Salt-resistant alkali-resistant high-yield indoleacetic acid bacillus, separation and identification method and application

Abstract

The invention discloses a salt-resistant alkali-resistant high-yield heteroauxin bacillus, a separation and identification method and application, belongs to the technical field of microorganisms, and is used for identifying bacillus and promoting growth of sorghum, the separation and identification method is a 16SrDNA identification method, an upstream primer is 27F, a downstream primer is 1492R, a PCR amplification system is 12.5 mu L of 2 * Taq PCR Master Mix (Green), the upstream primer is 1 mu L, and the downstream primer is 1 mu L. The template DNA is 2.5 [mu] L, and the ddH2O is 8 [mu] L; in the PCR amplification procedure, denaturation, annealing and extension are performed for 30 cycles, and finally extension is performed at 72 DEG C for 10 minutes. When salt stress exists, the bacillus LB3201 provided by the invention has an obvious growth promoting effect, can obviously promote the growth and development of sorghum seedlings, and simultaneously reduces the adverse effect of the salt stress on the growth of the sorghum seedlings.

Description

Technical Field

[0001] The invention discloses a salt-resistant and alkali-resistant high-yield indoleacetic acid bacillus, a separation and identification method and application thereof, and belongs to the technical field of microorganisms. Background Art

[0002] Plant growth-promoting bacteria grow, develop and reproduce in the soil around the plant roots. They can fix nitrogen, release nutrients from insoluble minerals, repair and treat contaminated soil, and enhance the plant's tolerance to harsh environments and resist the invasion of pathogenic microorganisms by secreting various metabolites. Through these effects, soil structure is improved, plant growth is promoted, and yield is increased, making crop production green, environmentally friendly, and long-lasting. Bacillus megaterium is a Gram-positive bacterium, an aerobic bacterium and a spore-forming bacillus. It can degrade phosphorus-containing organic compounds, so it is used in agricultural production to produce phosphorus bacterial fertilizers and to produce phosphorus-solubilizing and potassium-fixing fertilizers. Because it can effectively degrade organic phosphorus in the soil, it is often used to make biological organic fertilizers. By forming a biofilm on the surface of the seeds, a protective layer is provided to resist the invasion of pathogens and harmful microorganisms. At present, a large number of Bacillus megaterium have been found, but most of them are concentrated in one or a few growth-promoting functions, which are very small compared with the rich microbial resources in nature. This also makes the application of Bacillus megaterium still have certain limitations. The present invention separates and screens Bacillus with multiple growth-promoting functions in the soil of salt field reclamation, so as to obtain more Bacillus resources and lay a foundation for the application of Bacillus in various soil environments to play a growth-promoting role. Summary of the invention

[0003] The invention discloses a salt-resistant and alkali-resistant high-yield indoleacetic acid bacillus, an isolation and identification method and an application thereof, so as to solve the problem in the prior art that growth-promoting bacteria having multiple growth-promoting functions and being salt-resistant and alkali-resistant cannot be screened.

[0004] The salt-tolerant and alkali-tolerant high-yielding indoleacetic acid Bacillus is Bacillus LB3201, the nucleotide sequence is shown in SEQ ID NO: 1, and it is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is November 8, 2024, the deposit number is CGMCC No.32540, and the classification name is Bacillus megaterium.

[0005] The invention discloses a method for separating and identifying salt-tolerant and alkali-tolerant indoleacetic acid high-yielding Bacillus. The salt-tolerant and alkali-tolerant indoleacetic acid high-yielding Bacillus is used. The separation and identification method is a 16SrDNA identification method. The 16SrDNA identification method includes PCR amplification primers. The PCR amplification primers include upstream primers and downstream primers.

[0006] The upstream primer is 27F, and the nucleotide sequence is shown in SEQ ID NO:2.

[0007] The downstream primer is 1492R, and the nucleotide sequence is shown in SEQ ID NO:3.

[0008] The PCR amplification system for the 16SrDNA identification method was 12.5 μL of 2×Taq PCR Master Mix (Green), 1 μL of upstream primer, 1 μL of downstream primer, 2.5 μL of template DNA, and 8 μL of ddH2O.

