Bacillus cereus CBBL-18 and application thereof

Abstract

The invention relates to bacillus cereus (Bacillus cereus) CBBL-18, and the preservation number of the bacillus cereus CBBL-18 is CGMCC (China General Microbiological Culture Collection Center) No.30805. The strain not only has the capabilities of releasing silicon, dissolving phosphorus, dissolving potassium and fixing nitrogen, but also has the capabilities of producing siderophore and IAA (Indoleacetic Acid). The strain can well grow under the conditions that the pH is 5-9, the temperature is 20-40 DEG C and the NaCl concentration is 0-2%, and meanwhile, the strain grows in a culture medium containing 1 mM of copper ions, 2 mM of lead ions and 2 mM of chromium ions, so that the strain has the characteristics of better acid and alkali resistance, heavy metal resistance and wide temperature adaptability. In addition, the strain also has an antagonistic effect on fusarium oxysporum of cotton. The invention also discloses application of the strain in promoting plant growth and improving soil quality. According to the growth characteristics of the strain, the strain has a good application prospect in the aspects of barrier soil improvement, tailing area soil ecological reclamation, novel bio-organic fertilizer development and the like.

Description

Technical Field

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

[0002] The environmental pollution caused by the accumulation of large amounts of iron tailings and the resource treatment of large amounts of tailings are the focus of current attention. In the treatment of soil contaminated by iron tailings, microbial combined with plant remediation is widely used as a high-efficiency, low-cost and environmentally friendly soil remediation technology. For example, silicon can improve the drought resistance, heavy metal resistance 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 SiO2 in iron tailings. At this time, SiO2 can be decomposed and transformed by microorganisms into an available effective state, so that it can be absorbed by plants. Therefore, exploring multifunctional microbial strains and developing low-cost, effective and environmentally friendly microbial preparations are of great significance to the development and utilization of various element resources in iron tailings and soil, and the development of green ecological agriculture.

[0003] At present, scholars at home and abroad have discovered a variety of microorganisms that have the function of decomposing silicate minerals and dissolving silicon, such as: Bacillus mucilaginosus, Bacillus megaterium, Bacillus amyloliquefaciens, Aspergillus niger, Penicillium, etc., which are mainly used in general soil remediation or agricultural production. It is difficult to apply them to the resource treatment of iron tailings containing a large amount of heavy metal components and poor acid-base conditions and the ecological reclamation of tailings areas. Therefore, screening multifunctional strains with efficient silicon release and strong environmental adaptability from tailings areas can not only provide strains and technical support for the improvement of soil ecological environment in tailings areas and the resource utilization of tailings sand, but also have important significance for barrier soil remediation and agricultural green production. Summary of the invention

[0004] The purpose of the present invention is to provide a Bacillus cereus CBBL-18 which can efficiently release silicon, dissolve phosphorus, dissolve potassium, and fix nitrogen, and has the ability to produce IAA and siderophore, and also has the characteristics of acid and alkali resistance, heavy metal resistance, wide temperature adaptability, etc. and its application.

[0005] The present invention adopts the following technical solution: A Bacillus cereus ( Bacillus cereus )CBBL-18, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, Beijing, China, with the deposit number CGMCC No. 30805 and the deposit date May 29, 2024.

[0006] The Bacillus cereus CBBL-18 can grow well on PB culture medium, and the colonies are white, round, with complete edges, opaque, sticky, and slightly convex on the surface. The bacteria are Gram-positive, elongated, have spores, and have blunt ends.

[0007] The Bacillus cereus CBBL-18 can utilize glucose, sucrose, maltose, galactose, soluble starch, glycerol, mannitol, cellobiose and other carbon sources to ferment and produce acid, and can hydrolyze gelatin and starch and reduce nitrate.

[0008] Furthermore, Bacillus cereus CBBL-18 has the ability to release silicon, dissolve phosphorus, dissolve potassium, and fix nitrogen.

[0009] Furthermore, Bacillus cereus CBBL-18 has the ability to produce IAA and siderophore.

[0010] 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.

[0011] Furthermore, Bacillus cereus CBBL-18 can tolerate 1 mM copper ions, 2 mM lead ions, and 2 mM chromium ions.

[0012] Furthermore, Bacillus cereus CBBL-18 has an antagonistic effect on cotton Fusarium wilt.

[0013] An application of the Bacillus cereus CBBL-18 in promoting plant growth and improving soil quality.

