Application of Bacillus pumilus AD14 in improving saline-alkali soil and enhancing soybean resistance to saline-alkali stress

Abstract

The invention discloses an application of Bacillus pumilus AD14 in improving saline-alkali soil and improving the resistance of soybean to saline-alkali stress, which belongs to the technical field of agricultural microorganisms. In order to screen Bacillus pumilus that can improve saline-alkali soil and significantly improve the resistance of soybean to saline-alkali stress, the invention screens a strain AD14 with good salt-alkali resistance from saline-alkali soil. The strain is identified as Bacillus pumilus ( Bacillus pumilus ), the experiment found that the strain has the growth-promoting properties of dissolving phosphorus, fixing nitrogen and producing pectinase; it can change the physical and chemical properties of saline-alkali soil, increase the activities of urease, sucrase and catalase in saline-alkali soil; improve the stress resistance of soybean in saline-alkali soil and promote the growth of soybean in saline-alkali soil. Therefore, Bacillus pumilus AD14 has broad application prospects in improving saline-alkali soil, improving the resistance of soybean to saline-alkali stress and promoting the growth and development of soybean under saline-alkali stress.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and particularly relates to application of Bacillus pumilus AD14 in improving saline-alkali soil and improving the salt-alkali stress resistance of soybean. Background Art

[0002] Land salinization has always been a global problem, and with global climate change and unreasonable agricultural production activities, soil degradation and salinization are becoming increasingly serious, which seriously affects the growth and yield of crops. As an important economic crop, soybeans are particularly affected by saline-alkali stress in their growth environment. Salt-alkali stress can lead to increased osmotic pressure of soil solution, affect water absorption and nutrient utilization by plant roots, and thus affect plant growth and yield. At present, traditional soil improvement methods include physical improvement, chemical improvement and biological improvement. Physical improvement, such as deep plowing of soil and covering with organic matter, is effective but has high cost and limited effect; chemical improvement relies on the application of chemical substances, which may cause environmental pollution; biological improvement improves the soil environment by introducing beneficial microorganisms. This method has the advantages of low cost, environmental friendliness and high efficiency, and has been better promoted than other improvement methods.

[0003] Bacillus brevis ( Bacillus subtilis ) is a Gram-positive bacterium widely found in nature, with strong salt-alkali tolerance and plant growth promotion effects. Studies have shown that Bacillus pumilus can improve the stress resistance and growth performance of plants through multiple mechanisms such as producing plant hormones, dissolving insoluble nutrients in the soil, and enhancing the balance of plant rhizosphere microecology. However, existing research on the application of Bacillus pumilus mainly focuses on the single plant growth promotion aspect, and there is little research on how to further improve the resistance of crops such as soybeans to salt-alkali stress. Therefore, screening a Bacillus pumilus that can not only improve saline-alkali soil, but also significantly improve the resistance of crops such as soybeans to salt-alkali stress is of great significance to ensure the safety and sustainable development of agricultural production. Summary of the invention

[0004] In order to screen a Bacillus pumilus that can improve saline-alkali soil and significantly improve the resistance of crops such as soybeans to salt-alkali stress, the present invention screened a strain AD14 with good salt-alkali resistance from saline-alkali soil, and identified the strain as Bacillus pumilus ( Bacillus pumilus ), experiments have found that the strain has the characteristics of solubilizing phosphorus, fixing nitrogen and producing pectinase; it can change the physical and chemical properties of saline-alkali soil, including reducing soil pH and soluble salt content, and increasing the activity of urease, sucrase and catalase in saline-alkali soil; it can also improve the resistance of soybean to salt-alkali stress and promote the growth of soybean in saline-alkali soil.

[0005] In order to solve the above technical problems and achieve corresponding technical effects, the present invention specifically provides the following technical solutions:

[0006] The first object of the present invention is to provide Bacillus ( Bacillus sp.) AD14, the application of which is to use Bacillus AD14 for improving saline-alkali soil, the Bacillus AD14 is deposited in the China Center for Type Culture Collection, the deposit number is CCTCC NO: M 2024621, the deposit date is April 3, 2024, and the depository address is Wuhan University, Wuhan, China.