[0009] The PCR amplification program included pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min. Denaturation, annealing, and extension were performed for 30 cycles, and finally extension was performed at 72°C for 10 min.

[0010] The invention discloses an application of the salt-tolerant and alkali-tolerant indoleacetic acid high-yielding bacillus, and uses the salt-tolerant and alkali-tolerant indoleacetic acid high-yielding bacillus to promote the growth of sorghum.

[0011] Compared with the prior art, the present invention has the following beneficial effects: Bacillus LB3201 promotes the development of sorghum seedlings in terms of plant number, plant height and stem thickness; the seedlings inoculated with Bacillus LB3201 grow more vigorously than the sorghum seedlings not inoculated with growth-promoting bacteria at the corresponding concentration, have a large leaf area, and grow a large number of plants; the seedlings not inoculated with growth-promoting bacteria have obvious wilting at the leaf tips; when salt stress exists, Bacillus LB3201 has a significant growth-promoting effect, and Bacillus LB3201 can significantly promote the growth and development of sorghum seedlings, while reducing the adverse effects of salt stress on the growth of sorghum seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the phylogenetic tree of strain LB3201 based on 16SrDNA;

[0013] Figure 2 It is the growth curve and sporulation curve of Bacillus megaterium LB3201;

[0014] Figure 3 is the blank control group C0 of germination of sorghum seeds under different salt concentrations;

[0015] Figure 4 This is the germination of sorghum seeds under different salt concentrations, the bacterial group B0;

[0016] Figure 5 It is the salt-added group C200 showing the germination of sorghum seeds under different salt concentrations;

[0017] Figure 6 This is the germination of sorghum seeds under different salt concentrations in the salt-added bacteria group B200. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] The invention discloses a salt-tolerant and alkali-tolerant high-yielding indoleacetic acid bacillus, wherein the bacillus is bacillus LB3201, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the bacillus is deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection with a deposit number of CGMCC No. 32540, and the classification name is Bacillus megaterium.

[0020] The invention discloses a method for separating and identifying salt-tolerant and alkali-tolerant indoleacetic acid high-yielding Bacillus. The salt-tolerant and alkali-tolerant indoleacetic acid high-yielding Bacillus is used. The separation and identification method is a 16SrDNA identification method. The 16SrDNA identification method includes PCR amplification primers. The PCR amplification primers include upstream primers and downstream primers.

[0021] The upstream primer is 27F, and the nucleotide sequence is shown in SEQ ID NO:2.

[0022] The downstream primer is 1492R, and the nucleotide sequence is shown in SEQ ID NO:3.

[0023] The PCR amplification system for the 16SrDNA identification method was 12.5 μL of 2×Taq PCR Master Mix (Green), 1 μL of upstream primer, 1 μL of downstream primer, 2.5 μL of template DNA, and 8 μL of ddH2O.

[0024] The PCR amplification program included pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min. Denaturation, annealing, and extension were performed for 30 cycles, and finally extension was performed at 72°C for 10 min.

[0025] The invention discloses an application of the salt-tolerant and alkali-tolerant indoleacetic acid high-yielding bacillus, and uses the salt-tolerant and alkali-tolerant indoleacetic acid high-yielding bacillus to promote the growth of sorghum.

[0026] The nucleotide sequence of the salt-tolerant and alkali-tolerant high-yielding indoleacetic acid Bacillus is shown in SEQ ID NO: 1:

[0027]

[0028] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2:

[0029] AGAGTTTGATCCTGGCTCAG.

[0030] The nucleotide sequence of the downstream primer is shown in SEQ ID NO: 3:

[0031] TACGGTTACCTTGTTACGACTT.

[0032] The present invention uses a genetic resource named Bacillus LB3201, and extracts the coding gene of bacterial ribosomal RNA. The genetic resource is taken from a microorganism and collected by itself in Changyi City, Weifang City, Shandong Province on June 6, 2019. It is specifically located in the abandoned salt field reclamation area of ​​Zaohu Village, Liutuan Town, Changyi City. The nucleotide sequences in the present invention are all sorted from the 5' end to the 3' end. In an embodiment of the present invention, two culture media are set, LB seawater culture medium is used as a screening culture medium, including peptone 10g / L, yeast powder 5g / L, NaCl 100g / L, distilled water 1000mL, pH10.0, and the culture medium can simultaneously have the screening conditions of high salt and high alkali dual pressure; LB liquid culture medium includes peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, distilled water 1000mL, pH7.0.

