Lysinibacillus sp. YCL-7 and application thereof

Abstract

The invention discloses lysinibacillus sp.YCL-7 and application thereof, and belongs to the technical field of microorganisms, the lysinibacillus sp.YCL-7 is preserved in the China General Microbiological Culture Collection Center, the preservation number is CGMCC NO: 32174, and the lysinibacillus sp.YCL-7 can be widely applied and is particularly suitable for the fields of agricultural planting, stress resistance and yield increase of saline-alkali soil and soil remediation.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a Lysinibacillus sp. YCL-7 and its application. Background Art

[0002] Plants have a symbiotic relationship with soil microorganisms during their growth and development. Symbiotic soil microorganisms live around the rhizospheres of many different plants and have many beneficial effects on the host plants. They competitively colonize the plant roots through different mechanisms of action, promoting plant growth, including phosphorus solubilization, nitrogen fixation, production of indole-3-acetic acid (IAA), siderophores, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, etc. Nitrogen (N) and phosphorus (P) are essential nutrient elements for plant growth and development. However, 80% of the nitrogen in the air cannot be directly utilized by plants, and phosphorus in the soil mostly exists as Al-P and Fe-P, which is also difficult to be directly utilized by plants. Although plant growth-promoting bacteria have many advantages, the existing strain resources are still very scarce. Therefore, it is necessary to vigorously explore strains that can utilize elements such as phosphorus and iron, and promote the production of IAA, ACC, and EPS to solve the problems of slow plant growth and difficult utilization of nutrient elements. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a new microbial agent for promoting plant growth in agriculture.

[0004] The present invention solves the above technical problems through the following technical means:

[0005] A Lysinibacillus sp. YCL-7, the Lysinibacillus sp. YCL-7 (Lysinibacillus sp.) is deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC NO: 32174, the address is No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is October 11, 2024.

[0006] Furthermore, a microbial agent containing the Lysinibacillus sp. YCL-7 described in claim 1.

[0007] Furthermore, any of the following applications of the Lysinibacillus sp. YCL-7 or the microbial agent:

[0008] 1) Application in phosphorus solubilization or phosphorus decomposition;

[0009] 2) Application in siderophore production;

[0010] 3) Application in IAA production;

[0011] 4) Application in ACC secretion;

[0012] 5) Application in EPS secretion;

[0013] 6) Application in alleviating saline-alkali stress of plants;

[0014] 7) Application in promoting crop growth performance;

[0015] 8) Application in improving and enhancing soil fertility.

[0016] Furthermore, the application of the Lysinibacillus sp. YCL-7 or the microbial agent in preparing microbial fertilizers.

[0017] Beneficial effects:

[0018] The strain YCL-7 disclosed in the present invention simultaneously has the characteristics of phosphorus solubilization, siderophore production, salt-alkali tolerance, IAA secretion, high ACC deaminase activity and high EPS production, belonging to multifunctional plant growth-promoting bacteria, and is widely applicable, especially in the fields of saline-alkali land agriculture, stress resistance and yield increase, and soil remediation. It can promote the growth of crops in high-salt and high-pH soils, alleviate saline-alkali stress, and synergistically improve crop production performance and yield through the effects of phosphorus solubilization, promoting iron absorption, IAA promoting root growth, ACC deaminase stress resistance, EPS water retention, etc. At the same time, it can also improve saline-alkali land or barren soil, and enhance soil fertility and microbial activity. Description of the drawings

[0019] Figure 1 : The cell and colony morphology of strain YCL-7;

[0020] Figure 2 : The salt tolerance growth curve of strain YCL-7;

[0021] Figure 3 : The alkali tolerance growth curve of strain YCL-7;

[0022] Figure 4 : The diagrams of indole acetic acid IAA production, ACC deaminase activity, phosphorus solubilization ability, siderophore production ability, and extracellular polymer content of strain YCL-7. Specific embodiments

[0023] The present invention will be described in detail below in conjunction with specific embodiments and drawings:

[0024] The present invention provides a Lysinibacillus sp. YCL-7, which was deposited at the China Center for Microbial Culture Collection on October 11, 2024, with the deposit number CGMCC NO. 32174 and the taxonomic name Lysinibacillus sp.

