A Bacillus brevis ZLP-152 and its application in soil remediation

Abstract

The present invention relates to a Brevibacillus ( Brevibacillus brevis ) ZLP‑152, with the deposit number CGMCC No. 32228, can continuously produce alkali throughout the growth cycle and antagonize various pathogens such as cotton red rot. The composite bacterial agent formed by it, phosphate-solubilizing bacteria HM0332 and silicate bacteria HM8841 can improve soil acidification and / or soil microecological environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of soil remediation, and in particular relates to Bacillus brevis ZLP-152 and a composite microbial agent and application thereof. Background Art

[0002] Due to factors such as continuous cultivation, rainwater leaching and excessive application of chemical fertilizers, the pH of the soil in farmland has been declining year by year, leading to soil acidification. Soil acidification not only affects the plant's ability to absorb K, but also affects the plant's ability to absorb K. + , Ca 2+ Mg 2+ It will also damage the physical structure of the soil, reduce the number of microbial populations, cause soil compaction and imbalance of microbial populations, thereby affecting the normal growth and development of crops and having an adverse effect on crop yield and quality.

[0003] Currently, acidic soils are improved through the application of acidic soil conditioners. Traditionally, lime is used, which not only increases soil pH but also increases soil calcium content. However, lime application can easily create dust and pollute the environment, and improper application methods can also exacerbate soil compaction. Soil acidification seriously hinders sustainable agricultural development and food safety, necessitating the development of cost-effective, environmentally friendly, and crop-compatible soil remediation agents. Summary of the Invention

[0004] The present invention aims to provide a Bacillus brevis ZLP-152 and a composite microbial agent thereof and applications thereof in improving the quality and efficiency of acidified soil and in developing a microbial soil remediation agent.

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

[0006] A species of Bacillus brevis ( Brevibacillus brevis ) ZLP-152, with the deposit number CGMCC No. 32228, was deposited on October 16, 2024 at the General Microbiology Center of China Culture Collection Administration, in Beijing, China.

[0007] Furthermore, Bacillus brevis ZLP-152 can continuously produce alkali throughout the entire growth cycle.

[0008] Furthermore, Bacillus brevis ZLP-152 can antagonize cotton red rot, cotton wilt, cotton damping-off, cotton black rot, corn sheath blight, potato dry rot, potato early blight, potato wilt, potato ring rot, pear black spot, tomato gray mold, pepper anthracnose, pepper bacterial wilt, tomato root rot, cucumber wilt, wheat smut, grape white rot, grape gray mold, and tobacco brown spot.

[0009] A composite microbial agent comprises the above-mentioned Bacillus brevis ZLP-152, phosphate-solubilizing bacteria HM0332 and silicate bacteria HM8841.

[0010] Furthermore, the composite microbial agent comprises a fermentation broth of Bacillus brevis ZLP-152, a fermentation broth of phosphate-solubilizing bacteria HM0332, and a fermentation broth of silicate bacteria HM8841 in a volume ratio of 1-7:1-4:0.5-3; the number of viable bacteria in the fermentation broth of Bacillus brevis ZLP-1521 is ≥1.6×10 10 cfu / mL, the number of viable bacteria in the fermentation broth of silicate bacteria HM8841 is ≥1.0×10 9 cfu / mL, the number of viable bacteria in the fermentation liquid of phosphate-solubilizing bacteria HM0332 is ≥1.0×10 8 cfu / mL.

[0011] More preferably, the composite microbial agent comprises fermentation broth of Bacillus brevis ZLP-152, fermentation broth of phosphate-solubilizing bacteria HM0332 and fermentation broth of silicate bacteria HM8841 in a volume ratio of 3.5:2:1.5.

[0012] An application of the above-mentioned composite microbial agent in improving soil acidification and / or improving soil microecological environment.

[0013] A microbial soil remediation agent comprises the composite microbial agent, biochar, fungus chaff and bran in a mass ratio of 1-6.5:3.0-20.0:2.0-14.0:2.0-13.0.

[0014] An application of the above-mentioned microbial soil remediation agent in improving soil acidification and / or improving soil microecological environment.

[0015] Furthermore, the soil microecological environment includes the soil microbial community structure, the diversity and abundance of dominant bacteria, the number and abundance of pathogenic fungi and soil enzyme activity.

