Saline-alkali tolerant strain capable of producing IAA and application thereof

Abstract

The invention discloses and relates to a strain capable of resisting saline and alkaline and producing IAA (Indoleacetic Acid). The strain is classified and named as bacillus atrophaeus, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) NO.29799. The strain is preserved in China General Microbiological Culture Collection Center on January 29, 2024. The bacillus atrophaeus HHQGTS13-1 can grow under the condition that the salt content is higher than 3%, can generate indoleacetic acid, cellulase, amylase, protease and ligninase, can inhibit various plant pathogenic bacteria, such as banana anthracnose, tomato gray mold, cotton verticillium wilt, rice sheath blight disease, wheat head blight, banana fusarium wilt, capsicum white spot and the like, and can be used for preparing the bacillus atrophaeus HHQGTS13-1. Particularly, the compound plays an important role in preventing and treating wheat scab, banana anthracnose and capsicum white spot. The strain can promote plant growth in a saline-alkali environment, and can be used for improving soil quality, developing special microbial fertilizers, microbial agents and biopesticides for saline-alkali soil, relieving crop salt stress damage and increasing crop yield.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically, to a strain capable of tolerating salt and alkali and producing indole-3-acetic acid and its application. Background Art

[0003] Salt-alkali stress is one of the main abiotic stresses affecting the development and production of agriculture in China. High concentrations of salts and high pH values in the soil can cause phenomena such as changes in the osmotic potential in plants, ion concentration imbalance, metabolic disorders in plants, and severe damage to cell membranes. These adverse factors will inhibit the growth and development of plants and even directly lead to plant death.

[0004] Salt-tolerant and alkali-tolerant microorganisms have important application values in many fields. For example, in the agricultural field, they can be used to improve soil quality and increase crop yields. However, existing salt-tolerant and alkali-tolerant microorganisms often do not have the ability to produce indole-3-acetic acid (IAA), thus limiting their application scope. Therefore, it is of great practical value to develop a strain that is salt-tolerant and alkali-tolerant and can produce IAA. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain capable of tolerating salt and alkali and producing IAA and its application. This strain has strong stress resistance, can tolerate 12% sodium chloride, can reproduce within the range of pH 4.5 - 10.0, can produce indole-3-acetic acid, cellulase, amylase, protease, and ligninase, has a strong effect of inhibiting plant pathogens, and can promote the growth of plants in a salt-alkali environment.

[0006] To achieve the above purpose, in the first aspect of the present invention, a strain capable of tolerating salt and alkali and producing IAA is provided. This strain was deposited on January 29, 2024, at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the address being No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 29799, and the taxonomic name being Bacillus atrophaeus.

[0007] In the second aspect of the present invention, it is provided that the strain described in the first aspect has the ability to produce indole-3-acetic acid, cellulase, amylase, protease, and ligninase.

[0008] In the third aspect of the present invention, it is provided the application of the strain described in the first aspect in inhibiting plant pathogens.

[0009] In the fourth aspect of the present invention, it is provided the application of the strain described in the first aspect in promoting plant growth.

[0010] Selecting a location includes the following steps: in a saline-alkali or non-saline-alkali environment, applying the growth-promoting bacteria or the growth-promoting bacteria agent to the seeds of the plant, or applying it to the planting substrate of the plant.

