Bacillus atrophaeus and application thereof in plant salt tolerance and growth promotion
By using Bacillus atrophus CGMCC No.37219, the problems of plant salt tolerance and heavy metal wastewater treatment in high-concentration saline-alkali land were solved, achieving the effects of promoting plant growth and purifying water sources.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- LANZHOU UNIV
- Filing Date
- 2026-01-30
- Publication Date
- 2026-07-31
AI Technical Summary
Existing salt-tolerant microbial strains have poor improvement effects in high-concentration saline-alkali land, making it difficult to effectively enhance the salt tolerance of plants and promote plant growth in high-salt environments. At the same time, their ability to treat heavy metal wastewater is limited.
Bacillus atrophaeus CGMCC No. 37219 was used. It has high salt tolerance and multiple growth-promoting functions, such as nitrogen fixation, phosphorus solubilization, phosphorus dissolution, silicate solubilization, iron carrier production, and IAA production. It was applied to the salt-containing environment of wild barley cultivation to improve the plant's salt stress tolerance and remove heavy metals.
It significantly improves the growth performance of wild barley under high salt stress, promotes the absorption of nutrients, purifies water sources polluted by heavy metals, and provides technical means for planting crops in high-concentration saline-alkali land and treating heavy metal wastewater.
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Figure CN121780385B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a strain of Bacillus atrophus and its application in promoting salt tolerance in plants. Background Technology
[0002] Soil salinity in agricultural soils refers to the high concentration of soluble salts in the root soil moisture. These soluble salts create high osmotic pressure, which limits plant water absorption and the balanced absorption of basic nutrient ions by the roots, thus affecting plant growth. The formation of saline-alkali land is the result of multiple factors, commonly including long-term over-cultivation leading to soil structure damage, excessive fertilization causing salt accumulation in the soil, and drought causing groundwater salts to rise to the surface with evaporation. The most direct impact of soil salinization is reduced grain yields; annual crop failures due to salinization threaten food security. Improving and rationally utilizing saline-alkali land can not only transform previously low-yield "stagnant soil" into arable land but also directly increase the cultivated land area, playing an irreplaceable role in ensuring stable food supply and maintaining ecological security.
[0003] Beneficial microbial communities in microbial inoculants, through a series of decomposition and synthesis processes such as nitrogen fixation and phosphorus solubilization, can transform soil substances into various nutrients, improving soil fertility and promoting plant growth. Beneficial microorganisms can also secrete various antibiotics and other antibacterial substances, inhibiting the growth and reproduction of pathogens, inducing systemic disease resistance in plants, reducing disease occurrence, and enhancing plant stress resistance. In recent years, the application of microbial technology in saline-alkali soil remediation has received increasing attention and strengthening, mainly focusing on research into enhancing plant salt and alkali tolerance through microorganisms. Existing studies have found that salt-tolerant microorganisms can improve the rhizosphere environment of plants, reduce the inhibitory effect of salt on crop growth, and achieve the goal of improving saline-alkali soil. Studies have also found that strains can promote the emergence rate, increase biomass, and reduce disease incidence of mung beans under NaCl stress. Therefore, microbial activity can, to a certain extent, improve the survival rate and retention rate of vegetation in saline-alkali soils, playing an important role in the long-term improvement of saline-alkali soils, restoring soil physicochemical properties, and reconstructing the ecological environment of saline-alkali soils. Although there are many salt-tolerant microbial strains, their tolerance to salt stress concentrations is still limited, generally between 100-300 mmol / L. However, the salt concentration in saline-alkali land varies greatly depending on the region, soil depth, and season, with some high-salinity soils far exceeding 300 mmol / L. As a result, the current salt-tolerant microbial strains are not very effective in improving high-salinity soils. Therefore, it is of great significance to discover new microbial strains to adapt to high-concentration saline-alkali lands and promote the growth of plants in saline-alkali lands. Summary of the Invention
[0004] The purpose of this invention is to provide a strain of Bacillus atrophus and its application in promoting salt tolerance in plants, thereby solving the problems existing in the prior art. This Bacillus atrophus has a significant effect on improving the growth of wild barley plants under salt stress and their ability to tolerate salt stress, especially under high salt concentration stress of 400 mmol / L or higher, it still shows a growth-promoting effect on plants. At the same time, this bacterium can also remove heavy metals from high-salt wastewater. This invention provides a new growth-promoting bacterium for improving the salt tolerance of plants and purifying water sources, and provides a new technical means for the planting of crops in high-concentration saline-alkali land and the treatment of heavy metal wastewater pollution.