[0007] In one embodiment of the present invention, the Bacillus AD14 can reduce the pH and soluble salt content of the soil during the application.

[0008] In one embodiment of the present invention, the Bacillus AD14 in the application can increase the activities of urease, sucrase and catalase in the soil.

[0009] In one embodiment of the present invention, the method for improving saline-alkali soil in the application is to mix the bacterial solution of Bacillus AD14 with the saline-alkali soil.

[0010] The second object of the present invention is to provide an application of Bacillus AD14, wherein the application is to use Bacillus AD14 to improve the salt-alkali stress resistance of soybeans. The deposit number of the Bacillus AD14 is CCTCC NO: M 2024621.

[0011] In one embodiment of the present invention, the application of Bacillus AD14 can increase the activities of superoxide dismutase, peroxidase and catalase in soybean leaves and roots under saline-alkali stress, and reduce the malondialdehyde content in soybean leaves and roots under saline-alkali stress.

[0012] In one embodiment of the present invention, the application of Bacillus AD14 can increase the soluble sugar and soluble protein content of soybean plants under saline-alkali stress.

[0013] In one embodiment of the present invention, the method for improving the salt-alkali stress resistance of soybean in the application is to add a bacterial solution of Bacillus AD14 into the saline-alkali soil where soybean is planted.

[0014] The third object of the present invention is to provide an application of Bacillus AD14, wherein the application is to use Bacillus AD14 to promote the growth of soybean in saline-alkali soil. The deposit number of Bacillus AD14 is CCTCC NO: M 2024621.

[0015] In one embodiment of the present invention, the method for promoting the growth of soybeans in saline-alkali soil in the application is to add a bacterial solution of Bacillus AD14 to the saline-alkali soil where soybeans are grown.

[0016] The fourth object of the present invention is to provide an application of a microbial agent or biological fertilizer containing Bacillus AD14, wherein the application is to use the microbial agent or biological fertilizer to improve saline-alkali soil, and the preservation number of the Bacillus AD14 is CCTCC NO: M 2024621.

[0017] The fifth object of the present invention is to provide an application of a microbial agent or biological fertilizer containing Bacillus AD14, wherein the application is to use the microbial agent or biological fertilizer to improve the resistance of soybean to salt-alkali stress, and the preservation number of the Bacillus AD14 is CCTCC NO: M 2024621.

[0018] The sixth object of the present invention is to provide an application of a microbial agent or biological fertilizer containing Bacillus AD14, wherein the application is to use the microbial agent or biological fertilizer to promote the growth of soybeans in saline-alkali soil, and the preservation number of the Bacillus AD14 is CCTCC NO: M 2024621.

[0019] Beneficial effects of the present invention:

[0020] The present invention uses saline-alkali soil as soil sample, from which strain AD14 with good salt-alkali resistance is screened, and the strain is identified as Bacillus pumilus (Bacillus pumilus) through morphological, physiological and biochemical and molecular biological identification. Bacillus pumilus ). The present invention proves through multiple experiments that strain AD14 has the growth-promoting properties of dissolving phosphorus, fixing nitrogen and producing pectinase; it can improve the physical and chemical properties of saline-alkali soil (reduce soil pH and soluble salt content), and can increase the activity of urease, sucrase and catalase in saline-alkali soil; in addition, strain AD14 can improve the stress resistance of soybean in saline-alkali soil and promote the growth of soybean in saline-alkali soil, which is specifically manifested in increasing the activity of superoxide dismutase, peroxidase and catalase in soybean leaves and roots, reducing malondialdehyde content, and increasing soluble sugar and soluble protein content; increasing soybean plant height, root length, root number and root weight. Therefore, Bacillus AD14 has broad application prospects in improving saline-alkali soil, improving soybean resistance to salt-alkali stress, and promoting the growth and development of soybean under salt-alkali stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the result of 16S rDNA phylogenetic tree analysis of strain AD14;

