Bacillus cereus CBBL-18 and its application

Abstract

The present invention relates to a Bacillus cereus ( Bacillus cereus )CBBL‑18, with a deposit number of CGMCC No.30805. This strain not only has the ability to release silicon, solubilize phosphorus, solubilize potassium, and fix nitrogen, but also has the ability to produce siderophores and IAA. This strain can grow well at pH 5-9, temperature 20-40°C, and NaCl concentration 0-2%. It can also grow in a culture medium containing 1 mM copper ions, 2 mM lead ions, and 2 mM chromium ions, indicating that it has good acid and alkali resistance, heavy metal resistance, and wide temperature adaptability. In addition, the bacterium also has an antagonistic effect on cotton wilt pathogens. The present invention also discloses the application of the strain in promoting plant growth and improving soil quality. According to the growth characteristics of the strain, it has good application prospects in barrier soil improvement, soil ecological reclamation in tailings areas, and the development of new bio-organic fertilizers.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and particularly relates to Bacillus cereus CBBL-18 and applications thereof. Background Art

[0002] The environmental pollution caused by the accumulation of large amounts of iron tailings and the resource-based treatment of large amounts of tailings are currently the focus of people's attention. In the treatment of soil contaminated by iron tailings, microbial combined with plant remediation is widely used as a highly efficient, low-cost, and environmentally friendly soil remediation technology. For example, silicon can improve the drought resistance, heavy metal tolerance, and disease resistance of plants, and plays a key role in improving plant yield and quality. Iron tailings are rich in SiO2, but plants cannot directly use the SiO2 in iron tailings. In this case, microbial decomposition and conversion can convert SiO2 into an effective and usable state, which can then be absorbed by plants. Therefore, the development of multifunctional microbial strains and low-cost, effective, and environmentally friendly microbial preparations are of great significance to the development and utilization of various elemental resources in iron tailings and soil, and the development of green ecological agriculture.

[0003] Currently, domestic and foreign scholars have discovered a variety of microorganisms that have the ability to decompose silicate minerals and dissolve silicon, such as: Bacillus mucilaginosus, Bacillus megaterium, Bacillus agglomerans, Bacillus amyloliquefaciens, Aspergillus niger, Penicillium, etc. These are mainly used in general soil remediation or agricultural production, and it is difficult to apply them to the resource treatment of iron tailings and the ecological reclamation of tailings areas, which contain large amounts of heavy metal components and have poor acid-base conditions. Therefore, screening multifunctional strains with high silicon release efficiency and strong environmental adaptability from tailings areas can not only provide strains and technical support for improving the soil ecological environment in tailings areas and resource utilization of tailings sand, but also have important significance for barrier soil remediation and green agricultural production. Summary of the Invention

[0004] The present invention aims to provide a Bacillus cereus CBBL-18 and its application, which can not only efficiently release silicon, dissolve phosphorus, dissolve potassium, and fix nitrogen, but also has the ability to produce IAA and siderophore, and also has the characteristics of acid and alkali resistance, heavy metal resistance, and wide temperature adaptability.

[0005] The present invention adopts the following technical solutions:

[0006] A Bacillus cereus ( Bacillus cereus ) CBBL-18, deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, Beijing, China, with the deposit number CGMCC No. 30805 and the deposit date of May 29, 2024.

[0007] The Bacillus cereus CBBL-18 can grow well on PB culture medium, with white, round colonies, complete edges, opaque, sticky, and slightly convex surfaces. The bacteria are Gram-positive, elongated, spore-bearing, and have blunt ends.

[0008] The Bacillus cereus CBBL-18 can ferment and produce acid using a variety of carbon sources such as glucose, sucrose, maltose, galactose, soluble starch, glycerol, mannitol, cellobiose, etc., and can hydrolyze gelatin and starch and reduce nitrate.

[0009] Furthermore, Bacillus cereus CBBL-18 has the ability to release silicon, solubilize phosphorus, solubilize potassium, and fix nitrogen.

[0010] Furthermore, Bacillus cereus CBBL-18 has the ability to produce IAA and siderophore.

[0011] Furthermore, Bacillus cereus CBBL-18 can grow well at pH 5-9, NaCl concentration 0-2%, and temperature 20-40°C, and has good acid and alkali resistance and wide temperature adaptability.

[0012] Furthermore, Bacillus cereus CBBL-18 can tolerate 1 mM copper ions, 2 mM lead ions, and 2 mM chromium ions.

