Korla Bacillus CBK-5 and its application

Abstract

The present invention relates to a Korla Bacillus ( Bacillus korlensis ) CBK‑5, with a deposit number of CGMCC No. 33651. This strain not only has the ability to release silicon, potassium, produce siderophores and IAA, but also has a broad-spectrum resistance to plant pathogenic fungi. In addition, the strain can grow well at a pH of 5-9, a temperature of 20-45°C, and a NaCl concentration of 0-6%, and has a certain resistance to heavy metals. The present invention also discloses the application of the strain in promoting plant growth and improving soil quality. The strain has good application prospects in the improvement of barrier soils, ecological reclamation of soil in tailings areas, the development of new bio-organic fertilizers, biocontrol agents, and cultivation substrates.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and particularly relates to a Korla Bacillus CBK-5 and an application thereof. Background Art

[0002] Soil is rich in silicate minerals, containing significant amounts of essential plant nutrients such as silicon, potassium, and phosphorus. These minerals are mostly present in stable forms such as aluminosilicates and apatites, making them inaccessible to plants. Silicate bacteria are a class of bacteria that decompose silicate minerals by metabolizing them to produce organic acids and polysaccharides, releasing potassium, silicon, phosphorus, and other elements for plant absorption and utilization. Currently, domestic and international researchers have discovered a variety of microorganisms capable of decomposing silicate minerals, including Bacillus mucilaginosus, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus amyloliquefaciens, Pseudomonas, Burkholderia, Rhizobium, and Enterobacter.

[0003] Silicate bacteria have a wide range of applications in agriculture, particularly in the production of bio-organic fertilizers. By combining silicate bacteria with organic fertilizers, fertilizer utilization efficiency can be significantly improved, crop growth can be promoted, and the use of chemical fertilizers can be reduced. In addition to releasing effective silicon, which significantly promotes plant growth and enhances plant mechanical strength and stress resistance (such as resistance to pests and diseases, drought, and salt), the extracellular polymeric substances (EPS) they secrete can promote the aggregation of soil particles, improving soil aeration and water retention.

[0004] In addition, silicate bacteria can dissolve silicate minerals in tailings, releasing effective silicon, fixing heavy metals, reducing their bioavailability, and promoting soilification of tailings, which can then be used for ecological remediation of tailings and heavy metal-contaminated soils. Therefore, screening multifunctional strains with high silicon release, phosphorus and potassium solubility, disease resistance, and strong environmental adaptability can not only help in the ecological remediation of obstructed soils and the development of new bio-organic fertilizers, but also have important significance for the green and sustainable development of industry and agriculture. Summary of the Invention

[0005] The purpose of the present invention is to provide a Korla Bacillus CBK-5 which can efficiently release silicon and potassium, has the ability to produce siderophores and IAA, and has the characteristics of acid and alkali resistance, heavy metal resistance, wide temperature adaptability, etc. and its application.

[0006] The present invention adopts the following technical solutions:

[0007] A Korla Bacillus ( Bacillus korlensis )CBK-5, deposited in the China General Microorganism Culture Collection Center, Beijing, China, with the deposit number CGMCC No.33651 and the deposit date February 25, 2025.

[0008] The Korla Bacillus CBK-5 can grow well on PB culture medium, with milky white or light yellow colonies, smooth surfaces, neat edges, and opaque bacteria. The bacteria are Gram-positive, rod-shaped, motile, spore-bearing, and have blunt ends.

[0009] Korla Bacillus CBK-5 can use glucose, sucrose, maltose, galactose, glycerol, mannitol and other carbon sources to ferment and produce acid. It is negative in gelatin liquefaction and indole tests, does not produce urease and hydrogen sulfide, can hydrolyze starch and cellulose, and reduce nitrates, and is positive in catalase, amylase, protease and catalase tests.

[0010] Furthermore, Korla Bacillus CBK-5 has the ability to release silicon and potassium.

[0011] Furthermore, Korla Bacillus CBK-5 has the ability to produce IAA and siderophore.

[0012] Furthermore, Korla Bacillus CBK-5 can grow well at pH 5-9, NaCl concentration 0-6%, and temperature 20-45°C, and has good acid and alkali resistance and wide temperature adaptability.

[0013] Furthermore, Korla Bacillus CBK-5 can tolerate 0.5 mM copper ions, 2.0 mM lead ions, and 2.0 mM chromium ions.

[0014] Furthermore, Korla Bacillus CBK-5 has antagonistic effects on potato scab, potato late blight, cotton Verticillium dahliae, cotton Fusarium wilt, Fusarium graminearum, corn leaf blight, corn leaf blight, corn rot, tomato gray leaf spot, and tomato gray mold.