[0022] Figure 2This is the result of the phosphate solubilization ability test of strain AD14 on NBRIP medium;

[0023] Figure 3 This is a graph showing the nitrogen fixation ability test result of strain AD14 on Ashubei nitrogen-free medium;

[0024] Figure 4 This is a graph showing the results of the pectinase production ability test of strain AD14 on pectin culture medium;

[0025] Figure 5 The results of superoxide dismutase activity detection in soybean leaves and roots before and after treatment with bacterial solution of strain AD14 in different saline-alkali soils; Figure 5 A in the figure is the result of superoxide dismutase activity test in soybean leaves before and after bacterial solution treatment in different saline-alkali soils. Figure 5 B in the figure is the result of superoxide dismutase activity detection in soybean root system before and after bacterial solution treatment in different saline-alkali soils; different lowercase letters indicate that the superoxide dismutase activity under bacterial solution treatment is significantly different. P <0.05;

[0026] Figure 6 The results of peroxidase activity detection in soybean leaves and roots before and after treatment with bacterial solution of strain AD14 in different saline-alkali soils; Figure 6 A in the figure is the result of peroxidase activity test in soybean leaves before and after bacterial solution treatment in different saline-alkali soils. Figure 6 B in the figure is the result of peroxidase activity detection in soybean root system before and after bacterial solution treatment in different saline-alkali soils; different lowercase letters indicate significant differences in peroxidase activity under bacterial solution treatment. P <0.05;

[0027] Figure 7 The results of catalase activity detection in soybean leaves and roots before and after treatment with bacterial solution of strain AD14 in different saline-alkali soils; Figure 7 A in FIG. 7 is a graph showing the results of catalase activity detection in soybean leaves before and after bacterial solution treatment in different saline-alkali soils, and B in FIG. 7 is a graph showing the results of catalase activity detection in soybean roots before and after bacterial solution treatment in different saline-alkali soils; different lowercase letters indicate significant differences in catalase activity under bacterial solution treatment. P <0.05;

[0028] Figure 8 The results of the detection of malondialdehyde content in soybean leaves and roots before and after treatment with bacterial solution of strain AD14 in different saline-alkali soils; Figure 8 A in the figure is the detection result of malondialdehyde content in soybean leaves before and after bacterial liquid treatment in different saline-alkali soils. Figure 8B in the figure is the detection result of malondialdehyde content in soybean root system before and after bacterial solution treatment in different saline-alkali soils; different lowercase letters indicate that the malondialdehyde content under bacterial solution treatment is significantly different. P <0.05. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific examples and drawings. The following examples are convenient for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The drugs, reagents, and materials used in the following examples can be purchased through commercial channels unless otherwise specified.

[0030] The Bacillus ( Bacillus sp.) AD14 is deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO: M 2024621, the deposit date being April 3, 2024, and the depository address being Wuhan University, Wuhan, China.

[0031] The culture medium involved in the present invention is as follows:

[0032] LB solid and liquid medium (for strain culture): 10 g peptone, 5 g yeast extract powder, 10 g NaCl, 15 g agar (liquid without agar), and finally distilled water to 1000 mL, pH 7.0-7.2.

[0033] Ashuber nitrogen-free medium (used for testing the nitrogen fixation ability of strains): mannitol 15 g, CaCl2·2H2O 0.2 g, K2HPO4 0.2 g, MgSO4·7H2O 0.2 g, MoO3 0.001 g, FeCl3 0.005 g, agar 15 g (agar is not added to the liquid), and finally distilled water to make up to 1000 mL, pH 7.2±0.2.