[0014] The invention discloses an application of the Bacillus cereus CBBL-18 in preparing biological organic fertilizer, soil conditioner and artificial matrix.

[0015] A biological organic fertilizer comprising the Bacillus cereus CBBL-18.

[0016] A soil conditioner comprising the above-mentioned Bacillus cereus CBBL-18.

[0017] An artificial substrate comprising the above-mentioned Bacillus cereus CBBL-18.

[0018] In the above-mentioned biological organic fertilizer and soil conditioner, the bacterial count of Bacillus cereus CBBL-18 is not less than 10 8 Pieces / g.

[0019] An application of the Bacillus cereus CBBL-18 in preventing and controlling cotton wilt.

[0020] A biological pesticide for preventing and treating cotton wilt, comprising the above-mentioned Bacillus cereus CBBL-18.

[0021] An application of the Bacillus cereus CBBL-18 in soil restoration under heavy metal pollution and / or acid-base environmental conditions.

[0022] The beneficial effects of the present invention are as follows: First, the strain CBBL-18 provided by the present invention not only has the effect of releasing silicon, but also can release phosphorus, potassium, fix nitrogen, produce siderophore and IAA, which not only promotes plant growth, but also can improve 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, and can also provide excellent strain resources for the development of new biological organic fertilizers, soil conditioners, artificial substrates, etc., and has practical economic and social benefits and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the phylogenetic tree of strain CBBL-18.

[0024] Figure 2 The effect of temperature on the growth of strain CBBL-18.

[0025] Figure 3 The effect of pH concentration on the growth of strain CBBL-18.

[0026] Figure 4 This is the effect of NaCl on the growth of strain CBBL-18. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with 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.

[0028] Example 1 Isolation, identification and preservation of silicon-releasing Pantoea CBBL-18 The soil samples were obtained 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 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 solubility index of strain CBBL-18 was the largest at 2.8.

[0029] The single colonies obtained from the initial screening were screened for silicon release ability using iron tailings as substrate and the increase in effective silicon content in the fermentation broth as the standard. During the rescreening process, the seed liquid was fermented from PB medium to the logarithmic growth phase, and then inoculated into the leaching and desiliconization medium at 8% volume, and cultured in a shaking incubator at 30°C and 180 r / min. Samples were taken on the 5th day, and the fermentation broth was centrifuged to obtain the supernatant, and the effective silicon content was determined by the molybdenum blue colorimetric method. The results showed that the strain CBBL-18 still released the largest amount of silicon, which was 28.2 mg / L, so CBBL-18 was determined as the target strain for subsequent research.

[0030] Culture medium formula: (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.

[0031] (2) Mineral leaching and 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.

[0032] (3) PB 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.

[0033] The strain CBBL-18 grew on PB medium for 24 h, and the colonies were white, round, with complete edges, opaque, sticky, and slightly convex on the surface. The bacteria were Gram-positive, elongated, with spores, and blunt ends. The results of physiological and biochemical characteristics showed that the strain CBBL-18 could ferment and produce acid using a variety of carbon sources such as glucose, sucrose, maltose, galactose, soluble starch, glycerol, mannitol, and cellobiose, and could hydrolyze gelatin and starch and reduce nitrates (see Table 1 for details).

[0034] Table 1 Physiological and biochemical characteristics of strain CBBL-18 .

[0035] The total DNA of CBBL-18 strain was extracted using a bacterial genomic DNA extraction kit, and amplified using the total DNA as a template and universal 16SrDNA primers: 27 F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492 R (5'-GGTTACCTTGTTACGACTT-3'). A 1430 bp band was obtained by agarose electrophoresis. The PCR amplification product was sent to Genewise Biotechnology Co., Ltd. for sequencing.

[0036] 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 the 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%.

[0037] Based on the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, strain CBBL-18 was identified as Bacillus cereus, i.e. Bacillus cereus The strain was deposited at the General Microbiology Center of the China Microbiological Culture Collection Administration on May 29, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 30805.