[0013] Furthermore, Bacillus cereus CBBL-18 has an antagonistic effect on cotton Fusarium wilt.

[0014] An application of the Bacillus cereus CBBL-18 in promoting plant growth and improving soil quality.

[0015] The invention discloses an application of the Bacillus cereus CBBL-18 in the preparation of biological organic fertilizer, soil conditioner and artificial matrix.

[0016] A biological organic fertilizer containing the Bacillus cereus CBBL-18.

[0017] A soil conditioner comprising the above-mentioned Bacillus cereus CBBL-18.

[0018] An artificial substrate comprising the above-mentioned Bacillus cereus CBBL-18.

[0019] In the above-mentioned biological organic fertilizer and soil conditioner, the bacterial count of Bacillus cereus CBBL-18 is not less than 10 8 pcs / g.

[0020] An application of the Bacillus cereus CBBL-18 in preventing and treating cotton wilt.

[0021] A biological pesticide for preventing and controlling cotton wilt disease, comprising the above-mentioned Bacillus cereus CBBL-18.

[0022] A use of the Bacillus cereus CBBL-18 in soil remediation under heavy metal pollution and / or acid-base environmental conditions.

[0023] The beneficial effects of the present invention are as follows: First, the strain CBBL-18 provided by the present invention not only has a silicon-releasing effect, but can also solubilize phosphorus and potassium, fix nitrogen, produce siderophore and IAA, which not only promotes plant growth, but also improves soil quality. Secondly, the strain CBBL-18 of the present invention has an antagonistic effect on cotton wilt pathogens and can be used for the development of biocontrol agents. Thirdly, the strain CBBL-18 of the present invention is not only salt- and alkali-tolerant, but also heavy metal-tolerant, and can be used for the improvement and quality improvement of saline-alkali soil, heavy metal-contaminated soil, and tailings area soil. It can also provide excellent strain resources for the development of new bio-organic fertilizers, soil conditioners, artificial substrates, etc., and has tangible economic and social benefits and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the phylogenetic tree of strain CBBL-18.

[0025] Figure 2 The effect of temperature on the growth of strain CBBL-18.

[0026] Figure 3 The effect of pH concentration on the growth of strain CBBL-18.

[0027] Figure 4 This is the effect of NaCl on the growth of strain CBBL-18. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the embodiments and drawings. The protection scope of the present invention is not limited to the embodiments, and any changes made by those skilled in the art within the scope defined by the claims also fall within the protection scope of the present invention.

[0029] Example 1 Isolation, identification and preservation of silicon-releasing Pantoea CBBL-18

[0030] The soil samples were collected from the rhizosphere of plants around a tailings pond in Chengde City, Hebei Province. The soil samples were diluted to 10 -5 ~10 -7 After concentration, it was evenly spread on the silicon-releasing culture medium and inverted in a constant temperature incubator at 30℃ for 2 days. Single colonies with transparent circles were selected and repeatedly streaked for purification, and more than 20 silicon-releasing strains were obtained, among which the strain CBBL-18 had the largest solubility index of 2.8.

[0031] Using iron tailings as the substrate, the single colonies obtained in the initial screening were rescreened for their silicon release capacity, using the increase in available silicon content in the fermentation broth as the criterion. During the rescreening process, the seed solution was fermented in PB medium until the logarithmic growth phase, then inoculated with 8% of the volume of leaching and desiliconization medium. The culture was shaken at 30°C and 180 rpm. Samples were collected on the fifth day, and the supernatant was centrifuged and measured for available silicon content using the molybdenum blue colorimetric method. The results showed that strain CBBL-18 again released the highest amount of silicon, at 28.2 mg / L. Therefore, CBBL-18 was selected as the target strain for further research.

[0032] Culture medium formula:

[0033] (1) Silicon-releasing culture medium: sucrose 5.0 g, Na2HPO4 2.0 g, MgSO4·7H2O 0.5 g, FeCl3 0.005 g, CaCO3 0.1 g, potassium feldspar 1.0 g, agar 15-20 g, water 1000 mL, pH adjusted to 7.0-7.4.

[0034] (2) Mineral desiliconization culture medium: glucose 10 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaCl2·2H2O 0.2 g, iron tailings 1.0 g, CaCO3 5 g, water 1000 mL, pH 7.0-7.2.

[0035] (3) PB culture medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl. For solid culture medium, add 15-20 g agar and 1000 mL water. pH 7.2-7.5.