[0005] To achieve the above objectives, the present invention provides the following solution:
[0006] This invention provides a strain of Bacillus atrophaeus, which has the accession number CGMCC No. 37219.
[0007] The present invention also provides a microbial agent, including the aforementioned Bacillus atrophus.
[0008] The present invention also provides the application of the aforementioned Bacillus atrophus in the preparation of microbial agents that enhance the salt stress tolerance of wild barley.
[0009] The present invention also provides the application of the aforementioned Bacillus atrophus or the aforementioned bacterial agent in improving the salt stress tolerance of wild barley.
[0010] The present invention also provides the application of the aforementioned Bacillus atrophus or the aforementioned bacterial agent in promoting the growth of wild barley under salt stress.
[0011] Preferably, the salt concentration of the saline environment is ≥400 mmol / L.
[0012] The present invention also provides the application of the aforementioned Bacillus atrophus or the aforementioned bacterial agent in the removal of heavy metals from wastewater.
[0013] The present invention also provides the use of the aforementioned Bacillus atrophus in any of the following:
[0014] (1) Application in nitrogen fixation;
[0015] (2) Application in the degradation of inorganic and organic phosphorus;
[0016] (3) Application in the degradation of silicates;
[0017] (4) Application in iron-producing carriers;
[0018] (5) Application in high-yield IAA.
[0019] The present invention also provides a method for improving the salt stress tolerance of barley, comprising the step of applying the aforementioned Bacillus atrophus or the aforementioned inoculum to a saline environment in which wild barley is grown.
[0020] The present invention also provides a method for promoting the growth of wild barley in a saline environment, comprising the step of applying the aforementioned Bacillus atrophus or the aforementioned inoculum to the saline environment in which wild barley is grown.
[0021] The present invention discloses the following beneficial effects:
[0022] This invention screened a Bacillus atrophicus strain CZJ6 that promotes plant growth and enhances plant salt stress tolerance. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37219. Experimental results show that Bacillus atrophicus CZJ6 possesses salt tolerance characteristics and functions such as nitrogen fixation, IAA production, phosphorus solubilization, phosphorus dissolution, silicate solubilization, and siderophore production. It can effectively promote the absorption and utilization of nitrogen, phosphorus, and iron by plants, thereby promoting plant growth. Bacillus atrophicus CZJ6 significantly enhances the growth of wild barley plants under salt stress and their tolerance to salt stress. Furthermore, this Bacillus atrophicus can remove heavy metals from high-salt heavy metal wastewater. This invention provides a new growth-promoting bacterium for improving the salt tolerance of plants in high-salt environments and purifying water sources contaminated with heavy metals, and offers a new technical means for the cultivation of crops in high-concentration saline-alkali land and the treatment of heavy metal-contaminated wastewater. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0024] Figure 1 This is a colony morphology diagram of Bacillus atrophus CZJ6;
[0025] Figure 2 The growth curves of Bacillus atrophus CZJ6 at different salt concentrations are shown.
