Bacillus velezensis CMF18 and application thereof

Abstract

The invention discloses bacillus velezensis CMF18 and application thereof, and belongs to the technical field of microorganisms, the strain is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20251028. The strain disclosed by the invention has phosphorus dissolving capacity, potassium dissolving capacity and medium element magnesium dissolving capacity. The strain can generate xylanase and has the function of promoting organic matter decomposition. The strain of the invention has the ability to synthesize and secrete plant growth promoting substances, including auxin hormone gibberellin (GA3) and cytokinin plant hormone zeatin (Z). And the microbial fertilizer has excellent comprehensive capability and can be used for preparing microbial fertilizer strains for promoting the growth of crops.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a Bacillus velezensis strain and application thereof. Background Art

[0002] Plant growth promoting rhizobacteria (PGPR) dominate soil life by regulating nutrient cycling, decomposing organic matter, influencing soil structure, inhibiting plant diseases, and improving plant productivity, thereby fulfilling a range of important soil functions. Bacillus is one of the most common bacterial species in PGPR. Extensive research has shown that it plays an important role in soil health, including improving nutrient use efficiency, inhibiting soil pathogens, and regulating the soil microbiome. Therefore, screening Bacillus for potential agricultural applications has important research and application value. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a Bacillus velezensis strain with excellent comprehensive agricultural performance and application thereof.

[0004] The technical solution of the present invention is: a Bacillus velezensis CMF18, which is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 20251028.

[0005] A microbial agent containing the above-mentioned Bacillus velezensis CMF18.

[0006] The use of the above-mentioned Bacillus velezensis CMF18 or microbial agent in promoting plant growth.

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] The strain of the present invention has the ability to dissolve phosphorus, potassium, and the medium element magnesium; can produce xylanase, which has the function of promoting the decomposition of organic matter; and has the ability to synthesize and secrete plant growth-promoting substances, including the plant growth hormone gibberellin (GA3) and the cytokinin plant hormone zeatin (Z). With its excellent comprehensive capabilities, it can be used to produce microbial fertilizer for promoting the growth of crops.

[0009] Preservation Information

[0010] Bacillus velezensis CMF18 was deposited in the China Center for Type Culture Collection on May 12, 2025, with the deposit number CCTCC NO: M 20251028. The address of the collection center is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. DETAILED DESCRIPTION

[0011] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0012] This strain was isolated in 2018 from farmland soil collected at the Fengqiu Agricultural Ecological Experimental Station in Henan Province, China, using TSB culture medium and streak isolation. Whole-genome sequencing identified the strain as Bacillus velezensis CMF18. The full-genome sequence of this strain is registered in NCBI GenBank as CP085388.

[0013] Example 1 Determination of the Phosphate Solubilizing Ability of the Strain

[0014] (1) Preparation of seed solution: The strain was cultured on a plate to obtain a single colony. A single colony was randomly picked and inoculated into 50 mL of NB liquid medium. The culture was shaken at 28°C and 180 rpm until the logarithmic phase. The seed solution was obtained with a bacterial cell content of not less than 2 × 10 8 cfu / mL. NB liquid culture medium formula is glucose 10g·L -1 , peptone 5g·L -1 , beef extract 3g·L -1 , yeast powder 0.5g·L -1 , pH was adjusted to 7.0 and sterilized at 120℃ for 15min.

[0015] (2) Preparation of fermentation broth: Centrifuge the seed solution and adjust the OD 600Adjust the concentration to about 1, inoculate the activated test strain into the calcium phytate organophosphate medium at a 5% inoculum size, culture at 28°C and 150r / min with shaking, and take samples every 24 hours to obtain the fermentation product of the test strain. At the same time, add an equal amount of sterile water to the fermentation broth as a blank control. The formula of the calcium phytate organophosphate medium is: 10g glucose, 0.4048g NH4Cl, 0.2477g MgCl2·6H2O, 0.3g NaCl, 0.3g KCl, 0.0320g FeCl2·4H2O, 0.0267g MnCl2·4H2O, 10g CaSO4·2H2O, 2.0g organic phosphorus source calcium phytate, 1.0L distilled water, adjust the pH to 7.0, and sterilize at 120°C for 15min.