[0015] A use of Korla Bacillus CBK-5 in promoting plant growth and / or improving soil quality.

[0016] The invention discloses an application of Korla Bacillus CBK-5 in the preparation of biological organic fertilizer, soil conditioner and artificial matrix.

[0017] A biological organic fertilizer containing the Korla Bacillus CBK-5.

[0018] A soil conditioner comprising the Korla Bacillus CBK-5.

[0019] An artificial matrix comprising the Korla Bacillus CBK-5.

[0020] Among them, the bacterial count of Korla Bacillus CBK-5 in the above-mentioned biological organic fertilizer and soil conditioner is not less than 2×10 8 pcs / g.

[0021] An application of the Korla Bacillus CBK-5 in preventing and controlling plant diseases.

[0022] A biological pesticide comprising the above-mentioned Korla Bacillus CBK-5.

[0023] Furthermore, the biological pesticide can be used to prevent and control potato scab, potato late blight, cotton Verticillium dahliae, cotton wilt, Fusarium graminearum, corn leaf blight, corn leaf blight, corn rot, tomato gray leaf spot, and tomato gray mold.

[0024] An application of the Korla Bacillus CBK-5 in soil remediation in tailings areas and heavy metal polluted environments.

[0025] The beneficial effects of the present invention are:

[0026] First, the strain CBK-5 provided by the present invention has the functions of releasing silicon, solubilizing phosphorus, solubilizing potassium, and producing siderophores. It can convert insoluble silicon, phosphorus, and potassium in the soil into forms that can be absorbed by plants, greatly reducing the use of chemical fertilizers while promoting plant growth and improving soil quality.

[0027] Secondly, the strain CBK-5 of the present invention has a broad-spectrum resistance to plant pathogens, and has antagonistic effects on a variety of plant pathogens, including potato scab, potato late blight, cotton wilt, cotton fusarium wilt, Fusarium graminearum, corn leaf blight, corn leaf blight, corn rot, tomato gray leaf spot, and tomato gray mold, and can be used in the development of new biocontrol agents.

[0028] In summary, the strain CBK-5 of the present invention is not only salt- and alkali-tolerant, but also heavy metal-tolerant. It can be used to improve and enhance the quality of saline-alkali soil, heavy metal-contaminated soil, and soil in tailings areas. It can also provide excellent strain resources for the development of new bio-organic fertilizers, biocontrol agents, soil conditioners, artificial substrates, etc., thereby reducing the use of chemical fertilizers and pesticides, and achieving significant economic, social, and ecological benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the phylogenetic tree of strain CBK-5.

[0030] Figure 2 The effect of temperature on the growth of strain CBK-5.

[0031] Figure 3 The effect of pH concentration on the growth of strain CBK-5.

[0032] Figure 4 This is the effect of NaCl on the growth of strain CBK-5. DETAILED DESCRIPTION

[0033] 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.

[0034] Example 1 Isolation, identification and preservation of strain CBK-5

[0035] The soil samples were obtained from the rhizosphere soil of plants around an iron tailings pond in Hebei Province, China. The samples were diluted to 10 -4 ~10 -6 After the concentration was determined, the strain was evenly spread on the silicon-releasing culture medium and inverted in a constant temperature incubator at 30°C for 2 days. Single colonies with transparent circles were selected and repeatedly streaked for purification to obtain a strain CBK-5 with silicon-releasing function.

[0036] The CBK-5 seed solution was fermented in PB medium to the logarithmic growth phase and inoculated into a leaching and desiliconizing medium (mineral sand particle size 0.15 mm, mineral sand concentration 2.5%) at an inoculum volume of 8% (v / v). At the same time, 8% (v / v) blank PB culture solution was inoculated into the leaching and desiliconizing medium as a control. The volume of the solution was 30 mL (100 mL in a shake flask) and the culture was placed at 30°C and 150 r / min on a shaker for 7 days with three replicates. The fermentation broth sample was shaken at 8000 r·min. -1 After centrifugation for 10 minutes, the supernatant was filtered through a 0.22 μm membrane. The available silicon content in the fermentation broth was determined using the NYT1121.15-2006 silicomolybdenum blue colorimetric method. The results showed that after 7 days of culture, the available silicon content in the fermentation broth of strain CBK-5 reached 21.7 mg / L, 2.02 times that of the control.

[0037] Culture medium formula:

[0038] (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.

[0039] (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.