[0036] Strain CBBL-18, grown on polystyrene (PB) medium for 24 hours, produces white, round colonies with intact, opaque, sticky, and slightly convex surfaces. The bacteria are Gram-positive, elongated, spore-bearing, and have blunt ends. Physiological and biochemical characterization revealed that strain CBBL-18 can ferment and produce acid from a variety of carbon sources, including glucose, sucrose, maltose, galactose, soluble starch, glycerol, mannitol, and cellobiose. It can also hydrolyze gelatin and starch and reduce nitrates (see Table 1 for details).

[0037] Table 1 Physiological and biochemical characteristics of strain CBBL-18

[0038] .

[0039] Total DNA from the CBBL-18 strain was extracted using a bacterial genomic DNA extraction kit and used as a template for amplification using universal 16S rDNA primers: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). A 1430-bp band was detected by agarose gel electrophoresis. The PCR amplification product was sent to Genewise Biotechnology Co., Ltd. for sequencing.

[0040] The sequencing results were submitted to NCBI for homology comparison analysis with the existing 16S rDNA sequences in the database, and the 16S rDNA gene sequences of closely related strains were selected from Genebank and a phylogenetic tree was constructed using MEGA7.0. Figure 1 As shown, CBBL-18 and Bacillus cereus strain D8639 was the closest relative, with a similarity of 99%.

[0041] Based on the comprehensive morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, the strain CBBL-18 was identified as Bacillus cereus, i.e. Bacillus cereus The strain was deposited on May 29, 2024, at the General Microbiology Center of the China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 30805.

[0042] Example 2 Stress resistance of strain CBBL-18

[0043] PB liquid medium was used as the basic medium, and the growth rate was OD 600 The effects of different initial pH, temperature, and NaCl concentration on the growth of strain CBBL-18 were investigated using the pH value as an indicator. The initial pH of the culture medium was set at 4, 5, 6, 7, 8, 9, 10, and 11, the culture temperature was 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C, and the NaCl concentration in the culture medium was 0, 0.5%, 1%, 2%, and 4%. The CBBL-18 seed liquid was inoculated at a 2% (v / v) inoculum into a 30 mL PB liquid shake flask. At different initial pH and NaCl concentrations, the culture was shaken at 30°C and 180 r / min for 24 h, and the OD of the fermentation liquid was measured. 600 The stress resistance of strain CBBL-18 was determined by the PCR.

[0044] The results showed that (see Figure 2 、 Figure 3 and Figure 4), strain CBBL-18 has a certain salt tolerance and grows normally in a medium containing 0-2% NaC. Strain CBBL-18 shows good acid and alkali resistance and can grow normally at pH 5-9. When pH is 10, the growth state decreases slightly, but it can still survive and grow. When pH is 6, the growth performance is the best, and OD 600 The value reached 2.304, indicating that it has strong acid and alkali resistance and can be used in acidic or alkaline soils, which has obvious advantages in developing soil conditioners. The strain CPBS-16 also has a good wide temperature adaptability and can grow normally when cultured at 20℃~40℃, with the best growth at 25℃. 600 The value is 1.863.

[0045] Example 3 Silicon Release Characteristics of Strain CBBL-18

[0046] Prepare the CBBL-18 seed solution and adjust the concentration of the bacterial solution to 1×10 9 CFU / mL, the seed liquid was inoculated into the leaching and desiliconization medium (mineral sand particle size 0.15 mm, mineral sand concentration 2.5%) at an inoculum size of 2% (v / v). At the same time, 2% (v / v) blank PB culture medium was inoculated into the leaching and desiliconization medium as a control. The liquid volume was 50 mL (100 mL in a shake flask) and cultured on a shaker at 30°C and 120 r / min for 7 days. There were 3 replicates. The fermentation broth samples were taken on the 1st, 3rd, 5th and 7th days. The fermentation broth samples were shaken at 8000 r·min -1 After centrifugation for 10 min, the supernatant was filtered through a 0.22 μm membrane and the effective silicon content in the fermentation broth was determined using the NYT1121.15-2006 molybdenum blue colorimetric method.

[0047] The results showed that the effective silicon content in the fermentation broth of strain CBBL-18 reached 30.6 mg / L after culturing for 7 days, which was 2.45 times that of the control group.