[0016] Furthermore, a method for repairing acidified soil using the above-mentioned microbial soil remediation agent comprises the following steps: applying the microbial soil remediation agent to the soil to be modified, plowing and mixing the soil, the plowing depth being 15 to 30 cm, applying topdressing once every 45 days and plowing the soil.

[0017] The beneficial effects of the present invention are that the Brevibacillus ZLP-152 not only has a strong alkali-producing ability but also inhibits multiple pathogens. The combination of the phosphate-solubilizing bacteria HM0332 and the silicate bacteria HM8841 can dissolve calcium, phosphorus, potassium, iron-phosphorus, aluminum-phosphorus, and other compounds in the soil that are difficult for crops to absorb. This significantly improves the utilization rate of organic matter in the soil while increasing the pH of acidified soil. It also increases the diversity and abundance of beneficial bacteria in the soil, reduces the number and abundance of pathogenic fungi, and enhances soil enzyme activity, thereby alleviating soil compaction.

[0018] In addition, the microbial soil remediation agent of the present invention also uses biomass charcoal to improve soil structure, increase soil water retention and fertility, reduce soil erosion, and enhance soil biodiversity. It uses mushroom bran to provide sufficient nutrition and growth space for microorganisms, and can also improve soil structure. It uses bran containing rich nutrients such as protein and vitamins to provide nutrition for bacteria. At the same time, its porous structure is also conducive to the adsorption of bacteria.

[0019] The present invention has good application prospects in improving the quality and efficiency of acidified soil and developing it into a biological soil repair agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The spore morphology of Bacillus brevis ZLP-152 under a microscope.

[0021] Figure 2 This is the distribution map of the cucumber greenhouse experimental group and control group. DETAILED DESCRIPTION

[0022] The following is a detailed description of the technical solution of the present invention in conjunction with preferred embodiments. The following embodiments are only used to illustrate and explain the present invention and do not constitute a limitation to the technical solution of the present invention.

[0023] Example 1 Isolation and screening of strain ZLP-152

[0024] (1) Soil sampling

[0025] Soil samples were collected from Chengde, Hebei Province, with each sample weighing 50-100 g. The samples were placed in sterilized kraft paper bags, which were sealed, and the sampling location, environment, and date were recorded.

[0026] (2) Bacillus isolation and purification

[0027] The soil samples were separated by plate dilution method on PB medium. The specific operation was as follows: 10 g of soil sample was accurately weighed and added to a 90 mL conical flask of 0.9% deionized water containing glass beads. The suspension was shaken at 180 rpm for 30 min to make the soil sample evenly distributed and the concentration was 10 -1 ; Dilute to a concentration of 10-5 , 10 -6 , 10 -7 Incubate each plate in an 80°C water bath for 30 minutes. Spread 100 µL of each dilution onto a PB plate, which was then incubated upside down at 32°C for 48 hours. Single colonies of varying morphology were selected and identified using spore staining. Bacillus spores were purified by streak transfer and transferred to the slant of a PB culture tube. After incubation at 32°C for 48 hours, the plates were stored in a refrigerator at 4°C until use.

[0028] (3) Screening of disease-preventive strains

[0029] Using cucumber gray mold as an indicator, a plate standoff method was used for initial screening. In a sterile operating room, gray mold pathogens grown on PDA medium were punched into 5 mm cakes using a microporous tool. The cakes were then transferred to the center of PDA plates. Sterile filtrates of each antagonistic bacteria obtained in the initial screening were then inoculated 2.5 cm from the pathogens. Three replicates were set up, and a plate containing only the pathogen cake served as a control. The plates were incubated at 26°C. When the CKs filled the medium, colony diameters were measured and the average inhibition rate was calculated. This identified strain ZLP-152, which exhibited strong antagonism against the pathogen.

[0030] Example 2 Identification of strain ZLP-152

[0031] According to the experimental methods in the "Common Bacteria Systematic Identification Manual", the strain ZLP-152 was identified by morphology, physiology and biochemistry. The strain was cultured on PB medium plates at 32±1°C for 14-16 hours. The colonies of strain ZLP-152 were round, white, opaque, with a smooth and moist surface, a raised center, neat edges, and no soluble pigment. Under optical microscopy, the bacteria were bacilli, spore-forming, and oval (e.g. Figure 1 Its physiological and biochemical identification indicators are shown in Table 1.