[0040] (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.

[0041] Strain CBK-5, grown on PB medium for 24 hours, produces milky white or pale yellow colonies with smooth, neatly margined, and opaque colonies. The bacteria are Gram-positive, rod-shaped, motile, and spore-bearing with blunt ends. Physiological and biochemical characterization revealed that strain CBK-5 can ferment and produce acid using a variety of carbon sources, including glucose, sucrose, maltose, galactose, glycerol, and mannitol. It tested negative for gelatin liquefaction and indole, did not produce urease, and did not produce hydrogen sulfide. However, it hydrolyzed starch and cellulose and reduced nitrates, and tested positive for catalase, amylase, protease, and catalase (see Table 1).

[0042] Table 1 Physiological and biochemical characteristics of strain CBK-5

[0043] .

[0044] Total DNA from strain CBK-5 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 single band of approximately 1500 bp was detected by agarose gel electrophoresis. The PCR amplification product was sent to Genewise Biotechnology Co., Ltd. for sequencing.

[0045] 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, CBK-5 and Bacillus korlensis strain RH-11 is the closest relative.

[0046] Based on the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, the strain CBK-5 was identified as Korla Bacillus, i.e. Bacillus korlensis The strain was deposited on February 25, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number CGMCC No. 33651.

[0047] Example 2 Stress resistance of strain CBK-5

[0048] 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 CBK-5 were investigated using the pH as an indicator. The initial pH of the culture medium was set at 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, the culture temperature was 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C, and the NaCl concentration in the culture medium was 0, 1%, 2%, 4%, 6%, 8%, 10%, and 12%. The CBK-5 seed solution was inoculated with 2% (v / v) into a 30 mL PB liquid shake flask (100 mL 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 CBK-5 was determined by the PCR.

[0049] The results showed that (see Figure 2 、 Figure 3 and Figure 4 Strain CBK-5 exhibits excellent salt tolerance and grows normally in media containing 0-6% NaC. Strain CBK-5 exhibits excellent acid and alkali tolerance, growing normally at pH levels of 5-9, but failing at pH 10 and above. This demonstrates its strong acid and alkali tolerance, making it suitable for use in both acidic and alkaline soils, offering significant advantages in developing soil conditioners. Strain CBK-5 also exhibits excellent temperature tolerance, growing normally at temperatures between 20°C and 45°C, with optimal growth at 25-35°C.

[0050] Example 3 Determination of potassium-solubilizing ability of strain CBK-5

[0051] 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 12 g, water 1000 mL, pH 7.0-7.2.

[0052] Prepare the seed solution of strain CBK-5, inoculate it on potassium-dissolving medium, and culture it in a constant temperature incubator at 30℃ for 2 days. Then observe its growth and whether there is a dissolution zone.

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

[0054] The results showed that in the potassium-solubilizing medium, obvious transparent zones appeared around the CBK-5 colonies, and the D / d value was 1.55, indicating that it had a good potassium-solubilizing ability.

[0055] Example 4 Siderophore and IAA Production Capacity of Strain CBK-5

[0056] Prepare the CBK-5 strain seed solution, inoculate it onto the CAS test medium, and culture it at 30℃ for 2 days. If a yellow-green halo appears, it indicates that the siderophore is produced. Adjust the bacterial solution concentration to 1×10 9 CFU / 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, and developed the color for 30 minutes in the dark. 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.

[0057] The results showed that when CBK-5 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 reaction was positive, indicating that it had the ability to produce IAA, indirectly indicating that it had the ability to promote plant growth.

[0058] Example 5 Antagonism of strain CBK-5 against plant pathogens

[0059] The plate standoff assay was used to determine whether strain CBK-5 truly possesses antagonistic effects against plant pathogens: a pathogen block was inoculated in the center of a PDA plate, and strain CBK-5 was inoculated equidistantly on either side of the block. The plates were incubated at 25°C for 48 hours, with three replicates. Failure of pathogen growth near strain CBK-5 indicated antagonism. The results (Table 2) showed that strain CBK-5 exhibited significant antagonistic effects against potato scab, potato late blight, cotton Verticillium wilt, cotton Fusarium wilt, Fusarium graminearum, northern corn leaf blight, southern corn leaf blight, corn rot, tomato gray leaf spot, and tomato gray mold.

[0060] Table 2 Antagonistic activity of strain CBK-5 against some pathogenic fungi

[0061] .