[0016] (3) Calculate the potassium solubility rate of the strain: Pipette 0mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL, 5.00mL, and 6.00mL of the phosphorus standard solution (A.4.8) into a 50mL volumetric flask, dilute to about 30mL with distilled water, add 2 drops of dinitrophenol indicator, and adjust the pH with sodium hydroxide solution or dilute sulfuric acid solution until the solution is just slightly yellow. Then add 5mL of molybdenum antimony anti-colorant, shake well, and adjust to volume to obtain a series of standard solutions of 0.0mg / L, 0.1mg / L, 0.2mg / L, 0.3mg / L, 0.4mg / L, 0.5mg / L, and 0.6mg / L. After placing at room temperature above 15℃ for 30 minutes, read the absorbance using a spectrophotometer at a wavelength of 700nm. Draw a standard curve or establish a regression equation with absorbance as the ordinate and phosphorus concentration as the abscissa. After centrifuging the sample at 4000 r / min for 20 minutes, aspirate 10 mL of the supernatant (containing 5 μg to 25 μg of phosphorus; if the sample exceeds this range, dilute appropriately) into a 50 mL volumetric flask and dilute with water to approximately 30 mL. Add 2 drops of dinitrophenol indicator and adjust the pH with sodium hydroxide solution or dilute sulfuric acid solution until the solution is just slightly yellow. Then add 5 mL of molybdenum antimony colorimetric reagent, shake well, and adjust to volume. After standing at room temperature above 15°C for 30 minutes, adjust the zero point using the blank test solution as a reference. Develop and compare the color under the same conditions as the standard solution series and read the absorbance. Find the mass concentration (P) of phosphorus corresponding to the measured absorbance from the working curve. Phosphorus content in the culture medium (mg / L) = (P (mg / L) × volume of colorimetric solution V1 (mL) × dilution factor) / volume of aspirated supernatant V2 (mL). See Table 1 for the results:

[0017] Table 1 Ability of the tested strains to dissolve organic phosphorus

[0018]

[0019] After culturing the test strain CMF18 in a medium containing poorly soluble organophosphate for an appropriate period of time, the soluble phosphorus content increased by 95.1783 mg / L. According to the "General Technical Requirements for Quality Evaluation of Strains for Microbial Fertilizer Production" (NY / T1847-2010), if the soluble phosphorus content of an inoculated strain increases by at least 5 mg / L compared to an uninoculated strain, it is considered to have the ability to solubilize organophosphate. Therefore, the test strain CMF18 has the ability to solubilize organophosphate.

[0020] Example 2 Determination of potassium-solubilizing ability of strains

[0021] (1) Preparation of fermentation broth: 5 mL of seed solution was added to 95 mL of potassium-solubilizing fermentation broth. At the same time, a fermentation broth with an equal amount of inactivated seed solution was prepared as a control. The culture was conducted at 28°C and 180 rpm for 7 days. Three replicates were set up. The preparation of seed solution was the same as in Example 1. The potassium-solubilizing fermentation broth was formulated as follows: 10.0 g of sucrose, 0.5 g of MgSO4·7H2O, 0.2 g of (NH4)2·SO4, 0.1 g of NaCl, 0.1 g of CaCO3, 5.0 g of potassium feldspar powder, and 1.0 L of distilled water. The pH was adjusted to 7.2-7.5 and sterilized at 120°C for 15 min. The potassium feldspar powder was rinsed with 20% HCl solution and then rinsed with distilled water until neutral.

[0022] (2) Preparation of sample solution: Transfer the entire fermentation broth to an evaporating dish and concentrate in a water bath to approximately 10 mL. Add 2.0 mL of H₂O₂ solution and continue evaporation with constant stirring. Repeat the addition of 20% H₂O₂ solution several times until the viscous material is completely digested. Centrifuge at 3500 rpm for 10 min. Collect the supernatant in a 50 mL volumetric flask, dilute to volume with distilled water, and determine the available potassium content in the solution.

