Bacillus altitudinis strain k309 and application thereof

Abstract

The application belongs to the technical field of microorganisms, and specifically discloses a bacillus altitudinis strain K309 and application thereof. Bacillus altitudinis The preservation number of the bacillus altitudinis strain K309 is CGMCC No.39318, and the strain was preserved in the China General Microbiological Culture Collection Center on June 9, 2026. The strain K309 has excellent salt and alkali tolerance, has the abilities of IAA production and iron carrier production, and can significantly improve the salt tolerance of soybeans. When the strain K309 is used in combination with rhizobium under salt stress, the number of soybean root nodules can be obviously increased, and the nitrogen fixation effect can be enhanced, so that the strain K309 can be used for improving the growth of crops in saline-alkali land and improving the utilization rate of saline-alkali land, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus hygroscopicus 309 and its application in improving the salt stress tolerance of plants. Background Technology

[0002] Saline-alkali land improvement includes various methods such as physical, chemical, and biological approaches, among which biological improvement is the most economical, effective, and environmentally friendly. Salt-tolerant microorganisms and plant growth promoters (PGPRs) are widely used in saline-alkali land improvement and enhancing plant tolerance to salt and alkali stress. PGPRs are a class of beneficial bacteria that live freely in the soil or epiphytically on plant roots and can promote plant growth. They can enhance the growth potential of plants under salt stress through direct or indirect effects. Some PGPRs with strong salt and alkali tolerance can be used for saline-alkali land improvement and enhancing plant salt and alkali tolerance. Their main mechanisms of action include: (1) Synthesize a variety of osmotic regulators, such as proline, betaine, trehalose, glycine, phenols and flavonoids, to maintain cell osmotic potential, ensure normal metabolic processes, and improve the plant’s ability to adapt to salt and alkali stress. (2) It secretes plant growth hormones such as indoleacetic acid, promotes the development of plant roots, increases the total volume and surface area of ​​plant roots, improves the absorption and utilization rate of water by plant roots, and thus alleviates the osmotic stress caused by salt and alkali stress on plants. (3) Activate the antioxidant defense system and significantly improve the growth status of plants under salt and alkali stress by secreting antioxidant enzymes or promoting the accumulation of non-enzymatic antioxidants (such as carotenoids, proline and polyphenols). (4) It secretes a variety of volatile organic compounds, which enhance the plant’s salt tolerance by strengthening its antioxidant defense mechanism or increasing hormone levels; (5) Increase the photosynthetic rate, stomatal conductance, transpiration efficiency and intracellular CO2 concentration of plants, while increasing the content of carotenoids, chlorophyll, nitrogen and protein, and reducing the impact of salt stress on photosynthesis. (6) Provide iron carriers or dissolve phosphates to improve the availability of limiting nutrients in the soil, thereby enhancing the plant’s ability to cope with stress; (7) By regulating the expression of specific transcription factors and functional genes, the plant’s defense and adaptation mechanisms are activated, thereby improving the plant’s salt tolerance.

[0003] Soybean (Glycine max) is a moderately salt-tolerant crop and an important multi-purpose crop in my country, serving as grain, oil, and forage. It holds a crucial strategic position in my country's agricultural production and national economic development. Previous research has largely focused on screening salt-tolerant soybean germplasm resources and gene discovery, with limited studies on improving soybean salt tolerance through saline-alkali soil microorganisms. Transplanting salt-tolerant microbial communities into the rhizosphere of plant seedlings holds promise for altering the microbial community structure and function, thereby enhancing the salt tolerance of salt-sensitive plants and assisting their growth under salt stress, offering a potential solution for agricultural production in saline-alkali lands. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus hygroscopicus that has both strong salt tolerance and the ability to enhance the salt stress tolerance of plants, as well as the application of this strain in enhancing the salt stress tolerance of plants.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A strain of Highland Bacillus ( Bacillus altitudinis Strain K309, isolated from saline-alkali soil in Kenli District, Dongying City, Shandong Province, is salt-tolerant and can enhance the salt stress tolerance of plants. Its accession number is CGMCC No. 39318, and it was deposited at the China General Microbiological Culture Collection Center on June 9, 2026. Bacterial identification revealed that this strain is *Bacillus hygroscopicus* (Gaodi Bacillus). Bacillus altitudinis The 16S rRNA gene sequence of the strain is shown in SEQ ID NO.1.

