Bacillus korla CBK-5 and application thereof

Abstract

The invention relates to bacillus korlensis CBK-5, and the preservation number of the bacillus korlensis CBK-5 is CGMCC (China General Microbiological Culture Collection Center) No.33651. The strain not only has the capabilities of releasing silicon, dissolving potassium and producing siderophores and IAA, but also has the broad-spectrum plant pathogenic fungus resistance. In addition, the strain can well grow under the conditions that the pH is 5-9, the temperature is 20-45 DEG C and the NaCl concentration is 0-6%, and has certain heavy metal resistance. The invention also discloses application of the strain in promoting plant growth and improving soil quality. The strain has a good application prospect in the aspects of barrier soil improvement, tailing area soil ecological reclamation, novel bio-organic fertilizer, biocontrol microbial inoculum development, culture medium and the like.

Description

Technical Field

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

[0002] The soil is rich in silicate minerals, containing a large amount of essential nutrients for plants such as silicon, potassium, and phosphorus. Most of them exist in the form of stable aluminum silicate and apatite, which cannot be directly absorbed and utilized by plants. Silicate bacteria are a type of bacteria that can decompose silicate minerals by producing organic acids, polysaccharides and other substances through metabolism, and release potassium, silicon, phosphorus and other elements for plants to absorb and utilize. At present, scholars at home and abroad have discovered a variety of microorganisms that have the function of decomposing silicate minerals, such as: Bacillus mucilaginosus, Bacillus megaterium, Bacillus amyloliquefaciens, Pseudomonas, Burkholderia, Rhizobium, Enterobacter, etc.

[0003] Silicate bacteria have a wide range of applications in agriculture, especially in the production of bio-organic fertilizers. By combining silicate bacteria with organic fertilizers, the utilization efficiency of fertilizers can be significantly improved, crop growth can be promoted, and the use of chemical fertilizers can be reduced. In addition to the effective silicon released by silicate bacteria, which can significantly promote plant growth and enhance the mechanical strength and stress resistance of plants (such as resistance to pests and diseases, drought, and salt), the extracellular polymers (EPS) secreted by silicate bacteria can promote the aggregation of soil particles and improve the aeration and water retention of the soil.

[0004] In addition, silicate bacteria can dissolve silicate minerals in tailings, release effective silicon, fix heavy metals, reduce their biological effectiveness, promote soilification of tailings, and then be used for ecological restoration of tailings and heavy metal-contaminated soils. Therefore, screening multifunctional strains with high silicon release, phosphorus and potassium dissolution, disease resistance and strong environmental adaptability can not only provide ecological restoration of soil obstacles 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 solution: A Korla Bacillus ( Bacillus korlensis )CBK-5, deposited in the China General Microbiological Culture Collection Center, Beijing, China, with the deposit number CGMCC No.33651 and the deposit date February 25, 2025.

[0007] The Korla Bacillus CBK-5 can grow well on PB culture medium, and the colonies are milky white or light yellow, with smooth surface, neat edges, and opaque. The bacteria are Gram-positive, rod-shaped, motile, with spores, and blunt ends.

[0008] Korla Bacillus CBK-5 can utilize 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. It is positive in catalase, amylase, protease and catalase tests.

[0009] Furthermore, Korla Bacillus CBK-5 has the ability to release silicon and potassium.

[0010] Furthermore, Korla Bacillus CBK-5 has the ability to produce IAA and siderophore.

[0011] 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.

[0012] Furthermore, Korla Bacillus CBK-5 can tolerate 0.5 mM copper ions, 2.0 mM lead ions, and 2.0 mM chromium ions.

[0013] Furthermore, Korla Bacillus CBK-5 has antagonistic effects on potato scab, potato late blight, cotton Verticillium wilt, cotton Fusarium wilt, Fusarium graminearum, corn leaf blight, corn leaf blight, corn rot, tomato gray leaf spot, and tomato gray mold.

[0014] Application of Korla Bacillus CBK-5 in promoting plant growth and / or improving soil quality.

[0015] The invention discloses an application of Korla Bacillus CBK-5 in preparing biological organic fertilizer, soil conditioner and artificial matrix.