[0011] The beneficial effects of the present invention are as follows: The applicant of the present invention collected soil samples from an environment with a salt content higher than 3%, screened and isolated Bacillus atrophaeus HHQGTS13-1. After identification, this strain has strong stress resistance, can tolerate 12% sodium chloride, and can reproduce within the range of pH 4.5 - 10.0; it produces indole acetic acid, cellulase, amylase, protease, and ligninase; it has a strong effect of inhibiting plant pathogenic bacteria, can inhibit a variety of plant pathogenic bacteria, such as inhibiting banana anthracnose, tomato gray mold, cotton verticillium wilt, rice sheath blight, wheat scab, banana wilt, pepper white spot, wheat scab, potato common scab, rice bacterial leaf streak, etc. Especially, it plays an important role in controlling wheat scab, banana anthracnose, and pepper white spot. Through pot experiments, it was found that HHQGTS13-1 can improve the resistance of wheat to salt stress; it can significantly increase the stem length, root length, fresh weight, and dry weight of plants. Thus, it shows that the Bacillus atrophaeus HHQGTS13-1 of the present application has good salt and alkali tolerance characteristics, still has a growth-promoting effect on plants in saline-alkali land, and is a biocontrol strain with excellent traits. This strain has good applications in the fields of preparing saline-alkali land improvers, plant bio-fertilizers, biological pesticides, etc.

[0012] Figure 1 Colony morphology diagram of Bacillus atrophaeus HHQGTS13-1 Figure 2 Microscopic examination diagram of the cells of Bacillus atrophaeus HHQGTS13-1 Figure 3 Salt tolerance function identification diagram of Bacillus atrophaeus HHQGTS13-1 Figure 4 Alkali tolerance function identification diagram of Bacillus atrophaeus HHQGTS13-1 Figure 5 Indole acetic acid production function identification diagram of Bacillus atrophaeus HHQGTS13-1 Figure 6 Cellulase production function identification diagram of Bacillus atrophaeus HHQGTS13-1 Figure 7 Amylase production function identification diagram of Bacillus atrophaeus HHQGTS13-1 Figure 8 Protease production function identification diagram of Bacillus atrophaeus HHQGTS13-1 Figure 9 Ligninase production function identification diagram of Bacillus atrophaeus HHQGTS13-1 Figure 10 Effect diagram of Bacillus atrophaeus HHQGTS13-1 inhibiting plant pathogenic bacteria Figure 11Effect diagram of Bacillus atrophaeus HHQGTS13-1 inhibiting plant pathogens Specific implementation mode

[0013] In order to make the purpose, technical solutions and advantages of the present invention clearer, the following will detail the specific implementation modes of the present disclosure in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0014] Unless otherwise specified, the materials used in this embodiment are all commercially available products.

[0015] Nutrient agar medium: peptone 10 g / L, beef extract powder 3 g / L, sodium chloride 5 g / L, agar (solid) 15 g / L, pH adjusted to 7.0 - 7.2.

[0016] LB medium: tryptone 10.0 g, yeast extract powder 5.0 g, sodium chloride 10.0 g.

[0017] Indole acetic acid production medium: sucrose 5.0 g, tryptone 5.0 g, yeast powder 2.5 g, sodium chloride 2.5, tryptophan 500 mg.

[0018] Sodium carboxymethyl cellulose liquid culture medium: CMC-Na 20.0 g, (NH4)2SO4 2.0 g, MgSO4·7H2O 0.5 g, KH2PO4 1.0 g, NaCl 0.5 g, congo red 0.4 g, agar 20.0 g, water 1000 mL, sterilized at 121℃ for 30 min.

[0019] Amylase production medium: starch 20.0 g, yeast extract 5.0 g, peptone 10 g, Na2HPO4 5.0 g, MgSO4·7H2O 0.1 g, NaCl 0.1 g, agar 20.0 g, water 1000 ml, PH 7.0 - 7.4. Sterilized at 0.1 MPa for 20 min.

[0020] Protease production medium: beef extract 3.0 g / L, peptone 6.0 g / L, NaCl 3.0 g / L, agar 25.0 g / L, skimmed milk powder 20.0 g, deionized water 1000 mL, and sterilized at 120℃ for 15 min.

[0021] Aniline blue decolorization medium: lignin 10.0 g, (NH4)2SO4 4.3 g, MgSO4·7H2O 0.3 g, KH2PO4 4.3 g, CaCl2 0.3 g aniline blue 0.1 g, agar 18.0 g, distilled water 1000 mL.

