100% high homology. Subsequent experiments, including stress resistance tests, showed that its salt tolerance reached a maximum of 17%, and its maximum alkali tolerance was pH=12. Field verification in saline-alkali soil demonstrated that strain KY559 effectively promotes maize seed germination and plant growth. It also demonstrated the ability to degrade starch, cellulose, and lipids under conditions containing 15% NaCl.

[0040] In one aspect, this invention provides an isolated salt-tolerant and growth-promoting Bacillus subtilis strain KY559, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35395 on July 25, 2025. The Bacillus subtilis strain KY559 provided in this application has multiple functions. Compared with previously reported strains, this strain can promote plant growth in salt-tolerant environments, which is of great significance for improving crop stress resistance and improving saline-alkali soil.

[0041] In one embodiment, the 16S rDNA sequence of the Bacillus subtilis strain KY559 is the sequence shown in SEQ ID No. 1. 16S DNA sequencing of the strain of this application revealed 100% high homology with Bacillus subtilis in the NCBI database, confirming that this application is a Bacillus subtilis strain. However, the strain of this application differs significantly from existing Bacillus subtilis strains. The strain of this application has a salt tolerance of up to 17% and a maximum alkali tolerance of pH=12, which has not been reported in existing Bacillus subtilis strains. Furthermore, compared with existing strains, the strain of this application exhibits better plant growth-promoting functions and can also degrade starch, cellulose, and lipids.

[0042] In one specific embodiment, the strain KY559 of the present invention, when cultured on plate medium, can tolerate a maximum concentration of 17% NaCl, but grows better at concentrations of 15% and 16%. Therefore, the strain of the present invention is a rare, highly salt-tolerant strain.

[0043] In one specific embodiment, the strain KY559 of the present invention not only exhibits excellent salt tolerance on 15% NaCl plates, but also demonstrates a significant plant growth-promoting effect in saline-alkali soil cultivation. In one specific embodiment, the KY559 strain can promote plant (e.g., maize) growth in soil environments with EC values ​​ranging from approximately 6.95 ms / cm to 10.3 ms / cm.

[0044] In one specific implementation, strain KY559 can also promote plant growth in severely saline soils. For example, in soil environments with an EC value as high as EC ≈ 10.3 ms / cm, strain KY559 can still promote the growth of maize. It should be noted that the following embodiments of the present invention use maize as an example to present data and do not imply that the growth-promoting effect of the strain is limited to maize. The strain can also promote the growth of other crops in saline-alkali environments, including but not limited to millet and soybeans. Strain KY559 can promote plant growth in mildly to moderately saline, moderately saline, or severely saline soils.

[0045] In the context of this invention, "EC value" refers to Electrical Conductivity, a core indicator for measuring the total amount of soluble salts in soil or water, directly reflecting the degree of salinization and irrigation water quality. Soluble ions (Na+) in soil solution or water... + Cl - Ca 2+The higher the concentration of (etc.), the greater the EC value. The EC value of non-saline soil is generally 0-2 ms / cm, the EC value of slightly saline soil is generally 2-4 ms / cm, the EC value of moderately saline soil is generally 4-8 ms / cm, and the EC value of severely saline soil is generally >8 ms / cm.

[0046] In one specific embodiment, the strain KY559 of the present invention, when cultured on plate medium, can reach a maximum alkali tolerance of 12 and grows well at pH = 9, 10, and 11.

[0047] In one aspect, the present invention provides a microbial composition, said microbial composition being a culture of the aforementioned Bacillus subtilis strain KY559 or Bacillus subtilis strain KY559.

[0048] In one specific implementation, the "culture" refers to the fermentation product obtained after fermentation using the strain. This includes, but is not limited to, fermentation broth, bacterial suspension, and culture metabolites.

[0049] In one specific embodiment, the microbial composition is in liquid or solid form. In one specific embodiment, the microbial composition is a liquid inoculum obtained by activating and culturing a pure strain of Bacillus subtilis strain KY559. In one specific embodiment, the microbial composition is a solid inoculum obtained by activating and culturing a pure strain of Bacillus subtilis strain KY559, followed by drying the liquid inoculum. In one specific embodiment, the liquid inoculum can be applied using conventional methods in the art, such as root irrigation, seed soaking, or spraying, while the solid inoculum can be applied using conventional methods in the art, such as root application or hole application. The strains or microbial compositions of this application can be used alone or in combination with other excipients that have opposing effects or side effects.

[0050] In one specific embodiment, the dosage form of the microbial composition is a wettable powder, a water-dispersible granule, an aqueous suspension, or a dispersible oil suspension.

[0051] In one specific embodiment, the microbial composition further includes a pesticide-acceptable excipient selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers, and solvents.

[0052] In one specific embodiment, the microbial composition is a bio-fertilizer. The strains described in this application have promising applications in the preparation of plant growth promoters, saline-alkali soil conditioners, plant bio-fertilizers, and bio-pesticides.

