Brevibacillus brevis ZLP-152 and application thereof in soil remediation

Abstract

The invention relates to brevibacillus brevis ZLP-152, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.32228, the brevibacillus brevis ZLP-152 can continuously produce alkali in the whole growth cycle and can antagonize various pathogenic bacteria such as cotton red rot pathogen, and a complex microbial inoculant formed by matching the brevibacillus brevis ZLP-152 with phosphate solubilizing bacteria HM0332 and silicate bacteria HM8841 can improve soil acidification and / or improve the soil micro-ecological environment.

Description

Technical Field

[0001] The invention belongs to the technical field of soil remediation, and specifically 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 fertilizer application, the pH of the soil in farmland has been declining year after year, leading to soil acidification. Soil acidification not only 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] At present, there are two methods for improving acidic soil: applying acidic soil conditioners. The traditional method is to use lime, which can not only increase the soil pH but also increase the calcium content in the soil. However, the application of lime is likely to cause dust to fly, pollute the environment, and if the application method is not scientific, it will also aggravate soil compaction. Soil acidification seriously hinders the sustainable development of agriculture and food safety. There is an urgent need to develop economically efficient, environmentally friendly and suitable soil remediation agents for crops. Summary of the invention

[0004] The 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 solution: A brevis bacillus ( Brevibacillus brevis ) ZLP-152, with the deposit number CGMCC No.32228. The strain was deposited on October 16, 2024 at the General Microbiology Center of China Culture Collection Administration, with the address in Beijing, China.

[0006] Furthermore, Bacillus brevis ZLP-152 can continuously produce alkali throughout the entire growth cycle.

[0007] 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.

[0008] A composite microbial agent comprises the above-mentioned Bacillus brevis ZLP-152, phosphate-solubilizing bacteria HM0332 and silicate bacteria HM8841.

[0009] Furthermore, the composite microbial agent comprises a fermentation liquid of Bacillus brevis ZLP-152, a fermentation liquid of phosphate-solubilizing bacteria HM0332, and a 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 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.

[0010] 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.

[0011] An application of the above composite microbial agent in improving soil acidification and / or improving soil microecological environment.

[0012] A microbial soil remediation agent comprises the composite microbial agent, biochar, fungus bran and bran in a mass ratio of 1-6.5:3.0-20.0:2.0-14.0:2.0-13.0.

[0013] An application of the above-mentioned microbial soil remediation agent in improving soil acidification and / or improving soil microecological environment.

[0014] Furthermore, 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.

[0015] 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 a topdressing once every 45 days and plowing the soil.

[0016] The beneficial effects of the present invention are as follows: the brevis bacillus ZLP-152 of the present invention not only has a strong alkali production ability, but also has the characteristics of inhibiting multiple pathogens. The combination of phosphate-solubilizing bacteria HM0332 and silicate bacteria HM8841 can dissolve calcium, phosphorus, potassium, iron phosphorus, aluminum phosphorus and other compounds in the soil that are not easily absorbed by crops, and can significantly improve the utilization rate of organic matter in the soil on the basis of increasing the pH of the acidified soil, increase the diversity and abundance of beneficial bacteria in the soil, reduce the number and abundance of pathogenic fungi, and improve the soil enzyme activity to improve the soil compaction problem.

[0017] In addition, the microbial soil remediation agent of the present invention also utilizes biomass charcoal to improve soil structure, increase soil water retention and fertility, reduce soil erosion, and enhance soil biodiversity. Mushroom bran is used to provide sufficient nutrition and growth space for microorganisms, and can also improve soil structure. Bran contains 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.

[0018] The 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

[0019] Figure 1 This is the spore morphology of Bacillus brevis ZLP-152 under a microscope.

[0020] Figure 2 This is the distribution map of the cucumber greenhouse experimental group and the control group. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is described in detail below in conjunction with the preferred embodiments. The following embodiments are only used to illustrate and explain the present invention, and do not constitute a limitation on the technical solution of the present invention.

[0022] Example 1 Isolation and screening of strain ZLP-152 (1) Soil sampling 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.

[0023] (2) Bacillus isolation and purification The soil samples were separated by plate dilution method on PB medium. The specific operation was as follows: 10 g soil sample was accurately weighed and added to a 90 mL 0.9% deionized water conical bottle containing glass beads. The soil suspension was prepared by shaking at 180 rpm for 30 min to make the soil sample evenly distributed. -1 ; Dilute to a concentration of 10 -5 , 10 -6 , 10-7 . Place in a constant temperature water bath at 80℃ for 30min, take 100µL of the dilution solution and spread on PB plates, and incubate the plates in an incubator at 32℃ for 48h. Pick single colonies of different morphologies and identify them by spore staining. Bacillus is purified by streaking and transferred to the slant of PB medium test tubes. After culturing at 32℃ for 48h, store in a refrigerator at 4℃ for later use.