[0025] LB medium: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, ddH 2O 1L, pH = 7. (No additional NaCl added is considered 0%, and the salt content is determined by the mass fraction of the additional NaCl added. For example, adding 2g of NaCl to 100g of LB liquid medium is considered a salt concentration of 2%).

[0026] DF medium: First, prepare component one and component two solutions. Component one solution is prepared by dissolving 10mg of H 3 BO 3 , 11.19mg of MnSO 4 ·H 2 O, 124.6mg of ZnSO 4 ·7H 2 O, 78.22mg of CuSO 4 ·5H 2 O and 10mg of MoO 3 in 100mL of ddH 2 O; Component two is prepared by dissolving 100mg of FeSO 4 ·7H 2 O in 10mL of ddH 2 O. Then, prepare the medium according to the following ratio: 0.1mL / L of component one solution, 10mL / L of component two, KH 2 PO 4 4.0g / L, Na 2 HPO 4 6.0g / L, MgSO 4 ·7H 2 O 0.2g / L, glucose 2.0g / L, sodium gluconate 2.0g / L, citric acid 2.0g / L, and adjust the pH to 7.2 ± 0.05. The prepared medium is autoclaved at 121°C for 20 minutes and cooled for standby.

[0027] PKO medium: Glucose 10.0 / L, (NH 4 ) 2 SO 4 0.5g / L, NaCl 0.3g / L, NaCl 0.3g / L, MgSO 4 ·7H 2 O 0.3 / L, MnSO 4 ·H 2 O 0.03g / L, KCl 0.3g / L, FeSO 4 ·7H 2 O 0.03g / L, Ca 3 (PO 4 ) 2 5.0g / L. The prepared medium is autoclaved at 121°C for 15 minutes and cooled for standby.

[0028] MKB medium: 5.0 g / L of casein amino acids, K 2 HPO 4 2.5 g / L, MgSO 4 ·7H 2 O 2.5 g / L, autoclaved at 115 °C for 30 min.

[0029] CAS detection solution: Prepare solution A and solution B first. Solution A is prepared by dissolving 0.079 g of CAS in 50 mL of deionized water and adding 10 mL of FeCl 3 solution (1 mmol / L, this solution is prepared with hydrochloric acid); Solution B is prepared by dissolving 0.069 g of cetyltrimethylammonium bromide (HDTMA) in 40 mL of deionized water. Then, slowly add solution A to solution B and stir gently to mix evenly to obtain 100 mL of CAS detection solution.

[0030] Example 1: Collection and identification of Lysinibacillus sp. YCL-7

[0031] 1. Sample collection:

[0032] The soil sample was collected from a severely saline-alkali grassland around Zhaodong City, Heilongjiang Province (125°84′99″E, 46°03′61″N). The collected soil sample was sealed in a sterile bag and transported back to the laboratory under the storage condition of -20 °C, and the experiment was carried out immediately. The dilution coating separation method was used to screen for salt-tolerant bacteria. Weigh 5 g of soil and put it into a conical flask containing 45 ml of sterile water, and shake for about 20 min.

[0033] Perform serial 10-fold dilutions on the soil suspension. Select 60 μL of the soil dilutions with dilution factors of 10 4 、10 5 and 10 6 and spread them on the sterilized solid LB medium with a 2% salt concentration and pH = 8.0 by streaking. Incubate in an incubator at 28 °C for 48 h, pick out single colonies with different morphologies, and repeatedly streak and purify until pure cultures are obtained, and number the strains.