[0053] In one specific embodiment, the total viable count of the Bacillus subtilis strain KY559 contained in the microbial composition is at least ≥1×10⁻⁶. 7 cfu·mL -1 Or 1×10 7 cfu·g -1 In a preferred embodiment, the total viable count of the Bacillus subtilis strain KY559 in the microbial composition is at least 1 × 10⁻⁶. 8 cfu·mL -1 Or 1×10 8 cfu·g -1 .

[0054] In another aspect, the present invention provides the use of the aforementioned Bacillus subtilis strain KY559 or the aforementioned microbial composition in the following:

[0055] (a) for improving saline-alkali land; (b) for promoting plant growth; (c) for improving plant salt tolerance; and / or (d) for breaking down starch, cellulose and / or fats.

[0056] In one embodiment, the saline-alkali land improvement involves altering the soil environment of the saline-alkali land, such as changing the salt content.

[0057] In one specific embodiment, promoting plant growth includes promoting plant growth in saline-alkali or non-saline-alkali environments, including promoting plant growth in saline-alkali soils of varying degrees. Therefore, the present invention also provides applications in improving plant salt tolerance. In one specific embodiment, promoting plant growth includes promoting plant growth and development, particularly promoting the growth and development of plants in saline-alkali environments. The growth and development refers to the seed germination (germination) and post-emergence development process. In some cases, promoting plant growth and development includes increasing stem length, root length, fresh weight, or dry weight of the plant. The plant includes one or more of soybeans, corn, mung beans, and wheat; preferably, the plant is corn.

[0058] In one specific implementation, promoting plant growth includes promoting the growth of maize in a saline-alkali environment.

[0059] In a fourth aspect, the present invention provides a method for promoting crop growth under environmental stress, the method comprising applying the aforementioned Bacillus subtilis strain KY559 or the aforementioned microbial composition to the seeds of a plant or to a substrate in which the plant is grown.

[0060] In one specific implementation, the stress environment includes salt stress and / or alkali stress; the plant includes at least one of maize, soybean, and wheat.

[0061] In one specific implementation, the method includes mixing plant seeds with the mycelium powder, mycelium cake, fermentation broth, mycelium suspension, spore suspension and / or mycelium agent containing the strain KY559, or applying it as a base fertilizer or as a top dressing to the rhizosphere soil of the plant.

[0062] In one specific embodiment, the plant includes one or more of soybeans, corn, mung beans, and wheat; preferably, the plant is corn. In one specific embodiment, the strain KY559 of the present invention can improve the seed germination rate of corn and increase the fresh weight of corn.

[0063] The strain described in this application also has multiple functions, including the ability to break down fats, starches, and cellulose.

[0064] Example 1. Screening of salt- and alkali-tolerant strains

[0065] 1. Screening for Bacillus strains that can grow on 5%, 10%, and 15% NaCl plates.

[0066] 1.1 Source of strains

[0067] 575 strains of Bacillus were obtained from the rare microbial functional strain library of Kang Shengyuan (Zhaoqing) Biotechnology Co., Ltd. as screening targets.

[0068] 1.2 Culture medium

[0069] 1) R2A solid culture medium:

[0070] Yeast powder 0.50g, peptone 0.50g, tryptone 0.50g, glucose 0.50g, soluble starch 0.50g, dipotassium hydrogen phosphate 0.30g, sodium pyruvate 0.30g, magnesium sulfate heptahydrate 0.05g, agar powder 15.0g, water 1000mL;

[0071] 2) Salt and alkali tolerance screening medium:

[0072] Yeast powder 0.50g, peptone 0.50g, tryptone 0.50g, glucose 0.50g, soluble starch 0.50g, dipotassium hydrogen phosphate 0.30g, sodium pyruvate 0.30g, magnesium sulfate heptahydrate 0.05g, sodium chloride 50g / 100g / 150g, agar powder 15.0g, water 1000mL, pH=9;

[0073] 3) Sodium carboxymethyl cellulose (CMC) solid culture medium:

[0074] Dipotassium hydrogen phosphate 1.0g, magnesium sulfate heptahydrate 0.25g, yeast powder 2.0g, agar powder 10.0g, sodium carboxymethyl cellulose (CMC) 2.0g, sodium chloride 150g, water 1000mL;

[0075] 4) Starch-dissolving medium:

[0076] 10g peptone, 150g sodium chloride, 2g soluble starch, 20g agar, 1000ml water

[0077] 5) Lipid culture medium:

[0078] Yeast powder 0.50g, peptone 0.50g, tryptone 0.50g, glucose 0.50g, soluble starch 0.50g, dipotassium hydrogen phosphate 0.30g, sodium pyruvate 0.30g, magnesium sulfate heptahydrate 0.05g, LG lycerol 10g, sodium chloride 150g, agar powder 15.0g, water 1000mL

[0079] 1.3 Plate screening of salt-tolerant strains

[0080] 1.3.1 Screening for strains capable of growth in 5% NaCl

[0081] Bacillus strains from the rare strain library were activated at -80°C and streaked onto R2A solid medium, then incubated at 30°C for 24 hours. Single colonies were picked and streaked onto selection solid medium containing 5% NaCl and pH 9, then incubated upside down at 30°C for 48 hours. Strains that could grow single colonies were selected.