[0032] Table 1 Physiological and biochemical characteristics of strain ZLP-152

[0033] .

[0034] Through morphological observation and physiological and biochemical experiments, strain ZLP-152 was identified as a Bacillus genus. Molecular biological classification and identification were then conducted using 16S rDNA sequence analysis. Total genomic DNA extracted from the strain was used as a template for amplification of the 16S rDNA sequence. Universal primers were used to amplify the target fragment, and the amplified product was analyzed by 1% agarose gel electrophoresis. Gene sequencing was performed using bidirectional sequencing. Sequencing results were compared using BLAST sequence homology comparisons against the GenBank database, and strain ZLP-152 was identified as Bacillus brevis ( Brevibacillus brevis), named Bacillus brevis ZLP-152.

[0035] Bacillus brevis ZLP-152 was deposited in the General Microbiology Center of China Culture Collection Administration for Microorganisms, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The deposit date is October 16, 2024, and the deposit number is CGMCC No. 32228.

[0036] Example 3 Determination of pH during Brevibacillus fermentation

[0037] The pH changes during the fermentation of Bacillus brevis ZLP-152 were examined. The culture medium was NB liquid medium, initially at pH 7.0, and cultured in a shaking incubator at 32°C and 180 rpm for 40 hours until the fermentation endpoint (spore stage) was reached. The pH changes during the fermentation are shown in Table 2. The results showed that Bacillus brevis ZLP-152 produced alkali continuously throughout the fermentation process.

[0038] Table 2 pH changes during the fermentation process of Bacillus brevis ZLP-152

[0039] .

[0040] Example 4 Determination of the Antibacterial Spectrum of Brevibacillus ZLP-152

[0041] Common pathogens such as cotton red rot, corn sheath blight, tobacco brown spot, wheat smut, grape gray mold, grape white rot, cotton damping-off, cotton wilt, cotton black rot, potato early blight, potato wilt, potato ring rot, potato ring rot, potato dry rot, pear black spot, pepper anthracnose, pepper bacterial wilt, cucumber wilt, tomato gray mold, and tomato root rot were selected. The plate confrontation method was used to determine the antibacterial spectrum of the strain ZLP-152 and to determine the inhibitory ability of the ZLP-152 strain on the growth of pathogens.

[0042] According to the results in Table 3, strain ZLP-152 has a strong antagonistic effect on all 19 plant pathogens tested, among which the strongest inhibitory effect is on cotton wilt pathogen, potato wilt pathogen and cucumber wilt pathogen, with the diameters of the inhibition zones reaching 31.60 mm, 29.88 mm and 28.10 mm, respectively.

[0043] Table 3 Inhibitory effect of Bacillus brevis ZLP-152 on different plant pathogens

[0044] .

[0045] Example 5 Preparation of composite microbial agent

[0046] Bacillus brevis ( Brevibacillus brevis ) ZLP-152, the deposit number is CGMCC No.32228. Phosphate-solubilizing bacteria ( phosphate solubilizing microorganisms )HM0332 and silicate bacteria ( Silicate bacteria ) HM8841 was provided by Hebei Institute of Microbiology Co., Ltd.

[0047] (1) Inoculate Bacillus brevis ZLP-152 into 100 ml of NB liquid culture medium and incubate in a shaking incubator at 32°C at 180 rpm for 12 h. Inoculate phosphate-solubilizing bacteria HM0332 into 100 ml of liquid inorganic phosphate culture medium (sucrose 2 g / L, glucose 2 g / L, NH4Cl 1.5 g / L, KCl 0.3 g / L, MgSO4·7H2O 0.4 g / L, NaCl 0.2 g / L, calcium phosphate 20 g / L, pH 7.0) and incubate in a shaking incubator at 32°C for 12 h. Silicate bacteria HM8841 were inoculated into 100 ml of liquid culture medium (yeast extract 5.0 g / L, calcium carbonate 0.1 g / L, calcium chloride 0.04 g / L, K2HPO4·3H2O 1.67 g / L, KH2PO4 0.87 g / L, FeCl3·6H2O 0.004 g / L, MgSO4·7H2O 0.1 g / L, pH 6.9±0.1) and cultured in a shaking incubator at 25°C for 12 h.