[0034] 2. Colony morphology identification:

[0035] The physical form of strain YCL-7 is as Figure 1 shown: It is milky white, semi-transparent in color, dry and rough on the surface, convex inside, with a complete edge, and fast growth rate.

[0036] 3. Nucleotide sequence of strain YCL-7:

[0037] It was identified that strain YCL-7 is Lysinibacillus sp., and the nucleotide sequence is as follows:

[0038]

[0039] Example 2: Salt-alkali resistance of strain YCL-7

[0040] 1. Experiment on determining salt tolerance growth curve:

[0041] Take out the cryovial of strain YCL-7 (50% sterile glycerol: bacterial solution = 1:1 configuration) from the -80℃ refrigerator, and add 400μL of sterile liquid LB medium with pH = 7.0 and salt content of 0% for activation for 24h. Prepare 9 small conical bottles containing 30mL LB liquid medium, one of which is not treated, and add 0.6g (2%), 1.2g (4%), 1.8g (6%), 2.4g (8%), 3.0g (10%), 3.6g (12%), 4.2g (14%), 4.8g (16%) NaCl to the remaining 8 bottles, shake well and dispense into 3 15mL centrifuge tubes, 10mL each. After high temperature and high pressure sterilization at 121℃ for 15min, add 200μL of the prepared YCL-7 activated bacterial solution to each centrifuge tube.

[0042] Pipette 50 μL into a 96-well plate, shake well and use a microplate reader to determine the OD600 value of each group (determine the absorbance value of the LB liquid culture medium solvent without adding bacterial solution as the blank control, the actual OD600 value of the bacterial solution = OD600 reading - OD600 blank), record the situation of each group as the data at the 0th hour, place it in a shaker (28°C, 180 r / min) and record the data after 8h, 16h, 32h, 40h, and 48h of cultivation.

[0043] At the same time, the Logictis model was used to perform nonlinear fitting on the growth status of the strain after 48 hours to evaluate the effectiveness of the strain YCL-7 in salt tolerance in subsequent potted experiments in saline-alkali soil. The function is as follows:

[0044]

[0045] R 2 =0.9554,LD 50 =8.67, the median lethal dose of salt is 8.67%.

[0046] The data obtained is as follows Figure 2 As shown, analyzing the data, we can see that:

[0047] Under the salt concentration gradient, strain YCL-7 showed a trend of decreasing OD600 value with the increase of salt concentration. When growing for 24 h at 0-8% salt concentration, the strain could quickly go through the logarithmic phase, and reach the stationary phase between 24-48 h. When adding 10-16% salt concentration, the growth in the stationary phase was retarded and the number of strains decreased significantly. Growth stopped at 14% and 16% salt concentration. There was no significant difference in the total OD600 of the bacterial liquid between 4% salt concentration and the control group, indicating that YCL-7 had good salt tolerance. In summary, the strain YCL-7 of the present invention had salt tolerance and had application prospects in the environment of high-salt plots or high-salt wastewater.

[0048] 2. Experiment for measuring the growth curve of alkali tolerance:

[0049] Similarly, prepare 9 small conical flasks filled with 30 mL of LB liquid medium, and use NaOH (1M 0.1M) and HCl (1M 0.1M) to adjust the pH to 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, and 11.0 respectively, and dispense 10 mL into each of 3 15 mL centrifuge tubes. After autoclaving at 121 °C for 15 min, add 200 μL of the prepared activated bacterial liquid of YCL-7 to each centrifuge tube respectively.

[0050] After shaking well, pipette 50 μL into a 96-well plate, and use a microplate reader to measure the OD 600 value (measure the absorbance value of the LB liquid medium solvent without adding bacterial liquid as the blank control absorbance value, and the actual OD 600 value of the bacterial liquid = OD 600 reading - OD 600 blank), record the situation of each group as the data at the 0 h, and put it into a shaker (28 °C, 180 r / min), and record the data after culturing for 8 h, 16 h, 32 h, 40 h, and 48 h respectively.