[0023] (3) Calculate the potassium solubility rate of the strain: Take 0, 5.00, 10.00, 15.00, 20.00, 25.00, and 40.00 mL of potassium standard solution and place them in 6 50 mL volumetric flasks respectively. Add 10.00 mL of nitric acid solution and dilute to volume with water. This solution contains a series of standard solutions of potassium (K) of 0, 10.00, 20.00, 30.00, 40.00, and 80.00 μg in 1 mL. Use a blank solution to adjust the instrument zero point on the flame photometer, adjust the instrument full scale to 80 degrees with the standard solution with the highest concentration in the standard solution series, measure other standard solutions, and record the instrument readings. Draw a working curve based on the potassium concentration and the instrument readings. Preparation method of 0.1 mg / mL potassium standard solution: Weigh 0.1907 g of potassium chloride (GB 646, high purity) dried at 110℃ for 2 hours, dissolve it in water, dilute to 1 L, and store in a plastic bottle. The working curve was used to determine the available potassium content ρ1 (mg / L) in the seed fermentation broth and the available potassium content ρ0 (mg / L) in the inactivated seed fermentation broth control. This yielded the relative increase in available potassium a (%) for the strain, where a (%) = [(ρ1 - ρ0) / ρ0] × 100%. The results are shown in Table 2.

[0024] Table 2 Potassium solubilization ability of the tested strains

[0025]

[0026] The relative increase in available potassium in the test strain CMF18 was 45.80%. According to the technical specifications for silicate bacteria (NY882-2004), a relative increase in available potassium of ≥20% indicates that the strain has the ability to produce and dissolve potassium. Therefore, the test strain CMF18 has the ability to dissolve potassium.

[0027] Example 3 Determination of the ability of strains to dissolve insoluble magnesium

[0028] (1) Preparation of fermentation broth: 5 mL of seed solution was added to 95 mL of fermentation broth. At the same time, a fermentation broth with an equal amount of inactivated seed solution was prepared as a control. The culture was incubated at 28°C and 180 rpm for 7 days. Three replicates were set up. The preparation of seed solution was the same as in Example 1. The formula of calcium-dissolving fermentation broth was: glucose 10 g, NH4Cl 0.4048 g, MgCl2·6H2O 0.2477 g, NaCl 0.3 g, KCl 0.3 g, FeCl2·4H2O 0.0320 g, MnCl2·4H2O 0.0267 g, MgSiO3 10 g, distilled water 1.0 L, pH adjusted to 7.0, and sterilized at 120°C for 15 min.

[0029] (2) Preparation of sample solution: Weigh 0.2 g to 3 g of seed solution (accurate to 0.0001 g) into a 250 mL volumetric flask, dilute to volume with water, mix thoroughly, dry filter, and discard the first few mL of filtrate to obtain the sample solution. A blank solution is prepared using the same method without adding sample.

[0030] (3) Use plasma emission spectrometry to measure the increase in soluble magnesium: Pipette 0mL, 0.50mL, 1.00mL, 4.00mL, 8.00mL, and 10.00mL of magnesium standard solution into six 100mL volumetric flasks, dilute to volume with water, and mix well. The mass concentrations of magnesium in this standard series are 0μg / mL, 5.0μg / mL, 10.0μg / mL, 40.0μg / mL, 80.0μg / mL, and 100.0μg / mL, respectively. Before the measurement, the measurement conditions such as argon flow rate, observation height, RF generator power, and integration time were optimized according to the properties of the element to be measured and the performance of the instrument. Then, the radiation intensity of each standard solution was measured at a wavelength of 285.213nm using a plasma emission spectrometer. A working curve was drawn with the mass concentration of magnesium (μg / mL) of each standard solution as the horizontal axis and the corresponding radiation intensity as the vertical axis. The concentration of magnesium ions in the magnesium standard solution was 1mg / mL. The sample solution and blank solution are directly (or after appropriate dilution) measured under the same conditions as the standard series solutions, and the radiation intensity of magnesium is measured. The corresponding magnesium mass concentration (μg / mL) is found on the working curve. The results are shown in Table 3:

[0031] Table 3 Ability of the test strain CMF18 to dissolve the medium element magnesium

[0032] Sample name Mg (mg / L) CK 23.18±0.16b CMF18 108.34±0.66a

[0033] Note: a and b represent significant differences among treatments, P < 0.05

[0034] In a medium containing poorly soluble magnesium, the test strain CMF18 increased its soluble magnesium content by 85.16 mg / L. Compared to the control, this increase in soluble magnesium reached a significant level as determined by a t-test. According to the "General Technical Requirements for Quality Evaluation of Strains for Microbial Fertilizer Production" (NY / T1847-2010), if the corresponding increase in soluble elements in an inoculated strain compared to an uninoculated strain reaches a significant level as determined by a t-test, the strain is considered to have the ability to solubilize moderate-level elements. Therefore, the test strain CMF18 has the ability to solubilize moderate-level elements such as magnesium.