[0048] (2) The culture fluids of the three strains were inoculated into 800 ml of their respective liquid culture media. Brevibacillus ZLP-152 was cultured in a shaking incubator at 32°C at 180 rpm for 24 h. Phosphate-solubilizing bacteria HM0332 was cultured in a shaking incubator at 32°C for 24 h. Silicate bacteria HM8841 was cultured in a shaking incubator at 25°C for 48 h.

[0049] (3) The number of viable bacteria in the fermentation broth of Bacillus brevis ZLP-152 was 1.6×10 10 cfu / mL, the number of viable bacteria in the fermentation broth of silicate bacteria HM8841 was 1.0×10 9 cfu / mL, the number of viable bacteria in the fermentation broth of phosphate-solubilizing bacteria HM0332 was 1.0×10 8 cfu / mL. Prepare a single microbial inoculum.

[0050] (4) The fermentation broth of Bacillus brevis ZLP-152, the fermentation broth of phosphate-solubilizing bacteria HM0332 and the fermentation broth of silicate bacteria HM8841 were mixed in a volume ratio of 1:1:0.5 to prepare a composite microbial agent 1.

[0051] The fermentation broth of Bacillus brevis ZLP-152, the fermentation broth of phosphate-solubilizing bacteria HM0332 and the fermentation broth of silicate bacteria HM8841 were mixed in a volume ratio of 7:4:3 to prepare the composite microbial agent 2.

[0052] The fermentation broth of Bacillus brevis ZLP-152, the fermentation broth of phosphate-solubilizing bacteria HM0332 and the fermentation broth of silicate bacteria HM8841 were mixed in a volume ratio of 3.5:2:1.5 to prepare the composite microbial agent 3.

[0053] Example 6 Effects of Single or Composite Microbial Agents on Soil Acidification and Microecology Improvement (Pot Plant Experiment)

[0054] The test soil was severely acidified cucumber greenhouse soil. The single microbial agent group consisted of 20% vermiculite, 4%-8% single microbial agent, and the remainder of the test soil (by mass fraction). The combined microbial agent group consisted of 20% vermiculite, 20% combined microbial agent, and the remainder of the test soil (by mass fraction). After mixing, equal amounts (4.5 kg) were placed in 20 x 25 cm pots. Cucumbers were sown in pots on May 24th, and on June 12th, cucumbers with consistent growth were transplanted and maintained uniformly. On August 21st, the cucumber rhizosphere soil was sampled and tested for physical and chemical properties, microbial abundance, and enzyme activity. The treatment groups and their improvement results are shown in Tables 4 and 5.

[0055] Table 4 Effect of single microbial agent on soil pH improvement

[0056] .

[0057] Table 5 Effect of compound microbial agents on soil improvement

[0058] .

[0059] Tables 4 and 5 show that the soil pH increased by 0.4–1.10 after application of the composite microbial agent compared to the control, and the soil bulk density decreased. Furthermore, the contents of alkaline-hydrolyzable nitrogen, available phosphorus, and available potassium all increased to varying degrees. Furthermore, the abundance of bacteria and actinomycetes increased, while the abundance of fungi decreased. This suggests that the application of composite microbial agents can improve acidic soils, enhance soil aeration, increase the contents of alkaline-hydrolyzable nitrogen, available phosphorus, and available potassium, enhance soil enzyme activity, increase the abundance of bacteria and actinomycetes, and reduce the abundance of fungi, indirectly improving fertilizer utilization efficiency and having a significant effect on soil improvement.

[0060] Example 7 Preparation of microbial soil remediation agent

[0061] The composite microbial agent 3 obtained in Example 5 was mixed with biochar (Hebei Badu Biotechnology Co., Ltd.), mushroom bran (edible fungus cultivation base in Mengtuo Village, Lingshou County), and bran according to the mass ratio in Table 6 and processed into a powder suitable for agronomic operations, i.e., a microbial soil remediation agent.

[0062] Table 6 Specific ratios of microbial soil remediation agents

[0063] .

[0064] Example 8 Effect of microbial soil remediation agent on improving soil acidification and soil microecological environment

[0065] The experiment was conducted in a cucumber greenhouse with severe soil acidification. Four groups were set up, and the area of each treatment group was 36m 2 . Distribution as Figure 2 Experimental group: 150 kg / mu of the present invention's microbial soil remediation agent (Formulas 1-3) plus 200 kg / mu of organic fertilizer were applied to the planting soil, followed by tillage and mixing at a depth of 15-30 cm. The present invention's microbial soil remediation agent was applied once every 45 days, flushed with water.