[0082] 1.3.2 Screening for strains capable of growing in 10% NaCl

[0083] Select strains from 1.3.1 that can grow single colonies on 5% NaCl plates and streak them onto a screening solid medium containing 10% NaCl and pH=9. Incubate them upside down in an incubator for 48 hours and select strains that can grow single colonies.

[0084] 1.3.3 Screening for strains capable of growing in 15% NaCl

[0085] To screen for strains with higher salt tolerance, the pH of the culture medium was maintained at pH=9, and the NaCl concentration was increased to 15%. Strains from section 1.3.2 that could grow single colonies on 10% NaCl plates were selected and streaked onto a screening solid medium containing 15% NaCl at pH=9. The medium was then incubated upside down in an incubator for 48 hours. The growth of the strains was observed, and strains that could grow single colonies were selected.

[0086] 1.4 Screening Results

[0087] A total of 1695 functional microbial strains were collected from the rare microbial strain library of Kang Shengyuan (Zhaoqing) Biotechnology Co., Ltd., including 575 Bacillus strains. Among them, 306 strains were tolerant to 5% NaCl, and 183 strains were tolerant to 10% NaCl. Further screening revealed 8 strains tolerant to 15% NaCl. As shown in Table 1, 5 strains (KY40, KY53, KY151, KY207, and KY559) could grow single colonies on plates with 15% NaCl and pH=9; 3 strains (KY1178, KY1188, and KY1190) could grow but their growth was inhibited.

[0088] strain number Growth of 15% NaCl plate KY40 ++ KY53 ++ KY151 ++ KY207 ++ KY559 ++ KY1178 + KY1188 + KY1190 +

[0089] Table 1

[0090] Note: Evaluation criteria: "++" indicates single colony growth; "+" indicates growth, but with some inhibition (leading to small colonies / first-stage growth / small colonies); "-" indicates no growth.

[0091] 1.5 Growth-promoting effect of salt-tolerant strains on maize in saline-alkali soil

[0092] 1.5.1 Experimental Methods

[0093] To verify whether the eight strains screened in section 1.4 that can grow on 15% NaCl plates can protect plant growth under salt stress, the strains were prepared into a bacterial solution, and corn seeds were soaked in the solution before being planted in saline-alkali soil. The specific method is as follows:

[0094] The saline-alkali soil originated from Ordos City, Inner Mongolia; the corn variety was Zhengdan 958 (purchased online).

[0095] Eight strains selected in section 1.4 were streaked onto R2A plates from a -80℃ freezer and incubated upside down in a 30℃ incubator for 48 hours. The strains were then inoculated into R2A liquid medium and cultured at 30℃ with shaking at 200 rpm for one day. The OD values ​​were measured using a visible-ultraviolet spectrophotometer. 600 After measuring its OD value, prepare a bacterial culture with OD=0.1 into a 50mL centrifuge tube for later use.

[0096] Saline-alkali soil (pure saline-alkali soil EC value ≈ 4.29 ms / cm, pH ≈ 8) sent from Inner Mongolia was mixed in a specific ratio (high EC value saline-alkali soil was mixed with low EC value saline-alkali soil at a ratio of 1:0.5 until pure saline-alkali soil with EC value ≈ 4.29 ms / cm). The mixed soil was evenly poured into small paper cups, moistened with water, and selected corn seeds were added to centrifuge tubes with the prepared bacterial solution and soaked for 30 minutes before sowing 6 seeds per pot. Since the commercial strain Bacillus subtilis 92068 is a multifunctional and widely used commercial strain, it was used as a screening control. Preliminary screening was conducted to determine whether salt-tolerant strains provided growth-promoting protection for corn in saline-alkali soil. After corn seedlings emerged, they were irrigated once with the above-mentioned bacterial solution with OD = 0.1, and the corn growth was observed and photographed.

[0097] 1.5.2 Experimental Results

[0098] from Figures 1 to 4 It was found that, during the same period, compared with the commercial multifunctional strain 92068, corn seeds soaked in KY559 solution grew the tallest after germination, indicating that strain KY559 had a significantly better growth-promoting effect. Compared with multifunctional strain 92068, corn seeds of KY40, KY207, KY151, and KY1178 grew to almost the same height, showing slightly better growth. KY1188 and KY1190 were slightly inferior to multifunctional strain 92068; although one or two plants appeared to be taller than 92068, fewer corn seedlings emerged, indicating less uniform growth. Corn seeds soaked in KY53 solution did not grow in saline-alkali soil, indicating that although this strain showed good salt tolerance in 15% NaCl plates, it had no effect on promoting corn germination in saline-alkali soil.