[0035] Example 4 Determination of the ability of strains to produce xylanase

[0036] (1) Preparation of sample solution: Prepare seed solution as described in Example 1 and inoculate 1% of the seed solution into NB liquid medium until the logarithmic phase. Weigh 5.00 mL of the sample solution into a conical flask and add a certain volume of acetic acid-sodium acetate buffer as needed. Shake at 200 rpm for 30 min and centrifuge at 5000 rpm for 10 min. The supernatant is the enzyme solution to be tested. Dilute the solution appropriately with acetic acid-sodium acetate buffer (0.1 mol / mL, pH 5.50) (recommended concentration range: 6 u / mL to 15 u / mL enzyme activity).

[0037] (2) Plotting the standard curve: 4.0 mL of acetic acid-sodium acetate buffer (0.1 mol / mL, pH 5.50) was taken, 5.0 mL of DNS reagent was added, and the mixture was heated in a boiling water bath for 5 min. Cooled to room temperature with tap water, the volume was adjusted to 25.0 mL with water to prepare a standard blank. 1.00 mL, 2.00 mL, 3.00 mL, 4.00 mL, 5.00 mL, 6.00 mL, and 7.00 mL of xylose solution (10.0 mg / mL) were taken, and the volume was adjusted to 100 mL with acetic acid-sodium acetate buffer (0.1 mol / mL, pH 5.50) to prepare xylose standard solutions with concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, and 700 μg / mL.

[0038] Pipette 2.0 mL of each xylose standard solution from the above concentration series (perform two replicates) into stoppered colorimetric tubes. Add 2.0 mL of acetic acid-sodium acetate buffer (0.1 mol / mL, pH 5.50) and 5.0 mL of DNS reagent. Electromagnetic vibration is applied for 3–5 seconds, and the mixture is heated in a boiling water bath for 5 minutes. Cool the mixture to room temperature with tap water and dilute to 25 mL with water. Zero the sample using a standard blank as a control, and measure the absorbance (A) at 540 nm. Plot a standard curve with xylose concentration as the ordinate and absorbance (A) as the abscissa. A new standard curve should be plotted for each new preparation of DNS reagent.

[0039] (3) Quantification of xylanase activity of the test strain: First, prepare a xylan solution. Weigh 1.00 g of xylan (Sigma X0627), add 0.32 g of sodium hydroxide (NaOH), add 90 mL of water, stir magnetically, and slowly heat until completely dissolved. Then stop heating, continue stirring for 30 minutes, and add 0.5 mL of glacial acetic acid (C2H4O2). Continue magnetic stirring and measure the pH. If the pH is 5.50, dilute to 100 mL with acetic acid-sodium acetate buffer. If it deviates from 5.50, adjust to 5.50 with acetic acid solution or sodium acetate, and then dilute to 100 mL with acetic acid-sodium acetate buffer. Store at 4°C away from light. The shelf life is 12 hours. Pipette 10.0 mL of xylan solution and equilibrate at 50°C for 20 minutes. Pipette 10.0 mL of the appropriately diluted enzyme solution to be tested and equilibrate at 50°C for 20 minutes. Pipette 2.0 mL of the enzyme solution to be tested into a stoppered colorimetric tube. Add 5.0 mL of DNS reagent and shake the tube with an electromagnetic induction for 3–5 seconds. Add 2.0 mL of xylan solution and incubate at 50°C for 30 minutes. Heat in a boiling water bath for 5 minutes. Cool to room temperature with tap water, dilute to 25.0 mL with water, and shake the tube with an electromagnetic induction for 3–5 seconds. Use a standard blank as a reference for zero adjustment and measure the absorbance (A1) at 540 nm.