[0024] (3) Screening of disease-preventive strains Cucumber gray mold was used as the indicator bacteria, and the plate confrontation method was used for the initial screening. In the sterile operation room, the gray mold pathogens grown on the PDA medium were punched into d=5mm cakes, and then the cakes were transferred to the center of the PDA medium plate. Then the sterile filtrates of each antagonistic bacteria obtained in the initial screening were respectively connected to 2.5cm away from the pathogens, with 3 replicates. At the same time, the plate with only the pathogen cake was used as the control, and the plate was cultured at a constant temperature of 26℃. When the CK was cultured to fill the medium, the colony diameter was measured and the average inhibition rate was calculated. The strain ZLP-152 with strong antagonism to pathogens was screened out.

[0025] Example 2 Identification of strain ZLP-152 According to the experimental methods in the Manual of Identification of Common Bacteria Systems, the strain ZLP-152 was identified by morphology and physiological and biochemical methods. The strain was cultured on a PB medium plate at 32±1℃ for 14-16h. The colonies of strain ZLP-152 were observed to be round, white, opaque, smooth and moist on the surface, with a bulge in the middle and neat edges. No soluble pigment was produced. The bacterial morphology was observed under an optical microscope to be bacillus, producing spores, and the spores were oval (such as Figure 1 Its physiological and biochemical identification indicators are shown in Table 1.

[0026] Table 1 Physiological and biochemical characteristics of strain ZLP-152 .

[0027] Through morphological observation and physiological and biochemical experiments, strain ZLP-152 was determined to be Bacillus. Then molecular biological classification identification was carried out, and 16SrDNA sequence analysis was used to extract the total genomic DNA of the strain as a template to amplify its 16SrDNA sequence. Universal primers were used to amplify the target fragment, and the amplified product was detected by 1% agarose gel electrophoresis. The gene sequencing was performed using a bidirectional sequencing method. After the sequencing results were compared with the GenBank database by BLAST sequence homology, strain ZLP-152 was identified as Bacillus brevis ( Brevibacillus brevis ), named Bacillus brevis ZLP-152.

[0028] Bacillus brevis ZLP-152 was sent to the General Microbiology Center of China Microorganism Culture Collection Administration for preservation, the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The preservation date is October 16, 2024, and the preservation number is CGMCC No.32228.

[0029] Example 3 Determination of pH during fermentation of Bacillus brevis The pH changes during the fermentation of Bacillus brevis ZLP-152 were detected. The culture medium was NB liquid culture medium, with an initial pH of 7.0, and the fermentation end point (spore stage) was reached in a 32°C shaking incubator at 180 r / min for 40 hours. The pH changes during the fermentation process are shown in Table 2. The results show that Bacillus brevis ZLP-152 has the characteristic of continuous alkali production during the entire fermentation process.

[0030] Table 2 pH changes during fermentation of Bacillus brevis ZLP-152 .

[0031] Example 4 Determination of the antibacterial spectrum of Bacillus brevis ZLP-152 Common pathogens were selected 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. The plate confrontation method was used to determine the antibacterial spectrum of strain ZLP-152 to determine the inhibitory ability of ZLP-152 on the growth of pathogens.

[0032] According to the results in Table 3, strain ZLP-152 has a strong antagonistic effect on the 19 plant pathogens tested, among which the strongest inhibitory effect is on cotton wilt, potato wilt and cucumber wilt, with the diameters of the inhibition zones reaching 31.60 mm, 29.88 mm and 28.10 mm, respectively.

[0033] Table 3 Inhibitory effect of Bacillus brevis ZLP-152 on different plant pathogens .

[0034] Example 5 Preparation of composite microbial agent Bacterial strain: 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.

[0035] (1) Inoculate Bacillus brevis ZLP-152 into 100 ml of NB liquid culture medium and place it in a shaking incubator at 32°C at 180 r / min for 12 h. Inoculate phosphate-solubilizing bacteria HM0332 into 100 ml of liquid inorganic phosphorus 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 place it in a shaking incubator at 32°C for 12 h. The silicate bacteria HM8841 was inoculated into 100 ml of liquid culture medium (yeast extract powder 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.

[0036] (2) The culture fluids of the above three strains were inoculated into 800 ml of their respective liquid culture media. Among them, Bacillus brevis ZLP-152 was placed in a 32℃ shaking incubator and cultured at 180r / min for 24h. Phosphate solubilizing bacteria HM0332 was placed in a 32℃ shaking incubator and cultured for 24h. Silicate bacteria HM8841 was placed in a 25℃ shaking incubator and cultured for 48h.

[0037] (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 is 1.0×10 9 cfu / mL, the number of viable bacteria in the fermentation liquid of phosphate-solubilizing bacteria HM0332 was 1.0×10 8 cfu / mL. A single microbial inoculant was prepared.

[0038] (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.

[0039] The fermentation liquid of Bacillus brevis ZLP-152, the fermentation liquid of phosphate-solubilizing bacteria HM0332 and the fermentation liquid of silicate bacteria HM8841 were mixed in a volume ratio of 7:4:3 to prepare the composite microbial agent 2.

[0040] The fermentation liquid of Bacillus brevis ZLP-152, the fermentation liquid of phosphate-solubilizing bacteria HM0332 and the fermentation liquid of silicate bacteria HM8841 were mixed in a volume ratio of 3.5:2:1.5 to prepare the composite microbial agent 3.