[0038] Example 2 Stress resistance of strain CBBL-18 PB liquid medium was used as the basic medium, and the growth rate was OD 600 The effect of different initial pH, temperature and NaCl concentration on the growth of strain CBBL-18 was investigated using the value as an indicator. The initial pH of the culture medium was set to 4, 5, 6, 7, 8, 9, 10 and 11, the culture temperature was 15℃, 20℃, 25℃, 30℃, 35℃ and 40℃, the NaCl concentration in the culture medium was 0, 0.5%, 1%, 2% and 4%, and the CBBL-18 seed solution was inoculated with 2% (v / v) in a 30 mL PB liquid shake flask. At different initial pH and NaCl concentrations, the culture was shaken at 30℃ and 180 r / min for 24 h, and the fermentation broth OD was measured. 600 The stress resistance of strain CBBL-18 was determined by

[0039] 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, 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 the development of soil conditioners. The strain CPBS-16 also has a good wide temperature adaptability and can grow normally when cultured at 20℃~40℃. The temperature is 25℃ for the best growth. 600 The value is 1.863.

[0040] Example 3 Silicon release characteristics of strain CBBL-18 Prepare the seed solution of strain CBBL-18 and adjust the concentration of the solution to 1×10 9 CFU / mL, the seed solution was inoculated into the leaching and desiliconization medium (mineral sand particle size 0.15 mm, mineral sand concentration 2.5%) with an inoculum size of 2% (v / v), and 2% (v / v) blank PB culture solution was inoculated into the leaching and desiliconization medium as a control, with a liquid volume of 50 mL (100 mL in a shake flask), and cultured on a shaker at 30°C and 120 r / min for 7 days, with 3 replicates, and fermentation broth samples were taken at 1 d, 3 d, 5 d, and 7 d, and the fermentation broth samples were rotated at 8000 r·min -1 After centrifugation for 10 min, the supernatant was filtered with a 0.22 μm membrane and the effective silicon content in the fermentation broth after culture was determined by the NYT1121.15-2006 molybdenum blue colorimetric method.

[0041] The results showed that the effective silicon content in the fermentation broth of strain CBBL-18 reached 30.6 mg / L after 7 days of cultivation, which was 2.45 times that of the control group.

[0042] Example 4 Determination of Phosphorus Solubilization, Potassium Solubilization and Nitrogen Fixation Capacity of Strain CBBL-18 NBRIP phosphate solubilization medium: glucose 10 g, Ca3(PO4) 25 g, (NH4)2SO4 0.5 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.

[0043] 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.

[0044] Nitrogen fixation activity detection 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.

[0045] The seed solution of strain CBBL-18 was prepared and inoculated 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 whether there was a dissolution zone were observed.

[0046] The calculation method is: dissolution capacity = dissolution zone diameter D / colony diameter d.

[0047] The concentration of the CBBL-18 seed solution was adjusted to 1×10 9 CFU / mL, inoculated into nitrogen fixation activity detection medium at a volume ratio of 2%, cultured at 30℃ for 2 days, and observed its growth. After three transfers, strain CBBL-18 was still able to grow, indicating that it has the function of self-generated nitrogen fixation.

[0048] The results showed that in NBRIP phosphate-dissolving medium and potassium-dissolving medium, there were obvious transparent circles around the CBBL-18 colonies, and the D / d values ​​were greater than 1.6, indicating that it had good phosphate-dissolving and potassium-dissolving abilities. The strain CBBL-18 could grow after three consecutive transfers in nitrogen-fixing medium, indicating that the strain had nitrogen-fixing ability.

[0049] Example 5 Siderophore and IAA production capacity of strain CBBL-18 Prepare the seed solution of strain CBBL-18, inoculate it on CAS test medium, and culture it at 30℃ for 2 days. If a yellow-green halo appears, it means that the siderophore is produced. Adjust the concentration of the bacterial solution 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 d, 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 and protected from light for 30 minutes. If pink appears, it is positive, indicating that the strain can secrete IAA. The darker the color, the greater the intensity of secretion. No color change is negative, indicating that the strain cannot secrete IAA.

[0050] The results showed that when CBBL-18 was inoculated on the CAS detection medium, a clear yellow-green halo was produced around the colony, indicating that it had the ability to produce siderophores; the color development reaction was positive, indicating that it had the ability to produce IAA, which indirectly indicated that it had the ability to promote plant growth.

[0051] Example 6 Antagonism of strain CBBL-18 against plant pathogens The plate confrontation method was used to determine whether strain CBBL-18 had an antagonistic effect on plant pathogens: the pathogen block was inoculated in the center of the PDA plate, and the strain CBBL-18 was inoculated at equal distances on both sides of the block, and cultured at 25°C for 48 hours, repeated 3 times. The fact that the pathogen could not grow normally near the strain CBBL-18 indicated that there was an antagonistic effect. The results showed (see Table 2) that strain CBBL-18 had a significant antagonistic effect on cotton wilt pathogens.