[0016] A biological organic fertilizer comprising the Korla Bacillus CBK-5.

[0017] A soil conditioner comprising the Korla Bacillus CBK-5.

[0018] An artificial matrix comprising the above-mentioned Korla Bacillus CBK-5.

[0019] 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 Pieces / g.

[0020] An application of the Korla Bacillus CBK-5 in preventing and controlling plant diseases.

[0021] A biological pesticide comprising the above-mentioned Korla Bacillus CBK-5.

[0022] Furthermore, the biological pesticide can be used to prevent and control potato scab, potato late blight, cotton Verticillium wilt, cotton Fusarium graminearum, corn leaf blight, corn leaf blight, corn rot, tomato gray leaf spot, and tomato gray mold.

[0023] An application of the Korla Bacillus CBK-5 in soil restoration in tailings areas and heavy metal polluted environments.

[0024] The beneficial effects of the present invention are: Firstly, the strain CBK-5 provided by the present invention has the functions of releasing silicon, dissolving phosphorus, dissolving potassium and producing iron carriers, and can convert the insoluble silicon, phosphorus and potassium in the soil into forms that can be absorbed by plants, thereby greatly reducing the use of chemical fertilizers while promoting plant growth and improving soil quality.

[0025] Secondly, the strain CBK-5 of the present invention has a broad-spectrum resistance to plant pathogens, and has antagonistic effects on potato scab, potato late blight, cotton Verticillium wilt, cotton wilt, Fusarium graminearum, corn leaf blight, corn leaf blight, corn rot, tomato gray leaf spot, and tomato gray mold, and can be used for the development of new biocontrol agents.

[0026] 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 for improving and enhancing 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 matrices, etc., reduce the use of chemical fertilizers and pesticides, and has significant economic, social and ecological benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the phylogenetic tree of strain CBK-5.

[0028] Figure 2 The effect of temperature on the growth of strain CBK-5.

[0029] Figure 3 The effect of pH concentration on the growth of strain CBK-5.

[0030] Figure 4 This is the effect of NaCl on the growth of strain CBK-5. DETAILED DESCRIPTION

[0031] 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.

[0032] Example 1 Isolation, identification and preservation of strain CBK-5 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 concentration, it was evenly spread on the silicon-releasing 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.

[0033] The CBK-5 seed solution was fermented in PB medium to the logarithmic growth phase, and the seed solution was inoculated into the leaching and desiliconization medium (mineral sand particle size 0.15 mm, mineral sand concentration 2.5%) at an inoculation rate of 8% (v / v). At the same time, 8% (v / v) blank PB culture solution was inoculated into the leaching and desiliconization medium as a control. The liquid volume was 30 mL (100 mL in a shake flask) and cultured on a shaker at 30 °C and 150 r / min for 7 days with three replicates. The fermentation broth samples were cultured 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 cultivation was determined by the NYT1121.15-2006 silico-molybdenum blue colorimetric method. The results showed that the effective silicon content in the fermentation broth of strain CBK-5 reached 21.7 mg / L when it was cultivated for 7 days, which was 2.02 times that of the control group.

[0034] Culture medium formula: (1) Silicon-releasing medium: sucrose 5.0 g, Na 2 HPO 4 2.0 g, MgSO 4 7H 2 O 0.5 g, FeCl 3 0.005 g, CaCO 3 0.1 g, potassium feldspar 1.0 g, agar 15-20 g, water 1000 mL, pH adjusted to 7.0-7.4.

[0035] (2) Mineral desiliconization culture medium: glucose 10 g, KH 2 PO 4 0.2 g MgSO 4 7H 2 O 0.2 g, NaCl 0.2 g, CaCl 2 ·2H 2O 0.2 g, iron tailings 1.0 g, CaCO 3 5 g, water 1000 mL, pH 7.0~7.2.

[0036] (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.

[0037] The strain CBK-5 grew on PB medium for 24 h, and the colonies were milky white or light yellow, with smooth surface, neat edges, and opaque. The bacteria were Gram-positive, rod-shaped, motile, with spores, and blunt ends. The results of physiological and biochemical characteristics showed that the strain CBK-5 could ferment and produce acid using a variety of carbon sources such as glucose, sucrose, maltose, galactose, glycerol, and mannitol. The gelatin liquefaction and indole tests were negative, and urease and hydrogen sulfide were not produced. It could hydrolyze starch and cellulose and reduce nitrates. The catalase, amylase, protease, and catalase tests were positive (see Table 1 for details).