[0006] The primary objective of this invention is to protect the application of the aforementioned Bacillus subtilis K309 in enhancing the salt stress tolerance of plants, wherein the plant is soybean seedlings.

[0007] Experiments revealed that the strain possesses the ability to produce IAA and siderophores under salt stress, with the siderophore production capacity significantly increasing under salt stress; it also exhibits strong salt and alkali tolerance, and can grow normally in media containing 11% NaCl and in media with pH=9.

[0008] Salt tolerance experiments showed that the Bacillus hygroscopicus strain could increase the fresh weight of soybean seedlings, increase plant height, and improve their survival rate under salt stress. Furthermore, when the strain was applied in combination with rhizobia, the number of root nodules in the plants was significantly increased compared to the application of rhizobia alone.

[0009] Therefore, the present invention also provides a method for improving the salt and alkali tolerance of soybean seedlings. During the period from soybean emergence to the two-leaf and one-heart stage, 30 mL / plant of Bacillus hygroscopicus K309 bacterial suspension is added to the irrigation water during the watering process. The bacterial suspension uses sterile water as a solvent and the OD600 value is controlled at 0.1 to 0.3.

[0010] In addition, a method for increasing the number of root nodules in soybeans in saline-alkali soil is also provided. During the period from soybean emergence to the two-leaf stage, 30 mL / strain of Bacillus hygroscopicus K309 with OD600=0.1-0.3 and 50 mL / strain of slow-growing rhizobium with OD600=0.09 are added to the irrigation water during the watering process. The preservation number of K309 is CGMCC No.39318.

[0011] Advantages of this invention: (1) The strain K309 discovered in this invention comes from the saline-alkali soil of Kenli District, Dongying City. The strain has a high degree of salt and alkali tolerance and is suitable for survival in the saline-alkali environment.

[0012] (2) The strain K309 discovered in this invention has the ability to produce iron carriers and IAA, and can promote soybean germination under salt stress and improve the salt tolerance of seedlings.

[0013] (3) The strain K309 discovered in this invention can work together with rhizobia to increase the number of root nodules in soybean plants. Therefore, when applied in combination with rhizobia under salt stress conditions, it can significantly increase soybean root nodules and enhance nitrogen fixation. It can be used to improve the growth of crops in saline-alkali land and increase the utilization rate of saline-alkali land, and has good application prospects. Attached Figure Description

[0014] Figure 1 This is a colony morphology diagram of Bacillus hygroscopicus K309 on LB solid medium; Figure 2 This is an phylogenetic tree of Bacillus hygroscopicus strain K309 based on 16S rRNA sequencing; Figure 3 The figures show the salt-alkali tolerance growth curves of Bacillus hygroscopicus K309 in culture media with different salt concentrations and pH values. In the figure, A is the salt-alkali tolerance growth curve of Bacillus hygroscopicus K309 in culture media with different salt concentrations; B is the salt-alkali tolerance growth curve of Bacillus hygroscopicus K309 in culture media with different pH values. Figure 4 It is the IAA standard curve; Figure 5 This is a graph showing the growth-promoting characteristics of Bacillus hygroscopicus K309. In the graph, A is a comparison of the IAA production capacity of Bacillus hygroscopicus K309 in King's medium with different salt concentrations; B is a comparison of the siderophore production capacity of Bacillus hygroscopicus K309 in MKB medium with different salt concentrations. Figure 6 These are phenotypic diagrams of soybean germination and comparison diagrams of germination rates for each group; in the diagram, A is a phenotypic diagram of soybean germination after 7 days of cultivation; B is a comparison diagram of soybean germination rates for each group after 3-10 days of cultivation. Figure 7Phenotypic charts of soybean seedling growth in different groups after 7 days of salt stress; Figure 8 This is a statistical chart of soybean seedling survival rates in different groups after 7 days of salt stress. Figure 9 The results show the plant height of soybean seedlings in each group after 7 days of cultivation. Figure 10 The results are the statistical results of the fresh weight of soybean seedlings in each group after 7 days of cultivation; Figure 11 This is a phenotypic diagram of the number of root nodules per plant after 28 days of application of rhizobium and strain K309 under salt stress. Figure 12 This is a statistical chart showing the number of root nodules per plant after 28 days of application of rhizobium and strain K309 under salt stress.