[0033] When separating the strains, weigh 10g of soil samples collected at different stations, place the soil samples in conical flasks containing appropriate glass beads and 90mL sterile water, and place them in a shaking table at 30°C and 180rpm for 30min. After the soil suspension after shaking is left to stand for 5min, dilute it in different gradients, take 10-4, 10-5, and 10-6 dilutions of soil suspension 100μL each, apply them to the screening culture medium plate, place them at room temperature for 30min, then place the plate in a 30°C incubator and invert and culture for 3-5d, and observe and record the colony growth every day. Select the colonies with vigorous growth and different morphologies in the culture medium, purify the strains by plate streaking method, repeat 3 times for each group, so as to obtain purified strains and number them, inoculate the separated and purified strains on the slant culture medium for culture, and store them in a 4°C refrigerator. The separation method of salt-resistant and alkali-resistant bacteria of the present invention solves the problem that the separation and screening time of salt-resistant and alkali-resistant bacteria in the prior art is too long.

[0034] The growth-promoting properties of the strain were determined. The strain LB3201 had a strong ability to produce IAA (33.72 μg / mL) and ACC deaminase, as well as strong phosphate and potassium solubilization, protease, amylase, and pectinase activities (colony diameter / hydrolysis circle diameter). It could also produce iron carriers and fix nitrogen. As shown in Table 1, the growth-promoting properties of the strain were significant.

[0035] Table 1 Growth-promoting properties of strain LB3201

[0036] ;

[0037] In the table, IAA represents indoleacetic acid, and ACC represents ACC deaminase.

[0038] Salt tolerance was determined. Excessive salinity can affect the normal growth of microorganisms. Salt tolerance tests were conducted on the strain at salinities of 0%, 5%, 10%, 15%, and 20%. The results are shown in Table 2. When the salinity was 15%, the strain could grow normally. When it increased to 20%, its growth was inhibited. Overall, the strain had a strong salt tolerance.

[0039] Table 2 Effects of different salinities on the growth of strain LB3201

[0040] ;

[0041] DNA extraction includes the following 8 steps:

[0042] (1) Take 1.5 mL of bacterial solution and place it in an epoxy fire tube (EP tube). Centrifuge at 12,000 rpm for 2 minutes, discard the supernatant, and turn the EP tube upside down on absorbent paper to remove residual water. If there is mucus attached to the EP tube, add physiological saline to the EP tube and centrifuge again;

[0043] (2) Pipette 200 μL of lysis solution into the EP tube and pump vigorously to fill the EP tube with bubbles;

[0044] (3) Add 66 μL of 5 mol / L NaCl solution, mix well with a pipette, and centrifuge at 12,000 rpm for 10 min;

[0045] (4) Pipette the supernatant into a new EP tube and record the volume of the supernatant. Add an equal volume of Tris-saturated phenol reagent to the EP tube, invert it upside down to emulsify it, and centrifuge it at 12,000 rpm for 3 min.

[0046] (5) Take the aqueous phase from the EP tube and put it into a new EP tube. Record the volume of the supernatant. Add an equal volume of chloroform to the EP tube, mix by inverting it, and centrifuge it at 12,000 rpm for 3 min.

[0047] (6) Take the supernatant and put it into a new EP tube. Record the volume of the supernatant. Then add twice the volume of pre-cooled anhydrous ethanol. Let it stand at 4°C for 12 h. Centrifuge at 12,000 rpm for 15 min. Discard the ethanol and turn the EP tube upside down to drain the water.

[0048] (7) Vacuum dry the EP tube, then add 55 μL of ddH2O to dissolve the DNA;

[0049] (8) The extracted DNA was subjected to 1% agarose gel electrophoresis test. If it passed the test, it was stored in a -20℃ refrigerator for future use.