[0099] In summary, strain KY559 not only exhibits excellent salt tolerance on 15% NaCl plates but also demonstrates a significant growth-promoting effect when grown in saline-alkali soil. Therefore, this strain was selected as a key research subject for further investigation.

[0100] 1.5.3 Salt tolerance and growth promotion test of strain KY559 in saline-alkali soil with different EC values

[0101] As demonstrated in sections 1.4 and 1.5.2 above, strain KY559 not only exhibits good salt tolerance on salt-tolerant plates but also shows a significant salt-tolerance-promoting effect on maize in saline-alkali soil. To investigate whether strain KY559 still possesses salt-tolerance-promoting ability and enhances plant growth in saline-alkali environments in saline-alkali soil with higher EC values, the EC values ​​of the saline-alkali soil were proportionally increased to a certain range to verify whether strain KY559 exhibits salt tolerance-promoting ability and enhances plant growth in saline-alkali environments in higher-grade saline-alkali soil.

[0102] Using the bacterial solution preparation method described in 1.5.1 above, saline-alkali soils were collected from different regions of Inner Mongolia (soils with high salinity near the Yellow River basin, barren saline-alkali soils, and saline-alkali soils suitable for planting). These soils were mixed in a specific ratio to increase the EC values ​​of the saline-alkali soils to approximately 6.95 ms / cm and 10.3 ms / cm, respectively. The mixed soil was evenly poured into small flowerpots and watered. Selected corn seeds were added to centrifuge tubes containing the prepared bacterial solution and soaked for 30 minutes before sowing 7 seeds per pot. The commercial multifunctional strain Bacillus subtilis 92068 and CK (water) were used as controls. After the corn seedlings emerged, they were watered once with a bacterial solution containing OD=0.1, and the growth of the corn was observed and photographed.

[0103] 1.5.4 Results of Salt Tolerance and Growth Promotion Tests of Strain KY559 in Saline-Alkali Soils with Different EC Values

[0104] as follows Figure 5 As shown, when the EC value of saline-alkali soil is approximately 6.95 ms / cm, the number of corn seedlings soaked in KY559 bacterial solution during the same period was higher than that of the two control groups, and the seedlings also grew taller than those treated with CK and 92068. The corn plants treated with control strain 92068 showed signs of wilting. The corn plants in control CK were stunted, and the leaf tips were beginning to turn yellow. This indicates that both control corn plants were suffering from salt poisoning. In contrast, the corn plants treated with KY559 showed a high germination rate, vibrant green leaves, and no yellowing or wilting, indicating that this strain has a certain ability to promote salt growth and protect plant growth from salt damage in moderately saline soil.

[0105] as follows Figure 6 As shown, when the EC value of saline-alkali soil is approximately 10.3 ms / cm, only the corn soaked in KY559 bacterial solution grew and sprouted during the same period, while the corn treated with control CK (water) and 92068 did not germinate or sprout. This further demonstrates that the KY559 strain can also promote plant growth under severely saline soil conditions and has a good ability to improve the salt tolerance and growth promotion of corn.

[0106] In summary, strain KY559 can promote plant growth in mildly to moderately saline, moderately saline, and severely saline soils, reducing the stress on plants growing in saline environments and demonstrating excellent salt tolerance and growth-promoting effects.

[0107] Example 2. Determination of stress resistance of KY559 strain

[0108] 2.1 Determination of KY559's highest salt concentration tolerance

[0109] As can be seen from section 1.3.3 above, strain KY559 has the ability to tolerate up to 15% NaCl. To further verify whether strain KY559 has even higher salt tolerance, the NaCl concentration was increased to 16%, 17%, and 18%, while the pH was maintained at 9. Single colonies of KY559 were picked and streaked onto the above three different NaCl concentration plates, and then placed in an inverted incubator at 30°C to observe whether single colonies grew and to record the highest salt tolerance concentration.

[0110] 2.1.1 Results of the test on the highest salt concentration tolerance of KY559

[0111] From Table 2 and Figure 7 It can be seen that strain KY559 can grow single colonies on both 15% and 16% NaCl plates at pH=9. Growth inhibition begins on 17% NaCl plates after the first colony appears. The strain cannot grow on 18% NaCl plates. In summary, strain KY559 has a maximum salt tolerance of 17%, and grows well at 15% and 16% concentrations.