[0015] In the above figure, all lowercase letters such as "a", "b", and "c" are differential markers made by Fisher's LSD significance analysis when the confidence level is above 95%. Different letters indicate that there is a significant difference between the two groups. Detailed Implementation

[0016] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific test methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0017] Unless otherwise specified, the techniques used in the embodiments are conventional methods well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the reagents and materials used are commercially available. All quantitative experiments were performed in triplicate, and the results were averaged.

[0018] Example 1: Isolation, purification and identification of strain K309 1. Screening of strain K309 The sampling point was a saline-alkali land in Kenli District, Dongying City, Shandong Province (37°31' N, 118°45' W). The pH of this saline-alkali land was 8.25, and the salt content was 5‰. 5 g of saline-alkali soil was weighed into a 100 mL Erlenmeyer flask containing 45 mL of sterile deionized water. The shaker was set at 28 ℃ and 200 rpm for 30 min. After standing for sedimentation, 1 mL of the supernatant was transferred to a centrifuge tube containing 9 mL of sterile deionized water to prepare a 1 / 10 soil suspension. 1 mL of this 1 / 10 soil suspension was then transferred to a centrifuge tube containing 9 mL of sterile deionized water to prepare a 1 / 100 soil suspension. Similarly, 1 / 10 suspensions were prepared sequentially. 3 1 / 10 4 1 / 10 51 / 10 6 Soil suspension. Purification was performed using the triple-strike method on TSB solid medium. This purification process was repeated twice to obtain uniform, similarly shaped, white, dry, and wrinkled single colonies. Figure 1 Single colonies were further transferred to 6 ml LB liquid medium and incubated at 28°C and 200 rpm for 1 day until the culture became turbid. The bacteria were then preserved in 40% glycerol and stored at -80°C for later use.

[0019] The formula for liquid LB medium is: 10g sodium chloride, 10g tryptone, 5g yeast extract, 0.5g yeast extract, and 1L deionized water. Formula for solid LB medium: Add 12g of agar to the formula for liquid LB medium.

[0020] 2. Identification of strain K309 Single clones of strain K309 were picked from solid LB medium and added to a solution containing 25 μL of 2×Taq premixed PCR solution, 18 μL of ddH2O, 2 μL of primers (10 μM), and 2 μL of reverse primers (10 μM). The PCR amplification program used is shown in Table 1.

[0021] Table 1 PCR amplification program

[0022] After the PCR amplification procedure, 40 μL of sample was taken for agarose gel electrophoresis to confirm the correct band size. The gel was then excised and sent to Qingke Biotechnology for sequencing. The sequencing results were searched using 16S rRNA BLAST on the NCBI website. The genus with the highest alignment rate in the search results was […]. Bacillus altitudinis 41KF2b, NR_042337.1, alignment rate 99.72%. Therefore, K309 and... Bacillus altitudinis 41KF2b is the same species, therefore strain K309 is named Bacillus hygroscopicus K309. Figure 2 ).

[0023] 16S rRNA sequencing (sequence shown in SEQ ID NO.1), with the forward and reverse primers as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 2); 1492R: 5'-CTACGGCTACCTTGTTACGA-3' (SEQ ID NO. 3).