[0026] Figure 3 Plate dissolution zone for phosphorus solubilization of Bacillus atrophus CZJ6;
[0027] Figure 4 The plate dissolution zone for phosphate solubilization of Bacillus atrophus CZJ6;
[0028] Figure 5 Plate dissolution zone for silicate hydrolysis of Bacillus atrophus CZJ6;
[0029] Figure 6 Plate dissolution zone for Bacillus atrophus CZJ6 as an iron-producing carrier;
[0030] Figure 7 The figure shows the phenotypic observation results of Bacillus atrophus CZJ6 affecting the growth of wild barley under saline-alkali or non-saline-alkali conditions;
[0031] Figure 8 The statistical results show the phenotypic effects of Bacillus atrophus CZJ6 on the growth of wild barley under saline-alkali or non-saline-alkali conditions; A: plant height, B: fresh weight, C: dry weight;
[0032] Figure 9 The growth curve of Bacillus atrophus CZJ6 under heavy metal conditions is shown. Detailed Implementation
[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0038] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar, pH adjusted to 7.0-7.2.
[0039] LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH adjusted to 7.
[0040] Nitrogen-fixing medium (nitrogen-free medium): mannitol 10.0 g / L, CaCO3 5 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, CaSO4·2H2O 0.2 g / L, agar 20 g / L, pH adjusted to 7.0-7.2.
[0041] Monkina Inorganic Phosphorus Solid Culture Medium: Glucose 10.0 g / L, yeast extract 0.5 g / L, (NH4)2SO4 0.5 g / L, MgSO4·7H2O 0.3 g / L, MnSO4·4H2O 0.03 g / L, KCl 0.3 g / L, FeSO4·7H2O 0.03 g / L, NaCl 0.3 g / L, Ca3(PO4)2 10 g / L, agar 15 g / L, pH adjusted to 7.0-7.5.
[0042] Monkina Organic Phosphorus Solid Medium: Glucose 10.0 g / L, yeast extract 0.4 g / L, (NH4)2SO4 0.5 g / L, MnSO4·4H2O 0.03 g / L, KCl 0.3 g / L, FeSO4·7H2O 0.03 g / L, NaCl 0.3 g / L, CaCO3 5 g / L, egg yolk lecithin 0.2 g / L, agar 20 g / L, pH adjusted to 7.0.
[0043] Silicate-solubilizing bacteria culture medium: sucrose 5.0 g / L, MgSO4 0.5 g / L, CaCO3 0.1 g / L, Na2HPO4 2.0 g / L, FeCl3 0.005 g / L, glass powder 1.0 g / L, agar 15.0 g / L, pH adjusted to 7.0-7.2.
[0044] CAS detection medium: Chromium azurite S (CAS) 60.5 mg / L, hexadecyltrimethylammonium bromide 72.9 mg / L, FeCl3·6H2O 2.645 mg / L, NaH2PO4·2H2O 295.25 mg / L, Na2HPO4·12H2O 1213.5 mg / L, NH4Cl 125.0 mg / L, KH2PO4 37.5 mg / L, NaCl 62.5 mg / L, agar 9000.0 mg / L, pH adjusted to 6.7-6.9.
[0045] LB liquid medium containing L-tryptophan: 10 g / L tryptophan, 5 g / L yeast extract, 10 g / L sodium chloride, 5 mmol / L L-tryptophan, pH adjusted to 7.2.
[0046] Salkowski colorimetric reagent preparation: FeCl3 0.5 mol / L and 35% HClO4 were mixed and stored in a brown bottle at a ratio of 1:50.
[0047] Indoleacetic acid standard solution: 500 mg of indoleacetic acid was dissolved in ethyl acetate, and then diluted to 1000 mL with deionized water to obtain a 500 mg / L standard stock solution.
[0048] Example 1: Isolation, Identification and Preservation of Growth-Promoting Bacterial Strains
[0049] 1. Sample collection
[0050] Soil samples were collected from saline-alkali soil in Linze County, Zhangye City, Gansu Province. The soil collection method adopted was a five-point sampling method. Five sampling points were determined in the selected area. Soil samples of 10-20 cm were collected from saline-alkali soil of different degrees. Equal amounts of soil were collected from each point, mixed, and then placed in sterile bags and sealed. The collection number, collection location, date and other key information were marked. The samples were brought back to the laboratory and stored at 4℃.