[0112] strains 15% NaCl 16% NaCl 17% NaCl 18% NaCl KY559 ++ ++ + -

[0113] Table 2

[0114] Note: The evaluation criteria are: "++" indicates that a single colony can grow; "+" indicates that growth is possible, but there is some inhibition (growing of the head / growing of the first runner / small colony); "-" indicates that growth is impossible.

[0115] 2.2 Determination of the highest alkali resistance of KY559

[0116] As shown in 1.7 and 2.1 above, strain KY559 can grow normally under salt stress at pH=9. To further understand the maximum pH value that this strain can tolerate under salt stress, the NaCl concentration of the plates was set at 10%, and the original pH of 9 was increased to 10, 11, and 12, respectively. Single colonies of KY559 were picked and streaked onto the three different pH plates, and then incubated upside down in a 30°C constant temperature incubator to observe whether single colonies grew and to record the highest pH value.

[0117] 2.2.1 Results of the test for the highest alkali resistance of KY559

[0118] From Table 3 and Figure 8It can be seen that strain KY559 can grow normally under 10% NaCl conditions at pH=9, pH=10, and pH=11, and subtle differences can be observed in single colonies as the pH increases. At pH=12, KY559 can also grow normally and produce single colonies, but growth is significantly inhibited, and the single colonies are much smaller compared to those at pH=9, 10, and 11. Therefore, strain KY559 has a maximum alkali tolerance of 12, and grows better at pH=9, 10, and 11.

[0119] strains pH=9 pH = 10 pH=11 pH = 12 KY559 ++ ++ ++ +

[0120] Table 3

[0121] Note: Evaluation criteria: "++" indicates single colony growth; "+" indicates growth, but with inhibition (growth of the head / first colony / small colony); "-" indicates no growth. Example 3.16: sDNA determination and physiological morphological analysis of the strain:

[0122] 3.1 Determination of 16S DNA of strain KY559

[0123] 3.1.1 Extraction of bacterial DNA using the CTAB method

[0124] 1. Inoculate a single colony into 5 mL LR2A and incubate overnight at 30°C;

[0125] 2. Take 1 mL of seed culture medium and inoculate it into 100 mL of LR2A liquid, and incubate at 37℃ and 220 r / min for 16 hours;

[0126] 3. Centrifuge at 5000 r / min for 10 minutes and discard the supernatant.

[0127] 4. After centrifugation and washing with 10 mL TE, dissolve the bacterial cells with 10 mL TE, mix well, and store at -20℃ for later use.

[0128] 5. Take 3.5 mL of bacterial suspension, add 184 μL of 10% SDS, mix well, add 37 μL of 10 mg / mL proteinase K, mix well, and incubate at 37°C for 1 hour.

[0129] 6. Add 740 μL of 5 mol / L NaCl, then add 512 μL of LTAB / NaCl, mix well, and incubate at 65°C for 10 minutes.

[0130] 7. Add an equal volume of chloroform / isoamyl alcohol, mix well, centrifuge at 10000 r / min for 5 minutes, and retain the supernatant;

[0131] 8. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) to the supernatant, mix well, centrifuge at 10000 r / min for 5 minutes, and retain the supernatant;

[0132] 9. Add 0.6 times the amount of isopropanol, mix well, centrifuge at 10000 r / min for 5 minutes, collect the DNA precipitate, and wash the DNA precipitate with 70% ethanol by centrifugation.

[0133] 10. Dissolve the DNA in 1 mL TE buffer, add RNase A to a final concentration of 20 μg / mL, and store at 4°C.

[0134] 3.1.2 Amplification and Sequencing

[0135] PCR amplification of 16S rDNA was performed using universal primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID No. 2) and 1492r (5′-GGTTACCTTGTTACGACTT-3′, SEQ ID No. 3). PCR reaction conditions were: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 60 s, for 30 cycles. The PCR products were subjected to 2% agarose gel electrophoresis, and after agarose gel electrophoresis, the PCR products were recovered, purified, and sequenced (Beijing Meiyimei Biotechnology Co., Ltd.). Based on the obtained 16S rDNA sequence, homologous sequences were searched in GenBank using B1ast, and homologous sequence analysis was performed to construct a phylogenetic tree.

[0136] 3.1.3 16S sequencing results of strain KY559

[0137] The results of the 16sDNA sequence determination of strain KY559 (strain sequence) and comparison with the NCBI database showed that it has 100% high homology with Bacillus subtilis.

[0138] The 16SDNA sequence of strain KY559 is shown below:

[0139]

[0140] 3.2 Observation of strain morphology

[0141] The selected strains were inoculated onto R2A plates and cultured at 30°C for 2 days. The size, shape, color, gloss, viscosity, raised shape, transparency, edge characteristics, and presence or absence of spores of the colonies were observed.

[0142] Results of strain morphology observation: After 2 days of growth on R2A medium, the colonies were large, irregularly round, with serrated edges, milky white, and moist, rough, and non-sticky. With prolonged culture, yellow pigment was produced on the R2A plates, and spores were observed under a 100X microscope.