[0048] Example 4 Determination of Phosphate Solubilization, Potassium Solubilization and Nitrogen Fixation Abilities of Strain CBBL-18

[0049] NBRIP phosphate solubilization medium: glucose 10 g, Ca3(PO4)25 g, (NH4)2SO40.5 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, MgCl2·6H2O 5 g, 0.4% bromophenol blue 6 mL, agar powder 15 g, water 1000 mL, pH 7.0-7.2.

[0050] Potassium-dissolving medium: sucrose 10 g, Na2HPO4 1 g, (NH4)2SO4 0.5 g, MgSO4·7H2O 1 g, yeast powder 0.2 g, NaCl 0.1 g, CaCO3 0.1 g, FeCl3 0.005 g, potassium feldspar 5 g, agar powder 15 g, water 1000 mL, pH 7.0-7.2.

[0051] Nitrogen fixation activity assay medium (Ashby nitrogen-free medium): mannitol 10 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaSO4·2H2O 0.2 g, CaCO3 5 g, water 1000 mL, pH 7.0-7.2.

[0052] The seed liquid of strain CBBL-18 was prepared and spotted on NBRIP phosphate-dissolving medium and potassium-dissolving medium, respectively. After culturing in a constant temperature incubator at 30°C for 2 days, its growth and the presence of dissolution zones were observed.

[0053] The calculation method is: dissolution capacity = dissolution zone diameter D / colony diameter d.

[0054] The concentration of the strain CBBL-18 seed solution was adjusted to 1×10 9 CFU / mL, inoculated into nitrogen fixation activity assay medium at a volume ratio of 2%, cultured at 30°C for 2 days, and observed for growth. After three transfers, strain CBBL-18 was still able to grow, indicating that it has the ability to autogenously fix nitrogen.

[0055] The results showed that in both NBRIP phosphate-solubilizing and potassium-solubilizing media, CBBL-18 colonies exhibited distinct clear zones, with D / d values ​​exceeding 1.6, indicating excellent phosphate-solubilizing and potassium-solubilizing abilities. The strain CBBL-18 also continued to grow after three consecutive transfers to nitrogen-fixing media, demonstrating its ability to fix nitrogen.

[0056] Example 5 Siderophore and IAA Production Capacity of Strain CBBL-18

[0057] Prepare the CBBL-18 seed solution, inoculate it onto the CAS test medium, and culture it at 30°C for 2 days. If a yellow-green halo appears, it indicates that the siderophore is produced. Adjust the bacterial solution concentration to 1×10 9CFU / mL, inoculated into LB liquid culture medium (containing 100 mg / L L-tryptophan) at a volume ratio of 2%, placed on a shaker at 30℃ and 180 r / min for 1-2 days, took 50 μL of the supernatant after centrifugation at 8000 r / min, added 50 μL of Salkowski colorimetric solution, dropped it on a white porcelain plate in the dark for 30 minutes, and developed the color. If pink appears, it is positive, indicating that the strain can secrete IAA. The darker the color, the stronger the secretion intensity. No color change is negative, indicating that the strain cannot secrete IAA.

[0058] The results showed that when CBBL-18 was inoculated on CAS detection medium, a clear yellow-green halo was produced around the colony, indicating that it had the ability to produce siderophores; the color reaction was positive, indicating that it had the ability to produce IAA, indirectly indicating that it had the ability to promote plant growth.

[0059] Example 6 Antagonism of strain CBBL-18 against plant pathogens

[0060] The plate standoff assay was used to determine whether strain CBBL-18 exhibited antagonistic activity against plant pathogens. A pathogen block was inoculated in the center of a PDA plate, and strain CBBL-18 was inoculated equidistantly on either side of the block. The plates were incubated at 25°C for 48 hours, with three replicates. Failure of the pathogen to grow near strain CBBL-18 indicated antagonistic activity. The results (Table 2) showed that strain CBBL-18 exhibited significant antagonistic activity against Fusarium wilt.

[0061] Table 2 Antagonism of strain CBBL-18 against some pathogenic fungi

[0062] .

[0063] The growth rate method was used to determine the effect of CBBL-18 on cotton wilt pathogen ( Fusarium oxysporum To investigate the antibacterial activity of CBBL-18, the researchers cultured the PB liquid medium for 24 hours, centrifuged the supernatant, and sterilized it through a 0.22 μm sterile filter. A certain amount of the sterile filtrate was added to PDA medium at 40–45°C, mixed thoroughly, and then poured into 9 cm Petri dishes. A blank control was used without the sterile filtrate. After the medium solidified, a colony of Fusarium wilt pathogen was inoculated in the center of each plate. Three replicates were used for incubation at 28°C. Colony diameters were measured using the cross-hatch method, and the inhibition rate was calculated. The results showed that CBBL-18 had an inhibition rate of 62% against Fusarium wilt pathogen.