[0024] Example 2: Determination of salt and alkali tolerance and growth-promoting ability of strain K309 1. Salt and alkali tolerant growth curve of strain K309 Activated strain K309 was inoculated into 250 mL Erlenmeyer flasks containing 100 mL of LB medium. The strain was then inoculated at 1% (v / v) into LB liquid medium containing 0%, 1%, 3%, 5%, 7%, 9%, 10%, 11%, 12%, 13%, and 14% NaCl, and into LB liquid medium with pH values ​​of 6.0–11.0. The cultures were incubated at 28℃ and 160 r / min with shaking. The absorbance (OD600) was measured periodically to determine the salt and alkali tolerance of the strain, resulting in a salt and alkali tolerance curve. Figure 3 (A and B in the figure). In the figure, the vertical axis represents the bacterial concentration (OD600), and the horizontal axis represents time. Figure 3 It is known that strain K309 can tolerate a maximum NaCl concentration of 12% and can grow normally in media containing 11% NaCl and media with pH ≤ 9, thus belonging to highly salt-tolerant bacteria.

[0025] 2. Determination of IAA production capacity of strain K309 Indoleacetic acid (IAA) was used to prepare 0, 10, 20, 30, 40, and 50 mg·L⁻¹ solutions. -1 For the standard solution, mix 100 μL of supernatant and 100 μL of Salkowski colorimetric solution (Phygene), incubate in the dark for 30 min, and measure the OD600 value. Plot a standard curve with IAA concentration on the x-axis and absorbance on the y-axis. Activate the test bacteria, adjust their OD600 to 1.0 with sterile water, and add each bacterial suspension at a 1% (v / v) inoculation rate to a solution containing 100 mg·L⁻¹ of IAA. -1 L-tryptophan was added to LB liquid medium with salt concentrations of 1%, 2%, and 4%. The medium was placed in a shaker at 28 °C and 150 rpm for 24 h, and centrifuged at 12000 rpm for 5 min. 100 μL of supernatant and 100 μL of colorimetric solution were placed in centrifuge tubes and incubated in the dark for 30 min to allow for color development. Each treatment was repeated in triplicate. Sterile water added to a medium containing 1% (v / v) L-tryptophan was used as a control. OD530 values ​​were measured and substituted into the standard curve. Figure 4 In the study, the ability of the strain to produce indoleacetic acid under different salt concentrations was calculated. Figure 5 (A). As shown in the figure, strain K309 exhibits the strongest IAA production capacity in a medium without NaCl, and it can still produce IAA under salt stress after the addition of salt solution.

[0026] 3. Determination of the siderophore production capacity of strain K309 Activated strain K309 was inoculated into MKB liquid medium with different NaCl concentrations: 2%, 4%, 6%, 8%, and 10%. The culture was incubated at 28°C and 160 r / min for 2 days. Then, 2 mL of bacterial culture was centrifuged at 5500 rpm for 8 min from each bottle. 100 μL of the supernatant was transferred to a 96-well microplate, with three replicates per treatment. An equal volume of CAS detection solution (BIOSICO) was added to the supernatant and mixed thoroughly. After incubation in the dark for 2 h, the OD630 value (A) was measured using distilled water as the standard. An equal volume of MKB liquid medium and CAS detection solution was mixed as the reference value (Ar). The siderophore activity (SU) was calculated as: SU (%) = (1 - A / Ar) × 100%. A higher SU value indicates a stronger siderophore production capacity. MKB iron-limiting medium preparation method: First, prepare the following three solutions respectively: 1) Add 5 g of casein amino acids, 15 mL of glycerol (glycerol), and 785 mL of deionized water; 2) Dissolve 2.5 g of dipotassium hydrogen phosphate trihydrate in 100 mL of deionized water; 3) Dissolve 2.5 g of magnesium sulfate heptahydrate in 100 mL of deionized water; Before preparation, all glassware was soaked overnight in concentrated hydrochloric acid (about 10%) to remove residual iron.

[0027] After preparation, each solution is sterilized separately at 115 °C for 30 min, pH=7.2. When using, mix all three solutions together.

[0028] The relative content of siderophores in the culture medium of strain K309 at various salt concentrations is shown in the figure. Figure 5 Figure B shows that strain K309 exhibits a significant increase in siderophore production, increasing by tens of times, after the addition of different concentrations of NaCl.