[0051] Use the Logictis model to perform non-linear fitting on the growth status of the strain after 48 h, and use it to evaluate the effectiveness of the strain YCL-7 in terms of alkali tolerance when applying the bacterial agent in the subsequent saline-alkali soil pot experiment. The function is as follows:

[0052]

[0053] R 2 = 0.96377, LD 50 = 10.16, and the half-lethal dose of alkali is 10.16.

[0054] The obtained data is as Figure 3 shown. Analyzing the data, it can be seen that:

[0055] With the increase of pH, the time for strain YCL-7 to enter the logarithmic growth phase within 24 h is gradually delayed. Among them, the strain in the control group with pH = 7 enters the logarithmic phase first. Then, successively, under the conditions of pH 7.5 - 9.0 in the strain culture medium, strain YCL-7 can rapidly reach the stationary phase within 24 h. When pH = 10, strain YCL-7 can still maintain its viability and begins to enter the logarithmic phase after 24 h, showing good alkali tolerance. Under the condition of pH = 11, the bacterial liquid concentration always remains at the initial low concentration level. In summary, the strain YCL-7 of the present invention has alkali tolerance and has application prospects in saline-alkali land plots or environments with high-pH wastewater.

[0056] Example 3: Performance of strain YCL-7 in secreting indole-3-acetic acid (IAA)

[0057] (Salkowskis colorimetric method) Inoculate strain YCL-7 into DF liquid medium containing 200 mg / L L-tryptophan. The non-inoculated treatment is the control group. Incubate at 28 °C and 180 r / min for 48 h. Take 1 mL of the supernatant and add 4 mL of Salkowski color-developing solution and mix well (the operation of the control group is the same). Let it stand in the dark at room temperature for 20 min and measure the OD 530 value, and calculate the IAA content (mg / L) according to the standard curve.

[0058] The data obtained are as Figure 4 shown. Analyzing the data, it can be seen that:

[0059] Example 4: Performance of strain YCL-7 in secreting ACC

[0060] Extract the cell sample to be measured for protein determination, measure the amount of substance of α-ketobutyric acid, and determine the total protein content in the cell lysate according to the Bradford method. Define the amount of α-ketobutyric acid produced by the bacterial cells with unit protein content between units as the ACC deaminase activity.

[0061] Extract the cell sample to be measured: Take 7.5 mL of the strain culture solution and incubate at 28 °C and 180 r / min for 24 h. Centrifuge (12,000 r / min, 4 °C) for 5 min and collect the precipitate. Use 5 mL of DF nitrogen-free medium (without (NH 4 ) 2 SO 4) Resuspend the strain pellet, repeat twice. Resuspend the bacteria again with 7.5mL DF nitrogen-free medium, add 45μL 0.5mol / L ACC solution, and place it at 28℃, 180r / min constant temperature for 24h (induce the strain to secrete ACC deaminase). Centrifuge the culture solution at 5000r / min for 20min and collect the precipitate. Add 5mL 0.1mol / L Tris-HCl (pH=7.6) solution to suspend and wash the bacteria, repeat twice, and collect the precipitate. Dissolve in toluene and take 100μL of cell lysis solution and store the contents at 4℃ for subsequent protein determination.

[0062] Determination of enzyme activity: Take 200μL of the remaining cell lysis solution and put it in a 1.5mL centrifuge tube, add 20μL of 0.5mol / L and mix well. For the other treatment, do not add ACC solution, react at 30℃ for 15min, then add 1mL of 0.56mol / L HCl solution and mix well. Centrifuge (11000r / min, 4℃) for 10min and take 800μL of supernatant in 5mL centrifuge tube for use. Add HCl solution (800μL, 0.56mol / L) and 2,4-dinitrophenylhydrazine reaction solution (300μL) in turn, react at 30℃ for 30min, then add NaOH solution (22mL, 2mol / L) and mix well. After color development, measure OD 540 Value (Tris-HCl solution (0.1M, pH 8.5) was used as blank control). The absorbance value of the sample was substituted into the regression equation of the standard curve to obtain the content of α-ketobutyric acid therein, and the amount of substance (μmol) of α-ketobutyric acid was calculated, and then the total protein amount (mg) in the cell lysate was determined according to the Bradford method. Finally, the amount of α-ketobutyric acid produced by the bacterial cells per unit protein content per unit time was defined as ACC deaminase activity, and the ACC deaminase activity (U / mg) was calculated according to the formula, and the calculation formula is as follows. Three parallels were set for each strain.