[0034] NBRIP medium (for strain phosphate solubilization ability detection): glucose 10 g, Ca3(PO4)25 g, MgCl25 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO40.1 g, agar 15 g (liquid without agar), and finally distilled water to 1000 mL, pH 7.0±0.2.

[0035] Pectin culture medium (used for testing the ability of strains to produce pectinase): 4 g yeast extract powder, 20 g glucose, 2 g pectin, 3 g nitrate, 0.5 g KCl, 1 g K2HPO4, 0.5 g MgSO4, 0.01 g FeSO4, 15 g agar (no agar is added to the liquid), and finally distilled water to make up to 1000 mL, pH 6.0±0.2.

[0036] The soil samples used in the present invention were collected from the saline-alkali land area of ​​the Anda Experimental Station of the Daqing Branch of the Heilongjiang Academy of Agricultural Sciences in Anda City, Suihua City, Heilongjiang Province.

[0037] The soybean used in the present invention is "Dongnong Bean 252", which is stored and provided by the Botany Laboratory of Northeast Agricultural University.

[0038] Other reagents used in the present invention: plant superoxide dismutase (SOD) activity assay kit, peroxidase (POD) activity assay kit, catalase (CAT) activity assay kit, malondialdehyde (MDA) content assay kit, soluble sugar content assay kit, soluble protein content assay kit, soil urease activity assay kit, soil sucrase activity assay kit, soil catalase activity assay kit are all purchased from Taisge Biotechnology Co., Ltd.

[0039] Example 1: Isolation, screening and identification of Bacillus pumilus AD14

[0040] 1. Isolation of strain AD14

[0041] The five-point sampling method was used to collect soil samples in the saline-alkali land area of ​​the Anda Experimental Station of the Daqing Branch of the Heilongjiang Academy of Agricultural Sciences in Anda City, Suihua City, Heilongjiang Province. The specific method was as follows: two soils with different salinity and alkali levels and a depth of 0-20 cm were selected, and the gravel and grass roots were removed and sieved and packed in sterile plastic bags, and then brought back to a -20℃ refrigerator for storage. Under sterile conditions, 5 g of soil sample was weighed and dissolved in a conical flask containing 45 mL of sterile water, shaken at 180 r / min for 20 min, and then allowed to stand. The supernatant was taken as 1×10 -1 Dilution: 10 -1 1 mL of diluent was transferred into a test tube containing 9 mL of sterile water and pipetted to mix the bacterial solution evenly to obtain 1×10 -2 dilution; and so on, continuously dilute to 1×10 -7, the dilution was spread on LB solid medium, and three replicates were set for each concentration of dilution. After culturing at 37°C for 3-5 days, the colonies with different morphologies grown on the plate were picked respectively, and the colony morphology was observed and subcultured for more than three times. The strains were inoculated into LB liquid medium for further purification, and 15 strains were screened. The screened strains were inoculated into LB liquid medium, and cultured on a shaker at 37°C and 180 r / min for 12 h to obtain the fermentation broth for use.

[0042] 2. Screening of strains for salt and alkaline tolerance

[0043] The 15 strains screened were inoculated with LB solid medium with different NaCl concentrations and different pH values ​​for salt and alkali resistance tests. After culturing at 37°C for 3-7 days, the bacterial growth was observed and recorded. Based on the above experimental results, the salt concentration and pH value for the optimal growth of the strains were selected. In order to further screen out the strains with the strongest comprehensive salt and alkali resistance, a salt-alkali composite experiment was conducted. According to the screening results of the salt and alkali resistance tests, the optimal salt concentration range for the growth of the 15 strains was 4%-6%, and the optimal pH range was 8-9. The strain with the strongest salt and alkali resistance screened by the salt and alkali resistance composite test was AD14, and the results of its salt and alkali resistance composite test are shown in Table 1. Therefore, strain AD14 was used as the test strain for later salt and alkali resistance and growth promotion related tests.