[0051] 2. Soil sample enrichment treatment
[0052] Soil samples were sieved through a 20-mesh sieve (approximately 1 mm in diameter). 10 g of the sample was weighed and placed in an Erlenmeyer flask containing 90 mL of sterile aqueous solution (containing 10-15 sterile glass beads). The flask was incubated at 28°C and 150 rpm for 2-3 hours, then allowed to stand for 10 minutes to obtain a soil suspension diluted 10 times. This suspension was recorded as 10 g. -1 Diluent. Use a pipette to draw 1 mL of 10... -1 Add the diluent to a test tube containing 9 mL of sterile water, mix well by pipetting, and dilute to a 10:1 ratio. -2 Diluent, then dilute in the same manner to prepare 10. -3 10 -410 -5 10 -6 10 -7 Prepare a series of gradient dilutions; take 0.1 mL of each gradient dilution and spread it evenly on LB agar plates, repeating each concentration in 3 plates, and incubate the plates upside down in a 28℃ incubator for 2-3 days.
[0053] 3. Strains Isolation and Screening
[0054] Select plates with suitable colony density (30-300 / plate), pick single colonies of different types from the above LB agar plates and streak them onto LB agar plates for isolation, incubate at 28℃ for 2-3 days, pick single cells again for streaking, and repeat the single cell streaking 2-3 times to obtain single cell pure cultures of each strain; pick single cells of each strain and inoculate them into LB liquid medium, incubate at 28℃ and 150 rpm for 12-24 h with shaking, and screen to obtain strains that show obvious growth in the medium for strain purification and preservation.
[0055] 4. Strain purification and preservation
[0056] The pure culture was cryopreserved in 30% glycerol at low temperature. The preservation name was L43.
[0057] 5. Morphological identification of growth-promoting bacterial strains
[0058] The purified strain L43 was selected and cultured on LB agar using the streak plating method at 28°C for 2 days. Colony morphology was then observed; the colony morphology of the growth-promoting bacteria is shown below. Figure 1 As shown.
[0059] The results showed that the growth-promoting bacteria strain L43 exhibited irregular, slightly rough edges and a grayish-white or brownish colony morphology.
[0060] 6. Molecular biological identification and preservation of growth-promoting bacterial strains
[0061] The isolated and purified strain L43 was subjected to molecular biological identification. This was achieved through DNA extraction, PCR amplification, and 16S rRNA gene sequencing.
[0062] Forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1;
[0063] Reverse primer 1492R: 5'-TACGGCTACCTTACGACTT-3', SEQ ID NO.2.
[0064] The PCR product of the amplified 16S rRNA was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.
[0065] By performing nucleic acid sequence homology alignment (Blastn) in the GenBank database on NCBI, strain L43 was preliminarily identified as Bacillus atrophaeus.
[0066] Bacillus atrophaeus L43 has been renamed CZJ6. Bacillus atrophaeus CZJ6 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37219, deposited on December 26, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0067] Example 2 Salt tolerance of Bacillus atrophus CZJ6
[0068] Preparation of seed culture: A single colony of Bacillus atrophus CZJ6 was picked and inoculated into a test tube of LB liquid medium and cultured at 28℃ and 180 rpm for 24 h to prepare the seed culture.
[0069] Saline-alkaline culture media (NaCl:Na₂SO₄ = 1:1) containing concentrations of 0 mmol / L, 200 mmol / L, 400 mmol / L, 600 mmol / L, and 800 mmol / L were prepared. *Bacillus atrophicus* CZJ6 seed culture was inoculated at a 3% inoculum and cultured for 40 h. The growth curves of *Bacillus atrophicus* CZJ6 at different salt concentrations were then determined. The growth curves are shown below. Figure 2 As shown.