[0063] α-ketobutyric acid standard samples were prepared by preparing α-ketobutyric acid standard solutions (0, 0.2, 0.4, 0.6, 0.8, 1 mmol / L) with 0.1 mol / L Tris-HCl buffer (pH 8.5), and 300 μL of 2,4-dinitrophenylhydrazine (2 mol / L HCl dissolved, mass concentration of 2 g / L) was added thereto respectively. After thorough mixing, the mixture was placed in a 30°C water bath for reaction for 30 min. Then 2 mL of NaOH (2 mol / L) was added, and the OD was measured after the color change. 540 value (using Tris-HCl buffer at pH 8.5 as blank control); a standard curve was prepared according to the concentration of α-ketobutyric acid solution and its corresponding absorbance value.

[0064]

[0065] The obtained data are as follows Figure 4 As shown, by analyzing the data, it can be seen that:

[0066] Example 5: Phosphorus-solubilizing or phosphorus-dissolving performance and siderophore-producing performance of strain YCL-7

[0067] 1. Determination of phosphorus content (using the molybdenum-antimony anti-colorimetric method)

[0068] The activated strain (1 mL) was inoculated into PKO medium (50 mL). The control group was inoculated with an equal amount of sterile water and incubated at 30 °C and 150 r / min for 7 d; then centrifuged at 11000 r / min for 5 min to obtain the supernatant. 1-2 drops of dinitrophenol indicator were added to the supernatant (30 mL), and the solution was adjusted to just turn slightly yellow by micro-dropwise addition of NaOH solution (1 M, 10 M) and HCl solution (1 M, 5 M). Then, 5 mL of molybdenum-antimony anti-mixed colorimetric solution was accurately added, shaken well, and made up to 50 mL with deionized water. After reacting at room temperature above 15 °C for 30 min (within 8 h), the OD 700 value was measured and recorded. The phosphorus content (mg / L) was calculated based on the standard curve.

[0069] The obtained data are as follows Figure 4 As shown, by analyzing the data, it can be seen that:

[0070] The phosphorus content in the bacterial supernatant was determined by the molybdenum-antimony anti-colorimetric method. The results showed that the phosphorus-dissolving amount of strain YCL-7 was 68.24 μg / ml, indicating strong phosphorus-dissolving ability. Strain YCL-7 can convert phosphorus that is difficult for plants to absorb into a form that can be absorbed and utilized, improve soil fertility, and thus improve the availability of phosphorus in the soil.

[0071] 2. Determination of siderophore performance

[0072] To determine the siderophore synthesis ability of the strain, two groups of MKB liquid media were prepared, one inoculated with the strain and the other not. After culturing, the culture solution was mixed with the CAS detection solution, and the OD 630 was measured. Finally, the siderophore-producing ability of the strain was evaluated based on the ratio of the experimental group to the control group.

[0073] The activated strain (0.5 mL) was inoculated into MKB liquid medium (5 mL). At the same time, when measuring the reference value (Ar), an equal amount of MKB liquid medium without inoculated strain (1 mL) was added. After culturing at 30 °C and 150 r / min for 48 h, 1 mL of the culture solution was taken and mixed with the CAS detection solution at a ratio of 1:1 (v / v).