[0038] Table 1 Physiological and biochemical characteristics of strain CBK-5 .

[0039] The total DNA of strain CBK-5 was extracted using a bacterial genomic DNA extraction kit and used as a template. The universal 16SrDNA primers: 27 F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492 R (5'-GGTTACCTTGTTACGACTT-3') were used for amplification. A band of about 1500 bp was obtained by agarose gel electrophoresis. The PCR amplification product was sent to Jinweizhi 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 to construct a phylogenetic tree using MEGA7.0. Figure 1 As shown, CBK-5 and Bacillus korlensis strain RH-11 is the closest relative.

[0041] Based on the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, strain CBK-5 was identified as Bacillus subtilis, 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.

[0042] Example 2 Stress resistance of strain CBK-5 PB liquid medium was used as the basic medium, and the growth rate was OD 600 The value was used as an indicator to investigate the effects of different initial pH, temperature, and NaCl concentration on the growth of strain CBK-5. The initial pH of the culture medium was set to 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, the NaCl concentration in the culture medium was 0, 1%, 2%, 4%, 6%, 8%, 10%, and 12%, and the strain CBK-5 seed solution was inoculated with 2% (v / v) in 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 fermentation liquid OD was measured. 600 The stress resistance of strain CBK-5 was determined by

[0043] The results showed that (see Figure 2 , Figure 3 and Figure 4 ), strain CBK-5 has good salt tolerance and grows normally in a medium containing 0-6% NaC. Strain CBK-5 shows good acid and alkali resistance, and can grow normally at pH 5-9, but cannot grow normally at pH 10 and above, 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. Strain CBK-5 also has good wide temperature adaptability, and can grow normally when cultured at 20℃~45℃, and grows best at 25~35℃.

[0044] Example 3 Determination of potassium-solubilizing ability of strain CBK-5 Potassium-dissolving medium: sucrose 10 g, Na 2 HPO 4 1 g, (NH 4 ) 2 SO 4 0.5 g MgSO 4 7H 2 O 1 g, yeast powder 0.2 g, NaCl 0.1 g, CaCO 3 0.1 g, FeCl 3 0.005 g, potassium feldspar 5 g, agar powder 12 g, water 1000 mL, pH 7.0~7.2.

[0045] The seed solution of strain CBK-5 was prepared and inoculated on potassium-dissolving medium. 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 results showed that in the potassium-solubilizing medium, obvious transparent zones appeared around the colonies of CBK-5, and the D / d value was 1.55, indicating that it had a good potassium-solubilizing ability.

[0048] Example 4 Siderophore and IAA production capacity of strain CBK-5 Prepare the CBK-5 seed solution, inoculate it on the 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 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 d, 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 greater the intensity of secretion. No color change is negative, indicating that the strain cannot secrete IAA.

[0049] 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 has the ability to produce siderophores; the color reaction was positive, indicating that it has the ability to produce IAA, which indirectly indicates that it has the ability to promote plant growth.

[0050] Example 5 Antagonism of strain CBK-5 against plant pathogens The plate confrontation method was used to determine whether strain CBK-5 really had an antagonistic effect on plant pathogens: the pathogen block was inoculated in the center of the PDA plate, and the strain CBK-5 was inoculated at equal distances on both sides of the block, and cultured at 25℃ for 48 hours, repeated 3 times. The fact that the pathogen could not grow normally near strain CBK-5 indicated that there was an antagonistic effect. The results showed (see Table 2) that strain CBK-5 had a significant antagonistic effect on potato scab, potato late blight, cotton wilt, cotton wilt, Fusarium graminearum, corn leaf blight, corn leaf blight, corn rot, tomato gray leaf spot, and tomato gray mold.

[0051] Table 2 Antagonism of strain CBK-5 against some pathogenic fungi .