[0041] Example 6 Effects of single or composite microbial agents on soil acidification and microecology improvement (pot plant experiment) The test soil was a severely acidified cucumber greenhouse soil. Single microbial agent group: 20% vermiculite, 4%~8% single microbial agent and the rest of the test soil (by mass fraction). Composite microbial agent group: 20% vermiculite and 20% composite microbial agent and the rest of the test soil (by mass fraction). After mixing, put the same amount (4.5kg) into a 20×25 cm flower pot. Sowing in the nutrient pot on May 24, and selecting cucumbers with consistent growth on June 12 for transplanting and planting, and unified management. On August 21, the cucumber rhizosphere soil was taken to determine the soil physical and chemical properties, microbial count and soil enzyme activity. The treatment groups and improvement results are shown in Tables 4 and 5.

[0042] Table 4 Effect of single microbial agent on soil pH improvement .

[0043] Table 5 Effect of compound microbial agents on soil improvement .

[0044] According to Tables 4 and 5, the soil pH increased by 0.4-1.10 compared with the control group after the application of the composite microbial agent, and the soil bulk density decreased compared with before. At the same time, the contents of alkaline nitrogen, available phosphorus, and available potassium increased to varying degrees. At the same time, the number of bacteria and actinomycetes increased, and the number of fungi decreased. This shows that the application of composite microbial agents can improve acidic soil, improve soil permeability, increase the content of alkaline nitrogen, available phosphorus, and available potassium in the soil, increase soil enzyme activity, increase the abundance of bacteria and actinomycetes, and reduce the abundance of fungi, which indirectly improves the fertilizer utilization rate of the soil and has a significant effect on soil improvement.

[0045] Example 7 Preparation of microbial soil remediation agent The composite microbial agent 3 obtained in Example 5 was mixed with biochar (Hebei Badu Biotechnology Co., Ltd.), mushroom bran (edible fungus planting 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.

[0046] Table 6 Specific ratio of microbial soil remediation agent .

[0047] Example 8 Effect of microbial soil remediation agent on improving soil acidification and soil microecological environment 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 microbial soil remediation agent (Formula 1 to Formulation 3) of the present invention + 200 kg / mu of organic fertilizer were applied to the planting soil, and the soil was tilled and mixed, with a tillage depth of 15 to 30 cm. The microbial soil remediation agent of the present invention was applied once every 45 days, and applied with water.

[0048] Control group (CK): Apply 200 kg of organic fertilizer per mu before planting and till the land after application.

[0049] In the whole experiment, except for the microbial soil remediation agent, the field management and agronomic measures of Formulation 1 to Formulation 3 and the control group were the same. After 90 days, the five-point sampling method was used to collect cucumber rhizosphere soil to determine soil physical and chemical properties, soil enzyme activity and microbial count. The test results are shown in Tables 7 to 9.

[0050] Table 7 Effects of microbial soil remediation agents on soil physical and chemical properties .

[0051] Table 8 Effects of microbial soil remediation agents on soil enzyme activity .

[0052] Table 9 Effects of microbial soil remediation agents on the number of soil microorganisms .

[0053] According to Tables 7 to 9, after applying the microbial soil remediation agent (Formula 1 to Formulation 3) of the present invention, the soil pH of each treatment group increased by 0.5 to 1.1; the soil bulk density increased by 5.00% to 11.00% compared with the control group; the alkaline nitrogen, available phosphorus and available potassium content in the soil increased by 0.95% to 3.80%, 3.87% to 8.02% and 1.90% to 4.89% compared with the control group. The contents of urease, catalase, phosphatase and sucrase increased by 9.81% to 17.88%, 7.35% to 14.20%, 7.96% to 14.35% and 6.83% to 11.30% compared with the control group; the number of bacteria and actinomycetes increased by 10.10% to 17.15% and 4.03% to 7.00% respectively compared with the control group, and the number of fungi decreased by 2.81% to 4.60% compared with the control group. It shows that the microbial soil remediation agent of the present invention can significantly improve the soil acidification phenomenon. At the same time, it can improve soil fertility, vitality and air permeability, improve the soil microecological environment, and have a significant effect on improving soil quality and efficiency.

Claims

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

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

3. The Bacillus 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 Bacillus brevis ZLP-152, the phosphate-solubilizing bacteria HM0332 and the silicate bacteria HM8841 as claimed in claim 1.

5. The composite microbial agent according to claim 4, characterized in that: The invention comprises a fermentation liquid of Bacillus brevis ZLP-152, a fermentation liquid of phosphate-solubilizing bacteria HM0332 and a fermentation liquid of silicate bacteria HM8841 in a volume ratio of 1-7:1-4:0.5-3; the number of live bacteria in the fermentation liquid 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.

6. Use of the composite microbial agent as claimed in 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 bran 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 as claimed in claim 7 in improving soil acidification and / or improving soil microecological environment.

9. The use according to claim 8, characterized in that: The soil microecological environment includes soil microbial community structure, diversity and abundance of dominant bacteria, quantity and abundance of pathogenic fungi and soil enzyme activity.

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