[0062] The antibacterial activity of CBK-5 against several plant pathogens was determined using a growth rate assay. CBK-5 was cultured in PB liquid medium for 24 hours, centrifuged, and the supernatant sterilized by passing 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, plant pathogen colonies were inoculated in the center of each plate, replicated three times, and incubated at 28°C. Colony diameters were measured using the cross-hatch method, and inhibition rates were calculated. The results showed that CBK-5 exhibited an inhibition rate of over 60% against the aforementioned plant pathogens.

[0063] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial block diameter) × 100.

[0064] Example 6 Tolerance of strain CBK-5 to copper, lead, chromium, and cadmium

[0065] 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 CBK-5 was inoculated on different culture media, and its tolerance to heavy metal ions was determined based on the growth of the strain.

[0066] The results are shown in Table 3. The strain CBK-5 grew normally in the medium containing 0.5 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 CBK-5 is sensitive to Pb 2+ Cr 6+ It has a certain tolerance, and when using strain CBK-5 for soil remediation, the adverse effects of these two heavy metal ions can be avoided.

[0067] Table 3 Tolerance of strain CBK-5 to heavy metal ions

[0068] .

[0069] Example 7 Effects of strain CBK-5 on corn plant growth and soil nutrients

[0070] The CBK-5 strain was cultured in PB liquid medium until the spore-forming stage, and the concentration of the fermentation liquid was adjusted to 1×10 8 CFU / mL, based on 100 mL·kg -1A matrix composed of 50% garden soil, 10% iron ore tailings, 20% coconut coir, and 20% vermiculite was mixed into the soil. For the control, an equal volume of PB medium was mixed into the matrix. Potted corn plants were grown at 25°C. After the seeds germinated and seedlings emerged, three best-growing seedlings were retained from each pot. After 35 days, soil and plant parameters were measured. Three replicates were performed for each experiment.

[0071] Table 4 Effects of strain CBK-5 on corn plant growth

[0072] .

[0073] Table 4 shows the growth of corn. Strain CBK-5 significantly promoted corn growth. Compared with the CK treatment, the plant height, stem diameter, fresh weight, and dry weight of corn treated with the bacterial solution increased by 20.26%, 29.79%, 28.61%, and 36.36%, respectively. Chlorophyll content in the treated group was significantly higher than that in the CK group, increasing by 13.65%.

[0074] Table 5 Effects of strain CBK-5 on soil fertility (unit: mg / kg)

[0075] .

[0076] Table 5 shows that application of the CBK-5 bacterial solution in the corn pot experiment significantly increased soil nitrate nitrogen, ammonium nitrogen, available silicon, available phosphorus, available potassium, and organic matter contents by 12.25%, 28.50%, 10.91%, 13.81%, 16.16%, and 8.15%, respectively, compared to the control. This further demonstrates that the application of the CBK-5 bacterial solution can effectively increase soil nutrient content and improve soil nutrient status.

[0077] 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 Korla Bacillus ( Bacillus korlensis ) CBK-5, characterized in that, The deposit number is CGMCC No.33651.

2. A use of the Korla Bacillus CBK-5 according to claim 1 in promoting plant growth and / or improving soil quality.

3. Use of the Korla Bacillus CBK-5 according to claim 1 in the preparation of bio-organic fertilizer, soil conditioner and / or artificial matrix.

4. A use of the Korla Bacillus CBK-5 according to claim 1 in preventing and controlling plant diseases caused by potato scab, potato late blight, cotton Verticillium dahliae, cotton Fusarium wilt, Fusarium graminearum, corn leaf blight, corn leaf blight, corn rot, tomato gray leaf spot, and tomato gray mold.

5. A use of the Korla Bacillus CBK-5 according to claim 1 in the preparation of a biocontrol agent, wherein the biocontrol agent is used to antagonize potato scab pathogen, potato late blight pathogen, cotton Verticillium dahliae, cotton wilt pathogen, Fusarium graminearum, corn leaf blight pathogen, corn leaf blight pathogen, corn rot, tomato gray leaf spot pathogen, and tomato gray mold pathogen.

6. A use of the Korla Bacillus CBK-5 according to claim 1 in the preparation of a biopesticide, wherein the biopesticide is used to control potato scab, potato late blight, cotton Verticillium dahliae, cotton wilt, Fusarium graminearum, corn leaf blight, corn leaf blight, corn rot, tomato gray leaf spot, and tomato gray mold.

7. A use of the Korla Bacillus CBK-5 according to claim 1 in remediating soil in a heavy metal-contaminated environment, wherein the heavy metal is lead or chromium.

8. Use of the Korla Bacillus CBK-5 according to claim 1 in repairing soil in tailings areas.

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