[0050] Molecular biological identification was carried out, and PCR amplification was performed using the extracted total DNA as a template. The universal primers 27F and 1492R were selected as PCR primers. The PCR amplification reaction system is shown in Table 3. The PCR amplification program was: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1.5 min, 30 cycles, and extension at 72°C for 10 min. The PCR product was detected by agarose gel electrophoresis, and then the PCR product was purified and DNA sequenced. The sequencing results were compared and analyzed on the NCBI database using Blast (biological macromolecule sequence alignment search tool), and the phylogenetic tree was constructed using MEGAX software (evolutionary tree tool) with the Neighbor-joining statistical method (neighbor joining method) after 1000 bootstrappings. Figure 1 As shown in the figure, multiple strain information is included, and the strain LB3201 is preliminarily determined to be Bacillus megaterium based on the comparison results.

[0051] Table 3 PCR amplification reaction system

[0052] ;

[0053] In the optimization of the spore production conditions of the strain, the single-factor method was used to optimize the spore production conditions of Bacillus. The results showed that the optimal spore production medium composition of the strain was: molasses 7g / L, corn steep liquor 4g / L, manganese sulfate 0.04g / L, potassium dihydrogen phosphate 0.5g / L; the optimal spore production culture conditions were: temperature 30°C, rotation speed 200r / min, liquid volume 40mL / 250mL, initial pH value 5, inoculation amount 3.5%. Bacillus megaterium was fermented and cultured under the optimal conditions, and the OD600nm value (absorbance value) and spore number of the sample were measured every 4 hours, and the fermentation lasted for 48 hours. With the fermentation time as the horizontal axis and the OD600nm value and spore number of the sample as the vertical axis, the growth curve and spore production curve of Bacillus megaterium were plotted as shown in the figure. Figure 2 As shown. Figure 2It can be seen that the spore production curve of Bacillus megaterium LB3201 is not synchronized with the growth curve. Since spores are only produced in large quantities when the bacteria reproduce to a certain extent, the nutrients in the culture environment are scarce, or the metabolic waste increases, the spore production curve has a hysteresis effect. The delay period of the growth curve is between 0-4h, the logarithmic period is 4h-20h, of which 12h-20h is the late logarithmic period, and it enters the stable period after 20h. The spore production curve shows a significant upward trend within 8h-28h, and then it is still rising slowly. It enters the stable period after 44h, and the number of spores reaches the maximum, which is 6.2 billion / mL. Therefore, the harvest time of spores is selected at about 44h.

[0054] When preparing bacterial suspension, 50 ml of LB liquid medium was inoculated with strain LB3201 at a 3.5% inoculation rate, and cultured on a shaker at 30.0°C and 180 rpm for 24-28 hours. The bacterial suspension was transferred to a 50 ml centrifuge tube, centrifuged at 7000 rpm for 8 minutes, washed twice with sterile distilled water, centrifuged at 7000 rpm for 8 minutes, removed the supernatant, and retained the bacterial precipitate. An appropriate amount of sterile distilled water was added, shaken and mixed, and the OD value (600 nm) of the bacterial suspension was adjusted to 1.0.

[0055] The effects of growth-promoting bacteria on the growth and development of sorghum seedlings under different salt stresses were investigated. Peat organic nutrient soil, perlite, and vermiculite were mixed in a ratio of 3:1:1, and 80 mL of deionized water and 200 mmol / L of NaCl solution were added to every 40 g of mixed soil. The mixture was stirred evenly. Ten groups were prepared according to this standard, with five replicates in each group. The treated soil from each group was placed in a plastic cup.

[0056] After 3 days of cultivation, the sorghum seeds with the same growth conditions and germinated were moved into plastic cups filled with soil. The seeds were buried to a depth of about 1.5 cm, and 10 seeds were planted in each cup. Transfer to a light incubator and culture for 15 days at 27°C and 60% light intensity. During the culture period, distilled water was poured every two days. After 7 days, 3 mL of growth-promoting bacterial suspension with OD600 = 1.0 was evenly added to the soil where the seeds in each group treated with growth-promoting bacteria were located. After continuing to culture for 15 days, the biomass and related physiological indicators of sorghum seedlings were measured.