[0070] Depend on Figure 2 It can be seen that: with the increase of salt concentration in the culture medium, the time required for Bacillus atrophus CZJ6 to enter the logarithmic phase is longer; the CZJ6 strain exhibits the best growth at a salt concentration of 200 mmol / L, entering the logarithmic phase after 2 hours of culture and reaching the stationary phase after 16 hours; the CZJ6 strain can tolerate a salt concentration of 600 mmol / L, and at a salt concentration of 600 mmol / L, the bacterial concentration OD reaches the stationary phase. 600 With a value above 0.4, it can be concluded that *Bacillus atrophicus* CZJ6 can grow normally in culture media with salt concentrations not exceeding 600 mmol / L, maintaining stable viability and tolerance. Furthermore, *Bacillus atrophicus* CZJ6 can still maintain its growth in culture media with a salt concentration of 800 mmol / L, indicating that it can grow and reproduce normally and maintain its viability in high-salt culture media. This demonstrates that *Bacillus atrophicus* CZJ6 can adapt to high-salt, high-osmotic-pressure environments, and has a wider range of applications, including the biological treatment of high-salt wastewater and the improvement of saline-alkali soils.
[0071] Example 3: Nitrogen fixation, phosphorus solubilization, phosphorus lysis, silicate solubilization, and siderophore production characteristics of Bacillus atrophus CZJ6
[0072] 1. Nitrogen fixation detection experiment
[0073] Nitrogen-free medium test: Pick a small number of single colonies of Bacillus atrophus CZJ6, streak them on a nitrogen-free medium plate, and observe whether the strain can grow and form colonies on the nitrogen-free medium.
[0074] Nitrogenase activity assay (acetylene reduction method): Seed culture of Bacillus atrophus CZJ6 was inoculated into LB liquid medium at a 3% inoculum volume and cultured at 28℃ and 180 rpm for 24 h with shaking. The culture medium was then transferred to centrifuge tubes and centrifuged at 4℃ and 8000 rpm for 10 min to collect the bacterial cells. The cells were washed 2-3 times with physiological saline and resuspended, and the OD concentration was adjusted. 600 =1.0, prepare a bacterial suspension; take 1 mL of bacterial suspension and add it to a centrifuge tube containing 9 mL of nitrogen-free medium and react for 1 h and send it to Shanghai Zhuocai Biotechnology Co., Ltd.; the nitrogenase activity of Bacillus atrophus CZJ6 of the present invention is 50.7908 nmol / (mL×h).
[0075] This indicates that the fermentation products of Bacillus atrophus CZJ6 have high nitrogenase activity.
[0076] 2. Phosphorus solubility (inorganic phosphorus) detection experiment
[0077] Take 5 μL of Bacillus atrophus CZJ6 seed culture and inoculate it into the center of a Monkina inorganic phosphorus medium plate. Repeat the process 3 times and incubate at 28℃ for 3-5 days. Observe whether a clear zone is formed around the colony.
[0078] The results are as follows Figure 3 As shown, Bacillus atrophus CZJ6 produces a clear phosphorus-solubilizing zone on inorganic phosphorus medium, indicating that this strain has a significant effect on solubilizing inorganic phosphorus.
[0079] 3. Detection experiment of dissolved phosphorus (organic phosphorus)
[0080] Take 5 μL of Bacillus atrophus CZJ6 seed culture and inoculate it into the center of a Monkina organophosphate medium plate. Repeat the process 3 times and incubate at 28℃ for 3-5 days. Observe whether a clear zone is formed around the colony.
[0081] The results are as follows Figure 4 As shown, Bacillus atrophicus CZJ6 produces a clear phosphorus-solubilizing zone on organic phosphorus culture medium, indicating that Bacillus atrophicus CZJ6 has a significant effect on solubilizing organic phosphorus.
[0082] 4. Silicate detection experiment
[0083] Take 5 μL of Bacillus atrophus CZJ6 seed culture and spot it onto the center of a silicate-solubilizing bacteria culture medium plate. Repeat 3 times and incubate at 28℃ for 3-5 days. Observe whether oil droplets are produced around the colonies.
[0084] The results are as follows Figure 5 As shown, Bacillus atrophus CZJ6 can produce oil droplets on silicate-solubilizing bacteria culture medium, indicating that Bacillus atrophus CZJ6 has obvious silicate-solubilizing properties.