[0029] Example 3: Effects of strain K309 on soybean seed germination rate and seedling survival rate under salt stress Germination promotion experiment of salt-tolerant bacteria K309 Strain K309 was inoculated into LB liquid medium and cultured in a constant temperature incubator at 28℃ and 200 rpm for 12 h. The bacterial suspension was then collected, centrifuged at 8000 rpm for 10 min to obtain bacterial cells. The bacterial cells were then washed with sterile deionized water and finally resuspended in sterile 150 mM sodium chloride solution to prepare a bacterial suspension with OD600 = 0.1-0.3.

[0030] Undamaged, plump Wm82 (Williams 82) soybean seeds were placed in a sealed plastic box. 25 mL of concentrated hydrochloric acid and 75 mL of sodium hypochlorite were added to a beaker, and the seeds were surface-sterilized using chlorine gas. After sterilization for 12 hours, the seeds were removed and placed in a clean bench for 30 minutes to remove the chlorine. The soybean seeds were then placed in 12 cm × 12 cm germination boxes with two layers of sterile filter paper, 30 seeds per box, with three groups of three boxes per group. 30 mL of sterile water, 30 mL of sterile 150 mM NaCl solution, and 30 mL of K309 bacterial salt resuspension solution (OD600 = 0.1–0.3, this solvent is 150 mM NaCl) were added to each box, respectively. Germination rates were recorded on days 3, 5, 7, 9, and 10. Figure 6 Figure A shows the phenotypic diagram of soybean germination in each group after 7 days of cultivation. Germination rate was statistically analyzed using... Figure 6 As shown in Figure B, compared with NaCl treatment, the germination rate of strain K309 increased by 5.5% after treatment.

[0031] The above germination experiments show that exogenous application of strain K309 can improve the germination rate of soybean seeds under 150mM salt stress.

[0032] 2. Salt tolerance experiment of salt-tolerant bacteria K309 seedlings Mix nutrient soil, 2-4mm vermiculite, and field soil (previous soybean crop) in a 1:1:1 ratio, sterilize thoroughly at 121℃ for 60 minutes, and then package into mushroom bags, so that each mushroom bag contains 340 g of substrate.

[0033] Strain K309 was inoculated into LB liquid medium and incubated at 28℃ and 200 rpm for 12 h. The bacterial suspension was then collected and centrifuged at 8000 rpm for 10 min to obtain bacterial cells. The cells were then washed with sterile deionized water to prepare a bacterial suspension with an OD600 of 0.1–0.3. Four groups were set up: a control group (H2O, 4‰ NaCl), and experimental groups (K309 + H2O and K309 + 4‰ NaCl), with 40 bags in each group. 50 mL of the bacterial suspension was poured into mushroom bags and incubated at 28℃ for 28 days. For the H2O and 4‰ NaCl treatments, the same volume of sterile water was used instead of the bacterial suspension for incubation.

[0034] After incubation is complete, sterilized soybeans (using the same method as above) are planted in mushroom bags, with 3 soybeans per bag. Five days later, when the soybeans reach the two-leaf-one-heart stage, thinning is performed, leaving one seedling of similar growth in each mushroom bag. At this time, 50 mL of sodium chloride solution (4‰ of the substrate weight) is poured into the mushroom bags of the 4‰ NaCl control group and the K309 + 4‰ NaCl experimental group, while the others are irrigated with 50 mL of sterilized water. The plant growth is observed after 5 days. During this period, the experimental group is irrigated with K309 bacterial suspension with OD600 = 0.1-0.3, while the control group is irrigated with the same volume of sterilized water to maintain soil moisture.