[0022] Implementation case 1 This implementation case is used to illustrate the isolation and identification of Bacillus atrophaeus HHQGTS13-1 (1)Isolation of Bacillus atrophaeus HHQGTS13-1 Sample collection: Soil samples were collected from the soil with a salt content higher than 3% on the Taotiao Mountain Farm, 486th Township Road, Jingtai County, Baiyin City, Gansu Province. The soil collection used the five-point sampling method. In the selected area, five sampling points were determined. Select plants with good growth, remove the surface soil, and collect the near-root soil at 5-15 cm. Collect equal amounts of soil at each point, mix well, put it into a sterilized bag and seal it, mark the key information such as the collection number, collection location, and date, and bring it back to the laboratory for storage at 4°C.

[0023] Soil sample treatment: The soil sample was sieved through a 20-mesh sample sieve (aperture about 1 mm); 10 g of the test sample was weighed and put into a sterile physiological saline solution of 90 mL. Shake at 28°C and 200 rpm for 2-3 h, and let it stand for 10 min to obtain a soil suspension diluted 10 times, denoted as 10 -1 dilution. Use a pipette to aspirate 1 mL of 10 -1 dilution and add it to a test tube containing 9 mL of sterile physiological saline, blow and suck evenly to dilute it into 10 -2 dilution, and then dilute it successively according to this method to make 10 -3 、10 -4 、10 -5 、 10-6 and other series of gradient dilutions; Aspirate 0.1 mL of each gradient dilution and spread it evenly on an Ashby nitrogen-free medium plate. Repeat 3 plates for each concentration. Incubate the plates upside down in a 35°C constant temperature incubator for 3-4 d.

[0024] Strain isolation and screening: Select plates with appropriate colony growth density (30-300 / plate), pick different types of single colonies on the above nitrogen-free medium plates and streak them on nitrogen-free medium plates for isolation. Incubate at 28°C for 2-3 d, pick single cells and streak them again for culture. After 2-3 times of single-cell streaking, obtain the single-cell pure culture of each strain; Pick single cells of each strain and inoculate them into LB liquid medium containing 8%, 10%, and 12% NaCl respectively. Shake at 35°C and 200 rpm for 2-3 d. Screen the strains that grow normally on the 12% sodium chloride LB plate for strain purification and preservation.

[0025] Strain purification and preservation: Judge whether the strain is a single strain according to colony characteristics and combined with staining microscopy. Finally, store the pure strain at low temperature with 20% glycerol. Select the strain that grows normally on the 12% sodium chloride LB plate and store it named Bacillus atrophaeus HHQGTS13-1.

[0026] Implementation case 2 This example is used to illustrate the morphological identification of Bacillus atrophaeus HHQGTS13-1.

[0027] (1) Pick the purified Bacillus atrophaeus HHQGTS13-1 and culture it on a nutrient agar medium by the streaking method for 2 days at 35°C. Then observe the colony morphology, and the colony morphology is as Figure 1 shown.

[0028] The strains with the preservation number of CGMCC NO.29799 all showed colony morphologies that were white, nearly circular, slightly wrinkled on the surface, dry, and serrated at the edges.

[0029] (2) Observe the cell morphology of the strain through an oil immersion microscope (oil 100×10), and the results are as Figure 2 shown.

[0030] The cells of the strain were rod-shaped; after Gram staining, they showed purple and were identified as Gram-positive bacteria; and a large number of spores could be formed after the strain was cultured on a plate. Example 3

[0031] This example is used to illustrate the salt and alkali tolerance of Bacillus atrophaeus HHQGTS13-1.

[0032] Preparation of the seed solution: Pick a single colony of Bacillus atrophaeus HHQGTS13-1 and inoculate it into a test tube of LB liquid medium, and culture it at 35°C and 200 rpm for 24 h to prepare the bacterial seed solution.