[0057] The effects of growth-promoting bacteria on sorghum seed germination under different salt stresses were investigated. An appropriate amount of sorghum seeds with full grains and the same size were selected. The sorghum seeds were first soaked in a 75% ethanol solution for 1 minute, washed 5 times with sterile water, and then soaked in a 5% sodium hypochlorite solution for 1 minute. The seeds were washed 5 times with sterile water. After disinfection, bleaching, and rinsing, sterile sorghum seeds were obtained. The sterile sorghum seeds were placed in a bacterial suspension with an OD600 value of 1.0 and soaked in a dark place at room temperature for 24 hours. Deionized water soaking was used as a blank control.

[0058] The soaked seeds were placed in plates containing 200 mmol / L NaCl and sterile water (covered with 2 filter papers inside) for cultivation. Thirty seeds were evenly placed on each plate, with 3 replicates in each group. The plates were placed in a 26°C constant temperature and light incubator for cultivation.

[0059] The experimental design is as follows:

[0060] (1) Blank control group: Sorghum seeds soaked in deionized water (control group seeds) were added to a plate containing sterile water as the blank control group, marked as C0.

[0061] (2) Bacteria-added group: Sorghum seeds (experimental group seeds) soaked in a suspension of growth-promoting bacteria were added to a plate containing sterile water as the bacteria-added group, marked as B0.

[0062] (3) Salt-added control group: Sorghum seeds soaked in deionized water (control group seeds) were added to plates containing 200 mmol / L NaCl solution as the salt-added group, marked as C200.

[0063] (4) Salt-added bacteria group: Sorghum seeds (experimental group seeds) soaked in a suspension of growth-promoting bacteria were added to plates containing 200 mmol / L NaCl solution, as the salt-added bacteria group, marked as B200.

[0064] On the third day after incubation, the number of sorghum seeds that germinated in each plate was counted and the corresponding germination rate was calculated. The formula for calculating the seed germination rate in each plate is as follows:

[0065] Germination rate = (N3÷30) × 100%, N3 is the total number of sorghum seeds germinated within 3 days.

[0066] The results and analysis of the effects of growth-promoting bacteria on sorghum seed germination under different salt stresses were conducted. The germination rate of seeds is an important indicator for judging their economic value and quality in the agricultural production process. A higher seed germination rate can ensure consistency when germinating, thereby ensuring that the planting density can meet the standard, reducing the amount and area of ​​secondary replanting, and saving costs. The germination rate of seeds can more intuitively reflect the growth-promoting effect of growth-promoting bacteria on sorghum seed germination. Under salt stress conditions, the high seed germination rate proves that growth-promoting bacteria can alleviate the inhibitory effect of salt stress on seed germination and enhance the resistance of seeds to salt.

[0067] In order to preliminarily explore the growth-promoting effect of salt-tolerant rhizosphere growth-promoting bacteria, the growth-promoting bacteria were inoculated on sorghum seeds and cultured under different salt stress conditions. Figure 3 , Figure 4 , Figure 5 Figure 6 shows the effect of growth-promoting bacteria on the average germination rate of sorghum seeds after 3 days of cultivation under salt stress. The group inoculated with growth-promoting bacteria had more seeds germinated and longer buds. Figure 3 The germination rate of the blank control group C0 was 68.89%. Figure 4 The germination rate of the bacteria-added group B0 was 76.67%. Figure 5 The germination rate of C200 in the salt-added group was 53.33%. Figure 6 The germination rate of the salt-added bacteria group B200 was 71.11%. Under no salt stress, compared with the blank control group C0, the growth-promoting bacteria in the bacteria group B0 increased the germination rate of sorghum seeds by 7.78%; compared with the blank control group, the germination rates of the salt-added group (C200) decreased by 15.56% and 16.67%, respectively; compared with the salt-added groups of the corresponding salt concentrations, the seed germination rate of the salt-added bacteria group B200 increased by 17.78%. It can be seen that the seed germination rate is the highest when there is no salt, and the seed germination rate shows a decreasing trend with the increase of salt concentration, indicating that salt stress within this concentration range will inhibit the germination of sorghum seeds, and the growth-promoting bacteria play a significant role in promoting the germination and germination of sorghum seeds.