[0074] The obtained data are as follows Figure 4 As shown, by analyzing the data, it can be seen that:

[0075] After the supernatant of strain YCL-7 was mixed with the CAS detection solution, it showed an orange-yellow color, indicating that the strain could produce a certain amount of siderophores. By measuring the reference value, the siderophore-producing ability of strain YCL-7 was determined to be 47.83%. The ability of the strain to produce siderophores determines the content of iron obtained. The strain YCL-7 disclosed in the present invention has a high ability to produce siderophores, which can significantly improve the iron nutrition of plants and promote the growth and development of plants.

[0076] Example 6: Performance of strain YCL-7 in secreting extracellular polymeric substances (EPS)

[0077] The Congo red agar method was used to determine the EPS content. The activated strain (1 mL) was inoculated into LB liquid medium (40 mL) with a salinity of 5%, and incubated at 37 °C and 150 r / min overnight for 72 h; then centrifuged at 8000 r / min for 10 min to remove bacterial cells. The supernatant was mixed with absolute ethanol in a ratio of 1:2 (v / v) and incubated overnight at 4 °C. Then, the precipitate was separated by centrifugation to obtain EPS. The obtained EPS was dried in hot air at 40 °C for 2-3 d, and the dry weight obtained was the EPS content.

[0078] The data obtained are as Figure 4 shown. Analyzing the data, it can be seen that:

[0079] The EPS content of strain YCL-7 under stress conditions was determined by the Congo red agar method. After measurement, it was found that the EPS content secreted by strain YCL-7 under salt stress was 31.48 mg / mL, indicating that the strain could secrete a large amount of extracellular polysaccharides under salt stress, which could encapsulate the bacterial cells, enabling the strain to possibly have functions such as resisting drought, salinity, and other stresses. It could also improve the soil structure through the cementing effect of secreting a large amount of EPS, enhance the water and fertilizer retention capacity. At the same time, the extracellular polysaccharides on the surface of the strain could form a viscous biofilm, attach to the plant roots to form a symbiosis, and promote plant growth. Generally speaking, the strain YCL-7 disclosed in the present invention has practical significance in the directions of anti-salinity, soil improvement, and promoting plant symbiosis.

[0080] In summary, the strain YCL-7 disclosed in the present invention simultaneously possesses the characteristics of phosphorus solubilization, siderophore production, salt tolerance, IAA secretion, high ACC deaminase activity, and high EPS production. It belongs to a multifunctional plant growth-promoting bacterium and has a wide range of applications, especially suitable for saline-alkali land agricultural planting, stress resistance and yield increase, and soil remediation fields. It can promote the growth of crops in high-salt and high-pH soils, relieve salt stress, and synergistically improve the crop production performance and yield through the effects of phosphorus solubilization, promoting iron absorption, IAA promoting root growth, ACC deaminase stress resistance, and EPS water retention. At the same time, it can also improve saline-alkali land or barren soil, and enhance soil fertility and microbial activity.

[0081] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention. The technologies, shapes, and structures not described in detail in the present invention are all well-known technologies.

Claims

1. A lysinic Bacillus YCL-7, characterized in that The lysinibacillus sp. YCL-7 is deposited in the China Center for Microbiological Culture Collection with a deposit number of CGMCCNO:32174.

2. A microbial agent containing the lysinic Bacillus YCL-7 according to claim 1.

3. Any of the following uses of the lysinic Bacillus YCL-7 according to claim 1 or the bacterial agent according to claim 2, characterized in that: 1) Application in phosphate dissolution or solution; 2) Application in the production of siderophores; 3) Application in producing IAA; 4) Application in secretion of ACC; 5) Application in secretion of EPS; 6) Application in alleviating saline-alkali stress of plants; 7) Application in promoting crop growth performance; 8) Application in improving and enhancing soil fertility.

4. Use of the lysinic Bacillus YCL-7 according to claim 1 or the bacterial agent according to claim 2 in the preparation of microbial fertilizer.

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