[0033] Prepare media containing 10% and 12% NaCl by mass concentration respectively, inoculate the seed solution of Bacillus atrophaeus HHQGTS13-1 at an inoculation amount of 2%, and culture for 48 h. Bacillus atrophaeus HHQGTS13-1 grew normally on the LB plate containing 12% sodium chloride, as Figure 3 shown.

[0034] Configure a liquid LB medium with a pH of 9.5 / 10, inoculate the seed solution of Bacillus atrophaeus HHQGTS13-1 at an inoculation amount of 2%, and culture for 48 h. It can be seen from Figure 4 that the control group's liquid LB medium was clear, and the liquid LB medium with a pH of 9.5 / 10 was turbid, indicating that Bacillus atrophaeus HHQGTS13-1 could grow normally in the liquid LB medium with a pH of 9.5 / 10, as Figure 4 shown.

[0035] It can be seen from this that the strain can grow and reproduce normally in the salt and alkali concentration culture solution and maintain its vitality. It shows that the strain can adapt to the high-salt and high-osmotic pressure environment and has a wider application scenario, and can be used for biological treatment of high-salt wastewater and improvement of saline-alkali soil, etc. Example 4

[0036] This test example is used to illustrate the ability of Bacillus atrophaeus HHQGTS13-1 to produce indole-3-acetic acid, cellulase, amylase, protease, and ligninase.

[0037] (1)Determination of indole-3-acetic acid production ability: Inoculate 1-2 loops of Bacillus atrophaeus HHQGTS13-1 stored at low temperature in Example 1 into LB medium and culture at 35 °C for 24 h to obtain the primary seed liquid. Inoculate it into the high-yield indole-3-acetic acid liquid medium at an inoculation amount of 2%, set three replicates, and the shaking fermentation temperature is 30 °C, and the rotation speed is 180 r•min -1 conditions, and shake culture for 24 h. Take 100 μL of the bacterial suspension on a white ceramic plate, and then drop 100 μL of Salkowski colorimetric solution (1 mL of 0.5 mol / L FeCl3 + 50 mL of 35% HClO4). Use the LB liquid culture medium without inoculation as a control and add an equal volume of Salkowski colorimetric solution. Place the white ceramic plate in the dark for 30 min, take it out and observe. If the color turns red, it indicates the ability to produce indole-3-acetic acid, and the darker the color, the higher the content of indole-3-acetic acid. The test results show that the strain turns red and can produce indole-3-acetic acid. From Figure 5 it can be seen that Bacillus atrophaeus HHQGTS13-1 secreting indole-3-acetic acid is preliminarily screened.

[0038] (2)Determination of cellulase production ability: Use an inoculation loop to aseptically pick a single colony from the plate and place it in 1 mL of carboxymethyl cellulose sodium liquid culture medium, set three replicates, and culture it overnight in a shaker at 37 °C. Then centrifuge for 10 min at a rotation speed of 8000 r / min and take the supernatant. Use the sampling punching method to place 100 μL of the supernatant on the Congo red medium plate and culture at 37 °C for 3 d, and observe the diameter of the transparent circle. From the preliminary screening results of Congo red Figure 6 it can be seen that Bacillus atrophaeus HHQGTS13-1 secreting cellulase is preliminarily screened, and the size of the hydrolysis circle is 2.30 cm.

[0039] (3)Determination of amylase production ability: Inoculate 1-2 loops of Bacillus atrophaeus HHQGTS13-1 stored at low temperature in Example 1 into LB medium, and the shaking fermentation temperature is 30 °C, and the rotation speed is 180 r•min -1 conditions, and shake culture for 24 h. Inoculate the cultured strains on the amylase production medium by streaking, set three replicates, and culture at 37 °C for 48 h. Stain with iodine solution. If a transparent circle appears around the colony, it proves that the bacillus produces amylase. From the preliminary screening results of iodine solution Figure 7 it can be seen that Bacillus atrophaeus HHQGTS13-1 secreting amylase is preliminarily screened, and the size of the hydrolysis circle is 1.00 cm.