[0085] 5. Iron carrier characteristic testing experiment
[0086] Take 5 μL of Bacillus atrophus CZJ6 seed culture and spot it in the center of a CAS detection medium plate. Repeat the process 3 times and incubate at 28℃ for 3-5 days. Observe whether an orange-yellow halo appears around the colony.
[0087] The results are as follows Figure 6 As shown, Bacillus atrophus CZJ6 produces a yellow halo on the siderophore detection medium, indicating that Bacillus atrophus CZJ6 has siderophore production characteristics.
[0088] 6. Product IAA Characteristic Testing Experiment
[0089] Bacillus atrophicus CZJ6 was added to LB liquid medium containing L-tryptophan, while the control group received no bacterial strain. Both cultures were incubated for 5 days in a shaker at 120 rpm and 28°C. Then, 1 mL of the bacterial culture was added to a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 5 min. 200 μL of the supernatant was mixed with 200 μL of the colorimetric solution. Simultaneously, 200 μL of indoleacetic acid (IAA) standard solution was added, and the mixture was incubated in the dark for 30 min. The color change was then observed. If the color remained unchanged, the strain did not have the potential to secrete IAA. Immediately afterward, 200 μL of the colorimetric reaction mixture was added to a 96-well plate. Three replicates of the Bacillus atrophicus CZJ6 bacterial culture were performed, and the values were read at OD530 using a microplate reader. A standard curve of IAA concentration was constructed with the IAA value as the ordinate and the concentration as the abscissa, thus obtaining the IAA content of the bacteria. The IAA production of Bacillus atrophus CZJ6 of the present invention is 19.2352 mg / L.
[0090] In summary, the Bacillus atrophus CZJ6 isolated by this invention has the characteristics of nitrogen fixation, phosphorus solubilization, phosphorus dissolution, silicate solubilization, and acting as an iron carrier and IAA-producing strain. It can promote the decomposition and transformation of nutrients (nitrogen, phosphorus, silicate and iron) in the soil, which is beneficial to promoting the absorption of nutrients by plants.
[0091] Example 4: Bacillus atrophus CZJ6 promotes the growth of wild barley
[0092] Simulated saline-alkali planting system: A mixed salt solution was added to the loess culture system to simulate the saline-alkali planting environment, and the growth-promoting effect of Bacillus atrophus CZJ6 on wild barley seedlings was verified by an experiment.
[0093] (1) Disinfect the surface of wild barley seeds, soak them in 95% alcohol for 2 min, stir them in 1% sodium hypochlorite solution for 3 min, rinse them 10 times with sterile water, keep them moist, germinate them in the dark at 25℃ for 2 days, and select germinated seeds with a sprout length of about 1 cm for sowing.
[0094] (2) Select seeds with relatively uniform sprout length after germination and sow them in loess pots. Inoculate and do not inoculate the seeds, with 9 seedlings planted for each treatment. A salt-free control system was also set up, and inoculate and do not inoculate the seeds in the same way. After sowing, wild barley was conventionally cultivated until the seedling stage.
[0095] (3) Experimental strain: Bacillus atrophus CZJ6; LB liquid medium, 28℃, 180 rpm, cultured for 36 h; the bacterial culture was centrifuged at 8000 rpm for 10 min to obtain bacterial cells, washed once with physiological saline and then resuspended, and the OD was adjusted. 600 Within the range of 0.6-0.8, the inoculation amount is 50 mL / pot, and it is repeated twice every 3 days.
[0096] A control group was set up by watering wild barley plants with 50 mL of sterile water per pot.
[0097] (4) Prepare salt solutions with salt concentrations of 0 mmol / L, 200 mmol / L, 400 mmol / L, 600 mmol / L, and 800 mmol / L (NaCl:Na2SO4=1:1). After adding the bacterial culture, add 50 mL of salt solution per basin each time.