[0044] Table 1 Growth of strain AD14 on the optimal saline-alkali plate

[0045]

[0046] Note: "+++" indicates that the colonies grow vigorously on the culture medium, "++" indicates that the colonies grow more on the culture medium, "+" indicates that the colonies grow less on the culture medium, and "-" indicates that the colonies do not grow on the culture medium; Treatment group 1 is salt concentration 4%, pH value 8, treatment group 2 is salt concentration 6%, pH value 8, treatment group 3 is salt concentration 4%, pH value 9, treatment 4 is salt concentration 6%, pH value 9

[0047] 3. Identification of strain AD14

[0048] (1) Morphological identification: The colony morphology of strain AD14 grown on LB plates was obtained by plate spreading method, and then observed and recorded.

[0049] The morphological identification results showed that strain AD14 grew rapidly on the LB plate, and growth could be observed within 12 hours; the colony color was milky white, the colony edge was smooth, and the center was slightly convex. The cell morphology of strain AD14 was observed under a microscope, and it was found that the cell morphology of strain AD14 was straight rod-shaped.

[0050] (2) Physiological and biochemical identification: The physiological and biochemical indicators of the strain were identified by referring to the methods in the Manual of Identification of Common Bacterial Systems, and the strain AD14 was subjected to Gram staining test, catalase test, VP test, MR test, and indole test.

[0051] The results of physiological and biochemical identification showed that strain AD14 was a Gram-positive bacterium with catalase activity and was negative in VP, MR and indole tests.

[0052] (3) Molecular biological identification: The 16S rDNA molecular identification method was used as follows: After extracting the genomic DNA of strain AD14, PCR amplification was performed using the universal primers 27F (nucleotide sequence such as SEQ ID NO.1: 5'-GAGAGTTTGATCCTGGCTCAG-3') and 54lR (nucleotide sequence such as SEQ ID NO.2: 5'-AAGGAGGTGATCCAGCCGCA-3') of the bacterial 16S rDNA sequence. The PCR product was detected by agarose gel electrophoresis and sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing. The sequence obtained by sequencing was compared with the NCBI database and sequence homology analysis was performed. The phylogenetic tree was constructed using MEGA5.0 software, and the species of strain AD14 was molecularly identified.

[0053] The 16S rDNA sequence of strain AD14 was found to be as shown in SEQ ID NO.3. The comparison results of 16S rDNA sequence showed that the 16S rDNA sequence of strain AD14 was similar to Bacillus pumilus The homology of strain AD14 to Bacillus pumilus CP027116.1, HQ218989.1 and CP054310.1 was 100%, and the phylogenetic tree analysis showed that strain AD14 belonged to the same branch as Bacillus pumilus CP027116.1, HQ218989.1 and CP054310.1 ( Figure 1 Therefore, combined with the morphological and physiological and biochemical identification results of strain AD14, it was identified as Bacillus pumilus ( Bacillus pumilus ) and deposited in the China Center for Type Culture Collection, the deposit number is CCTCC NO: M2024621, the deposit date is April 3, 2024, and the depository address is Wuhan University, Wuhan, China.

[0054] SEQ ID NO.3:

[0055]

[0056] Example 2: Determination of the growth-promoting properties of Bacillus pumilus AD14

[0057] (1) Determination of phosphate solubilization ability of strain AD14

[0058] After activating the strain AD14 stored in the glycerol tube, 5.0 µL was inoculated on the NBRIP plate. After culturing in the dark at 37°C for 7 days, the growth of the strain was observed. The ability of the strain AD14 to dissolve phosphorus was determined by whether it could produce a transparent phosphorus-dissolving ring on the culture medium. The phosphorus-dissolving ability of the strain was determined by the ratio of the phosphorus-dissolving ring diameter (D) / colony diameter (d). The larger the ratio, the stronger the phosphorus-dissolving ability of the strain, the smaller the ratio, the weaker the phosphorus-dissolving ability of the strain, and a ratio of 1 indicates that the strain has no phosphorus-dissolving ability.