[0040] (4) Determination of protease production ability: Inoculate 1 - 2 loops of Bacillus atrophaeus HHQGTS13 - 1 preserved at low temperature in Example 1 into LB medium, with the shaking flask fermentation temperature at 30 °C and the rotation speed at 180 r•min -1 condition, and shake - culture for 24 h. Inoculate the cultured strains on the protease - producing medium by streaking, set three replicates, and culture at 37 °C for 48 h. The production of protease by the bacillus is proved by the appearance of a clear zone around the colony. It can be Figure 8 seen that Bacillus atrophaeus HHQGTS13 - 1 secreting protease is initially screened, and the size of the hydrolysis zone is 2.55 cm.

[0041] (5) Determination of lignin - enzyme production ability: Inoculate 1 - 2 loops of Bacillus atrophaeus HHQGTS13 - 1 preserved at low temperature in Example 1 into LB medium, with the shaking flask fermentation temperature at 30 °C and the rotation speed at 180 r•min -1 condition, and shake - culture for 24 h. Inoculate the cultured strains on the aniline blue decolorization medium by streaking, set three replicates, and culture at 37 °C for 10 d. Observe and record every day. The production of lignin - enzyme by Bacillus atrophaeus HHQGTS13 - 1 is proved by the appearance of a decolorization zone around the colony. It can be Figure 9 seen that Bacillus atrophaeus HHQGTS13 - 1 secreting amylase is initially screened, and the size of the hydrolysis zone is 1.85 cm.

[0042] Test Example 5 This test example is used to illustrate the effect of Bacillus atrophaeus HHQGTS13 - 1 on inhibiting plant pathogenic bacteria.

[0043] Confrontational culture of Bacillus atrophaeus HHQGTS13 - 1 with 11 tested pathogenic fungi. After culturing in a constant - temperature incubator at 35 °C for 7 d, measure the colony diameter. It has an inhibitory effect on pathogens such as banana anthracnose, tomato gray mold, cotton verticillium wilt, rice sheath blight, wheat scab, banana wilt, pepper white spot, wheat head blight, potato common scab, and rice bacterial leaf streak. Among them, it has a better inhibitory effect on wheat head blight, banana anthracnose, and pepper white spot. ( Figure 10 , Table 1) Table 1 Inhibitory zone width (cm) of Bacillus atrophaeus HHQGTS13 - 1 against different pathogenic bacteria

[0044] Experimental Example 6 This example is used to illustrate the ability of Bacillus atrophaeus HHQGTS13 - 1 to improve wheat's resistance to salt stress and promote wheat growth.

[0045] Simulated saline - alkali planting system: Add a mixed salt solution to the vermiculite culture system to simulate the saline - alkali planting environment and conduct an experiment to verify the growth - promoting effect of the strain on wheat.

[0046] Prepare a salt solution with a salt content of 6 g / kg (NaCl:Na2CO3 = 5:1), measure its pH value to be 10.57, and EC = 10.97 mS / cm; moisten vermiculite with the salt solution to an appropriate humidity, put it into pots to prepare a saline-alkali system, and set up a salt-free system as a control.

[0047] For the seed germination test with the bacterium Bacillus atrophaeus HHQGTS13-1 bacterial liquid dressing, set 8 treatments, with 4 replicates for each treatment and 100 seeds for each replicate. During the test period, keep the soil humidity appropriate, and count the emergence number of each replicate on the 10th day; count the fresh weight of the above-ground part of each replicate on the 10th day.