[0068] The results and analysis of the effects of growth-promoting bacteria on the number of sorghum seedlings, plant height and stem diameter were carried out. Pot experiments were carried out under different salt stress conditions to investigate the effects of growth-promoting bacteria on the average number of sorghum seedlings, plant height and stem diameter under different salt stress conditions. C0 grew an average of 9.4 plants, B0 grew an average of 9.4 plants, C200 grew an average of 7.6 plants, and B200 grew an average of 9.0 plants. Compared with C0, B0 did not have a significant growth-promoting effect, but when salt stress existed, B200 grew 18.42% more plants than C200. Table 4 shows the growth of sorghum seedlings under different treatments.

[0069] Table 4 Growth of sorghum seedlings under different treatments

[0070] ;

[0071] The results in Table 4 show that growth-promoting bacteria can promote the development of sorghum seedlings in terms of plant number, plant height, and stem thickness. The growth of seedlings inoculated with growth-promoting bacteria is significantly more vigorous than that of sorghum seedlings not inoculated with growth-promoting bacteria at the corresponding concentration, with a larger leaf area and more growing plants. The wilting phenomenon at the tip of the leaves of seedlings not inoculated with growth-promoting bacteria is obvious. With the increase of salt concentration, the number of germination plants, plant height and stem thickness of sorghum seedlings showed a downward trend regardless of whether the growth-promoting bacteria were sprayed. However, in terms of plant number and plant height, B0 and C0 were not much different. However, when salt stress existed, the number of plants in B200 increased by 18.42% and the plant height increased by 17.37% compared with C200, and the growth-promoting effect was significant. It shows that growth-promoting bacteria can significantly promote the growth and development of sorghum seedlings and reduce the adverse effects of salt stress on the growth of sorghum seedlings. Although inoculation with growth-promoting bacteria cannot completely eliminate the inhibitory effect of salinity, it can alleviate the damage caused by salt stress to the growth and development of sorghum seedlings to a certain extent.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features may be replaced by equivalents, and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A salt-tolerant and alkali-tolerant high-yielding indoleacetic acid Bacillus, characterized in that: The bacillus is Bacillus LB3201, the nucleotide sequence of which is shown in SEQ ID NO: 1, and is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 32540, and a classification name of Bacillus megaterium.

2. A method for isolating and identifying a salt-tolerant and alkali-tolerant high-yielding indoleacetic acid Bacillus, characterized in that: The salt-tolerant and alkali-tolerant high-yield indoleacetic acid Bacillus described in claim 1 is used, and the separation and identification method is a 16SrDNA identification method, the 16SrDNA identification method includes PCR amplification primers, and the PCR amplification primers include upstream primers and downstream primers.

3. The method for separating and identifying the salt-tolerant and alkali-tolerant high-yielding indoleacetic acid Bacillus according to claim 2, characterized in that: The upstream primer is 27F, and the nucleotide sequence is shown in SEQ ID NO:

2.

4. The method for separating and identifying the salt-tolerant and alkali-tolerant high-yielding indoleacetic acid Bacillus according to claim 2, characterized in that: The downstream primer is 1492R, and the nucleotide sequence is shown in SEQ ID NO:

3.

5. The method for separating and identifying the salt-tolerant and alkali-tolerant high-yielding indoleacetic acid Bacillus according to claim 3 or 4, characterized in that: The PCR amplification system for the 16SrDNA identification method was 12.5 μL of 2×Taq PCR Master Mix (Green), 1 μL of upstream primer, 1 μL of downstream primer, 2.5 μL of template DNA, and 8 μL of ddH2O.

6. The method for separating and identifying the salt-tolerant and alkali-tolerant high-yielding indoleacetic acid Bacillus according to claim 5, characterized in that: The PCR amplification program included pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min. Denaturation, annealing, and extension were performed for 30 cycles, and finally extension was performed at 72°C for 10 min.

7. The application of salt-tolerant and alkali-tolerant high-yielding indoleacetic acid Bacillus, characterized in that: The salt-tolerant and alkali-tolerant high-yield indoleacetic acid Bacillus according to claim 1 is used to promote the growth of sorghum.

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