[0059] Figure 2 The results showed that the D / d ratio of strain AD14 was 2.6, indicating that strain AD14 had a strong phosphate solubilizing ability.

[0060] (2) Determination of nitrogen fixation ability of strain AD14

[0061] After the strain AD14 stored in the glycerol tube is restored from the refrigerated state to room temperature, it is inoculated into LB solid medium for cultivation. This step is called strain activation. The activation step is repeated 2-3 times, and a single colony is picked and inoculated into Asbestos nitrogen-free medium by streaking method. After 7 days of dark cultivation at 37°C, the growth of the strain is observed. Whether the strain AD14 can grow on the medium is judged to determine whether it has the ability to fix nitrogen.

[0062] from Figure 3 It can be seen that strain AD14 has strong nitrogen fixation activity.

[0063] (3) Determination of the pectinase production capacity of strain AD14:

[0064] After the strain AD14 preserved in the glycerol tube was activated, it was inoculated in pectin culture medium, with three replicates for each treatment. After being placed in a 37°C incubator for dark culture for 3-5 days, the culture medium was stained with Gram iodine solution. The ability of strain AD14 to produce pectinase was judged by whether it could produce a transparent circle on the culture medium, and the strength of the strain's pectinase production ability was judged by the ratio of transparent circle diameter (D) / colony diameter (d). The larger the ratio, the stronger the strain's ability to produce pectinase, the smaller the ratio, the weaker the strain's ability to produce pectinase, and a ratio of 1 indicates that the strain has no ability to produce pectinase.

[0065] Figure 4 The results showed that the D / d ratio of strain AD14 was 3.8, indicating that strain AD14 had a good ability to produce pectinase.

[0066] Example 3: Determination of the effect of Bacillus pumilus AD14 on the physicochemical properties and enzyme activity of saline-alkali soil

[0067] The experimental site was set up in the plant culture room of the College of Life Sciences of Northeast Agricultural University. The culture room conditions were 25±2℃ and the light cycle was 18 h / 6 h (day / night). The two saline-alkali soils collected in Anda City in the early stage were used as the cultivation medium for potted planting. The pots used were 15 cm in diameter, 12 cm in height, and 2 kg in soil. The pH value of soil 1 was 7.82, the salt content was 0.73%, and it was classified as slightly saline-alkali soil; the pH value of soil 2 was 8.44, the salt content was 1.12%, and it was classified as moderately saline-alkali soil. The soybean variety was Dongnong 252, and the bacterial solution was Bacillus pumilus AD14 bacterial solution. The untreated saline-alkali soil was used as the control group, and the Bacillus pumilus AD14 bacterial solution was used as the treatment group. Each group was set up with 10 replicates. After 5 days of soybean germination, the treatment group began to irrigate the bacterial solution (OD 600 =0.8), 10 mL was evenly irrigated to each pot, and an equal volume of distilled water was added to the control group. During the experiment, the bacterial solution was irrigated every 5 days, and the daily management was watering once every 2 days. 30 days after inoculation, soybean plants and rhizosphere soil samples of each group were collected, sealed in sterile bags, and stored in a -20℃ refrigerator for subsequent determination of soybean-related indicators, soil physical and chemical properties, and enzyme activity.

[0068] (1) Determination of the effects of strain AD14 on the physical and chemical properties of saline-alkali soil

[0069] The soil pH value and soluble salt content were determined by referring to the kit method. The kit was purchased from Taisge Biotechnology Co., Ltd. and the method is detailed in the instructions.

[0070] The results of the effect of the bacterial solution of strain AD14 on the physical and chemical properties of saline-alkali soil are shown in Table 2. Before the bacterial solution treatment, the pH values ​​of the two saline-alkali soil samples were 7.82 and 8.44, and the salt contents were 0.73% and 1.12%, respectively. After the treatment, the pH values ​​of the two soil samples decreased by 12.15% and 10.90%, respectively, compared with those before the treatment, both showing significant differences; the soluble salt content of soil 1 also showed a decreasing trend after the bacterial solution treatment, but it was not significant, and the salt content of soil 2 decreased by 0.15% after the bacterial solution treatment, which was a more significant effect.