[0143] Example 4. Stress resistance test of Bacillus subtilis KY559 plate.

[0144] As confirmed in section 3.1.3 above, strain KY559 is *Bacillus subtilis*. To demonstrate that not all *Bacillus subtilis* strains possess strong salt and alkali tolerance and growth-promoting capabilities, strain KY559 was compared with salt- and alkali-tolerant *Bacillus subtilis* strains or strains very similar to *Bacillus subtilis* from the rare functional strain library of Kang Sheng Yuan (Zhaoqing) Biotechnology Co., Ltd. Therefore, the following experiment was conducted.

[0145] 4.1 Salt and Alkali Tolerance Test Method for Bacillus subtilis Plates

[0146] All Bacillus subtilis strains very similar to Bacillus subtilis from the rare strain library were activated at -80°C, resulting in 30 strains. These 30 strains were streaked on R2A solid medium and incubated at 30°C for 24 to 48 hours. Single colonies were then picked and streaked on selection solid media containing 5% NaCl, 10% NaCl, and 15% NaCl at pH 9, and incubated upside down at 30°C. Strains that produced single colonies were selected and recorded.

[0147] 4.2 Experimental Results

[0148] Table 4 shows that 28 strains could grow single colonies on 5% NaCl, pH=9 plates, while 2 strains, KY547 and KY634, were not tolerant to 5% NaCl. On 10% NaCl, pH=9 plates, 19 strains could grow single colonies, while 6 strains, KY203, KY454, KY547, KY634, KY774, and KY777, were not tolerant to 10% NaCl. Two of these strains were not salt-tolerant on 5% NaCl plates. Six strains showed inhibited growth under 10% NaCl conditions: KY81, KY231, KY384, KY697, KY776, and KY889. Only one strain, KY559, could grow a single colony on a 15% NaCl, pH=9 plate; the remaining 28 strains did not exhibit tolerance to 15% NaCl. This indicates that most Bacillus subtilis species, which are very similar to it, generally have a maximum salt tolerance of around 10%, and KY559 is the only strain among these 30 with relatively high salt tolerance.

[0149]

[0150]

[0151] Table 4

[0152] Note: Evaluation criteria: "++" indicates single colony growth; "+" indicates growth, but with some inhibition (leading to small colonies / first-stage growth / small colonies); "-" indicates no growth.

[0153] In summary, among the 30 Bacillus strains very similar to Bacillus subtilis from the rare strain library of Kang Shengyuan (Zhaoqing) Biotechnology Co., Ltd., only one strain, KY559, grew a single colony on a 15% NaCl plate after stress resistance experiments with different NaCl concentrations. This indicates that strain KY559 has the strongest salt tolerance among the many strains very similar to Bacillus subtilis and is unique compared to other Bacillus subtilis strains.

[0154] Example 5. Detection of how salt-tolerant Bacillus subtilis strains enhance the salt and alkali tolerance of maize in saline-alkali soil.

[0155] As demonstrated in sections 1.5.2 and 1.5.4, strain KY559 outperforms the commercially available multifunctional strain Bacillus subtilis 92068 in saline-alkali soil cultivation. To verify the uniqueness of KY559 among Bacillus subtilis strains, 10 randomly selected Bacillus subtilis strains capable of producing single colonies on 10% NaCl plates, or strains very similar to Bacillus subtilis, from section 4.2 above, will be compared with KY559 strain in corn cultivation in saline-alkali soil. This will verify whether KY559 exhibits the best salt tolerance and growth-promoting ability compared to salt-tolerant Bacillus subtilis strains in saline-alkali cultivation environments.

[0156] 5.1 Experimental Methods

[0157] Using the bacterial solution preparation method described in 1.5.1 above, saline-alkali soil sent from Inner Mongolia was mixed in a specific ratio, with an EC value of approximately 5.15 ms / cm. The mixed soil was evenly poured into small flowerpots, watered, and the selected corn seeds were added to the prepared bacterial solution centrifuge tubes and soaked for 30 minutes before sowing 7 seeds per pot. CK (water) and the commercial multifunctional strain 92068 were used as controls. After the corn seedlings emerged, each seedling was watered once with the bacterial solution containing OD=0.1, and the growth of the corn was observed and photographed.

[0158]

[0159] 5.1.2 Experimental Results

[0160] Table 5 shows that strain KY559 achieved the highest germination rate (100%) on the seventh day, making it the fastest and most complete germination strain among all maize strains. Table 6 shows that maize seeds soaked in KY559 solution had the heaviest above-ground parts after emergence compared to other strains. This indicates that strain KY559 not only performed best among maize strains grown in saline-alkali soil, but also outperformed strains closely related to *Bacillus subtilis* in saline-alkali soil.