[0035] Seven days after salt treatment, physiological indicators such as plant height, root length, fresh weight, and dry weight were measured. Significant differences were observed in plant height and fresh weight. Figure 7 and Figure 8 It can be seen that the soybeans treated with the bacterial suspension generally showed better growth than the negative control, with a 10% increase in survival rate. Soybeans treated with sodium chloride were stunted, with yellowing and wilting leaves and weak stems, resulting in a survival rate of 44%; while soybeans inoculated with the bacterial suspension generally grew normally, with greener leaves, and experienced less salt stress, achieving a survival rate of 55%. Furthermore, from... Figure 9 and Figure 10 It can be seen that, with the addition of bacteria, the plant height of soybean seedlings under salt stress was significantly increased compared with that under pure NaCl treatment, and the fresh weight of the plants was also significantly increased. All the results of the above salt tolerance experiment at the seedling stage indicate that exogenous application of strain K309 can improve the salt stress tolerance of soybean seedlings.

[0036] Example 4: Effect of strain K309 on the number of root nodules in soybean plants under salt stress This experiment used 8cm×8cm gallon pots. Sterilized soybean seeds (using the same method as above) were planted in 2-4mm vermiculite (sterilized at 121℃ for 20min). Five treatment groups were set up: water, NaCl, rhizobium + NaCl, K309 + NaCl, and K309 + rhizobium + NaCl. One plant was planted per gallon pot, with 20 plants per treatment. When the plants reached the stage of two leaves and one bud, all treatment groups with rhizobium were first watered with 50 mL of slow-growing rhizobium with an OD600 of 0.09, while the others were not watered. Four hours later, the rhizobium + NaCl treatment group was treated with 50 mL of 75 mM NaCl solution; the K309 + NaCl treatment group and the K309 + rhizobium + NaCl treatment group were treated with 50 mL of K309 resuspension (OD600 = 0.1–0.3) and 50 mL of 75 mM NaCl solution; the water control group was treated with 50 mL of sterile water; and the NaCl control group was treated with 50 mL of 75 mM NaCl solution. Subsequently, the rhizobium-containing treatment groups were treated with rhizobium (concentration and volume as above) every week, but no longer with salt or K309 suspension; all groups without rhizobium were treated with the same amount of water. Rhizobium phenotype was observed 28 days after salt application.

[0037] The method for preparing K309 bacterial suspension is the same as above.

[0038] The results show that applying K309 alone can significantly increase the number of root nodules in soybean plants. Figure 11 Compared to water and pure salt treatment, the number of bacteria per plant increased by about 10. Furthermore, the simultaneous application of K309 and rhizobium was more effective than applying either bacterium alone, increasing the number of bacteria per plant by about 25 compared to water and pure salt treatment. Figure 12 ).

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus hygroscopicus, characterized in that, The strain is Bacillus hygroscopicus (Bacillus hygroscopicus) Bacillus altitudinis Strain K309 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39318.

2. The *Bacillus hygroscopicus* strain according to claim 1, characterized in that, The 16S rRNA gene coding sequence of the strain is shown in SEQ ID NO.

1.

3. The application of the Bacillus hygroscopicus strain according to claim 1 or 2 in improving the salt and alkali stress tolerance of soybeans.

4. The application according to claim 3, characterized in that, The strain has the ability to produce IAA and siderophores under salt stress, and the siderophore production ability increases significantly under salt stress; it also has strong salt and alkali tolerance and can grow normally in medium containing 11% NaCl and medium with pH=9.

5. The application according to claim 3, characterized in that, The Bacillus hygroscopicus strain can increase the fresh weight of soybean seedlings, increase plant height, and improve their survival rate under salt stress. When the strain is applied in combination with rhizobia, the number of root nodules in the plants is significantly increased compared with the application of rhizobia alone.

6. A method for improving the salt and alkali tolerance of soybean seedlings, characterized in that, During the soybean seedling emergence to the two-leaf-one-heart stage, add 30 mL / strain of Bacillus hygroscopicus K309 with OD600=0.1~0.3 to the irrigation water during the watering process. The preservation number of K309 is CGMCC No.39318.

7. A method for increasing the number of soybean root nodules in saline-alkali land, characterized in that, During the soybean seedling emergence to the two-leaf-one-heart stage, add 30 mL / strain of Bacillus hygroscopicus K309 with OD600=0.1-0.3 and 50 mL / strain of Slow-growing Rhizobium with OD600=0.09 to the irrigation water during the watering process. The preservation number of K309 is CGMCC No.39318.

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