[0071] Table 2 Results of the test on the effects of strain AD14 on the physical and chemical properties of saline-alkali soil

[0072]

[0073] Note: * indicates that there are significant differences in the indicators of the same soil before and after treatment. P <0.05

[0074] (2) Determination of the effect of strain AD14 on enzyme activity in saline-alkali soil

[0075] The soil urease, sucrase and catalase activities were determined according to the kit method. The kit was purchased from Taisge Biotechnology Co., Ltd. and the method is detailed in the instructions.

[0076] The results of the effect of the bacterial solution of strain AD14 on the enzyme activity of saline-alkali soil are shown in Table 3. After the application of the bacterial solution, the activities of urease, sucrase and catalase in the two saline-alkali soils increased. The urease activity of soil 1 and soil 2 increased by 23.27% and 23.56% respectively compared with the control group, the sucrase activity increased by 29.71% and 20.56% respectively compared with the control group, and the catalase activity increased by 16.65% and 25.36% respectively compared with the control group. This shows that the application of the bacterial solution has enhanced the soil fertility of saline-alkali soil to a certain extent, providing excellent environmental conditions for the growth and development of soybean plants. Overall, the bacterial solution has a higher effect on improving the enzyme activity of soil 1 than soil 2.

[0077] Table 3 Results of the determination of the effect of strain AD14 on enzyme activity in saline-alkali soil

[0078]

[0079] Note: * indicates that there is a significant difference in the enzyme activity of the same soil before and after treatment. P <0.05.

[0080] Example 4: Determination of the effect of bacterial solution of strain AD14 on improving the ability of soybean to resist salt-alkali stress

[0081] The experimental method for obtaining soybean plants is the same as that in Example 3.

[0082] (1) Determination of the effect of strain AD14 on soybean growth

[0083] The soybean plant height, root length, root number and root weight were measured after planting.

[0084] The results of the effect of the bacterial solution of strain AD14 on the growth of soybean seedlings are shown in Table 4. The growth indicators of soybeans grown in the two soils treated with bacterial solution were significantly improved compared with the control without bacterial solution. In particular, the root length and root weight of soybean plants grown in soil 1 increased by 75.25% and 91.34% compared with the control group; the soybean plants grown in soil 2 were significantly lower than those in soil 1. This shows that the use of bacterial solution can enable soybeans to grow in different degrees of saline-alkali stress environments, especially reduce the inhibition of plant growth in mild saline-alkali soils, increase the colonization ability of soybean roots, and improve the survival rate of soybean plants in saline-alkali environments.

[0085] Table 4 Results of the test on the effect of strain AD14 on soybean growth

[0086]

[0087] Note: * indicates that there are significant differences in soybean growth indicators in the same soil before and after treatment. P <0.05

[0088] (2) Determination of the effect of strain AD14 on the soybean antioxidant system

[0089] The SOD, POD, CAT, and MDA contents of soybean plants were determined using a kit purchased from Taisge Biotechnology Co., Ltd.

[0090] Effects of strain AD14 on soybean antioxidant system Figure 5 , Figure 6 , Figure 7 and Figure 8 shown. Figure 5 A in Figure 6 A and Figure 7 A in the figure shows the changes in the activities of SOD, POD, and CAT in the leaves of soybean plants after inoculation with Bacillus pumilus AD14. 30 days after the application of the bacterial solution, the antioxidant enzyme activities in the leaves showed significant differences, especially in the soybeans grown in soil 2 containing the bacterial solution, the SOD activity increased by 30.81% compared with the control group without the bacterial solution. Figure 5 B in Figure 6 B and Figure 7 As shown in Figure B, the bacterial solution significantly increased the antioxidant enzyme activity in the roots of soybean plants, especially in the soybeans grown in soil 2 containing bacterial solution, where the CAT activity increased by 36.80% compared to the control group without bacterial solution. Figure 8 It can be seen that the MDA content of soybean leaves and roots in the treatment group was significantly lower than that in the control group, indicating that the bacterial solution, on the basis of improving the physical and chemical properties of the soil, alleviated the adverse effects of saline-alkali stress on soybeans. Overall, the effect of soil 1 was more significant.