[0098] After treatment, the plants were placed in a smart greenhouse, with appropriate moisture replenished every two days. After 7 days of cultivation, the plant height, fresh weight, and dry weight of wild barley seedlings were measured to evaluate the effect of Bacillus atrophus CZJ6 on the growth phenotype of wild barley. Results are as follows: Figure 7 As shown.
[0099] Depend on Figures 7-8 It was found that under normal conditions (0 mmol / L saline solution), the barley plants treated with CZJ6 (L43) were taller than the control group. The control group of barley plants with saline solution concentrations greater than 400 mmol / L all showed significant wilting, yellowing, and reduced plant height; while at saline concentrations of 600 and 800 mmol / L, the phenotypes of the barley plants treated with CZJ6 (L43) were significantly better than those of the control group at the corresponding concentrations.
[0100] Example 5: Effects of Bacillus atrophus CZJ6 on heavy metals in high-salt wastewater
[0101] Seed culture preparation: A single colony of *Bacillus atrophus* CZJ6 was picked and inoculated into a test tube containing LB liquid medium. The culture was incubated at 28°C and 180 rpm for 24 h to prepare the seed culture. The concentration was 1.0 × 10⁻⁶. 8 CFU / mL bacterial suspension;
[0102] Prepare simulated high-salinity wastewater with a salinity of 10% (NaCl) by adding K2Cr2O. 7， Set Cr 6+ Concentration gradients of 5, 10, and 20 mg / L were used, and the pH was adjusted to 7.0-7.5.
[0103] Two control groups were set up: a treatment group (simulated wastewater + 10% bacterial suspension) and a control group (simulated wastewater + 10% sterile saline), with three replicates in each group. The cultures were incubated at 25℃ with constant temperature shaking at 180 r / min for 36 h, and samples were taken at 6 h, 12 h, 24 h, and 36 h. After sampling, the cultures were centrifuged at 8000 r / min for 10 min, and the OD of the bacterial suspension was measured using the supernatant. 600 Value. The growth of the strain under simulated wastewater conditions is as follows: Figure 9 As shown.
[0104] Experimental analysis revealed that the strain grew stably in a heavy metal environment with a 10% salt concentration, and its OD... 600 The value continued to increase with the culture time, Cr 6+ Growth was inhibited at concentrations ≥20 mg / L. In the control group, OD... 600 The lack of significant change in the values ruled out interference from abiotic factors on the heavy metal concentration, corroborating that the decrease in heavy metal concentration in the experimental group was due to the adsorption effect of the bacterial strain.
[0105] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A strain of Bacillus atrophus ( Bacillus atrophaeus ), characterized in that, The Bacillus atrophus has the accession number CGMCC No. 37219.
2. An inoculant characterized in that, Includes the Bacillus atrophus as described in claim 1.
3. The application of Bacillus atrophus as described in claim 1 in the preparation of a microbial agent to improve the salt stress tolerance of wild barley.
4. The application of Bacillus atrophus as described in claim 1 or the bacterial agent as described in claim 2 in improving the salt stress tolerance of wild barley.
5. The application of Bacillus atrophus as described in claim 1 or the bacterial agent as described in claim 2 in promoting the growth of wild barley under salt stress.
6. Use according to claim 4 or 5, wherein the compound is ###0002### Salt stress concentration ≥400 mmol / L.
7. The use of Bacillus atrophus as described in claim 1 in any of the following: (1) Application in nitrogen fixation; (2) Application in the degradation of inorganic and organic phosphorus; (3) Application in the degradation of silicates; (4) Application in iron-producing carriers; (5) Application in high-yield IAA.
8. A method for improving salt stress tolerance in barley, characterized by, The method includes the step of applying the Bacillus atrophus of claim 1 or the inoculum of claim 2 to a saline environment in which wild barley is grown.
9. A method for promoting the growth of wild barley in a saline environment, characterized in that, The method includes the step of applying the Bacillus atrophus of claim 1 or the inoculum of claim 2 to a saline environment in which wild barley is grown.