[0064] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial block diameter) × 100.

[0065] Example 7 Tolerance of strain CBBL-18 to copper, lead, chromium, and cadmium

[0066] Lead acetate (Pb 2+ ), copper sulfate (Cu 2+ ), potassium dichromate (Cr 6+ ), cadmium chloride (Cd 2+ ) were prepared into metal ion solutions (all with a concentration of 100 mM) as stock solutions. In subsequent experiments, each metal ion stock solution was added to the PB culture medium at a certain amount to make the culture medium contain 0.5 mM, 1.0 mM, 2.0 mM, and 5.0 mM Cu, respectively. 2+ , Pb 2+ Cr 6+ and Cd 2+ , sterilized at 121℃, poured into plates, strain CBBL-18 was inoculated on different culture media, and its tolerance to heavy metal ions was determined based on the growth of the strain.

[0067] The results are shown in Table 3. The strain CBBL-18 grew normally in the medium containing 1.0 mM copper, 2.0 mM lead ion, and 2.0 mM hexavalent chromium ion, but could not grow normally in the medium containing 0.5-10.0 mM cadmium ion. This indicates that the strain CBBL-18 is sensitive to Cu. 2+ , Pb 2+ Cr 6+ It has a certain tolerance, and when using strain CBBL-18 for soil remediation, the adverse effects of these three heavy metal ions can be avoided.

[0068] Table 3 Tolerance of strain CBBL-18 to heavy metal ions

[0069] .

[0070] Example 8 Effects of strain CBBL-18 on alfalfa growth and soil nutrients

[0071] The CBBL-18 seed solution was prepared using PB liquid medium and the concentration of the bacterial solution was adjusted to 1×10 8 CFU / mL, based on 100 mL·kg -1 A substrate composed of 45% garden soil, 10% iron tailings sand (sourced from Qian'an, Tangshan, Hebei), and 45% vermiculite was mixed into the soil. For a control, an equal volume of PB medium was mixed into the substrate. A potted alfalfa experiment was conducted. After seed germination in a 25°C greenhouse, the five best-growing seedlings from each pot were retained. Soil and plant aboveground parameters were measured 30 days later. Three replicates were performed in each experiment.

[0072] Table 4 Effects of strain CBBL-18 on alfalfa growth

[0073] .

[0074] The growth of alfalfa is shown in Table 4. The strain CBBL-18 significantly promoted the growth of alfalfa. Compared with the control group, the plant height, fresh weight, dry weight and chlorophyll content of alfalfa treated with CBBL-18 solution increased by 20.92%, 61.54%, 55.56% and 15.48%, respectively.

[0075] Table 5 Effect of strain CBBL-18 on soil fertility (unit: mg / kg)

[0076] .

[0077] As shown in Table 5, the application of CBBL-18 bacterial solution in the alfalfa pot experiment can significantly increase the contents of nitrate nitrogen, ammonia nitrogen, available silicon, available phosphorus, available potassium and organic matter in the soil. Compared with the control, the increases were 12.10%, 24.80%, 15.63%, 16.39%, 9.67% and 13.21%, respectively. This shows that the strain CBBL-18 can effectively increase the nutrient content of soil and improve soil nutrients.

[0078] The present invention has been described in detail based on the above embodiments. It should be noted that the above embodiments are merely for the purpose of illustrating the invention. Without departing from the spirit and substance of the present invention, those skilled in the art may devise various alternatives and improvements of the present invention, all of which should be understood to be within the scope of protection of the present invention.

Claims

1. A Bacillus cereus ( Bacillus cereus ) CBBL-18, characterized in that The deposit number is CGMCC No.30805.

2. A use of the Bacillus cereus CBBL-18 according to claim 1 in promoting plant growth and improving soil quality.

3. Use of the Bacillus cereus CBBL-18 according to claim 1 in the preparation of bio-organic fertilizers, soil conditioners and artificial substrates.

4. Use of the Bacillus cereus CBBL-18 according to claim 1 in preventing and treating cotton wilt.

5. Use of the Bacillus cereus CBBL-18 according to claim 1 in soil remediation under heavy metal pollution and / or acid-base environmental conditions, wherein the heavy metal is copper, lead or chromium.

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