[0040] Pipette 2.0mL of the enzyme solution to be tested, add it to a stoppered colorimetric tube, then add 2.0mL of xylan solution, electromagnetically oscillate for 3s to 5s, and keep warm at 50℃ for 30min. Add 5.0mL of DNS reagent, electromagnetically oscillate for 3s to 5s to terminate the enzymatic reaction. Heat in a boiling water bath for 5min. Cool to room temperature with tap water, dilute to 25.0mL with water, and electromagnetically oscillate for 3s to 5s. Use the standard blank sample as a control to adjust to zero, and measure the absorbance A2 at 540nm. Sample xylanase activity [u / g (mL)]X = [absorbance A2 of the enzyme blank sample - absorbance A1 of the enzyme reaction solution × slope of the standard curve + intercept of the standard curve] × total dilution multiple of the sample / reaction time (min). The results are shown in Table 4:

[0041] Table 4 Xylanase secretion ability of the tested strains

[0042]

[0043] The calculated xylanase activity of the test strain CMF18 was 2001.43 u / g(mL). According to the "General Technical Requirements for Quality Evaluation of Microbial Fertilizer Production Strains" (NY / T1847-2010), strains with a xylanase activity of 700 u / g(mL) or higher are considered to have xylanase production capacity. Therefore, the test strain CMF18 has xylanase production capacity.

[0044] Example 4 Determination of the ability of strains to secrete plant hormones

[0045] (1) Preparation of standard samples: Preparation method of plant auxin standard solution: Weigh 0.0020g (±0.0001) of plant auxin standard (gibberellic acid GA3, indoleacetic acid IAA) respectively, dissolve in methanol and adjust the volume to 25.0mL. Preparation method of cytokinin standard solution: Weigh 0.0040g (±0.0001) of cytokinin standard (zeatin Z, isopentenyl adenine IPA, etc.) respectively, dissolve in methanol and adjust the volume to 25.0mL. Preparation of sample solution: Prepare seed solution according to Example 1, inoculate in NB liquid culture medium at a 1% inoculation ratio and culture to the logarithmic phase. Take 10.00mL of fermentation broth in a 50mL conical flask, concentrate to dryness under reduced pressure, add 50mL of cold methanol, and ultrasonically oscillate at about 5℃ for 2h. Filter through a 0.5μm filter membrane, and the filtrate is tested.

[0046] (2) Preparation of sample solution: Prepare seed solution as described in Example 1. Inoculate 1% of the seed solution into NB liquid medium and culture until the logarithmic phase. Place 10.00 mL of the fermentation broth in a 50 mL conical flask, concentrate to dryness under reduced pressure, add 50 mL of cold methanol, and ultrasonically vibrate at approximately 5°C for 2 h. Filter through a 0.5 μm filter membrane, and the filtrate is tested.

[0047] (3) Determination of Standard Samples: Under the following chromatographic conditions, inject 10 μL of the plant auxin standard solution into the sample and analyze it. Obtain chromatograms of gibberellins and auxins, and record the retention time and peak area of ​​each component. Repeat the injection three times, and take the average peak area of ​​each component. Chromatographic conditions: Column: C18, 0.4 cm × 25 cm; Mobile phase: 15% CH3CN, 40% CH3OH, 45% H2O (adjust pH to 4.0 with H3PO4); Detector: UV 254 nm × 0.1 AUFS; Injection volume: 10 μL; Flow rate: 0.7 mL / min.

[0048] (4) Determination of sample solution: Under the same chromatographic conditions, 10 μL of the sample solution was injected for analysis, and the content of each component in the sample was calculated using the external standard method.

[0049] (5) External standard one-point method to quantify the ability of strains to secrete plant hormones:

[0050] Total amount of auxin in the sample:

[0051]

[0052] Total amount of cytokinin in the sample:

[0053]

[0054] The total amount of plant hormones is the sum of plant growth hormones and cytokinins. The results are shown in Tables 5 and 6:

[0055] Table 5 Peak areas of plant hormone standards

[0056]

[0057]

[0058] Table 6 Plant hormone production ability of the tested strains

[0059]

[0060] The test strain CMF18 secreted 32.948 mg / L of plant hormones. According to the "General Technical Requirements for Quality Evaluation of Microbial Fertilizer Production Strains" (NY / T1847-2010), a strain producing a total of 5 mg / L or more of active substances that promote crop growth is considered capable of producing these substances. Therefore, the test strain CMF18 possesses the ability to produce active substances that promote crop growth.

Claims

1. A strain of Bacillus velezensis CMF18, deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO: M 20251028.

2. A microbial agent containing the Bacillus velezensis CMF18 according to claim 1.

3. Use of the Bacillus velezensis CMF18 according to claim 1 or the microbial agent according to claim 2 in promoting plant growth.

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