[0091] (3) Determination of the effect of strain AD14 on soybean osmotic substances

[0092] The soluble sugar and soluble protein contents of soybean plants were determined using a kit purchased from Taisge Biotechnology Co., Ltd.

[0093] The effect of strain AD14 on the permeability of soybean plants is shown in Table 5. After exogenous application of bacterial solution, the permeability of soybean plants grown in two saline-alkali soils was significantly improved compared with the control group. The increase in the soluble sugar and soluble protein content of the leaves of soybean plants grown in soil 1 reached 11.19% and 35.90%, respectively, and the soluble sugar and soluble protein content of the roots increased by 46.83% and 47.87%, respectively; the increase in the soluble sugar and soluble protein content of the leaves of soybean plants grown in soil 2 reached 25.87% and 37.17%, respectively, and the soluble sugar and soluble protein content of the roots increased by 14.22% and 34.71%, respectively. Based on the above results, the permeability of soybean plants cultivated in soil 1 after being treated with strain AD14 was higher than that in soil 2, and the effect was more obvious.

[0094] Table 5 The results of the determination of the effect of strain AD14 on soybean osmotic substances

[0095]

[0096] Note: * indicates that there are significant differences in the permeability of soybean plants grown in the same soil before and after treatment. P <0.05.

[0097] Under adverse conditions, plants can resist saline-alkali stress by increasing the synthesis of osmotic substances. The research results of the present invention found that Bacillus pumilus AD14 bacterial solution can significantly increase the soluble sugar and soluble protein content of soybean leaves and roots in mild and moderate saline-alkali soils, and osmotic regulating substances can be accumulated and synthesized, thereby maintaining the balance of cellular osmotic potential inside the plant and reducing the damage caused by saline-alkali stress to soybean plants.

[0098] The above experiments show that the bacterial solution of Bacillus microsporus AD14 can regulate the soil pH, reduce the accumulation of salt in the soil, and increase the activity of enzymes in the soil; it can also alleviate the adverse effects of salt-alkali stress on soybean plants, such as significantly increasing the activity of antioxidant enzymes in the leaves and roots of soybean plants, significantly increasing the content of osmotic substances in soybean plants, and promoting the growth of soybeans under salt-alkali stress.

[0099] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Bacillus ( Bacillus sp.) application of AD14, characterized in that, The application is to use Bacillus AD14 to improve the resistance of soybean to salt-alkali stress. Improving the resistance of soybean to salt-alkali stress refers to improving the activities of superoxide dismutase, peroxidase and catalase in soybean leaves and roots under salt-alkali stress, reducing the malondialdehyde content in soybean leaves and roots under salt-alkali stress, and increasing the soluble sugar and soluble protein content of soybean plants under salt-alkali stress. The preservation number of the Bacillus AD14 is CCTCC NO: M 2024621.

2. An application of a microbial agent or biological fertilizer containing Bacillus AD14, characterized in that: The application is to use the microbial agent or biological fertilizer to improve the resistance of soybean to salt-alkali stress. Improving the resistance of soybean to salt-alkali stress refers to improving the activities of superoxide dismutase, peroxidase and catalase in soybean leaves and roots under salt-alkali stress, reducing the malondialdehyde content in soybean leaves and roots under salt-alkali stress, and increasing the soluble sugar and soluble protein content of soybean plants under salt-alkali stress. The preservation number of the Bacillus AD14 is CCTCC NO: M 2024621.

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