[0048] (I) Experimental treatments Table 2. Test treatments

[0049] (II) Experimental results and analysis ① Effects of different treatments on emergence rate In the saline-alkali planting system, the emergence rates of each treatment with the bacterial liquid increased by 5.30% - 7.77% compared with the control. The emergence rate of the 1:2000 treatment with the bacterial liquid increased by 5.30% compared with the control; the emergence rate of the 1:1000 treatment with the bacterial liquid increased by 6.36% compared with the control; the emergence rate of the 1:1000 treatment with the bacterial liquid increased by 7.77% compared with the control. In the non-saline-alkali planting system, the emergence rates of each treatment with the bacterial liquid increased by 4.71% - 6.65% compared with the control. The emergence rate of the 1:2000 treatment with the bacterial liquid increased by 4.71% compared with the control; the emergence rate of the 1:1000 treatment with the bacterial liquid increased by 5.54% compared with the control; the emergence rate of the 1:1000 treatment with the bacterial liquid increased by 6.65% compared with the control.

[0050] Table 3 Effects of different treatments on emergence rate (unit: 1)

[0051] (2) Effects of different treatments on wheat stem length, root length, fresh weight and dry weight In the saline-alkali planting system, the stem lengths of each treatment with the bacterial solution increased by 30.87% - 90.00% compared with the control, the root lengths of each treatment with the bacterial solution increased by 50.00% - 77.33% compared with the control, the fresh weights of each treatment with the bacterial solution increased by 52.80% - 71.68% compared with the control, and the dry weights of each treatment with the bacterial solution increased by 42.98% - 64.26% compared with the control. In the non-saline-alkali planting system, the stem lengths of each treatment with the bacterial solution increased by 18.67% - 62.13% compared with the control, the root lengths of each treatment with the bacterial solution increased by 37.28% - 57.78% compared with the control, the fresh weights of each treatment with the bacterial solution increased by 40.73% - 55.35% compared with the control, and the dry weights of each treatment with the bacterial solution increased by 21.37% - 62.19% compared with the control.

[0052] Table 4 Effects of Different Treatments on the Stem Length, Root Length, Fresh Weight and Dry Weight of Wheat

[0053] As can be seen from Table 4, the plant phenotype of the treatment with clear water control H2O is better than that of the saline-alkali control NaCl treatment, and there are significant differences between the two treatments, indicating that the prepared saline-alkali system has an obvious inhibitory effect on wheat growth.

[0054] In the non-saline system, the phenotype of the inoculated treatment is better than that of the non-inoculated treatment, indicating that in the normal planting system, the strain can promote wheat growth. In the saline-alkali system, the phenotype of the inoculated treatment is better than that of the non-inoculated treatment, indicating that the strain can still promote wheat growth in the saline-alkali environment.

[0055] The above description is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A strain of salt-tolerant and IAA-producing Bacillus atrophaeus HHQGTS13-1, characterized in that, Isolated from a soil environment with a salt content higher than 3%, and preserved in the General Microbiology Center of the China Microbial Culture Collection Center, with the preservation number: CGMCC NO. 29799, and the classification name is Bacillus atrophaeus.

2. The salt-tolerant and IAA-producing strain Bacillus atrophaeus HHQGTS13-1 according to claim 1 has the ability to produce indole acetic acid, cellulase, amylase, protease, and ligninase.

3. The salt-tolerant and IAA-producing strain Bacillus atrophaeus HHQGTS13-1 according to claim 1 has the effect of inhibiting plant pathogenic bacteria, and has an inhibitory effect on various pathogenic bacteria such as banana anthracnose, tomato gray mold, cotton verticillium wilt, rice sheath blight, wheat scab, banana wilt, pepper white spot, wheat scab, potato scab, and rice bacterial leaf streak. Among them, it has a better inhibitory effect on wheat scab, banana anthracnose, and pepper white spot.

4. Use of the halotolerant and IAA-producing strain Bacillus atrophaeus HHQGTS13-1 according to claim 1 in promoting plant growth, characterized in that, In a saline-alkali or non-saline-alkali environment, the said strain is applied to the seeds of plants, or applied to the planting substrate of the said plants.

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