[0161]

[0162] Table 5

[0163]

[0164]

[0165] Table 6

[0166] In summary, compared with corn grown using the other 10 Bacillus subtilis strains, strains very similar to Bacillus subtilis, CK, and the multifunctional strain 92068, corn grown using KY559 not only germinated the fastest and most completely, but also had the heaviest fresh weight of the above-ground parts among all the compared strains. This indicates that strain KY559 has strong salt and alkali tolerance in saline-alkali environments and the ability to promote plant growth in saline-alkali environments, thus having practical application value in agricultural production.

[0167] Example 5. Multifunctional assay of strain KY559

[0168] In summary, strain KY559 exhibits salt tolerance and growth-promoting effects in saline-alkali soils. Therefore, strain KY559 can be a key component in products that enhance plant salt tolerance and growth. To further explore its functions, its potential for expanding applications, such as the resource utilization of livestock manure, can be investigated. In livestock farms in saline-alkali areas, these bacteria can decompose starch, cellulose, and fats in livestock manure, converting them into organic fertilizer. This addresses manure pollution and produces fertilizer suitable for saline-alkali soils. Therefore, expanding the application of this product primarily involves exploring whether strain KY559 has the ability to degrade starch, cellulose, and lipids under salt stress.

[0169] 6.1 Determination of the starch-dissolving ability of strain KY559 on 15% NaCl, pH=9 plates

[0170] Strain strain KY559, grown on R2A medium for 1 day, was needled into a starch solid medium (10g peptone, 150g sodium chloride, 2g soluble starch, 20g agar, 1000ml water) containing 15% NaCl and pH=9. A commercial multifunctional strain, Bacillus subtilis 92068, was used as a control. The strain was cultured at 30℃ for 3 days. After growth, the diameter of the starch-degrading zone was measured. The strain was then fumigated with iodine solution, and the hydrolysis zone was measured again. A larger transparent zone indicated a better starch-degrading ability. The starch-degrading ability of the strain was thus determined, with units of mm.

[0171] Starch-dissolving capacity = (Diameter of the starch-dissolving ring in millimeters) + X

[0172] (Note: X is a weighting coefficient, corresponding to -1, 0, 1, and 2 based on the transparency of the hydrolysis zone of the bacterial strain. A number 2 represents a completely transparent hydrolysis zone; a number 1 represents a semi-transparent hydrolysis zone; a number 0 represents an opaque hydrolysis zone, but with traces of hydrolysis on the culture medium surface, barely visible to the naked eye, but after rinsing the colony with water, faint traces of hydrolysis are visible at the inoculation site; -1 represents no hydrolytic activity. This method has also been used to test the lipolysis and CMC-degrading activities of bacteria.)

[0173] 6.1.2 Results of starch-dissolving ability determination of strain KY559 on 15% NaCl, pH=9 plates

[0174] Table 7 below shows the starch-dissolving ability of strain KY559 and the control strain. Figure 9 The results show that strain KY559 has a significantly stronger ability to dehydrate starch on 15% NaCl and pH=9 plates than the control commercial multifunctional strain Bacillus subtilis 92068, and strain 92068 does not grow and has no ability to dehydrate starch under 15% NaCl conditions.

[0175] Function 92068 KY559 The ability to break down starch -1 2.8

[0176] Table 7

[0177] 6.2 Determination of the lipolysis ability of strain KY559 on 15% NaCl, pH=9 plates

[0178] The strain KY559, which was cultured on R2A medium for 1 day, was then needled into a lipid solid medium (R2A + 10 g / L Glycerol, 20 g agar, 1000 ml water) containing 15% NaCl and pH=9. The commercial multifunctional strain Bacillus subtilis 92068 was set as a control. The strain was cultured at 30℃ for 3 days. After the strain grew, the diameter of the lipid-degrading zone was measured to determine the strain's ability to degrade lipids, in mm.

[0179] Fat-dissolving ability = (diameter of fat-dissolving area in millimeters) + X

[0180] (X is a weighting coefficient, which is -1, 0, 1, or 2 depending on the transparency of the lipolysis zone of the strain.)

[0181] 6.1.2 Results of the determination of the lipolysis ability of strain KY559 on 15% NaCl, pH=9 plates

[0182] Table 8 (showing the lipolysis ability of strain KY559 and control 92068) and Figure 10 The results showed that strain KY559 had a significantly stronger ability to deliquinate on plates with 15% NaCl and pH=9 than the control commercial multifunctional strain Bacillus subtilis 92068, and strain 92068 did not grow and had no ability to deliquinate under 15% NaCl conditions.