[0052] Table 2 Antagonism of strain CBBL-18 against some pathogenic fungi .

[0053] The growth rate method was used to determine the activity of CBBL-18 against cotton wilt pathogen ( Fusarium oxysporum ) was cultured in PB liquid medium for 24 h, centrifuged, and the supernatant was sterilized by 0.22 μm sterile filter membrane. A certain amount of sterile filtrate was added to PDA medium at 40-45 °C, mixed and poured into a 9 cm culture dish, and no sterile filtrate was added as a blank control. After the culture medium solidified, cotton wilt bacteria were inoculated in the center of each plate, repeated 3 times, and cultured at 28 °C. The colony diameter was measured by the cross method, and the inhibition rate was calculated. The results showed that the inhibition rate of CBBL-18 against cotton wilt bacteria reached 62%.

[0054] Inhibition rate (%) = (colony diameter of control bacteria - colony diameter of treated bacteria) / (colony diameter of control bacteria - diameter of bacterial block) × 100.

[0055] Example 7 Tolerance of strain CBBL-18 to copper, lead, chromium and cadmium Lead acetate (Pb2+ ), 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 in 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°C, 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.

[0056] 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 strain CBBL-18 is used for soil remediation, the adverse effects of these three heavy metal ions can be avoided.

[0057] Table 3 Tolerance of strain CBBL-18 to heavy metal ions .

[0058] Example 8 Effects of strain CBBL-18 on alfalfa growth and soil nutrients 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 The matrix was mixed with 45% garden soil, 10% iron tailings sand (from Qian'an, Tangshan, Hebei) and 45% vermiculite. The same volume of PB culture medium was mixed into the matrix for the control, and the alfalfa pot test was carried out. In a 25℃ greenhouse, the five best-growing seedlings in each pot were retained after seed germination, and the aboveground indicators of the soil and plants were measured after 30 days. Three parallel treatments were performed in each group of experiments.

[0059] Table 4 Effects of strain CBBL-18 on alfalfa growth .

[0060] The growth of alfalfa is shown in Table 4. The strain CBBL-18 has a significant promoting effect on 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 bacterial solution increased by 20.92%, 61.54%, 55.56% and 15.48%, respectively.

[0061] Table 5 Effect of strain CBBL-18 on soil fertility (unit: mg / kg) .

[0062] As shown in Table 5, the application of CBBL-18 bacterial solution in the alfalfa pot experiment can significantly increase the content of nitrate nitrogen, ammonia nitrogen, available silicon, available phosphorus, available potassium and organic matter in the soil. Compared with the control, they increased by 12.10%, 24.80%, 15.63%, 16.39%, 9.67% and 13.21%, respectively, indicating that the strain CBBL-18 can effectively increase the soil nutrient content and improve soil nutrients.

[0063] The present invention has been described in detail according to the above-mentioned embodiments. It should be noted that the above embodiments are only for the purpose of illustrating the invention. Without departing from the spirit and essence of the present invention, those skilled in the art can design various alternatives and improvements of the present invention, which should all 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. The Bacillus cereus CBBL-18 according to claim 1, characterized in that It has the ability to release silicon, dissolve phosphorus, dissolve potassium and fix nitrogen.

3. The Bacillus cereus CBBL-18 according to claim 1, characterized in that It has the ability to produce IAA and siderophore.

4. The Bacillus cereus CBBL-18 according to claim 1, characterized in that It can grow well at pH 5~9, NaCl concentration 0~2%, and temperature 20~40℃.

5. The Bacillus cereus CBBL-18 according to claim 1, characterized in that It can tolerate 1 mM copper ions, 2 mM lead ions, and 2 mM chromium ions.

6. The Bacillus cereus CBBL-18 according to claim 1, characterized in that It has antagonistic effect on cotton wilt pathogen.

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

8. Use of the Bacillus cereus CBBL-18 as claimed in claim 1 in the preparation of biological organic fertilizer, soil conditioner and artificial matrix.

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

10. Use of the Bacillus cereus CBBL-18 as claimed in claim 1 in soil remediation under heavy metal pollution and / or acid-base environmental conditions.

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