[0052] The antibacterial activity of CBK-5 against several plant pathogens was determined by the growth rate method. That is, CBK-5 was cultured in PB liquid medium for 24 h, centrifuged, and the supernatant was sterilized by 0.22μm sterile filter membrane. A certain amount of sterile filtrate was added to PDA medium at 40~45℃, mixed and poured into a 9 cm culture dish, and no sterile filtrate was added as a blank control. After the culture medium solidified, plant pathogenic bacteria were inoculated in the center of each plate, repeated 3 times, and cultured at 28℃. The colony diameter was measured by the cross method, and the inhibition rate was calculated. The results showed that the inhibition rate of CBK-5 against the above-mentioned plant pathogens was more than 60%.

[0053] Inhibition rate (%) = (colony diameter of control bacteria - colony diameter of treated bacteria) / (colony diameter of control bacteria - diameter of bacterial block) × 100.

[0054] Example 6 Tolerance of strain CBK-5 to copper, lead, chromium and cadmium 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 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 CBK-5 was inoculated on different culture media, and its tolerance to heavy metal ions was determined based on the growth of the strain.

[0055] 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 strain CBK-5 is used for soil remediation, the adverse effects of these two heavy metal ions can be avoided.

[0056] Table 3 Tolerance of strain CBK-5 to heavy metal ions .

[0057] Example 7 Effect of strain CBK-5 on corn plant growth and soil nutrients The CBK-5 strain was cultured in PB liquid medium until the spore-forming stage, and the concentration of the fermentation broth was adjusted to 1×10 8 CFU / mL, based on 100 mL·kg -1 Mix in the matrix, which is a composite of 50% garden soil, 10% iron tailings sand, 20% coconut bran and 20% vermiculite. For the control, mix the same volume of PB culture medium into the matrix and conduct a corn pot test. At room temperature of 25°C, after the seeds germinate and grow seedlings, keep three best-growing seedlings in each pot, and measure the relevant indicators of soil and plants after 35 days. Three parallel treatments were performed in each group of experiments.

[0058] Table 4 Effects of strain CBK-5 on corn plant growth .

[0059] The growth of corn is shown in Table 4. The strain CBK-5 has a significant promoting effect on corn growth. Compared with the CK treatment group, the plant height, stem diameter, fresh weight and dry weight of corn treated with bacterial solution increased by 20.26%, 29.79%, 28.61% and 36.36% respectively. The chlorophyll content of the treatment group was significantly higher than that of the CK group, increasing by 13.65%.

[0060] Table 5 Effect of strain CBK-5 on soil fertility (unit: mg / kg) .

[0061] As shown in Table 5, the application of CBK-5 bacterial solution in the corn pot experiment can significantly increase the content of nitrate nitrogen, ammonium nitrogen, available silicon, available phosphorus, available potassium and organic matter in the soil, which increased by 12.25%, 28.50%, 10.91%, 13.81%, 16.16% and 8.15% respectively compared with the control. This further shows that the application of CBK-5 bacterial solution can effectively increase the nutrient content of soil and improve soil nutrients.

[0062] 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 Korla Bacillus ( Bacillus korlensis ) CBK-5, characterized in that, The deposit number is CGMCC No.33651.

2. The Korla Bacillus CBK-5 according to claim 1, characterized in that It has the ability to release silicon and potassium.

3. The Korla Bacillus CBK-5 according to claim 1, characterized in that It has the ability to produce siderophores and IAA.

4. The Korla Bacillus CBK-5 according to claim 1, characterized in that It can tolerate 0.5 mM copper ions, 2 mM lead ions, and 2 mM chromium ions.

5. The Korla Bacillus CBK-5 according to claim 1, characterized in that It has antagonistic effects on potato scab, potato late blight, cotton Verticillium wilt, cotton Fusarium graminearum, corn leaf blight, corn leaf blight, corn rot, tomato gray leaf spot, and tomato gray mold.

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

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

8. Use of the Korla Bacillus CBK-5 according to claim 1 in preventing and controlling plant diseases.

9. Use of the Korla Bacillus CBK-5 according to claim 1 in the preparation of biocontrol agents and / or biopesticides.

10. Use of the Korla Bacillus CBK-5 as claimed in claim 1 in soil restoration in tailings areas and heavy metal polluted environments.

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