[0183] Function 92068 KY559 Fat-dissolving ability -1 3.1

[0184] Table 8

[0185] 6.3. Determination of the CMC degrading ability of strain KY559 on 15% NaCl, pH=9 plates

[0186] The strain KY559, which had been cultured on R2A medium for 1 day, was then needled into a CMC solid medium containing 15% NaCl and pH=9 (1 g dipotassium hydrogen phosphate, 0.25 g magnesium sulfate heptahydrate, 2 g yeast, 2 g sodium carboxymethyl cellulose, 20 g agar, and 1000 mL water). A commercial multifunctional strain, Bacillus subtilis 92068, was set as a control. The strain was cultured at 30°C for 3 days. After the strain had grown, the diameter of the CMC-degrading rings was measured to determine the strain's ability to degrade CMC, in mm.

[0187] CMC solving capability = number of millimeters of CMC ring diameter solved + X

[0188] (X is a weighting coefficient, which is -1, 0, 1, or 2 depending on the transparency of the CMC loop of the strain.)

[0189] 6.3.1 Results of the determination of the CMC degrading ability of strain KY559 on 15% NaCl, pH=9 plates

[0190] From Table 9 (CMC-dissolving ability of strain KY559 and control 92068) and Figure 11 The results showed that strain KY559 had a significantly stronger ability to decompose CMC on a plate with 15% NaCl and pH=9 than the control commercial multifunctional strain Bacillus subtilis 92068, and strain 92068 did not grow and had no ability to decompose CMC under 15% NaCl conditions.

[0191] Function 92068 KY559 CMC decoding capability -1 2.5

[0192] Table 9

[0193] in conclusion

[0194] This application selected 575 Bacillus strains from a rare microbial resource library of 1695 strains constructed through functional strain screening of different soil samples. From these, strain KY559 was selected as the best performing strain in saline-alkali soil, capable of growing on 15% NaCl plates at pH=9 and exhibiting the best salt tolerance and growth-promoting effect. 16SDNA sequence analysis of this strain showed 100% homology with Bacillus subtilis. Compared to the commercially available multifunctional Bacillus subtilis strain 92068, strain KY559 showed better corn growth in saline-alkali soil experiments at different EC values. Further investigation revealed that strain KY559 has a maximum salt tolerance of 17% and a maximum alkali tolerance of pH=12. Furthermore, to demonstrate the uniqueness of this strain, subsequent experiments compared it with Bacillus subtilis strains in the strain library that are tolerant to 10% NaCl and strains very similar to Bacillus subtilis through salt-alkali plate tests and saline-alkali soil planting tests. The results showed that only strain KY559 produced single colonies on 15% NaCl plates, and it exhibited the best germination rate and highest fresh weight in the saline-alkali soil planting experiment. Simultaneously, it also demonstrated the ability to degrade starch, lipids, and CMCs on 15% NaCl plates at pH 9. In saline-alkali soil planting, the corn grown with KY559 showed better growth than corn grown with other strains, indicating that strain KY559 differs from other Bacillus subtilis strains in that it exhibits stronger salt-alkali tolerance, promotes plant growth in saline-alkali environments, and simultaneously possesses the ability to degrade starch, lipids, and CMCs, thus demonstrating high application value in agricultural production.

Claims

1. A salt- and alkali-tolerant, growth-promoting Bacillus subtilis strain KY559, characterized in that... The strain KY559 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35395; the deposit date is July 25, 2025.

2. The Bacillus subtilis strain KY559 according to claim 1, characterized in that, The 16S rDNA sequence of strain KY559 is shown in SEQ ID No.

1.

3. A microbial composition, characterized in that, The microbial composition contains the Bacillus subtilis strain KY559 or a culture of Bacillus subtilis strain KY559 as described in claim 1 or claim 2.

4. The microbial composition according to claim 3, characterized in that, The microbial composition may be in liquid or solid form.

5. The microbial composition according to claim 4, characterized in that, The total viable count of the Bacillus subtilis strain KY559 contained in the microbial composition is at least 1 × 10⁻⁶. 7 cfu·mL -1 Or 1×10 7 cfu·g -1 .

6. Use of the Bacillus subtilis strain KY559 according to claim 1 or claim 2, or the microbial composition according to any one of claims 3 to 5, in the following: (a) for improving saline-alkali land; (b) for promoting plant growth; (c) for improving plant salt tolerance; and / or (d) for breaking down starch, cellulose and / or fats.

7. The use according to claim 6, characterized in that, The promotion of plant growth includes promoting plant growth in saline-alkali or non-saline-alkali environments.

8. The use according to claim 6, characterized in that, The promotion of plant growth includes promoting the growth of corn in saline-alkali environments.

9. A method for promoting crop growth under stress conditions, characterized in that, The method includes applying the Bacillus subtilis strain KY559 of claim 1 or claim 2, or the microbial composition of any one of claims 3 to 5, to the seeds of a plant, or to the substrate in which the plant is grown.

10. The method according to claim 9, characterized in that, The stress environment includes salt stress and / or alkali stress; the plant includes at least one of maize, soybean and wheat.

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