[0066] Control group (CK): Apply 200 kg of organic fertilizer per mu before planting and till the land after application.

[0067] Formulations 1–3 and the control group used the same field management and agronomic practices throughout the experiment, except for the microbial soil remediation agent. After 90 days, the cucumber rhizosphere soil was sampled using a five-point sampling method to measure soil physical and chemical properties, soil enzyme activity, and microbial abundance. The results are shown in Tables 7–9.

[0068] Table 7 Effects of microbial soil remediation agents on soil physical and chemical properties

[0069] .

[0070] Table 8 Effects of microbial soil remediation agents on soil enzyme activity

[0071] .

[0072] Table 9 Effects of microbial soil remediation agents on the number of soil microorganisms

[0073] .

[0074] Tables 7-9 show that after application of the microbial soil remediation agents described herein (Formulas 1-3), soil pH in each treatment group increased by 0.5-1.1; soil bulk density increased by 5.00%-11.00% compared to the control group; soil alkaline nitrogen, available phosphorus, and available potassium contents increased by 0.95%-3.80%, 3.87%-8.02%, and 1.90%-4.89% compared to the control group. Urease, catalase, phosphatase, and sucrase contents increased by 9.81%-17.88%, 7.35%-14.20%, 7.96%-14.35%, and 6.83%-11.30%, respectively, compared to the control group. Bacterial and actinomycete counts increased by 10.10%-17.15% and 4.03%-7.00%, respectively, compared to the control group, while fungal counts decreased by 2.81%-4.60%. This demonstrates that the microbial soil remediation agents described herein can significantly improve soil acidification. At the same time, it can improve the fertility, vitality and air permeability of the soil, improve the soil microecological environment, and have a significant effect on improving the quality and efficiency of the soil.

Claims

1. A Brevibacillus brevis ( Brevibacillus brevis ) ZLP-152, characterized in that The deposit number is CGMCC No.32228.

2. The Brevibacillus brevis ZLP-152 according to claim 1, characterized in that It produces alkali continuously throughout its growth cycle.

3. The Brevibacillus brevis ZLP-152 according to claim 1, characterized in that It can antagonize cotton red rot, cotton wilt, cotton damping-off, cotton black rot, corn sheath blight, potato dry rot, potato early blight, potato wilt, potato ring rot, pear black spot, tomato gray mold, pepper anthracnose, pepper bacterial wilt, tomato root rot, cucumber wilt, wheat smut, grape white rot, grape gray mold, and tobacco brown spot.

4. A composite microbial agent, characterized in that: The method comprises the Brevibacillus ZLP-152 as claimed in claim 1, the phosphate-solubilizing bacteria HM0332 and the silicate bacteria HM8841.

5. The composite microbial agent according to claim 4, characterized in that The invention comprises fermentation liquid of Brevibacillus ZLP-152, fermentation liquid of phosphate-solubilizing bacteria HM0332 and fermentation liquid of silicate bacteria HM8841 in a volume ratio of 1-7:1-4:0.5-3; the number of viable bacteria in the fermentation liquid of Brevibacillus ZLP-1521 is ≥1.6×10 10 cfu / mL, the number of viable bacteria in the fermentation broth of silicate bacteria HM8841 is ≥1.0×10 9 cfu / mL, the number of viable bacteria in the fermentation liquid of phosphate-solubilizing bacteria HM0332 is ≥1.0×10 8 cfu / mL.

6. Use of the composite microbial agent according to claim 4 or 5 in improving soil acidification and / or improving soil microecological environment.

7. A microbial soil remediation agent, characterized in that: The invention comprises the composite microbial agent according to claim 4 or 5, biomass charcoal, fungus chaff and bran in a mass ratio of 1-6.5:3.0-20.0:2.0-14.0:2.0-13.

0.

8. Use of the microbial soil remediation agent according to claim 7 in improving soil acidification.

9. Use of the microbial soil remediation agent according to claim 7 in improving soil microecological environment.

10. The use according to claim 9, characterized in that The soil microecological environment includes soil microbial community structure, diversity and abundance of dominant bacteria, number and abundance of pathogenic fungi and soil enzyme activity.

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